Water-soluble substrates of the peptidoglycan-modifying enzyme O-acetylpeptidoglycan esterase (Ape1) from Neisseria gonorrheae

Article abstract of DOI:10.1021/jo102329c

Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-acetylglucosamine (GlcNAc) and N-acetylmuramic acid (Mur

Full text of DOI:10.1021/jo102329c

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Water-Soluble Substrates of the Peptidoglycan-Modifying Enzyme
O-Acetylpeptidoglycan Esterase (Ape1) from Neisseria gonorrheae
Timin Hadi,† John M. Pfeffer,‡ Anthony J. Clarke,‡ and Martin E. Tanner*,†
†Department of Chemistry, University of British Columbia, Vancouver, British Columbia,
Canada V6T 1Z1, and Department of Molecular and Cellular Biology, University of Guelph, Guelph,
Ontario, Canada N1G 2W1
‡
mtanner@chem.ubc.ca
Received November 23, 2010
Peptidoglycan is the component of the bacterial cell wall that is essential for maintaining the shape
and rigidity of the cell. As such, its polymeric structure, consisting of alternating units of N-
acetylglucosamine (GlcNAc) and N-acetylmuramic acid (MurNAc), is also a target for the action of
host defense enzymes, such as lysozymes. Many bacteria have developed methods of masking their
cell wall from these environmental dangers through the addition of aglycon moieties that prevent
recognition or sterically hinder the degradative action of exogenous enzymes that would otherwise
prove detrimental to the cell. Peptidoglycan acetyl-transferases (Pat’s) and O-acetylpeptidoglycan
esterases (Ape’s) are the enzymes responsible for the controlled addition and removal of acetate onto
the C-6 hydroxyl group of MurNAc residues in peptidoglycan. Studies on Ape1, an O-acetylpepti-
doglycan esterase found in Neisseria gonorrheae, have suggested that this enzyme is essential for
bacterial viability and thus presents an attractive target for antibacterial design. Previous studies on
Ape1 have been hindered by the fact that Ape1’s natural substrate is an insoluble polymer. In this
paper we outline the design, synthesis, and testing of the water-soluble di- and monosaccharide
substrate analogues 1 and 2. Both 1 and 2 serve as substrates of Ape1 with kcat/KM values of (5.1 (
1.7) ꢀ 103 M-1 s-1 and (3.1 ( 0.8) ꢀ 103 M-1 s-1, respectively. It was determined that the substitution
of the GlcNAc residue in compound 1 with an O-benzyl group in compound 2 did not significantly
decrease the enzyme’s affinity for the monosaccharide. These findings are important as they
demonstrate that the catalytic prowess of Ape1 is not dependent on its binding to a polymeric
substrate. This ensures that small molecule transition state/intermediate analogues can also capture
the transition state binding energy of Ape1 and potentially serve as potent inhibitors. The synthetic
route to compounds 1 and 2 could readily be modified to allow for the installation of a wide variety of
functional groups at the MurNAc C-6 position in both the mono- and disaccharide scaffolds. This
will serve as a general method for the construction of Ape1 substrates and inhibitors.
Introduction
their shape, and its rigid, polymeric structure protects the
cell from lysis due to high osmotic pressure. Its structure is
comprised of alternating N-acetylglucosamine (GlcNAc)
and N-acetylmuramic acid (MurNAc) residues connected
via β-(1f4)-glycosidic linkages (Figure 1). Each MurNAc
residue possesses a D-lactate group at the C-3 position that
Peptidoglycan is an essential component of the bacterial
cell wall found in both Gram-positive and Gram-negative
bacteria.1,2 Peptidoglycan allows eubacteria to maintain
(1) Park, J. T. The Murein Sacculus. In Escherichia coli and Salmonella:
Cellular and Molecular Biology; Neidhardt, F. C., Curtis, R., III, Ingraham,
J. L., Lin, E. C. C., Low, K. B., Magasanik, B., Resnikoff, W. S., Riley, M.,
Schaechter, M., Umbarger, H. E., Eds.; American Society for Microbiology:
Washington, DC, 1996; pp 48-57.
(2) Vollmer, W.; Blanot, D.; De Pedro, M. A. FEMS Microbiol. Rev.
2008, 32, 149–167.
1118 J. Org. Chem. 2011, 76, 1118–1125
Published on Web 01/18/2011
DOI: 10.1021/jo102329c
r
2011 American Chemical Society

Hadi et al.
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FIGURE 1. Peptidoglycan O-acetylation/deacetylation and the action of lytic transglycosylase.
bears a peptide side chain. Cross-linking of neighboring
strands through their respective stem peptides then gives rise
to the three-dimensional structure of peptidoglycan.3
groups on peptidoglycan must exist so that these activities
may continue.3,6
In Neisseria gonorrheae, the O-acetylation and deacetyla-
tion of MurNAc residues in peptidoglycan are governed by a
set of enzymes encoded in the poa (peptidoglycan O-ace-
tylation) gene cluster.7 Homologous poa gene clusters have
also been identified in other pathogenic bacteria includ-
ing Neisseria menningitidis, Proteus mirabilis, and species of
Helicobacter, Campylobacter, Bacillus, andTreponema.7 Twopep-
tidoglycan O-acetyltransferases (PatA and PatB) have been
proposed to translocate acetate from the cytoplasm to the
periplasm and finally onto peptidoglycan.8 In an opposing
fashion, the O-acetylpeptidoglycan esterase (Ape1) has been
shown to remove acetate from O-acetylated peptidoglycan
to allow for the action of LTs and other autolysins involved
in peptidoglycan metabolism (Figure 1).9,10 Previous studies
have shown that both of these enzymes play an important
and perhaps essential role in the metabolism of peptidogly-
can.9 Further characterization of the Pat’s and Ape’s found
in pathogenic bacteria will help to determine their viability as
antibacterial targets.
Ape1 belongs to the CE-3 family of carbohydrate esterases
and possesses a catalytic triad of Asp-His-Ser that is con-
served in many serine proteases/esterases.10 Ape1 from
N. gonorrheae was previously expressed in and purified from
Escherichia coli. Two N-terminally processed forms of the
wild-type protein were generated and were found to be
localized predominately to the perisplasm and the outer
membrane, areas where the maintenance of peptidoglycan
would be required.9 The cellular localization of Ape1 makes
it an attractive antibacterial target, as inhibitors would not
have to pass through the cytoplasmic membrane to reach
their target. Perhaps more importantly, Ape1 deletion strains
lacked viability, suggesting that Ape1 is an essential enzyme in
many pathogenic bacteria.9
Peptidoglycan is known to be an important factor govern-
ing virulence and toxicity by aiding in many pathogenic
bacteria’s ability to persist within the host. Because of its
essential nature, significant research effort has focused on
the design and discovery of molecules that inhibit its bio-
synthesis, as evidenced by the vast number of antibacterial
compounds that target the cell wall. For example, the β-
lactam penicillin binds to and irreversibly inhibits the peni-
cillin-binding proteins (PBPs) of target cells, the biosynthetic
enzymes that are responsible for the insertion and cross-
linking of new peptidoglycan strands in the cell wall.3
Bacteria, however, have rapidly developed resistance to both
the natural host mechanisms of defense and virtually all
known antibiotics, thus necessitating the discovery of new
methods for combating infection and virulence.4 As a re-
sponse to invading pathogenic bacteria, hosts will release
lysozymes as a first line of defense; lysozyme’s mode of action
involves hydrolysis of the glycosidic linkage between the
GlcNAc and MurNAc residues in bacterial peptidoglycan.
As a way to combat these host defenses, bacteria have
evolved methods of masking their peptidoglycan through
various covalent modifications. One such modification is the
O-acetylation of the C-6 hydroxyl group on the MurNAc
residues of peptidoglycan (Figure 1).5 This modification has
been shown to interfere with the action of lysozyme on
peptidoglycan substrates by preventing binding. While this
modification effectively blocks lysozymal degradation, it
also interferes with the action of bacterial autolysins, most
notably the lytic transglycosylases (LTs). Unlike lysozyme,
these LTs cleave the β-(1f4) linkages nonhydrolytically to
form 1,6-anhydromuramoyl residues and have a strict re-
quirement for a free C-6 hydroxyl group on the MurNAc
residues for activity (Figure 1).3,6 Because the LTs play an
important role in the metabolism of peptidoglycan during
cell growth and division, the function of bacterial secretion
systems, and the insertion of bacterial appendages, a mech-
anism for the controlled addition and removal of O-acetyl
Ape1 activity was demonstrated using O-acetylated pepti-
doglycan samples isolated from bacteria, but these experi-
ments were complicated by the fact that the substrate is an
insoluble polymer and required sonication for even dispersal.9
In order to obtain the kinetic parameters kcat and KM of Ape1,
(3) Holtje, J. V. Microbiol. Mol. Biol. Rev. 1998, 62, 181–203.
(4) Davies, J.; Davies, D. Microbiol. Mol. Biol. Rev. 2010, 74, 417–433.
(5) Clarke, A. J.; Dupont, C. Can. J. Microbiol. 1992, 38, 85–91.
(6) Koraimann, G. Cell. Mol. Life Sci. 2003, 60, 2371–2388.
(7) Weadge, J. T.; Pfeffer, J. M.; Clarke, A. J. BMC Microbiol. 2005, 5, 49.
(8) Moynihan, P. J.; Clarke, A. J. J. Biol. Chem. 2010, 285, 13264–13273.
(9) Weadge, J. T.; Clarke, A. J. Biochemistry 2006, 45, 839–851.
(10) Weadge, J. T.; Clarke, A. J. Biochemistry 2007, 46, 4932–4941.
J. Org. Chem. Vol. 76, No. 4, 2011 1119

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it was necessary to use either of the highly activated esters
p-nitrophenyl acetate or R-naphthyl acetate as model substr-
ates. Although these data provide some sense of Ape1’s cata-
lytic efficiency, many esterases would be expected to non-
specifically hydrolyze these highly labile acetate esters. In
order to obtain more meaningful kinetic data, analysis of
enzyme activity with a soluble, unactivated substrate that
more closely resembles peptidoglycan is required.
elements toward binding. The GlcNAc moiety at O-4 has
been replaced by a hydrophobic O-benzyl ether that is des-
igned to occupy the GlcNAc binding pocket in the enzyme
active site and also serves to prevent migration of the 6-O-acetyl
ester. In addition, the L-alanine moiety has been deleted from
the lactyl side chain to test whether it is a critical recognition
element for the enzyme. Because compound 2 lacks the free
hydroxyl groups contained in the GlcNAc residue of disac-
charide 1, the lactate was left as a free carboxylate to confer
greater water solubility. The testing of compounds 1 and 2,
both bearing an unactivated acetate ester at the C-6 position
of the MurNAc residue, will provide valuable information
with regard to the structural elements required for binding.
The results of these tests will also shape future strategies in
the design and synthesis of Ape1 substrates and inhibitors.
Synthesis of Disaccharide 1. The synthesis of oligosacchar-
ides containing D-glucosamine and, more commonly, its N-
acetyl derivative GlcNAc has been the subject of significant
research effort as a result of their prevalence in glycoconjuga-
tes.13 Glycoside bond formation using GlcNAc donors is
complicated, however, by the formation of an oxazolinium
intermediate via neighboring-group participation of the
2-acetamido group. This participation ensures the formation
of the β-anomeric linkage in coupling reactions. However,
the oxazolinium system is also relatively stable, resulting in
weak glycosyl donor properties.13,14 Several methods have
been developed to increase the electrophilicity of GlcNAc
donors, while still preferentially forming the β-linkage in the
coupled products.15 These methods rely primarily on in-
creasing the electron-withdrawing character of the N-acyl
substituents. This serves to decrease the stability of the
oxazolinium system and increases the rate of nucleophilic
attack by a glycosyl acceptor. For the synthesis of disaccha-
ride 1, we chose to mask the 2-acetamido group as a 2-tri-
chloroacetamido group in our GlcNAc donor system (comp-
ound 6, Scheme 1). This trichloroacetamido group can be
reduced back to the corresponding acetamido group using
tributyltin hydride and AIBN after the coupling has been
completed.14 Although the synthesis of related peptidogly-
can fragments has been previously reported,16 the require-
ment of the 6-O-acetate ester on the MurNAc moiety of
disaccharide 1 necessitates a number of changes to our syn-
thetic strategy, as O-acetate esters are traditionally employed
as protecting groups in oligosaccharide synthesis. In order to
selectively O-acetylate at the C-6 position on the MurNAc
residue, the hydroxyl groups at C-3, C-4, and C-6 on the
GlcNAc moiety must be masked until the O-acetate ester is
installed. We chose to protect the three hydroxyl groups on
the GlcNAc moiety as O-levulinate esters after coupling
because they can be selectively removed in the presence of
an O-acetate ester. The orthogonal protecting group strategy
detailed within also allows for the installation of different
functional groups at the C-6 position of MurNAc, a feature
that is very attractive for the future development of small
molecule inhibitors of Ape1.
To this end we designed the 6-O-acetylated disaccharide
and monosaccharide peptidoglycan analogues 1 and 2 that we
predicted would serve as water-soluble substrates of Ape1.
Since Ape1 acts on a polymeric substrate, it is likely that
both binding and catalysis is influenced by the number of
sugar residues present in the substrate. Consequently, Ape1’s
ability to catalyze the deacetylation of truncated fragments
of O-acetylated peptidoglycan could be significantly im-
paired. A marked dependency on the rate of reaction as a
function of the number of monomer units in the substrate has
been reported for other deacetylases that operate on
polysaccharides.11 The synthesis and testing of both the
mono- and disaccharide substrate analogues will serve to
identify whether Ape1 is able to accept truncated substrates
and, if so, examine whether there is a strict requirement for
the presence of the GlcNAc moiety. These substrates will
also serve to validate the use of mono- and disaccharide
fragments as the basic scaffold for the design of small-
molecule transition state/intermediate analogues of Ape1
and act as benchmarks for future experiments involving
the testing of these compounds as potential antibacterials.
Results and Discussion
We chose to start with the relatively complex disaccharide
substrate 1 to maximize our chances of observing a high level
of activity with Ape1. This substrate was designed to serve as
a benchmark to which other monosaccharide substrates
could be compared. The core structure of 1 is a β-(1f4)-
linked GlcNAc-MurNAc disaccharide that is elaborated
with an O-acetyl ester at the C-6 position of the MurNAc
moiety and an L-alanine methyl ester appended to the lactyl
side chain. The β-methoxy group at the anomeric carbon of
MurNAc was chosen to mimic the β-linkages found in
peptidoglycan, and it was anticipated that the three free
hydroxyl groups on the GlcNAc residue would increase the
water solubility of the final compound. It is important to
note that the presence of the β-linked GlcNAc at the C-4
position of MurNAc also serves to prevent any potential
migration of the 6-O-acetyl ester.12
The truncated monosaccharide substrate 2 was designed
to further examine the contributions of different structural
(13) Schmidt, R. R. Angew. Chem., Int. Ed. 1986, 25, 212–235.
(14) Blatter, G.; Beau, J. M.; Jacquinet, J. C. Carbohydr. Res. 1994, 260,
189–202.
(11) Hekmat, O.; Tokuyasu, K.; Withers, S. G. Biochem. J. 2003, 374,
369–380.
(15) Dullenkopf, W.; Castro-Palomino, J. C.; Manzoni, L.; Schmidt,
R. R. Carbohydr. Res. 1996, 296, 135–147.
(12) Chang, K. Y.; Wu, S. H.; Wang, K. T. Carbohydr. Res. 1991, 222,
121–129.
(16) Hesek, D.; Lee, M.; Morio, K.; Mobashery, S. J. Org. Chem. 2004,
69, 2137–2146.
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SCHEME 1. Synthesis of Disaccharide Substrate 1
SCHEME 2. Synthesis of Monosaccharide Substrate 2
The known 4,6-O-benzylidene-protected MurNAc deri-
vative 3 was prepared from N-acetylglucosamine using a
combination of literature methods (Scheme 1).17,18 Since
peptidoglycan monomers are linked in a β-(1f4) manner,
the methyl glycoside at C-1 was installed in the β-configuration.
1-Ethyl-3-(3-dimethylaminopropyl) carbodiimide (EDC) and
1-hydroxybenzotriazole (HOBt)-mediated coupling of the
methyl ester of L-alanine to 3 gave compound 4 in good yield.
A regioselective reductive ring opening of the benzylidene
acetal with Et3SiH and TfOH was then used to gen-
erate the 6-O-benzyl ether 5.19 The yield of this selective
reduction was lower than expected, most likely due to the
presence of the L-alanine attached to the C-3 lactate group
and its interaction with TfOH. Reactions involving the selec-
tive reduction of the MurNAc derivatives lacking the L-alanine
side chain gave significantly higher yields (data not shown).
The donor, 6, was synthesized using literature methodology
starting from D-glucosamine.14 As discussed previously, the
2-trichloroacetamido group was incorporated to mask the
2-acetamido moiety and to ensure successful coupling. The
MurNAc acceptor 5 was coupled with donor 6 using TMS-
OTf as a promoter. The modest yield of disaccharide 7 can be
partially accounted for by the lack of nucleophilicity of the
C-4 hydroxyl group in acceptors possessing an unmasked
2-acetamido group.20 Although previous strategies involving
masking of the 2-acetamido group in the acceptor have been
reported,16 we chose to leave the group intact to minimize the
number of manipulations required after the disaccharide
coupling step. The trichloroacetamido functionality of dis-
accharide 7 was then reduced with tributyltin hydride/AIBN
to unmask the 2-acetamido group on the “GlcNAc” portion
of disaccharide 8.14 To generate the final product 1, a prot-
ecting group for the hydroxyl groups at C-3, C-4, and C-6 of
the GlcNAc moiety that could be selectively cleaved in the
presence of an O-acetate ester was required. Accordingly, the
O-acetate groups on compound 8 were replaced with O-
levulinate esters through a two-step procedure involving
deprotection with NaOMe, and a subsequent N,N0-diisopro-
pylcarbodiimide (DIPC)-mediated coupling of the free hy-
droxyls with levulinic acid to give disaccharide 9.
(17) Hesek, D.; Suvorov, M.; Morio, K.; Lee, M.; Brown, S.; Vakulenko,
S. B.; Mobashery, S. J. Org. Chem. 2004, 69, 778–784.
(18) Cai, Y.; Ling, C. C.; Bundle, D. R. Org. Lett. 2005, 7, 4021–4024.
(19) Sakagami, M.; Hamana, H. Tetrahedron Lett. 2000, 41, 5547–5551.
(20) Crich, D.; Dudkin, V. J. Am. Chem. Soc. 2001, 123, 6819–6825.
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TABLE 1. Kinetic Parameters for the Ape1-Catalyzed Hydrolysis of
Synthetic Substrates 1 and 2
enzymatic activity. The disaccharide, 1, possesses a β-(1f4)-
linked GlcNAc moiety, as well as a methyl ester-protected L-
alanine appended to its lactyl side chain. Its more complex
design, with the inclusion of these recognition elements, was
pursued with the goal of obtaining a substrate with a high
level of activity. Indeed, compound 1 served as a slightly
better substrate than 2, but the small magnitude of this
difference was perhaps the more interesting finding. Because
Ape1 acts on polymeric O-acetylated peptidoglycan, it is not
unreasonable to assume that disaccharide substrates would
bind more tightly in the Ape1 active site than the correspond-
ing monosaccharide units. Replacing the GlcNAc moiety
with an O-benzyl ether at the C-4 position of MurNAc,
however, results in only a minimal decrease in the rate of
Ape1 hydrolysis of the acetate ester. It is likely that the
movement of the benzyl group from bulk water into the
relatively hydrophobic pocket of the GlcNAc binding site is
favored by the hydrophobic effect. This entropic driving
force could compensate for any loss of enthalpic interactions
due to the lack of the GlcNAc moiety. Compound 2 also
lacks the L-alanine that was appended to the lactyl side chain
in compound 1. The small decrease in activity with this
substrate also suggests that the peptides normally attached
to the lactyl moiety are not required for binding in the Ape1
active site. These findings provide important information
regarding the recognition elements required for productive
binding and bode well for the future development of sub-
strates and inhibitors of Ape1. The synthesis of monosac-
charide 2 is more efficient and requires fewer synthetic
transformations than that of disaccharide 1, so the future
development of Ape1 inhibitors will focus on monosaccha-
ride scaffolds.
substrate
kcat (s-1
)
KM (mM)
kcat/KM (M-1 s-1
)
1
2
2.36 ( 0.18
1.24 ( 0.07
0.46 ( 0.10
0.39 ( 0.08
(5.1 ( 1.7) ꢀ 103
(3.1 ( 0.8) ꢀ 103
With the proper protecting groups now in place, the
acetate ester was introduced selectively at the C-6 position
of the MurNAc residue through the hydrogenolysis of the
benzyl ether and acetylation of the newly formed primary
hydroxyl group. Deprotection of the O-levulinate esters was
accomplished selectively in the presence of the O-acetate
ester with the use of hydrazine acetate, generating disaccha-
ride substrate 1. The removal of the O-levulinate esters was
complete almost immediately after addition of the hydrazine
acetate, and no corresponding product lacking the C-6
O-acetyl ester was detected.
Synthesis of Monosaccharide 2. The synthesis of mono-
saccharide substrate 2 was accomplished in three steps from
the protected MurNAc derivative 10 (Scheme 2).21 The known
compound 10 was prepared from compound 3 using TMS-
diazomethane to protect the carboxylic acid as a methyl ester.
The protection of the free carboxylate as a methyl ester was
required to increase solubility in methylene chloride dur-
ing the subsequent regioselective reductive ring opening of
the benzylidene acetal with Et3SiH and PhBCl2. 4-O-Benzyl
ether 11 was generated in good yield (89%), leaving a
primary hydroxyl at C-6 for further functionalization. The
methyl ester was hydrolyzed with LiOH in a mixture of
MeOH and dioxane to give the free carboxylic acid, and the
crude compound was O-acetylated at the C-6 position. A
subsequent exchange from the triethylamine salt to the
sodium salt gave monosaccharide substrate 2 in a 63% yield.
Testing of Compounds 1 and 2 with Ape1. The esterase
activity with the alternate substrates 1 and 2 was determined
by incubating varying concentrations of the compounds with
a truncated form of the Ape1 enzyme lacking the N-terminal
signal peptide.10 The enzymatic reactions were terminated
with the addition of H2SO4, and the amount of acetate rele-
ased was quantified using a coupled enzymatic assay. It was
determined that Ape1 follows Michaelis-Menten kinetics
when acting on both the mono- and disaccharide substrates.
The parameters kcat, KM, and kcat/KM were determined for
both substrates, and the results are outlined in Table 1.
Conclusion
We have successfully synthesized and tested water-soluble
mono- and disaccharide substrate analogues of the O-acet-
ylpeptidoglycan esterase Ape1. Both 1 and 2 served as
substrates of Ape1, and interestingly, the substitution of
the GlcNAc residue for an O-benzyl ether at the C-4 position
of MurNAc did not greatly affect the activity on substrate 2.
These results validate the use of both MurNAc monosac-
charides and MurNAc-GlcNAc disaccharides as templates
for Ape1 substrate and inhibitor design. This is an important
finding as it demonstrates that the catalytic prowess of Ape1
is not dependent on its binding to a polymeric substrate. This
ensures that small molecule transition state/intermediate
analogues can also capture the transition state binding
energy of Ape1 and potentially serve as potent inhibitors.
Future efforts toward the design and synthesis of potential
inhibitors of Ape1 are aided by the abundance of literature
available outlining mechanism-based strategies for the in-
hibition of serine proteases and esterases.22 The conserved
catalytic triad of Asp-His-Ser found in this class of enzymes
provides a target that has a very well-characterized mechan-
ism of action. Our synthetic route to monosaccharide 2
introduces the O-acetyl group in the late stages of the synthesis
and allows for the installation of different functional groups at
the C-6 position. Therefore, the synthesis of a wide variety of
potential inhibitors of Ape1 could be easily accomplished
Both 1 and 2 served as substrates of Ape1, and their kcat
/
KM values were on the same order of magnitude as those
previously reported for the highly activated p-nitrophenyl
acetate (KM = 0.51 ( 0.21 mM, kcat/KM = 2.0 ꢀ 104 M-1
s-1).10 Each substrate analogue bears an unactivated acetate
ester at the C-6 position of the MurNAc residue, and their
acceptance as substrates of Ape1 demonstrates that binding
to a polymeric substrate is not a strict requirement for
€
(22) Powers, J. C.; Asgian, J. L.; Ekici, O. D.; James, K. E. Chem. Rev.
2002, 102, 4639–4750.
(21) Jeanloz, R. W.; Walker, E.; Sinay, P. Carbohydr. Res. 1968, 6,
184–196.
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using this synthetic scheme. Testing of these compounds for
bacteriocidal activity will eventually serve to determine
Ape1’s viability as an antibacterial target.
mmol) was added. After stirring for 1 h at 0 οC, the ice bath was
removed, and the mixture was warmed to rt. The reaction was
judged complete by monitoring the disappearance of acceptor 5
by TLC analysis (EtOAc) and was subsequently quenched with
0.4 mL of NEt3. The mixture was diluted with CH2Cl2 and
filtered through a pad of Celite, and the solvents removed in
vacuo, leaving a dark brown foam. Silica gel chromatography
(EtOAc, then 9:1 EtOAc/MeOH) gave compound 7 (355 mg,
0.388 mmol, 56% based on acceptor) as a white solid. 1H NMR
(400 MHz, MeOD) δ ppm 7.44-7.28 (m, 5H), 5.40 (dd, 1H, J=
9.2 Hz, 10.4 Hz), 4.98 (dd, 1H, J=9.6 Hz), 4.86 (obscured by
solvent, 1H), 4.67 (dd, 1H, J=11.6 Hz), 4.64 (dd, 1H, J=11.6
Hz), 4.39 (q, 1H, J=7.2 Hz), 4.33 (dd, 1H, J=4.0 Hz, 12.4 Hz),
4.27 (d, 1H, J=8.4 Hz), 4.23 (q, 1H, J=6.8 Hz), 4.02-3.94 (m,
2H), 3.89-3.76 (m, 4H), 3.73 (s, 3H), 3.58-3.45 (m, 3H), 3.43 (s,
3H), 2.02-1.96 (m, 9H), 1.92 (s, 3H), 1.46-1.41 (m, 6H); 13C
NMR (100 MHz, MeOD) δ ppm 174.1, 173.2, 172.2, 170.9,
170.3, 170.0, 163.0, 138.5, 128.2 (2ꢀC), 127.6(4) (2ꢀC), 127.5(7),
102.1, 98.6, 92.5, 79.5, 78.0, 75.0, 74.4, 73.0, 71.5, 71.4, 68.8,
68.3, 61.4, 56.5, 55.7, 54.8, 51.6, 48.0, 21.8, 19.5, 19.3 (2 ꢀ C),
17.8, 16.3; HRMS (ESI) m/z calcd for [C37H50N3Cl3O17Na]þ,
936.2104, found 936.2111.
Experimental Section
Compounds 3 and 6 were prepared by published literature
methods and their identities confirmed by MS and NMR spec-
troscopy.14,17
Methyl 4,6-O-Benzylidene-β-D-N-acetylmuramylpyranoside-
L-alanine Methyl Ester (4). Compound 3 (2.9 g, 7.34 mmol)
was dissolved in 160 mL of anhydrous DMF, and HOBt (1.35 g,
8.81 mmol), EDC (1.69 g, 8.81 mmol), and 1.25 mL of NEt3 were
added. The resultant mixture was allowed to stir for 5 min at rt
before L-alanine methyl ester hydrochloride (1.23 g, 8.81 mmol)
and an additional 1.25 mL of NEt3 were added.The reaction was
allowed to stir overnight at room temperature under an argon
atmosphere, after which it was judged complete by TLC analysis
(9:1 CH2Cl2/MeOH). The mixture was diluted with CH2Cl2 and
washed consecutively with 5% KHSO4, sat. NaHCO3, sat.
NaCl, and H2O. The organic layer was separated, dried over
MgSO4, and filtered. The solvents were removed in vacuo, and
the solid was purified via silica gel chromatography (40:1
CH2Cl2/MeOH) to give product 4 (3.37 g, 7.02 mmol, 95%) as
a white solid. 1H NMR (400 MHz, CDCl3) δ ppm 7.48-7.34 (m,
5H), 7.18 (d, 1H, J=6.8 Hz), 6.09 (d, 1H, J=8.0 Hz), 5.55 (s,
1H), 4.78 (d, 1H, J=8.0 Hz), 4.47 (qd, 1H, J=7.2 Hz), 4.36 (dd,
1H, J=4.8 Hz, 10.4 Hz), 4.19 (q, 1H, J=6.8 Hz), 4.13 (dd, 1H,
J=9.2 Hz), 3.79 (dd, 1H, J=10.4 Hz), 3.76 (s, 3H), 3.61 (dd, 1H,
J=9.2 Hz), 3.54-3.47 (m, 5H), 1.99 (s, 3H), 1.43 (d, 3H, J =
7.2 Hz), 1.39 (d, 3H, J=6.4 Hz); 13C NMR (100 MHz, CDCl3) δ
ppm 173.7, 173.1, 171.3, 137.2, 129.3, 128.5 (2 ꢀ C), 126.1 (2 ꢀ
C), 101.6, 101.5, 82.0, 78.6, 78.2, 68.9, 66.0, 57.7, 57.2, 52.7, 48.2,
23.9, 19.6, 18.3; HRMS (ESI) m/z calcd for [C23H32N2O9Na]þ,
503.2006, found 503.1994.
Methyl 2-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-β-D-glucopy-
ranosyl-(1f4)-6-O-benzyl-β-D-N-acetylmuramylpyranoside-
L-alanine Methyl Ester (8). Disaccharide 7 (270 mg, 0.295 mmol)
and AIBN (10 mg, 0.059 mmol) were dissolved in 10 mL of
DMA, and the resultant solution was stirred at 90 οC for 1 h with
argon gas bubbling directly through the mixture. Tributyltin
hydride (0.63 mL, 2.36 mmol) and additional AIBN (3 mg,
0.0183 mmol) were added to the reaction mixture, and the
stirring was continued at 90 οC. The reaction progress was
monitored for the disappearance of starting material by TLC
analysis (9:1 CH2Cl2/MeOH) and was judged complete after
approximately 2 h. The solvents were removed in vacuo, and the
residue purified by silica gel chromatography (EtOAc, then 19:1
EtOAc/MeOH, then 9:1 EtOAc/MeOH) to give compound 8
Methyl 6-O-Benzyl-β-D-N-acetylmuramylpyranoside-L-alanine
1
˚
Methyl Ester (5). Molecular sieves (4 A) were added to a
(190 mg, 0.234 mmol, 79%) as a white solid. H NMR (400
solution of compound 4 (247 mg, 0.514 mmol) in 40 mL of
distilled CH2Cl2, and the mixture stirred under an argon atmo-
sphere for 1 h. The reaction flask was cooled to -78 οC, and
Et3SiH (0.25 mL, 1.54 mmol) was added, followed 5 min later
by TfOH (0.15 mL, 1.75 mmol). The mixture was maintained
at -78 οC while stirring and was judged complete after 1.5 h
by TLC analysis (9:1 CH2Cl2/MeOH). NEt3 (2 mL) and
MeOH (2 mL) were added, and the solution was diluted with
CHCl3 and filtered through a pad of Celite. The filtrate was
washed consecutively with sat. NaHCO3, sat. NaCl, and H2O.
The organic layer was dried over MgSO4 and filtered, and the
solvents were removed in vacuo. The residue was purified via
silica gel chromatography (CH2Cl2, then 39:1 CH2Cl2/MeOH,
then 9:1 CH2Cl2/MeOH) to give compound 5 (170 mg, 0.353
mmol, 68%) as a white foam. 1H NMR (400 MHz, CDCl3) δ
ppm 7.38-7.24 (m, 5H), 6.90 (d, 1H, J=8.8 Hz), 4.61-4.53 (m,
2H), 4.46-4.37 (m, 2H), 4.25 (q, 1H, J=6.8 Hz), 3.86-3.70 (m,
4H), 3.70 (s, 3H), 3.66-3.46 (m, 3H), 3.43 (s, 3H), 2.64 (br s,
1H), 1.94 (s, 3H), 1.42 (d, 3H, J=6.8 Hz), 1.38 (d, 3H, J=
6.8 Hz); 13C NMR (100 MHz, CDCl3) δ ppm 173.9(6), 173.5(3),
171.5, 138.0, 128.6 (2ꢀC), 127.9(3), 127.8(7) (2 ꢀ C), 101.9,
82.2, 77.0, 74.6, 73.8, 71.5, 70.5, 56.7, 54.8, 52.6, 48.3, 23.6,
19.6, 17.6; HRMS (ESI) m/z calcd for [C23H34N2O9Na]þ,
505.2162, found 505.2166.
MHz, MeOD) δ ppm 7.45-7.29 (m, 5H), 5.20 (dd, 1H, J=9.2
Hz, 10.4 Hz), 4.94 (dd, 1H, J=9.6 Hz), 4.73-4.59 (m, 3H), 4.40
(q, 1H, J = 7.2 Hz), 4.32-4.20 (m, 3H), 4.00-3.90 (m, 2H),
3.88-3.79 (m, 4H), 3.73 (s, 3H), 3.53-3.40 (m, 3H), 3.44 (s, 3H),
2.01-1.96 (m, 9H), 1.93 (s, 3H), 1.91 (s, 3H), 1.44-1.39 (m, 6H);
13C NMR (100 MHz, MeOD) δ ppm 174.2, 173.3, 172.4, 172.2,
170.9(7), 170.5(2), 170.0(0), 138.5, 128.3 (2ꢀC), 127.7(1) (2ꢀC),
127.6(3), 102.3, 99.5, 79.3, 77.7, 75.1, 74.7, 73.0, 72.4, 71.4,
68.8(2), 68.4(7), 61.5, 55.7, 54.6(9), 54.5(5), 51.7, 48.0, 21.8(5),
21.6(2), 19.5(0), 19.3(3), 19.2(9), 17.7, 16.3; HRMS (ESI) m/z
calcd for [C37H54N3O17]þ, 812.3453, found 812.3436.
Methyl 2-Acetamido-2-deoxy-3,4,6-tri-O-levulinoyl-β-D-gluco-
pyranosyl-(1f4)-6-O-benzyl-β-D-N-acetylmuramylpyranoside-
L-alanine Methyl Ester (9). Disaccharide 8 (42 mg, 0.052 mmol)
was dissolved in 10 mL of distilled MeOH in a flame-dried
round-bottom flask, and NaOMe was added (108 mg, 2.0 mmol).
The reaction mixture was stirred at rt under an argon atmo-
sphere for 20 min, after which it was judged complete by TLC
analysis (9:1 CH2Cl2/MeOH). The reaction was neutra-
lized with Amberlite IR120(Hþ), filtered, and concentrated
in vacuo. The residue was dissolved in 9:1 CH2Cl2/MeOH, filtered
through a silica gel plug, and concentrated in vacuo to give the
deacetylated product (30 mg) that was used without further
purification.
Methyl 3,4,6-Tri-O-acetyl-2-deoxy-2-trichloroacetamido-β-D-
glucopyranosyl-(1f4)-6-O-benzyl-β-D-N-acetylmuramylpyrano-
side-L-alanine Methyl Ester (7). To a solution of acceptor 5 (330
mg, 0.684 mmol) and donor 6 (666 mg, 1.13 mmol) in 20 mL of
Levulinic acid (0.015 mL, 0.144 mmol) and DMAP (17 mg,
1.38 mmol) were dissolved in 5 mL of distilled CH2Cl2, and the
mixture was cooled to 0 οC under an argon atmosphere. DIPC
(0.023 mL, 0.144 mmol) was added, and the mixture was stirred
for 5 min before a solution of the deacetylated disaccharide
(30 mg) in 2 mL of CH2Cl2 was added. The mixture was allowed to
warm to rt and was stirred overnight under an argon atmosphere,
˚
distilled CH2Cl2 was added 4 A molecular sieves, and the
resultant mixture was stirred under argon atmosphere for 1 h.
The mixture was cooled to 0 C, and TMSOTf (0.19 mL, 1.03
ο
J. Org. Chem. Vol. 76, No. 4, 2011 1123

Hadi et al.
JOCArticle
after which TLC analysis (9:1 CH2Cl2/MeOH) confirmed the
disappearance of the starting material. The solution was diluted
with EtOAc and filtered through a plug of silica; the plug was
then flushed with 200 mL of 9:1 CH2Cl2/MeOH, and the eluent
concentrated in vacuo. The residue was subjected to silica gel
chromatography (40:1 CH2Cl2/MeOH then 20:1 CH2Cl2/
MeOH) to give the levulinate-protected disaccharide 9 (37.8
mg, 0.0386 mmol, 75%) as a white solid. 1H NMR (400 MHz,
MeOD) δ ppm 7.44-7.29 (m, 5H), 5.21 (dd, 1H, J=9.2 Hz, 10.4
Hz), 4.92 (dd, 1H, J=9.2 Hz, 10.4 Hz), 4.71-4.58 (m, 3H), 4.41
(q, 1H, J=7.2 Hz), 4.28-4.19 (m, 3H), 4.01 (dd, 1H, J=1.6 Hz,
12 Hz), 3.93 (dd, 1H, J=8.4 Hz), 3.88-3.78 (m, 4H), 3.73 (s,
3H), 3.52-3.42 (m, 3H), 3.43 (s, 3H), 2.84-2.66 (m, 6H),
2.58-2.42 (m, 6H), 2.16-2.12 (m, 9H), 1.93 (s, 3H), 1.92 (s,
3H), 1.43-1.37 (m, 6H); 13C NMR (100 MHz, MeOD) δ ppm
207.8, 207.6, 207.5, 174.1, 173.3, 172.6, 172.4(6), 172.4(1), 172.1,
171.9, 138.5, 128.3 (2ꢀC), 127.8 (2ꢀC), 127.6, 102.3, 99.6, 79.0,
77.4, 75.3, 74.7, 73.2, 72.3, 71.7, 69.1, 68.9, 62.1, 55.6, 54.7, 54.2,
51.6, 48.0, 47.7, 37.5, 37.2(8), 37.2(4), 28.4, 28.2, 27.7(8), 27.7(2),
27.6(8), 21.7(7), 21.7(2), 17.8, 16.3; HRMS (ESI) m/z calcd for
[C46H65N3O20Na]þ, 1002.4059, found 1002.4034.
Methyl 2-Acetamido-2-deoxy-β-D-glucopyranosyl-(1f4)-6-
O-acetyl-β-D-N-acetylmuramylpyranoside-L-alanine Methyl Es-
ter (1). Disaccharide 9 (22 mg, 0.022 mmol) was dissolved in 10
mL of distilled MeOH, and 10% Pd/C (20 mg) was added. The
reaction flask was placed under a H2 atmosphere (1 atm), and
the reaction was stirred at rt until TLC analysis (9:1 CH2Cl2/
MeOH) indicated that the starting material was completely
consumed and the hydrogenolysis was complete. The mixture
was filtered through a pad of Celite, and the solvents were
removed in vacuo. The crude residue was carried on without
further purification.
color in the solution persisted. The solution was stirred at rt for 1.5
h, after which the reaction was judged complete by TLC analysis
(6:3:1 EtOAc/MeOH/H2O). The solvents were removed in vacuo,
and the crude methyl ester was subjected to silica gel chromatog-
raphy (CH2Cl2 then 9:1 CH2Cl2/MeOH), affording compound 10
1
as a white solid (134 mg, 0.328 mmol, 94%). MS and H NMR
spectroscopy were used to confirm the identity of the known
compound.21
Methyl 2-Acetamido-4-O-benzyl-2-deoxy-3-O-((R)-1-(metho-
xycarbonyl)ethyl)-β-D-glucopyranoside (11). The known methyl
ester-protected MurNAc derivative 10 (170 mg, 0.415 mmol)
˚
was dissolved in distilled CH2Cl2, and 4 A molecular sieves were
added. The resultant mixture was stirred under an argon atmo-
sphere for 1 h and was subsequently cooled to -78 οC. Et3SiH
(0.33 mL, 2.08 mmol) was first added, followed 5 min later by
PhBCl2 (0.27 mL, 2.08 mmol), and the reaction was stirred
at -78 οC. The reaction was monitored for the disappearance of
starting material by TLC analysis (9:1 CH2Cl2/MeOH) and was
quenched with 1 mL each of NEt3 and MeOH after it was judged
complete. The mixture was then diluted with CHCl3 and washed
with saturated NaHCO3. The organics were dried over MgSO4,
filtered, and then concentrated in vacuo. The crude product was
purified via silica gel chromatography (40:1 CH2Cl2/MeOH) to
give compound 11 (152 mg, 0.37 mmol, 89%) as a white solid. 1H
NMR (400 MHz, CDCl3) δ ppm 7.38-7.28 (m, 5H), 6.93 (d, 1H,
J=5.6 Hz), 4.71 (m, 2H), 4.57 (q, 1H, J=6.8 Hz), 4.41 (d, 1H, J=
7.2 Hz), 3.87 (m, 1H), 3.76-3.59 (m, 4H), 3.71 (s, 3H), 3.48 (s,
3H), 3.37-3.31 (m, 1H), 2.32 (br s, 1H), 2.02 (s, 3H), 1.35 (d, 3H,
J=6.8 Hz); 13C NMR (100 MHz, CDCl3) δ ppm 175.2, 171.9,
137.7, 128.7(7) (2ꢀC), 128.2(2), 127.9(8) (2ꢀC), 103.0, 79.4(0),
79.2(0), 75.5 (2 ꢀ C), 75.1, 61.6, 57.1, 55.7, 52.2, 23.7, 19.3;
HRMS (ESI) m/z calcd for [C20H29NO8Na]þ, 434.1791, found
434.1782.
Methyl 2-Acetamido-6-O-acetyl-4-O-benzyl-3-O-((R)-1-car-
boxyethyl)-2-deoxy-β-D-glucopyranoside Monosodium Salt (2).
Compound 11 (152 mg, 0.369 mmol) was dissolved in 25 mL of
1:1 dioxane/MeOH, and 1 M LiOH was added until the solution
reached a pH of 10. The reaction mixture was left to stir
overnight at rt, after which it was judged complete by TLC
analysis (6:3:1 EtOAc/MeOH/H2O). The mixture was neutra-
lized by the addition of Amberlite IR-120(Hþ), filtered, and then
concentrated in vacuo. The crude solid was redissolved in H2O
and washed with CH2Cl2, and the aqueous layer was concen-
trated in vacuo and used without further purification.
The crude debenzylated disaccharide was dissolved in 5 mL of
distilled pyridine and cooled to 0 °C before acetic anhydride (0.5
mL, 5.3 mmol) was added. The reaction mixture was allowed to
warm to rt and was stirred under a blanket of argon. After 2.5 h,
TLC analysis (9:1 CH2Cl2/MeOH) showed the disappearance of
the starting material and the appearance of a new spot at a
higher Rf. Toluene was added, and the mixture was co-evapo-
rated in vacuo; this procedure was repeated three times to ensure
all of the residual pyridine was removed from the reaction
mixture. Acetylation of the disaccharide was confirmed by MS
1
and H NMR spectroscopy (data not shown), and the crude
product was used without further purification.
The crude 6-O-acetylated disaccharide was dissolved in 5 mL
of distilled pyridine, and hydrazine acetate (12 mg, 0.13 mmol)
was added. The reaction mixture was monitored by TLC
analysis (9:1 CH2Cl2/MeOH) for the disappearance of starting
material and was judged complete after 10 min. The reaction
was diluted with toluene, and the mixture was co-evaporated to
remove any residual pyridine. Silica gel chromatography (20:1
CH2Cl2/MeOH then 9:1 CH2Cl2/MeOH) gave compound 1,
which was dissolved in water, frozen, and lyophilized to produce
a white solid (5.6 mg, 0.0088 mmol, 39%). 1H NMR (400 MHz,
D2O) δ ppm 4.55 (m, 1H), 4.48-4.35 (m, 4H), 4.19 (dd, 1H, J=
5.2 Hz, 12.0 Hz), 3.98-3.92 (m, 2H), 3.84-3.72 (m, 4H), 3.79 (s,
3H), 3.64-3.52 (m, 2H), 3.48 (s, 3H), 3.44-3.38 (m, 2H), 2.17 (s,
3H), 2.06 (s, 3H), 1.98 (s, 3H), 1.46 (d, 3H, J=6.8 Hz), 1.41 (d,
3H, J=6.8 Hz); 13C NMR (100 MHz, D2O) δ ppm 175.3, 175.1,
174.8, 174.4, 173.9, 102.0, 100.5, 79.4, 78.3, 76.4, 74.9, 73.6, 72.8,
70.4, 62.8, 61.3, 57.2, 56.1, 54.6, 53.2, 48.7, 22.3(7), 22.3(0), 20.4,
18.3, 16.1; HRMS (ESI) m/z calcd for [C26H43N3O15Na]þ,
660.2592, found 660.2582.
The crude carboxylic acid was dissolved in 20 mL of distilled
pyridine, and the resulting solution was cooled to 0 °C. Acetic
anhydride (1.0 mL, 10.6 mmol) was added, and the mixture was
allowed to warm to rt before being left to stir overnight under an
argon atmosphere. After TLC analysis of the reaction mixture
(6:3:1 EtOAc/MeOH/H2O) confirmed the disappearance of
starting material, toluene was added, and the solvents were co-
evaporated in vacuo. The addition of toluene and its removal
in vacuo was repeated several times to ensure the removal of all
residual pyridine. The residue was redissolved in CH2Cl2, excess
NEt3 was added, and the resultant solution evaporated in vacuo.
This procedure was repeated twice to generate the triethylam-
monium salt of the 6-O-acetylated ester and was confirmed by
1H NMR spectroscopy (data not shown). The triethylammo-
nium salt was dissolved in H2O and passed through a 10 mL
column of Amberlite IR-120(Naþ); fractions containing pro-
duct were pooled, frozen, and lyophilized to generate the sodium
1
salt 12 as a white, fluffy solid (108 mg, 0.234 mmol, 63%). H
NMR (400 MHz, MeOD) δ ppm 7.37-7.26 (m, 5H), 4.83 (d,
1H, J=10.8 Hz), 4.61 (d, 1H, J=10.8 Hz), 4.42 (q, 1H, J=6.8
Hz), 4.38-4.28 (m, 2H), 4.16 (dd, 1H, J = 3.2 Hz, 11.6 Hz),
3.74-3.48 (m, 4H), 3.41 (s, 3H), 2.03 (s, 3H), 1.98 (s, 3H), 1.37
(d, 3H, J=6.8 Hz); 13C NMR (100 MHz, MeOD) δ ppm 176.8,
172.9, 171.3, 138.0, 128.3 (2ꢀC), 127.7(7) (2ꢀC), 127.7(1), 102.8,
Methyl 2-Acetamido-4,6-O-benzylidene-2-deoxy-3-O-((R)-
1-(methoxycarbonyl)ethyl)-β-D-glucopyranoside (10). Compound
3 (138 mg, 0.349 mmol) was dissolved in 72 mL of an 8:1 mixture
of MeOH/toluene. TMS-diazomethane (2 M solution in diethyl
ether) was added dropwise to the reaction mixture until the yellow
1124 J. Org. Chem. Vol. 76, No. 4, 2011

Hadi et al.
JOCArticle
80.8, 78.8, 77.2, 74.7, 73.1, 62.8, 55.7(7), 55.2(4), 22.0, 19.5, 18.6;
HRMS (ESI) m/z calcd for [C21H28NO9]-, 438.1764, found
438.1773.
150 mM imidazole. The eluted protein was then dialyzed for 16 h
against 2 ꢀ 4 L of 25 mM sodium phosphate buffer (pH 7.0).
His6-tagged Ape1 was further purified by cation-exchange
chromatography on MonoS. Protein was applied to the column
following its equilibration in running buffer (25 mM NaPO4, pH
7.0) at a flow rate of 0.7 mL/min. Elution of protein from the
column was accomplished by increasing the ionic strength of
the running buffer using a linear gradient of 0-1 M NaCl over
30 min. Eluted Ape1 was then collected and dialyzed against 6 L
of 50 mM sodium phosphate buffer, pH 7.0 for 16 h at 4 οC.
Enzyme Assays. For the routine detection of acetyl esterase
activity, 2 mM p-nitrophenylacetate (pNP-acetate) in 50 mM
sodium phosphate buffer (pH 6.5) was used as a substrate in a
96-well microtiter plate as previously described.9,10
The Michaelis-Menten parameters of Ape1 were determined
for 1 and 2 using concentrations ranging from 0.05 to 4 mM in 50
mM sodium phosphate buffer pH 6.5. Reaction mixtures,
performed in triplicate, were initiated by the addition of sub-
strate and incubated at 25 οC. Reactions were terminated by
acidifying with H2SO4 to a final concentration of 60 mM.
Samples of substrate incubated in the absence of enzyme were
used as controls for the spontaneous release of any acetate.
Quantification of released acetate was performed using the
Megazyme Acetic Acid Assay kit (Megazyme International
Ireland, Ltd., Wicklow, Ireland). Plots of initial reaction velo-
cities as a function of substrate concentration were analyzed by
nonlinear regression using Microcal Origin 7.5, assuming a one-
site binding model.
Bacterial Strains and Growth Media. The sources of plasmids
and bacterial strains used in this study are listed with their
respective genotypic descriptions in the Supporting Informa-
tion. Escherichia coli Rosetta(λDE3)pLysS, used for protein
expression, was maintained on LB agar containing 35 μg/mL
chloramphenicol. For expression of high levels of Ape1
(previously Ape1a),10 cells were always freshly transformed with
pACJW16 using standard procedures and grown in SuperBroth
(5 g NaCl, 20 g yeast extract and 32 g tryptone/L) containing 35
μg/mL chloramphenicol and 50 μg/mL kanamycin at 37 οC with
agitation. All reagents were from Sigma unless otherwise noted.
Production and Purification of Ape1. For the overexpression
of Ape1, E. coli Rosetta(λDE3)pLysS cells transformed with
pACJW16 were grown in SuperBroth at 37 οC to an OD600 of
∼0.6 and then induced for a minimum of 3 h at 18 οC with the
addition of IPTG to a final concentration of 1 mM. Cells were
isolated by centrifugation (5000g, 15 min, 4 οC), and pellets were
stored at -20 οC until needed.
To purify His6-Ape1, cell pellets were thawed and resus-
pended in a minimal volume of lysis buffer [50 mM NaPO4
and 500 mM NaCl (pH 8.0)]. Lysozyme (1 mg/mL), RNase A
(10 mg/mL), and DNase I (5 mg/mL) were added to aid in lysis,
and EDTA-free protease inhibitor tablets (1 per 15 mL of
suspension) were added to prevent protein degradation. The
suspension was incubated at 4 οC for 1 h before being subjected
to lysis in a French pressure cell (four passes at 18 000 psi). The
resulting cell lysates were clarified by centrifugation (20,000g, 20
min, 4 οC), and 1 mL of Ni2þNTA-agarose was added for every
10 mL of cleared lysate. The mixture was incubated overnight at
Acknowledgment. This work was supported by the Nat-
ural Sciences and Engineering Research Council of Canada
(NSERC) and the Canadian Institutes of Health Research
(CIHR). T.H. is supported by a Pacific Century Graduate
Fellowship from the University of British Columbia, and
J.M.P. by NSERC PGS D.
ο
4 C with shaking before being applied to a 15 mL disposable
plastic column. Unbound proteins were allowed to flow
through, and the matrix was washed with approximately 30
column volumes of lysis buffer. The matrix was subsequently
washed with approximately 20 column volumes of wash buffer
containing 20 mM imidazole and a further 10 column volumes
of wash buffer containing 30 mM imidazole. Bound Ape1 was
recovered by batch elution in 10 mL of wash buffer containing
Supporting Information Available: 1H and 13C NMR spec-
tra of new compounds and kinetic data. This material is avail-
able free of charge via the Internet at http://pubs.acs.org
J. Org. Chem. Vol. 76, No. 4, 2011 1125