Endolysins of Bacillus anthracis bacteriophages recognize unique carbohydrate epitopes of vegetative cell wall polysaccharides with high affinity and selectivity

Article abstract of DOI:10.1021/ja3069962

Bacteriophages express endolysins which are the enzymes that hydrolyze peptidoglycan resulting in cell lysis and release of bacteriophages. Endolysins have acquired stringent substrate specificities, which have been attributed to cell wall binding domains (CBD). Although it has been realized that CBDs of bacteriophages that infect Gram-positive bacteria target cell wall carbohydrate structures, molecular mechanisms that confer selectivity are not understood. A range of oligosaccharides, derived from the secondary cell wall polysaccharides of Bacillus anthracis, has been chemically synthesized. The compounds contain an α-d-GlcNAc-(1→4)-β-d-ManNAc-(1→4)-β-d-GlcNAc backbone that is modified by various patterns of α-d-Gal and β-d-Gal branching points. The library of compounds could readily be prepared by employing a core trisaccharide modified by the orthogonal protecting groups N^α-9-fluorenylmethyloxycarbonate (Fmoc), 2-methylnaphthyl ether (Nap), levulinoyl ester (Lev) and dimethylthexylsilyl ether (TDS) at key branching points. Dissociation constants for the binding the cell wall binding domains of the endolysins PlyL and PlyG were determined by surface plasmon resonance (SPR). It was found that the pattern of galactosylation greatly influenced binding affinities, and in particular a compound having a galactosyl moiety at C-4 of the nonreducing GlcNAc moiety bound in the low micromolar range. It is known that secondary cell wall polysaccharides of various bacilli may have both common and variable structural features and in particular differences in the pattern of galactosylation have been noted. Therefore, it is proposed that specificity of endolysins for specific bacilli is achieved by selective binding to a uniquely galactosylated core structure.

Full text of DOI:10.1021/ja3069962

Article
pubs.acs.org/JACS
Endolysins of Bacillus anthracis Bacteriophages Recognize Unique
Carbohydrate Epitopes of Vegetative Cell Wall Polysaccharides with
High Affinity and Selectivity
Kai-For Mo,† Xiuru Li,† Huiqing Li,† Lieh Yoon Low,‡ Conrad P. Quinn,§ and Geert-Jan Boons*,†
†Complex Carbohydrate Research Center, University of Georgia, 315 Riverbend Road, Athens, Georgia 30602, United States
‡Shire Human Genetic Therapies, Inc., 300 Shire Way, Lexington, Massachusetts 02421, United States
§Centers for Disease Control and Prevention, Microbial Pathogenesis and Immune Response Laboratory, Meningitis and Vaccine
Preventable Diseases Branch, NCIRD, DBD, 1600 Clifton Road, MS D-11, Atlanta, Georgia 30333, United States
S
* Supporting Information
ABSTRACT: Bacteriophages express endolysins which are the enzymes
that hydrolyze peptidoglycan resulting in cell lysis and release of
bacteriophages. Endolysins have acquired stringent substrate specificities,
which have been attributed to cell wall binding domains (CBD). Although
it has been realized that CBDs of bacteriophages that infect Gram-positive
bacteria target cell wall carbohydrate structures, molecular mechanisms that
confer selectivity are not understood. A range of oligosaccharides, derived
from the secondary cell wall polysaccharides of Bacillus anthracis, has been
chemically synthesized. The compounds contain an α-D-GlcNAc-(1→4)-β-D-ManNAc-(1→4)-β-D-GlcNAc backbone that is
modified by various patterns of α-D-Gal and β-D-Gal branching points. The library of compounds could readily be prepared by
employing a core trisaccharide modified by the orthogonal protecting groups Nα-9-fluorenylmethyloxycarbonate (Fmoc), 2-
methylnaphthyl ether (Nap), levulinoyl ester (Lev) and dimethylthexylsilyl ether (TDS) at key branching points. Dissociation
constants for the binding the cell wall binding domains of the endolysins PlyL and PlyG were determined by surface plasmon
resonance (SPR). It was found that the pattern of galactosylation greatly influenced binding affinities, and in particular a
compound having a galactosyl moiety at C-4 of the nonreducing GlcNAc moiety bound in the low micromolar range. It is known
that secondary cell wall polysaccharides of various bacilli may have both common and variable structural features and in particular
differences in the pattern of galactosylation have been noted. Therefore, it is proposed that specificity of endolysins for specific
bacilli is achieved by selective binding to a uniquely galactosylated core structure.
release of bacteriophages. Endolysins are usually chimaeric
proteins composed of a well-conserved catalytic domain fused
to a largely divergent cell wall binding domain that directs the
enzyme to a substrate. In many cases, the cell wall binding
domain recognizes polysaccharides that are essential for
viability. Molecular mechanisms that confer high affinity and
selectivity to these carbohydrate-binding domains are not
understood.
B. anthracis lysins such PlyB, PlyG and PlyL can specifically
lyse B. anthracis in vitro and in vivo and have attracted attention
for the development of novel antimicrobial agents and early
disease diagnostics.6 PlyB is bactericidal when applied as a full-
length protein, whereas the catalytic domain alone showed no
detectable lytic activity.7 On the other hand, the recombinant
catalytic domain of PlyL shows lytic activity, however, it
displays a much larger host range compared to the full-length
lysin.8 These and other findings highlight the importance of the
carbohydrate domain for selective targeting of host bacteria.
INTRODUCTION
■
Bacillus anthracis is a Gram-positive, spore-forming bacterium
that causes anthrax in humans and other mammals.1 The
relative ease by which B. anthracis can be weaponized and
difficulties associated with the early recognition of inhalation of
anthrax due to the nonspecific nature of its symptoms were
underscored by the deaths of five people who inhaled spores
from contaminated mail.2 As a result, there is a renewed interest
in anthrax vaccines and early disease diagnostics.3
Immunoassays are attractive for the rapid detection of B.
anthracis spores, vegetative cells and toxins; however, the
challenge of developing such assays is the identification of poly-
or monoclonal antibodies that do not cross react with antigens
of related species.4 It has been recognized that the inherent
binding specificity and lytic action of bacteriophage enzymes
called endolysins offer an attractive alternative to antibodies for
the detection of microbes.5
Bacteriophages, which are viruses that infect bacteria, have
coevolved with their hosts to infect with high efficiency and
specificity. During the final stage of infection, bacteriophages
express enzymes known as endolysins, which cleave glycosidic
or peptide bonds of peptidoglycan resulting in cell lysis and
Received: July 17, 2012
Published: August 30, 2012
© 2012 American Chemical Society
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The carbohydrate binding domain of B. anthracis endolysins
targets secondary cell wall polysaccharides.9 The vegetative cell
wall of bacteria B. anthracis is composed of a peptidoglycan
layer, which underlies an exterior coating of surface-layer (S-
layer) glycoproteins. It also contains a secondary cell wall
polysaccharide that is attached to muramic acid of peptidogly-
can through a phosphodiester linkage and is critically involved
in the organization of the cell wall by anchoring S-layer proteins
to the bacterial cell membrane through noncovalent inter-
actions.10,11 The polysaccharide is composed of a →6)-α-D-
GlcNAc-(1→4)-β-D-ManNAc-(1→4)-β-D-GlcNAc-(1→) back-
bone that is branched at C-3 and C-4 of the α-D-GlcNAc
moiety with α-D-Gal and β-D-Gal, respectively, and at C-3 of
the β-GlcNAc residue with a α-Gal (Figure 1).12,13 These
positions are, however, only partially substituted resulting in
considerable microheterogeneity.
moiety at each of the possible branching positions. Compound
6 is the backbone trisaccharide lacking the terminal N-acetyl-
glucosamine moiety and compound 7 is a derivative of
trisaccharide 2 in which the terminal N-acetyl-glucosamine
moiety is replaced by N-acetyl-galactosamine. This structure
represents the backbone of the secondary cell wall poly-
saccharide of Bacillus cereus, which is closely related to B.
anthracis.14 The compounds could readily be prepared by
employing a core trisaccharide modified by the orthogonal
protecting groups Nα-9-fluorenylmethyloxycarbonate (Fmoc),
2-methylnaphthyl ether (Nap), levulinoyl ester (Lev) and
dimethylthexylsilyl ether (TDS), which in a sequential manner
could be removed to install side chain galactosides or modify
the anomeric center. The binding studies demonstrated that
PlyL and PlyG exhibit similar structure activity relationship for
the synthetic compounds and exceptional high affinity binding
was observed for monogalactosylated tetrasaccharide 4.
Furthermore, the pattern of galactosylation greatly influenced
binding. A β-linked galactosyl moiety to C-4 of the non-
reducing GlcNAc significantly increased affinity whereas an α-
galactoside at C-3 of this residue greatly reduced binding. An α-
galactoside at C-3 of GlcNAc at the reducing end can be
accommodated by the PlyL and PlyG but does not significantly
increase the binding affinity. It is proposed here that the pattern
of galactosylation required for high affinity binding offers
specificity for specific bacilli species.
Figure 1. The structure of the secondary cell wall polysaccharide of B.
anthracis vegetative cells.
RESULTS
■
Chemical Synthesis. It was envisaged that compounds 1−
5 could be prepared from common trisaccharides 8, which at
key positions is modified by the orthogonal protecting groups
Nα-9-fluorenylmethyloxycarbonate (Fmoc),15 2-methylnaphth-
yl ether (Nap),16 levulinoyl ester (Lev),17 and dimethylthex-
ylsilyl ether (TDS) (Figure 3). It was expected that the Fmoc
To uncover molecular mechanisms by which B. anthracis
lysins target secondary cell wall polysaccharides, compounds
1−7 (Figure 2) were chemically synthesized for binding studies
with PlyL and PlyG using surface plasmon resonance (SPR).
Compound 1 is a hexasaccharide composed of the trisaccharide
backbone modified at all relevant positions with a galactosyl
moiety. Compound 2 represents the backbone trisaccharide
and tetrasaccharides 3, 4, and 5 have an additional galactosyl
Figure 3. Building blocks for assembly of oligosaccharides.
carbamate could be selectively cleaved by treatment with a non-
nucleophilic base such as triethylamine, whereas the Lev ester is
susceptible to treatment with hydrazine acetate18 and this
reagent should not affect any of the other protecting groups.
The Nap ether can be removed by oxidation with 2,3-dichloro-
5,6-dicyano-1,4-benzoquinone (DDQ) and these conditions
should be compatible with the other functions.16 The anomeric
center of 8 is protected as a dimethylthexylsilyl (TDS) ether,
which can be selectively unmasked by treatment with hydrogen
fluoride in pyridine. The resulting hemiacetal can then be
converted into the trichloroacetimidate19 for further glyco-
Figure 2. Synthetic targets for binding studies.
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a
Scheme 1
a
Reagents and conditions: (a) NIS, TMSOTf, DCM, −20 °C (86%); (b) Pd(PPh3)4, THF/H2O (91%) (c) Tf2O, DMAP, Py/DCM, 0 °C followed
by NaN3, DMF, 55 °C (86%, 2 steps); (d) Et3SiH, TfOH, DCM, −78 to −35 °C (65%); (e) 11, TMSOTf, Et2O/DCM, −55 to 0 °C (54%, α/β =
8/1).
a
sylation to provide selectively a β-anomer due to neighboring
participating by the 2,2,2-trichloroethyoxycarbamate (Troc)
function.20 In this way, an anomeric N-benzyl-N-benzylox-
ycarbonyl pentanol moiety was introduced, which provides an
opportunity for future conjugations.
Scheme 2
Sequential removal of the protecting groups of 8 and
galactosylation of the resulting acceptors with galactosyl donors
1321 or 1422 should, after global deprotection, provide target
hexasaccharide 1. On the other hand, complete deprotection of
8 should give the backbone trisaccharide 2, whereas selective
removal of one of the orthogonal protection groups followed by
the introduction of a single galactoside and deprotection should
give access to tetrasaccharides 3, 4, and 5.
Trisaccharide 8 was assembled from monosaccharide
building blocks 9, 10, and 11, which were readily be prepared
by standard procedures (Figure 3). Thus, the challenging β-
mannosamine moiety was introduced by a strategy whereby
initially a β-glucoside is installed by coupling acceptor 10 with
glucosyl donor 9 having a participating ester protecting group
at C-2 to control β-anomeric selectivity.23 Next, the C-2 Alloc
group was removed and the resulting hydroxyl converted into a
leaving group, which was then displaced by an azide to give a 2-
azido-β-D-mannoside.
Thus, condensation of thioglucoside 10 with acceptor 9
using NIS and TMSOTf as the promoter24 at −20 °C afforded
disaccharide 15 in a yield of 86% as only the β-anomer
(Scheme 1). The allyloxycarbonate moiety of 15 was removed
by treatment with catalytic amount of Pd(PPh3)4 in a mixture
of THF and water to provide 16 in a high yield. Compound 16
was treated with triflic anhydride (Tf2O) in the presence of
catalytic 4-dimethylaminopyridine (DMAP) in a mixture of
pyridine and dichloromethane and the resulting triflate was
displaced by sodium azide in DMF at 55 °C to afford azido-
mannoside 17 in a yield of 86% over two steps. Selective
reductive ring-opening of the benzylidene acetal of 17 was
achieved using Et3SiH and TfOH25 to provide acceptor 18,
which was coupled with trichloroacetimidate 11 in a mixture of
Et2O/DCM to give trisaccharide 8 in a yield of 54% as mainly
the α-anomer (α/β = 8/1).
Next, the anomeric aminopentyl linker was introduced by a
three-step process to give 21 by selective removal of the
anomeric TDS ether of 8 by treatment with hydrogen fluoride-
pyridine in THF to give hemiacetal 19, which was transformed
into the corresponding trichloroacetimidate 20 by reaction with
trichloroacetonitrile in the presence of K2CO3, and finally, a
TMSOTf-catalyzed glycosylation of 20 with aminopentyl linker
12 (Scheme 2).
a
Reagents and conditions: (a) HFPy, THF (92%); (b) Cl3CCN,
K2CO3, DCM (91%); (c) N-benzyl-N-benzyloxycarbonyl-5-amino-
pentanol (12), TMSOTf, DCM, −30 to −20 °C (87%); (d) Et3N/
DCM (98%); (e) 13, TMSOTf, DCM, −35 to −25 °C (88%); (f)
DDQ, DCM/H2O (78%); (g) 14, TMSOTf, Et2O/DCM, −55 to 0
°C (72%); (h) H2NNH2, HOAc, DCM/MeOH; (93%); (i) 14,
TMSOTf, Et2O/DCM, −55 to 0 °C (65%, α/β > 20/1); (j) Zn,
Ac2O/AcOH/THF, CuSO4(aq) (72%); (k) NaOMe, MeOH/DCM
followed by Pd(OH)2/C, H2, tBuOH/AcOH/H2O (69%, 2 steps).
Removal of the Fmoc carbamate of 21 was accomplished by
reaction with triethylamine in DCM without affecting the other
protecting group to give the corresponding trisaccharide
acceptor 22. A TMSOTf catalyzed glycosylation of trichlor-
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Figure 4. Sensorgram representing the concentration-dependent kinetic analysis of the binding of trisaccharide 2 with immobilized PlyL. It shows
the simultaneous kinetic analysis of 2-fold serial dilutions of 2 at concentrations of 100 to 6.25 μM, fitted with a Langmuir 1:1 binding model (black
lines).
oacetimidate 1319 with 22 afforded tetrasaccharide 23 in high
yield as only the β-anomer. The Nap ether of 23 was removed
oxidatively by reaction with DDQ16 to give the corresponding
tetrasaccharide acceptor 24. As expected, the other two
protecting groups remained intact. Coupling of acceptor 24
with trichloroacetimidate donor 1422 by using catalytic
TMSOTf in a mixture of Et2O/DCM provided pentasaccharide
25 in a yield of 72% as only the α anomer. Next, the Lev ester
of 25 was removed by reaction with hydrazine acetate,17,18 and
the resulting pentasaccharide acceptor 26 coupled with 14 by
employing TMSOTf in a mixture of Et2O/DCM provided the
desired hexasaccharide 27 as mainly the α anomer (65%, α/β >
20/1). Hexasaccharide 27 was treated with Zn in a solution of
AcOH/Ac2O/THF and aqueous CuSO426 to convert the azido
and Troc groups into acetimido moieties. Deacetylation of 28,
followed by hydrogenolysis over Pd(OH)2/C in a mixture of
tBuOH/AcOH/H2O furnished the hexasaccharide 1 in a yield
of 69% in two steps.
The cell wall binding domains of PlyL and PlyG were
immobilized on NHS-activated groups of a CM-5 research
grade sensor chip surface and titration experiments were
performed with the synthetic compounds 1−7. Good agree-
ments between the theoretically predicted and experimentally
observed maximal specific binding (Rmax) parameters indicated
that amine immobilization of the protein yielded fully active
surfaces. The high mass transport coefficients (>1010) obtained
from the kinetic model fit with mass transport limitation
(MTL) experiments using the MTL wizard were indicative of
insignificant mass transport effects and negligible rebinding of
the analyte during the post injection phase (data not shown).
For all SPR experiments, bulk refraction caused by the
difference in the refractive index of the running buffer and
sample injection and nonspecific binding were negated by using
a control cell functionalized by ethanolamine.
A representative sensorgram for the interaction of
trisaccharide 2 with immobilized PlyL is shown in Figure 4.
A 1:1 Langmuir binding model gave a good fit (χ2 = 1.26) and
provided a KD of 13.5 μM (Table 1). Interestingly,
Trisaccharide 2 was prepared by standard global depro-
tection of 21. Tetrasaccharides 3, 4 and 5 were prepared by
selective removal of Fmoc, Nap or Lev of 21 followed by
galactosylation with 13 or 14 and global deprotection (see SI
for details). Compounds 6 and 7 were assembled by a similar
approach as used for 8 (see SI for details).
Table 1. Dissociation Constants for the Binding of PlyL and
PlyG Measured by SPR
analyte
KD (μm) PlyL
KD (μm) PlyG
Binding Studies. SPR is a rapid and sensitive method for
the evaluation of affinities of biomolecular interactions27 that
has as a benefit on relying exclusively on mass changes, thus
allowing the study of interactions in real time without the need
for external labels such as fluorophores, which in some cases
can alter the nature of the interaction. Collecting SPR data for
low molecular weight analytes such as compounds 1−7 is
challenging because the refractive index monitored during a
binding event is relatively small, and thus results in responses
with much lower magnitudes than those observed in typical
protein−protein interactions. In spite of these challenges, the
high sensitivity and reproducibility of modern instruments
combined with proper experimental design permits the direct
monitoring of binding of low molecular weight analytes to
immobilized proteins.28
1
2
3
4
5
6
7
23.0 2.0
13.5 1.5
1.1 0.2
153 10
6.2 2.0
110.0 9.0
43 11
23.3 0.5
8.6 0.4
2.1 1.1
136.0
7
4.1 0.5
49 2.0
19.5 7.5
tetrasaccharide 3, which has an additional β-galactoside at C-
4 of the terminal GlcNAc moiety, exhibited significantly
stronger binding (KD = 1.1 μM) indicating that the β-Gal
moiety substantially contributes to binding. On the other hand,
tetrasaccharide 4 complexed poorly with the protein and this
result suggests that the α-galactoside at C-3 of the terminal
GlcNAc moiety cannot be readily accommodated by PlyL.
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Tetrasaccharide 5 showed a similar dissociation constant as
trisaccharide 2, which suggests that the α-Gal moiety at the
reducing GlcNAc moiety can be accommodated by the protein
but does not contribute to binding. Finally, hexasaccharide 1
was found to bind with a 10-fold reduced affinity compared to
tetrasaccahride 3, which is in agreement with the observation
that the α-Gal moiety at C-3 of the terminal GlcNAc residue of
the core trisaccharide cannot be properly accommodated by the
protein.
PlyG and PlyL exhibit similar affinities for the range of
synthetic compounds (Table 1), and thus, it appears that the
two endolysins have evolved in such a way that they recognize a
saccharide epitope with similar galactosylation pattern. On the
basis of the 68 C-terminal residues that constitute the cell-wall
binding domain, PlyL and PlyG share 64% identity and 78%
homology. The differences are mainly attributed to residues
with substitution of side chains with very similar chemical
properties. Therefore, the folds of these proteins are likely to be
similar, and may provide similar binding properties for cell wall
oligosaccharides. Further structural studies will be required to
establish the molecular mechanisms that confer high affinity
and selectivity for the glycans. Future studies should include
larger synthetic oligosaccharides to further validate that both
endolysins recognize a similar carbohydrate epitope as
oligosaccharide length may modulate binding.
entity simultaneously interact with multiple receptors on a
complementary entity provide high avidity and selectivity.30
Recently, several bacterial lectins have been described that
display micromolar range affinities for their saccharide
ligands.31 In one case, high affinity was achieved by a unique
involvement of two calcium ions in the binding site and
displacement of water molecules complex to the metals by
saccharide ligands resulted in a gain of entropy.32 In other
cases, the rigidity of branched oligosaccharides, such as the
blood group antigens, appear to improve the binding entropy
term.33 Interestingly, PlyL, which does not contain metal ions,
can complex a linear tetrasaccharide (3) with exceptional high
affinity. Structural studies will be required to uncover the
molecular features that account for the high affinity binding.
CONCLUSION
■
The high specificity and activity of endolysins makes them
attractive for in vitro and in vivo applications in food science,
microbial diagnostics, and for the treatment of infectious
diseases.5 They also provide potential tools for molecular
biology, and biotechnology. These promises will only be
realized if ligand requirements of endolysins can accurately be
defined. This paper demonstrates, for the first time, that such
information can be obtained when detailed knowledge is
available of structures of cell wall polysaccharides, which can
guide the chemically synthesis of part structures for binding
studies. A potential stumbling block in such an approach is the
difficulty of preparing libraries of well-defined oligosaccharides
and previous efforts to prepare the secondary cell wall
polysaccharide of B. anthracis had focused on the synthesis of
one compound at the time.34 Here we address this difficulty by
preparing a backbone trisaccharide that at key branching
positions is modified by orthogonal protecting groups.18,35 This
feature made it possible to prepare rapidly a library of
structurally related compounds for structure−activity relation-
ships. Specifically, we demonstrate here that the protecting
groups Nα-9-fluorenylmethyloxycarbonate (Fmoc), 2-methyl-
naphthyl ether (Nap), levulinoyl ester (Lev) and dimethylth-
exylsilyl ether (TDS) are orthogonal and provide chemical
flexibility. This set of protecting group should facilitate the
preparation of many other biologically important oligosacchar-
ides and glycoconjugates.
DISCUSSION
■
Although it is appreciated that cell wall binding domains of
endolysins of bacteriophages confer host selectivity,5 molecular
mechanisms that can account for unique targeting are not
understood. To address this deficiency, we have chemically
synthesized a range of well-defined oligosaccharides derived
from the secondary cell wall polysaccharides of B. anthraces and
employed these compounds to dissect ligand requirements for
the carbohydrate binding domain of the endolysins PlyL and
PlyG using SPR.
Secondary cell wall polysaccharides of B. anthracis have been
implicated as ligands for endolysins,9 however, this poly-
saccharide is structurally heterogeneous and contains structural
elements found in other bacillus species. In this respect, cell
wall polysaccharides from various bacilli have both common
and variable structural features.11,13,29 They contain an
oligosaccharide-repeating unit rich in aminoglycosyl residues
and contain at least a residue having manno- and gluco-
configuration. Furthermore, quantitative glycosyl analysis has
showed that strains belonging to the same lineage differ in
relatively quantities of various monosaccharides, indicating the
presence of strain- specific cell wall carbohydrates. It has also
been observed that B. anthracis strains cell wall glycosyl
compositions differ from one another in a plasmid-dependent
manner and in particular in the absence of the plasmid pXO2
the ManNAc/Gal ratio was significantly decreased.13 The
results reported here indicate that the carbohydrate binding
domain of PlyL and PlyG have evolved in such a way that they
require unique features of the trisaccharide backbone and
galactosyl substitution pattern for high affinity binding and it is
likely that these structural motifs confer selectivity for the host.
Another remarkably observation reported here is that a
simple tetrasaccharide can bind with exceptional high affinity
with the protein target. In this respect, complexation of glycan
bind proteins with monovalent ligands is in general of low
affinity and in the millimolar range. In biological systems,
multivalent binding events in which multiple ligands on one
EXPERIMENTAL SECTION
■
Reagents and General Procedures. Reagents were obtained
from commercial sources and used as purchase. Dichloromethane
(DCM) was freshly distilled using standard procedures. Other organic
solvents were purchased anhydrous and used without further
purification. Unless otherwise noted, all reactions were carried out at
room temperature in oven-dried glassware with magnetic stirring.
Molecular sieves were flame-dried under high vacuum prior to use.
Organic solutions were concentrated under diminished pressure with
bath temperatures <40 °C. Flash column chromatography was carried
out on silica gel G60 (Silicycle, 60−200 μm, 60 Å). Thin-layer
chromatography (TLC) was carried out on Silica gel 60 F254 (EMD
Chemicals, Inc.) with detection by UV absorption (254 nm) were
applicable, and by spraying with 20% sulfuric acid in ethanol followed
by charring at ∼150 °C or by spraying with a solution of
(NH4)6Mo7O24.H2O (25 g/L) in 10% sulfuric acid in ethanol followed
by charring at ∼150 °C. 1H and 13C NMR spectra were recorded on a
Varian Inova-300 (300/75 MHz), a Varian Inova-500 (500/125 MHz)
and a Varian Inova-600 (600/150 MHz) spectrometer equipped with
sun workstations. Multiplicities are quoted as singlet (s), broad singlet
(br s), doublet (d), doublet of doublets (dd), triplet (t), or multiplet
(m). Spectra were assigned using COSY, DEPT and HSQC
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experiments. All chemical shifts are quoted on the δ-scale in parts per
million (ppm). Residual solvent signals were used as an internal
reference. Reverse-Phase HPLC was performed on an Aglient 1200
series system equipped with an autosampler, fraction-collector, UV-
detector and eclipse XDB-C18 column (5 μm, 4.6 × 250 mm) at a
flow rate of 1.5 mL/min. Mass spectra were recorded on an Applied
Biosystems 5800 MALDI-TOF proteomics analyzer. The matrix used
was 2,5-dihydroxybenzoic acid (DHB) and ultamark 1621 as the
internal standard.
min.), the reaction mixture was filtered, and the filtrate was washed
with saturated NaHCO3, and brine. The organic phase was dried
(MgSO4), filtered and the filtrate was concentrated under reduced
pressure. The resulting yellow oil was purified by flash chromatog-
raphy over silica gel (MeOH/DCM) to give pure product.
General Procedure for Ester and Carbamate Deprotection.
To a stirred solution of starting material (2.5 mM solution) in a
mixture of MeOH/DCM (1/2, v/v) was added a methanolic NaOMe
solution (10 equivalents, 1.0 M). The resulting reaction mixture was
stirred for 12 h. after which it was neutralized with 10% AcOH in
MeOH. The resulting solution was diluted with DCM and washed
with saturated NaHCO3 and brine. The organic phase was dried
(MgSO4), filtered and the filtrate was concentrated under reduced
pressure to afford an amorphous white solid. The product used directly
in next step.
General Procedure for Reductive Opening of a 4,6-
Benzylidene. A mixture of the starting material (30 mM solution)
and 4 Å molecular sieves (1.5 times the weight of starting material) in
DCM was stirred under an atmosphere of Ar for 1 h. The reaction was
cooled (−78 °C) and triethylsilane (2.5 equiv) was added followed by
the addition of triflic acid (2.2 equiv). After the reaction was kept at
−78 °C or warmed to −40 °C over a period of time until TLC analysis
(EtOAc/hexanes) indicated disappearance of starting material, it was
quenched by the addition of MeOH. The mixture was filtered, and the
filtrate was washed with saturated NaHCO3, brine. The organic phase
was dried (MgSO4), filtered and the filtrate was concentrated under
reduced pressure. The resulting oil was purified by flash chromatog-
raphy over silica gel (EtOAc/hexanes) to give pure product.
General Procedure for Deprotection of Nap Ethers. To a
stirred solution of starting material (10 mM solution) in a mixture of
DCM/water (10/1, v/v) was added DDQ (3 equiv). The reaction
mixture was vigorously stirred in dark until TLC analysis (EtOAc/
hexanes) indicated disappearance of starting material (∼4 h). Next, the
reaction mixture was diluted with DCM and washed with saturated
NaHCO3 brine. The organic phase was dried (MgSO4), filtered and
the filtrate was concentrated under reduced pressure. The resulting
yellow oil was purified by flash chromatography over silica gel
(EtOAc/hexanes) to give pure product.
General Procedure for Deprotection of Lev Esters. To a
stirred solution of starting material (10 mM solution) in a mixture of
MeOH/DCM (1/8, v/v) was added hydrazine acetate (2 equiv). The
reaction mixture was stirred until TLC analysis (EtOAc/hexanes)
indicated disappearance of starting material (2 h), after which it was
diluted with DCM and washed with saturated NaHCO3, brine. The
organic phase was dried (MgSO4), filtered and the filtrate was
concentrated under reduced pressure. The resulting yellow oil was
purified by flash chromatography over silica gel (EtOAc/hexanes) to
give pure product.
General Procedure of TMSOTf-Mediated Glycosylation for
Synthesis of α-Anomers. A mixture of the acceptor (1 equiv) and
trichloroacetimidate (2 to 3 equiv), 4 Å molecular sieves (1.5 times the
combined weight of donor and acceptor) in a mixture Et2O/DCM (5/
1, v/v, reaction concentration at ∼0.03 to 0.05 M) was stirred under
an atmosphere of Ar for 1 h. The reaction was cooled (−55 °C) and
TMSOTf (0.2 equiv) was added. After the reaction temperature was
slowly increased to 0 °C over a period of 1.5−2 h, it was quenched by
the addition of pyridine (0.2 mL). The mixture was filtered, and the
filtrate was concentrated under reduced pressure. The resulting yellow
oil was purified by flash chromatography over silica gel (EtOAc/
hexanes or acetone/toluene) to give pure product.
General Procedure of TMSOTf-Mediated Glycosylation for
Synthesis of β-Anomers. A mixture of the acceptor (1 equiv) and
trichloroacetimidate (0.33−2.5 equiv), 4 Å molecular sieves (1.5 times
the combined weight of donor and acceptor) in DCM (reaction
concentration at ∼0.06 to 0.08 M) was stirred under an atmosphere of
Ar for 1 h. The reaction was cooled (−30 °C) and TMSOTf (0.2
equiv) was added. After stirring at −25 °C for 1 h, the reaction was
quenched by the addition of pyridine (0.2 mL). The mixture was
filtered, and the filtrate was concentrated under reduced pressure. The
resulting yellow oil was purified by flash chromatography over silica gel
(EtOAc/hexanes) to give pure product.
General Procedure for Hydrogenolysis. To a stirred solution of
t
starting material (∼3 mM solution) in a mixture of BuOH/AcOH/
H2O (40/1/1, v/v/v) and Pd(OH)2/C (2 times the weight of starting
material, 20 wt.%, Degussa type) was added. The resulting mixture was
placed under a hydrogen atmosphere (1 psi). After stirring for 24 h,
the catalyst was filtered off and washed thoroughly with MeOH. The
combined filtrates were concentrated under reduced pressure. The
resulting yellow solid was purified by reversed phase HPLC on an
analytical C-18 column using a gradient of 0 → 100% acetonitrile in
H2O over a 25 min period to give, after lyophilization of the
appropriate fractions, an amorphous white solid.
Cloning, Expression, and Purification of the PlyL and PlyG
Cell Wall Binding Domain. The preparation of plyL CWB was
performed according to a previously reported procedure. The gene of
the PlyG C-terminus was codon-optimized for Escherichia coli and
synthesized from amino acids Lys166 to Lys233 based on NCBI
accession DQ221100 for the PlyG full-length protein. The resulting
construct, with a methionine added in front of Lys166, was inserted
into the vector pET15b (Novagen) between NdeI and BamHI
restriction sites. BL21DE3 (Agilent) was used for expressing
theproteins following standard protocols. The lysis buffer (20 mM
Tris-Cl, 300 mM NaCl, and 0.5% Triton X-100, at pH 7.0) was used
to resuspend the cell pellet. Cells were lysed by French-press with 3
passes at 1000 PSI and clarified by centrifugation at 18 000 rpm
(Sorvall SS-34) for 1 h at 4 °C. The clarified lysate was loaded directly
onto a HITRAP Ni-chelating column (GE Healthcare) and
equilibrated with 50 mL of buffer A (20 mM Tris-Cl and 300 mM
NaCl at pH 7.0). Unbound protein was washed out by buffer A
containing imidazole (30 mM). The His-tagged protein was eluted by
imidazole (0.3 M) in buffer A. Superdex S200 16/60 (GE Healthcare)
gel filtration, equilibrated with PBS, was then used to further purify the
target protein. The molecular weight of the final protein product was
confirmed by MALDI-TOF/TOF.
SPR Measurements. Binding interactions between PlyL and PlyG
and oligosaccahrides 1−7 were examined using a Biacore T100
biosensor system (Biacore Inc., GE Healthcare). The protein was
immobilized by standard amine coupling using an amine coupling kit
(Biacore Inc., GE Healthcare). The surface was activated using freshly
mixed N-hydroxysuccimide (NHS; 100 mM) and 1-(3-dimethylami-
nopropyl)-ethylcarbodiimide (EDC; 391 mM) (1/1, v/v) in water.
Next, PlyL or PlyG (50 μg/mL) in aqueous NaOAc (10 mM, pH 5.5)
was passed over the chip surface until a ligand density of approximately
2500 RU was achieved. The remaining active esters were quenched by
aqueous ethanolamine (1.0 M; pH 8.5). The control flow cell was
activated with NHS and EDC followed by immediate quenching with
ethanolamine. HBS-EP (0.01 M HEPES, 150 mM NaCl, 3 mM
EDTA, 0.005% polysorbate 20; pH 7.4) and HBS-N (0.01 M HEPES,
150 mM NaCl; pH 7.4) were used as the running buffer for the
immobilization and kinetic studies, respectively. Analytes were
dissolved in running buffer and a flow rate of 30 μL/min was
employed for association and dissociation at a constant temperature of
25 °C. A sequential 60 s injection of aqueous glycine-HCl (10 mM;
pH 2.0) and 30 s injection of NaCl (1.5 M) at a flow rate of 50 μL/
min were used for regeneration and achieved prior baseline status.
With the use of the Biacore T100 evaluation software, the response
General Procedure for Reduction of Azido and Troc Groups.
To a stirred solution of starting material (3 mM solution) in AcOH/
Ac2O/THF (1/2/3, v/v/v) was added metallic zinc power (50 equiv)
followed by saturated CuSO4(aq) (0.2 mL). After stirring until HR
MALDI-TOF MS indicated disappearance of starting material (45
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dx.doi.org/10.1021/ja3069962 | J. Am. Chem. Soc. 2012, 134, 15556−15562

Journal of the American Chemical Society
Article
curves of various analyte concentrations were globally fitted to a one to
one or the two-state binding model.
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ASSOCIATED CONTENT
* Supporting Information
The preparation of the starting materials, assembly of the
oligosaccharides and copies of NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
■
S
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AUTHOR INFORMATION
Corresponding Author
gjboons@ccrc.uga.edu
■
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Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS
■
Research reported in this publication was supported by the
National Institute of General Medical Sciences of the National
Institutes of Health (R01GM065248, G.-J.B.).
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