Development of a plate-based scintillation proximity assay for the mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis

Article abstract of DOI:10.1016/j.bmc.2010.07.040

A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD have been characterized and implicated to be involved in the cell wall arabinan assembly. These arabinosyltransferases are essential for the viability of the organism and are logically valid targets for developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug, targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among these arabinosyltransferases, the terminal β-(1→2) arabinosyltransferase activity has been associated with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmembrane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide, α-d-Araf-(1→5)-α-d-Araf-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was identified as the β-(1→2) arabinofuranose adduct. When synthetic tri-and tetra-saccharides were used as substrates, a mixture of products containing both β-(1→2) and α-(1→5) linkages were formed. Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.

Full text of DOI:10.1016/j.bmc.2010.07.040

Bioorganic & Medicinal Chemistry 18 (2010) 7121–7131
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Development of a plate-based scintillation proximity assay for the
mycobacterial AftB enzyme involved in cell wall arabinan biosynthesis
Jian Zhang , Anita G. Amin, Alexandra Hölemann à, Peter H. Seeberger §, Delphi Chatterjee
⇑
Mycobacteria Research Laboratories, Department of Microbiology, Immunology and Pathology, Colorado State University, Fort Collins, CO 80523, USA
Laboratory for Organic Chemistry, Swiss Federal Institute of Technology (ETH) Zürich, Wolfgang-Pauli-Str. 10, 8093 Zürich, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A number of mycobacterial arabinosyltransferases, such as the Emb proteins, AftA, AftB, AftC, and AftD
have been characterized and implicated to be involved in the cell wall arabinan assembly. These ara-
binosyltransferases are essential for the viability of the organism and are logically valid targets for
developing new anti-tuberculosis agents. For instance, Ethambutol, a first line anti-tuberculosis drug,
targets the Emb proteins involved in the formation of the arabinan of cell wall arabinogalactan. Among
these arabinosyltransferases, the terminal b-(1?2) arabinosyltransferase activity has been associated
with AftB. The predicted topology of AftB in Mycobacterium tuberculosis has 10 N terminal transmem-
brane domains and a C terminal hydrophilic domain similar to the Emb proteins. It has a conserved
Received 12 May 2010
Revised 13 July 2010
Accepted 16 July 2010
Available online 5 August 2010
Keywords:
Mycobacteria
Assay
Arabinosyltransferase
AftB
GT-C motif and is difficult to express. In a cell free assay, synthetic disaccharide,
a-D-Araf-(1?5)-a-
D-Araf-octyl, has been used as a substrate to explore the function of AftB. In our work, the disaccharide
was synthesized in its pentenylated and biotinylated form, and the enzymatic product formed was
Carbohydrate synthesis
identified as the b-(1?2) arabinofuranose adduct. When synthetic tri- and tetra-saccharides were used
as substrates, a mixture of products containing both b-(1?2) and
a-(1?5) linkages were formed.
Therefore, the biotinylated disaccharide was selected to develop a scintillation proximity assay.
Ó 2010 Elsevier Ltd. All rights reserved.
1. Introduction
viability of the Mycobacterium tuberculosis (M. tuberculosis).4 The
mycobacterial cell envelope comprises of four macromolecules:
Tuberculosis (TB) is still a major public health problem.1 Partic-
ularly with the emergence of multi-drug-resistant (MDR-TB) tuber-
culosis and, more recently, extensively drug-resistant (XDR-TB)
tuberculosis clinical isolates.2 MDR-TB is defined as resistance to
at least two of the most powerful first-line anti-TB drugs, isoniazid
(INH) and rifampin. XDR-TB is defined as MDR-TB plus resistance to
any fluoroquinolone, and at least to one of the second-line anti-TB
drugs, capreomycin, kanamycin or amikacin. The recent reports of
XDR-TB in HIV co-infected patients have renewed global awareness
of drug-resistant TB3 and prompted the need for research on devel-
oping new anti-TB drugs and identify new drug targets.
lipoarabinomannan (LAM), mycolic acids, arabinogalactan (AG),
and peptidoglycan.5–8 The galactan domain of AG is linked to pep-
tidoglycan through a diglycosylphosphoryl linker of
-GlcNAc, and the arabinan domain is attached to mycolic acids,
forming the mycolyl-arabinogalactan-peptidoglycan (mAGP) com-
plex. The arabinan contains -(1?5), -(1?3), and b-(1?2) arabi-
L-Rhap-(1?3)-
a-D
a
a
nofuranosyl (Araf) linkages that forms different structural motifs of
arabinan.6,9 Enzymes associated with the synthesis of these mole-
cules have all been found to be essential.10–13
The Araf residues of the arabinan originate from the pentose
phosphate shunt pathway and the immediate donor is decaprenyl-
Two first-line drugs, INH and ethambutol (EMB), target the bio-
synthesis of the mycobacterial cell-wall, which is essential for the
phosphoryl-D
-arabinofuranose (DPA).14,15 The biosynthetic pathway
for the DPA formation has been recently elucidated. Phosphoribose-
pyrophosphate (pRpp) is converted to 5-phosphodecaprenylribose
(DPPR), which is dephosphorylated to form decaprenylphosphoryl-
⇑
Corresponding author.
E-mail addresses: jianz@adatech.com (J. Zhang), delphi.chatterjee@colostate.edu
(D. Chatterjee).
D
-ribose (DPR). Finally, DPR is epimerized to form DPA.16 For the
assembly of the arabinan from DPA, in the ten years of study that
focused on arabinosyltransferases (AraT), it has been established
that the Emb proteins (EmbA, EmbB, and EmbC) were required
for the arabinan synthesis and worked not only on the formation
of the non-reducing end of the arabinan but also in the assembly
of the internal arabinan.17–19 Recently, four additional AraTs have
been reported. As illustrated in Figure 1, one of the enzymes, AftA,
ADA Technologies, Inc., 8100 Shaffer Parkway, Littleton, CO 80127, USA.
Present address: Technische Universität Dortmund, Faculty of Chemistry, Organic
à
Chemistry, Otto-Hahn-Str. 6, 44227 Dortmund, Germany.
§
Present address: Max-Planck-Institute of Colloids and Interfaces, Department of
Biomolecular Systems, Am Mühlenberg 1, 14476 Potsdam-Golm, Germany and Freie
Universität Berlin, Institute of Chemistry and Biochemistry, Arnimallee 22, 14195,
Berlin.
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.040

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J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
AftB,Rv3805c
-1,2 arabinosyltransferase
Insensitive to Ethambutol
-Araf
-Araf
3,5- -Araf
2
EmbA,Rv3794; EmbB,Rv3795
5- -Araf
terminal -1,3 arabinosyltransferase
3,5- -Araf
AftC,Rv2673; AftD,Rv0236c
internal -1,3 arabinosyltransferase
5- -Araf
AftA,Rv3792
Figure 1. Biosynthesis of arabinan in AG in M. tuberculosis. There are three Ara22 mers (only one is shown) on the galactan backbone. All Araf residues are donated by DPA. Key
AraTs (six in total) identified till date are shown. These are only for the AG synthesis. It is interesting to note that both EmbA and EmbB have similar functions and AftC and
AftD also, have similar functions. AftA is unique in that it only recognizes galactan and donates the first Araf residues.
is responsible for recognizing the galactan and donating the initial
key
-Araf residues.10 AftB on the other hand, is involved in the
synthesis of the terminal b-Araf cap. The disaccharide with the oc-
tyl aglycon ( -Araf-(1?5)- -Araf-octyl) has been used as
AG contributes to the structural integrity of the bacterial cell.
Ongoing drug development efforts have recently identified benzo-
thiazinone, as an inhibitor of DprE1 (Rv 3790, an enzyme identified
in the epimerization of DPR to DPA).20 This enzyme is also not
inhibited by EMB. Thus the arabinan is validated as an attractive
target for developing new class of inhibitors not only on the pre-
mises of success of EMB, but also for the fact that many of these
newly identified enzymes are essential.10–13 Finding inhibitors for
separate enzymes in one essential biosynthetic pathway might
be beneficial to avert drug resistance.
In this work, we developed a plate-based assay to screen inhib-
itors for AftB. We chose M. smegmatis as the source of AftB since
studies have shown that AftB is a conserved protein although
Rv3805c is essential in M. tuberculosis. Biochemical phenotype of
the deletion mutant of an orthologue NCgl2780 in Corynebacterium
glutamicum could be fully complemented with AftB from M. tuber-
culosis.11 First, oligosaccharides with a pentenyl aglycon (Ara2-
Ara4, 1–3 in Fig. 2) were examined in a cell-free assay using
p[14C]Rpp as an indirect arabinosyl donor. Later, biotinylated
disaccharide (4 in Fig. 2) was synthesized and utilized in a
a
a
-D
a-D
acceptor in the functional characterization of AftB to confirm that
AftB was a terminal b-(1?2) AraT, and this activity was found to
be insensitive to EMB.11 Recently, two
a-(1?3) branching AraT en-
zymes, AftC (Rv2673) and AftD (Rv0236c), have been identified by
constructing a deletion mutant of MSMEG2785 (homolog of
Rv2673) in Mycobacterium smegmatis (M. smegmatis)12 and overex-
pression of Rv0236c in M. smegmatis,13 respectively. Whereas
EmbA and EmbB were suggested to be
ing terminal Arab1?2Ara 1?5(Arab1?2Ara
1? (Ara6) motif,17 Rv2673 has been shown to be a
for synthesizing internal arabinan in AG.
a
-(1?3) AraT for synthesiz-
1?3)Ara 1?5Ara-
-(1?3) AraT
a
a
a
(a
a
One of the primary criteria for selecting a protein for screening
for inhibitors is the essentiality of the encoding gene for growth.
AftB fits this criterion, as it is essential in M. tuberculosis. Moreover,
the function of AftB is unique in that, it is a retaining enzyme and
its function is to terminate growing arabinan chain in AG (Fig. 1).
OH
HO
OH
HO
O
O
OH
HO
OH
O
O
OH
OH
O
O
OH
O
OH
O
OH
OH
O
OH
O
OH
O
O
OH
O
O
OH
OH
O
O
OH
O
OH
2
3
O
1
OH
OH
HO
O
O
OH
O
O
HN NH
OH
O
S
OH
N
S
H
O
4
Figure 2. Acceptors 1–4 used in this study.

J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
7123
scintillation proximity assay (SPA).21,22 The plate-based SPA assay
was constructed and optimum conditions were developed.
tracted with ethanol and unincorporated p[14C]Rpp removed by
passing through an anion exchange column (SAX). The eluate from
the SAX column was dried and the residue was partitioned be-
tween 1-butanol and water. The 1-butanol layer was dried and
the residue was applied to TLC. TLC analysis revealed formation
of radioactive products (lanes 10, 20, and 30 in Fig. 3A), that
migrated slower compared to the corresponding acceptor (lanes
1, 2, and 3 in Fig. 3A).
2. Results and discussion
2.1. Chemical synthesis of acceptors 1, 2, and 3
The synthesis of arabinan acceptors 1, 2, and 3 was accom-
plished using a linear strategy. Glycosylation of known disaccha-
ride 523,24 with known trichloroacetimidate 623 using standard
TMSOTf-catalyzed activation (Scheme 1) proceeded smoothly with
To confirm that [14C]-arabinose had been added to the acceptor,
an aliquot of the material extracted with 1-butanol (2000 dpm)
was hydrolyzed with 2 M TFA and the hydrolysate was subjected
to TLC. Autoradiography showed that the radioactivity was
exclusively associated with arabinose ( Fig. 3B) indicating that
one extra Araf was transferred from DPA to the acceptors used in
this study.
complete
a-selectivity and the corresponding trisaccharide 7 was
obtained in 95% yield. Desilylation afforded 8 (96%), which was
subsequently coupled to glycosyl trichloroacetimidate 6 under
TMSOTf activation to give the desired tetrasaccharide 9 in 84%
yield. Removal of the TIPS protecting group gave 10.
Finally, global deprotection of the three linear arabinosides was
carried out using a three-step protocol (Scheme 2). Birch reduction
of 5, 8, and 10 followed by peracetylation of the intermediates
furnished the peracetylated pentenyl arabinosides that could be
easily purified by silica gel column chromatography. Deacetylation
under basic conditions finally gave the target arabinosyl acceptors
1, 2, and 3 in good overall yields.
When the disaccharide acceptor 1 was used (lane 1 of Fig. 3),
the band corresponding to the biosynthetic product b-
(1??)- -Araf-(1?5)-
-Araf-pentenyl (lane 10 of Fig. 3A)
migrated slower than the trisaccharide acceptor 2, -Araf-
(1?5)- -Araf-(1?5)- -Araf-pentenyl (lane 2 of Fig. 3A) indi-
cating that the enzymatic product was different and did not incor-
porate additional -(1?5) Araf onto the acceptor. Linkage analysis
D-Araf-
a-
D
a-D
a-D
a-
D
a-D
a
on the enzymatic product revealed the presence of t-Araf, 2-Araf
and a 5-Araf (Fig. 1S).
Therefore, combining the previous data that showed both
-(1?5) and b-(1?2) containing trisaccharide products were
2.2. Enzymatic assays using pentenyl aglycon acceptors
a
formed when a-D-Araf-(1?5)-a-D-Araf-octyl was used as an accep-
Compounds 1, 2, and 3 ( Fig. 2) with pentenyl aglycon were
tested for their ability to function as acceptors in a cell-free assay
using p[14C]Rpp (that was converted to DPA in situ) as an indirect
arabinosyl donor.16 After a typical reaction,25 products were ex-
tor,11 the disaccharide acceptor with pentenyl aglycon showed
more specific recognition of AftB arabinosyltransferase. Only the
product containing b-(1?2)-Araf formed in the cell-free assay.
Scheme 1.

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J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
Scheme 2.
Figure 3. (A) TLC analysis of biosynthetic products from enzymatic reaction with acceptors 1, 2, and 3. Acceptors were visualized after spraying with a-naphthol (lanes 1, 2,
and 3), and the radioactive products were revealed by autoradiography (lanes 10, 20, and 30). An aliquot of the product labeled with p[14C]Rpp from the eluate after the SAX
column followed by 1-butanol extraction was applied to TLC plate, developed in CHCl3/MeOH/NH4OH/H2O (65:25:0.4:3.6) and exposed for four days. The migration of the
enzymatically synthesized products was slower than the corresponding acceptors. (B) Radioactive AG (lane1) and the enzymatic product from acceptor 1 (lane 2) were
hydrolyzed with 2 M TFA, and applied to TLC. Chromatogram was developed in pyridine/ethyl acetate/acetic acid/water (5:5:1:3) and subjected to autoradiography.
For trisaccharide acceptor 2 (lane 2 of Fig. 3A), the major b-
(1?2) and minor -(1?5) product (tetrasaccharides) was formed
per TLC (lane 20 of Fig. 3A) analysis that showed that the acceptor
is recognized by both AftB and an -1,5 AraT in comparison to
acceptor 1. Tetrasaccharide acceptor 3 yielded a pentasaccharide
(lane 30 of Fig. 3). We reasoned that this substrate should not be
recognized by AftB since a similar four Araf linear stretch is not
present in AG. The enzymatic product was isolated, digested with
Cellulomonas endo-arabinanase25 and the product formed was
examined by Dionex High-Performance Anion Exchange Chroma-
tography (HPAEC). The product formed after the enzyme digestion
block 1125,26 as a functional spacer for further synthesis.27 The
n-pentenyl glycoside 12 was deprotected at 5-OH to obtain 13.
Radical elongation with 2-[(tert-butoxycarbonyl)amino]-1-etha-
nethiol in the presence of AIBN at 75 °C formed sulfide spacer
glycoside 14 in high yield (81%). Monosaccharide building block
a
a
14 was coupled with perbenzoylated a-D-arabinofuranosyl trichlo-
roacetimidate (15)28 to afford disaccharide 16 (87%).29 After depro-
tection of 16 by MeONa/MeOH and 30% TFA to remove benzoyl and
BOC groups, respectively, compounds 17 and 18 were obtained.
The spacer glycoside 18 could be linked with a commercial N-
hydroxysuccinimide ester reagent to form the biotinylated accep-
tor 4.30
co-chromatographed with
[(1?3,5)]2- -Araf-(1?5)- -Araf, suggesting AftC
ing AraT of acceptor 3 (Fig. 2S).
a
synthetic tetrasaccharide-
a-Araf-
a
a
a-(1?3) branch-
2.4. Enzymatic assays using biotinylated acceptor (4)
2.3. Chemical synthesis of biotinylated acceptor (4)
The biotinylated acceptor 4, S-
D-Biotinoyl-5-(2-amino-1-thio-
ethyl)-pentyl 5-O-( -arabinofuranosyl)-a-D
a
-
D
-arabinofuranoside,
During the chemical synthesis of the biotinylated acceptor
was used in the arabinosyltransferase assay with a similar proce-
(Scheme 3), an n-pentenyl group was introduced using building
dure as described for acceptors 1–3. The major enzymatic product

J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
7125
OBz
HO
OBz
OBz
HO
n-Pentenol
TBDPSO
OBz
TBAF
THF
63%
HSCH2CH2NHBOC
TBDPSO
O
O
O
O
O
OBz
BF3.Et2O
0oC, CH2Cl2
63%
AIBN
O
O
S
O
OBz
OBz
OBz
N
H
O
Dioxane, 75oC
OBz
13
12
11
80%
14
OBz
BzO
OBz
O
BzO
O
OH
HO
O
CCl3
O
15 OBz
OBz
O
MeONa
OBz
NH
O
OH
O
O
OH
O
Me3SiOTf
O
O
S
MeOH-CH2Cl2 (3:1)
82%
OBz
N
H
O
CH2Cl2, -20 to 0 o
87%
C
O
S
OH
N
H
O
16
17
OH
HO
OH
30% TFA
CH2Cl2
Biotin-NHS
DMF, TEA
HO
O
O
O
OH
O
O
OH
O
HN NH
OH
96%
S
O
OH
79%
O
S
O
OH
OH
N
H
NH2
S
O
18
4
Scheme 3. Chemical synthesis of the biotinylated acceptor (4).
was b-(1?2) Araf (Band I in Fig. 4A) and minor with
(Band II in Fig. 4A), both of the products were shown to contain
14C]Araf by TFA hydrolysis (Fig. 4B). In a separate experiment,
a
-(1??) Araf
2.5. Developing SPA assay for AftB
[
A schematic representation of a scintillation proximity assay
(SPA) for AftB enzyme is illustrated in Figure 5A. In this assay, AftB
catalyzes the addition of a 3H-labeled Araf to the biotinylated
acceptor 4 to generate biotinylated trisaccharide product with
b-(1?2) Araf residue. Once the streptavidin-coated SPA beads are
added to the reaction mixture, this 3H-labeled biotinylated product
is captured by the SPA beads as a result of the specific biotin/strep-
tavidin binding.34 Thus, the 3H is put to proximity of the scintillant
impregnated within the bead. Such proximity results in a b-emis-
sion that can be readily detected in MicroBeta scintillation count
plate reader. The unreacted p[3H] Rpp and its degradation products
(DPPR, DPR, DPA, etc.) remain in solution and are sufficiently dis-
tant from the scintillant so that no significant signal is observed.
Therefore, the SPA technology enables assays to be carried out
without the intermediate purification steps and can be used di-
rectly for microtitreplate assay.
on a streptavidin coated plate, when biotinylated acceptor 4, crude
mycobacterial cell lysate and pRpp, followed by monoclonal
antibody CS35 was added sequentially, positive color reaction
was obtained after adding the developing reagent. CS35 is known
to recognize b-Araf-(1?2)-a
-Araf.31–33 Positive reaction of the
enzymatic product to CS35 indicated that proper trisaccharide
was formed with the acceptor 4. However, since ELISA typically in-
volves several washing steps not favoured in assay, we moved on
to developing the assay for screening purpose.
A typical SPA assay consisted of incubating biotinylated accep-
tor 4, radio-labeled p[3H]Rpp, and membrane/P60 enzyme source
for a specific amount of time. P60 is the cell wall enriched partic-
ulate fraction which improved the efficiency of pRpp transforma-
tion to DPA. The resulting reaction was terminated with addition
of ethanol and SPA beads were incubated overnight with reaction
mixture. Typically, the assay reaction with acceptors resulted in
about 150 cpm more counts than the control reaction (reaction
without addition of biotinylated acceptor (4)). In this assay, both
the radio-labeled biotinylated product and unlabeled acceptor (4)
can be bound to the beads. A maximal SPA signal should be ob-
tained when the biotin-binding capacity of SPA beads exceeds
the amount of acceptor used in a typical assay. This prediction
was investigated by determining the optimal relationship between
the SPA signal and the SPA beads quantity (Fig. 5B). A maximal SPA
signal was obtained when the beads quantity was 1.0 mg/assay.
Another variable in the SPA assay was the amount of the enzyme
used. As shown in Figure 5C, the maximal SPA signal was achieved
at enzyme concentrations of 0.4 mg/assay (membrane/P60, 5:3). In
contrast, SPA assays conducted with enzyme quantity more than
0.4 mg resulted in significant incomplete binding of the radio-la-
beled product since the surface of SPA beads was covered by the
P60 particles that blocked biotin/streptavidin binding. Therefore,
Figure 4. Enzymatic assays using the biotinylated acceptor 4. (A) TLC analysis of
the biosynthetic products from biotinylated acceptor 4. Acceptor 4 was visualized
by a-naphthol (lane 1); the radioactive reactions without (lane 2) or with (lane 3)
the acceptor were detected by autoradiography. (B) Radioactive AG (lane1) and the
products (lane 2) were hydrolyzed with 2 M TFA, and applied to TLC.

7126
J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
Figure 5. The SPA assay using p[3H]Rpp. (A) Schematic representation of the principle of scintillation proximity assay (SPA) targeted at AftB. (B) Dependence of scintillation
signals on SPA beads concentration. The concentration of acceptor 4 was 300 M in the assay. The data was expressed as net CPM after subtracting the background (reaction
l
without adding SPA beads). (C) Effect of protein concentrations on net SPA signals after subtracting the background (reaction without membranes). The enzyme used in the
assay was a mixture of membrane and P60 at the ratio of 5:3.
the radioactive product formed was not proportional to the
amount of enzyme used in the assay.
rived at the maximum when using 1.0 mg of membranes in the
reaction. An optimal assay should include approximately 0.5 mg
of membrane proteins. The kinetic parameter of AftB was investi-
gated using different concentrations of the substrate (acceptor 4).
As shown in Figure 6B, the apparent Km for acceptor 4 was found
In order to improve the homogeneity in the system and increase
efficiency of the assay, DP[3H]A was used in the reaction instead of
p[3H]Rpp to avoid the incomplete binding of enzymatic product to
SPA beads. Igepal CA-630 (Sigma) was used as a detergent in this
assay to enhance DP[3H]A solubility. Compared to the assay using
p[3H]Rpp, DP[3H]A assay had a more homogenous appearance and
P60 was eliminated from the assay resulting in higher efficiency of
biotin/streptavidin binding. A variable quantity of membranes as
the enzyme source was used in the assay, and as can be seen in Fig-
ure 6A, the addition of increasing amounts of enzyme up to 0.5 mg
of membranes resulted in a proportional increase in product for-
mation. The product formation achieved a saturation starting at
the point of using 0.5 mg of membranes and the reaction rate ar-
to be 40.9 lM when 0.5 mg of membranes were used in assay.
On the basis of the SPA assay developed to measure the activity
of KasA and KasB enzymes in mycolic acid biosynthesis in M. tuber-
culosis22 we believe that the assay described in this work could be
transformed into a fast, sensitive, and plate-based assay for screen-
ing of inhibitors against AraT activities. Here, we have developed a
SPA-assay to evaluate an important and essential AraT (AftB) that
is functional in synthesizing b-(1?2) Araf, a terminal Araf required
to tether mycolic acid in cell wall. The method has potential for
developing into assay for screening of inhibitors.
Figure 6. The SPA assay using DP[3H]A. (A) Dependence of signal generation on enzyme amounts (0, 0.125, 0.25, 0.5, 1.0 mg). (B) Effect of acceptor 4 concentration (0, 5, 10,
50, 100, 200 M) on the formation of enzymatic product captured by SPA beads. The data obtained was subjected to analysis using GraFit 5.0, and expressed as net CPM after
subtracting the background (reaction without enzyme or acceptor 4).
l

J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
7127
(0.30 mL, 4.2 mmol) was added. After 4 h at 0 °C, the reaction
was quenched by addition of saturated aqueous NaHCO3-solution
(15 mL). The aqueous layer was extracted with CH2Cl2
(4 ꢀ 30 mL). The combined organic extracts were washed with
brine (50 mL), dried with Na2SO4, filtered and evaporated. Purifica-
tion by flash silica gel column chromatography (hexanes/EtOAc,
3:1) yielded 8 (0.434 g, 96%) as a colorless oil; Rf = 0.15 (hexanes/
3. Experimental procedures
3.1. General methods
Radioactive p[14C]Rpp (300 mCi/mmol) and p[3H]Rpp (60 mCi/
mmol) were generated from uniformly labeled
D
-[14C]glucose and
D
-[3H]glucose, respectively, (American Radiolabeled Chemicals
EtOAc, 3:1); [a]
+103.1 (c 1.2, CHCl3). 1H NMR (300 MHz, CDCl3):
Inc.) as described.15 Streptavidin-coated polyvinyl toluene SPA
scintillation beads were purchased from GE-Healthcare. Protein
concentrations were determined using the BCA assay (Pierce). All
chemicals were purchased from Sigma/Aldrich and were used
without further purification. Reagent grade dichloromethane
(CH2Cl2), tetrahydrofuran (THF), and toluene were passed through
an activated neutral alumina column prior to use. Reagent grade
methanol (MeOH) was dried over activated molecular sieves prior
to use. Pyridine and triethylamine (Et3 N) were distilled over CaH2
prior to use. Thin layer chromatography (TLC) was performed on
silica gel G60 aluminium-backed plates (Merck). Column chroma-
tography was performed using silica gel (70–230 mesh). Unless
otherwise stated, all reactions were performed at room tempera-
ture under nitrogen or argon. Glycosylation reactions were per-
formed in oven-dried glassware under an inert atmosphere.
Drying after organic extractions was done over anhydrous Na2SO4.
1H NMR spectra were recorded at 300 or 400 MHz and 13C NMR
spectra were recorded at 75 or 100 MHz.
D
d 8.07–7.98 (m, 6H), 7.61–7.52 (m, 3H), 7.46–7.17 (m, 21H), 5.89–
5.76 (m, 1H), 5.45 (d, J = 1.3 Hz, 1H), 5.40 (d, J = 1.6 Hz, 1H), 5.36 (d,
J = 1.3 Hz, 1H), 5.30 (s, 1H), 5.25 (s, 1H), 5.14 (s, 1H), 5.07–4.93 (m,
2H), 4.80 (d, J = 12.1 Hz, 1H), 4.67 (d, J = 12.1 Hz, 1H), 4.63 (d,
J = 11.7 Hz, 1H), 4.59 (d, J = 11.7 Hz, 1H), 4.51 (d, J = 12.1 Hz, 1H),
4.43 (d, J = 12.1 Hz, 1H), 4.37–4.33 (m, 1H), 4.24–4.20 (m, 1H),
4.19–4.16 (m, 1H), 4.12–4.10 (m, 1H), 4.04–4.00 (m, 1H), 3.97–
3.92 (m, 2H), 3.85 (dd, J = 4.0, 11.3 Hz, 1H), 3.80–3.68 (m, 4H),
3.57–3.45 (m, 2H), 2.19–2.12 (m, 2H), 1.77–1.68 (m, 3H). 13C
NMR (75 MHz, CDCl3): d 165.5, 165.3, 165.2, 138.2, 137.8, 137.6,
133.4, 133.3, 133.3, 129.8, 129.7, 129.5, 129.5, 129.4, 128.5,
128.3, 128.3, 127.8, 127.7, 127.7, 127.6, 127.5, 114.8, 106.2,
106.1, 106.0, 83.3, 83.2, 82.7, 82.1, 82.0, 81.7, 81.4, 72.3, 72.1,
66.8, 65.6, 65.5, 61.9, 30.2, 28.6. IR (CHCl3): mmax 2922, 1720,
1451, 1311, 1296, 1265, 1109, 1068, 1026 cmꢁ1. MALDI-HRMS:
m/z (M+Na)+ calcd for C62H64NaO16: 1087.4092, obsd 1087.4104.
3.2.3. Pent-4-enyl 5-O-(5-O-(5-O-(2-O-benzoyl-3-O-benzyl-5-O-
triisopropysilyl-
-arabinofuranosyl)-2-O-benzoyl-3-O-benzyl-
anosyl)-2-O-benzoyl-3-O-benzyl- -arabinofuranoside (9)
a
-
D
-arabinofuranosyl)-2-O-benzoyl-3-O-benzyl-
3.2. Chemical synthesis of acceptors 1, 2, and 3
a-D
a-D-arabinofur-
a-D
3.2.1. Pent-4-enyl 5-O-(5-O-(2-O-benzoyl-3-O-benzyl-5-O-triiso-
propysilyl-a-D-arabinofuranosyl)-2-O-benzoyl-3-O-benzyl-a-D-
arabinofuranosyl)-2-O-benzoyl-3-O-benzyl-a-D-arabinofuran-
oside (7)
Trisaccharide 8 (0.490 g, 0.460 mmol) and imidate 623 (0.386 g,
0.598 mmol) were combined, azeotroped with toluene (5 ꢀ 10 mL)
and dried in vacuo overnight. The mixture was dissolved in anhy-
drous CH2Cl2 (10 mL), cooled to ꢁ40 °C and TMSOTf (0.012 mL,
0.63 mmol) was added. After 45 min at ꢁ40 to ꢁ30 °C, the reaction
was quenched by addition of Et3N, the solution was stirred for
20 min at room temperature and the solvents were removed in va-
cuo. Purification by flash silica gel column chromatography (hex-
anes/EtOAc, 8:1 to 7:1) yielded tetrasaccharide 9 (0.432 g, 94%)
Disaccharide 523,24 (0.330 g, 0.447 mmol) and imidate 623
(0.390 g, 0.605 mmol) were combined, azeotroped with toluene
(5 ꢀ 20 mL) and dried in vacuo overnight. The mixture was dis-
solved in anhydrous CH2Cl2 (12 mL), cooled to ꢁ40 °C and TMSOTf
(0.012 mL, 0.063 mmol) was added. After 30 min at ꢁ40 to ꢁ30 °C,
the reaction was quenched by addition of Et3N, the solution was
stirred for 10 min at room temperature, and the solvents were re-
moved in vacuo. Purification by flash silica gel column chromatog-
raphy (pure toluene to toluene/EtOAc, 50:1) yielded trisaccharide 7
as a colorless oil; Rf = 0.10 (hexanes/EtOAc, 7:1); [
a
]
D
+88.9 (c
0.83, CHCl3). 1H NMR (300 MHz, CDCl3): d 8.04–7.97 (m, 8H),
7.60–7.50 (m, 4H), 7.44–7-14 (m, 28H), 5.87–5.74 (m, 1H), 5.42
(d, J = 1.3 Hz, 1H), 5.38–5.36 (m, 3H), 5.26 (s, 1H), 5.23 (s, 1H),
5.20 (s, 1H), 5.12 (s, 1H), 5.05–4.91 (m, 2H), 4.77 (d, J = 12.1 Hz,
1H), 4.62 (d, J = 12.1 Hz, 1H), 4.60 (d, J = 12.1 Hz, 1H), 4.57 (d,
J = 12.2 Hz, 1H), 4.55 (d, J = 12.1 Hz, 1H), 4.48 (d, J = 12.2 Hz, 1H),
4.44 (d, J = 12.1 Hz, 1H), 4.42 (d, J = 12.1 Hz, 1H), 4.34–4.29 (m,
1H), 4.20–4.00 (m, 7H), 3.91 (dd, J = 4.1, 11.3 Hz, 1H), 3.87–3.60
(m, 8H), 3.47 (td, J = 6.6, 9.6 Hz, 1H), 2.17–2.10 (m, 2H), 1.76–
1.66 (m, 2H), 1.04–0.93 (m, 21H). 13C NMR (75 MHz, CDCl3): d
165.2, 165.0, 164.9, 164.9, 137.9, 137.6, 137.6, 137.5, 133.1,
133.0, 129.6, 129.5, 129.3,129.3, 128.2, 128.1, 128.1, 128.0, 128.0,
127.6, 127.5, 127.5, 127.4, 127.4, 127.3, 127.3, 127.2, 114.6,
105.9, 105.8, 83.9, 83.1, 83.1, 83.0, 82.6, 82.0, 81.9, 81.8, 81.7,
81.6, 81.4, 72.2, 72.0, 71.9, 71.8, 66.6, 65.5, 65.3, 65.1, 62.5, 30.2,
28.6, 17.9, 11.8. IR (CHCl3): mmax 3005, 2944, 2862, 1718, 1446,
1292, 1267, 1113, 1072 cmꢁ1. MALDI-HRMS: m/z (M+Na)+ calcd
for C90H102NaO21Si: 1569.6581, obsd 1569.6546.
(0.520 g, 95%) as a colorless oil; Rf = 0.15 (hexanes/EtOAc, 7:1); [a]
D
+78.9 (c 0.90, CHCl3). 1H NMR (300 MHz, CDCl3): d 8.08–8.00 (m,
6H), 7.59–7.51 (m, 3H), 7.44–7.39 (m, 6H), 7.37–7.17 (m, 15H),
5.89–5.76 (m, 1H), 5.47 (d, J = 1.4 Hz, 1H), 5.43–5.42 (m, 2H),
5.31 (s, 1H), 5.27 (s, 1H), 5.16 (s, 1H), 5.07–4.94 (m, 2H), 4.81 (d,
J = 12.1 Hz, 1H), 4.65 (d, J = 12.1 Hz, 1H), 4.64 (d, J = 12.1 Hz, 1H),
4.62 (d, J = 12.1 Hz, 1H), 4.51 (d, J = 12.1 Hz, 1H), 4.48 (d,
J = 12.1 Hz, 1H), 4.39–4.34 (m, 1H), 4.26–4.14 (m, 3H), 4.09 (s,
2H), 3.96 (dd, J = 4.1, 11.2 Hz, 1H), 3.90 (dd, J = 3.9, 11.2 Hz, 1H),
3.82–3.68 (m, 5H), 3.53–3.44 (m, 1H), 2.20–2.13 (m, 2H), 1.78–
1.69 (m, 2H), 1.08–0.94 (m, 21H). 13C NMR (75 MHz, CDCl3): d
165.4, 165.1, 165.1, 138.1, 137.8, 137.7, 133.2, 133.1, 129.7,
129.6, 129.4, 128.4, 128.2, 128.2, 128.1, 127.7, 127.6, 127.5,
127.4, 127.4, 114.7, 106.0, 105.9, 83.9, 83.2, 83.2, 82.7, 82.0, 81.9,
81.8, 81.7, 81.4, 72.2, 72.0, 71.8, 66.6, 65.5, 65.2, 62.5, 30.1, 28.5,
17.8, 11.7. IR (CHCl3): mmax 2944, 1718, 1451, 1292, 1267, 1113,
1067, 1021 cmꢁ1
.
MALDI-HRMS: m/z (M+Na)+ calcd for
3.2.4. Pent-4-enyl 5-O-(5-O-(5-O-(2-O-benzoyl-3-O-benzyl-
arabinofuranosyl)-2-O-benzoyl-3-O-benzyl- -arabinofurano-
syl)-2-O-benzoyl-3-O-benzyl- -arabinofuranosyl)-2-O-benzo-
yl-3-O-benzyl- -arabinofuranoside (10)
a-D-
C71H84NaO16Si: 1243.5426, obsd 1243.5439.
a-D
a-D
3.2.2. Pent-4-enyl 5-O-(5-O-(2-O-benzoyl-3-O-benzyl-
nofuranosyl)-2-O-benzoyl-3-O-benzyl- -arabinofuranosyl)-2-
O-benzoyl-3-O-benzyl- -arabinofuranoside (8)
a-D-arabi-
a-D
a-D
A solution of 9 (0.310 g, 0.200 mmol) in anhydrous MeOH/
CH2Cl2 (4:1, 5 mL) was cooled to 0 °C and acetyl chloride
(0.14 mL, 1.9 mmol) was added. The mixture was stirred for 6 h,
while slowly warming to room temperature. The reaction was
a
-D
A solution of 7 (0.520 g, 0.426 mmol) in anhydrous MeOH/
CH2Cl2 (4:1, 10 mL) was cooled to 0 °C and acetyl chloride

7128
J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
quenched by addition of saturated aqueous NaHCO3-solution
(20 mL). The aqueous layer was extracted with CH2Cl2
(4 ꢀ 40 mL). The combined organic extracts were dried with
Na2SO4, filtered and evaporated. Purification by flash silica gel col-
umn chromatography (hexanes/EtOAc, 3:1–2:1) yielded 10
(0.255 g, 92%) as a colorless solid; mp 42–44 °C; Rf = 0.30 (hex-
HRMS: m/z (M+Na)+ calcd for C15H26NaO9: 373.1475, obsd
373.1467.
3.2.6. Pent-4-enyl 5-O-(5-O-(a-D-arabinofuranosyl)-a-D-arabi-
nofuranosyl)- -arabinofuranoside (2)
a-D
Trisaccharide 8 (0.055 g, 0.052 mmol) was dissolved in anhy-
drous THF (4.5 mL) under argon, cooled to ꢁ78 °C and ammonia
(about 20 mL) was condensed into the flask. Small pieces of sodium
(washed with n-pentane) were then added to the solution until the
color of the reaction mixture turned dark blue. After 35 min at
ꢁ78 °C, MeOH was added until the blue color disappeared. Small
pieces of sodium were again added to the mixture until the blue
color of the solution persisted. After 30 min at ꢁ78 °C, the reaction
was quenched by addition of MeOH (3 mL), the cooling bath was
removed and the ammonia was evaporated using a stream of nitro-
gen. The solution was diluted with MeOH (5 mL), neutralized with
Amberlite IR-120 and filtered. The resin was washed with MeOH
and the solution was concentrated in vacuo. The residue was dis-
solved in pyridine (2 mL) and acetic anhydride (1.5 mL) and the
solution was stirred at room temperature for 20 h. The solvents
were evaporated and the residue was purified by flash silica gel
column chromatography (hexanes/EtOAc, 1:1) to give the peracet-
ylated trisaccharide (0.037 g, 92% over two steps) as a colorless oil;
anes/EtOAc, 3:2); [a]
+115.8 (c 0.52, CHCl3). 1H NMR (300 MHz,
D
CDCl3): d 8.09–8.00 (m, 8H), 7.62–7.54 (m, 4H), 7.48–7.40 (m,
8H), 7.38–7.20 (m, 20H), 5.91–5.78 (m, 1H), 5.47 (d, J = 1.3 Hz,
1H), 5.44–5.43 (m, 2H), 5.38 (d, J = 1.3 Hz, 1H), 5.31 (s, 1H), 5.29
(s, 1H), 5.25 (s, 1H), 5.16 (s, 1H), 5.09–4.95 (m, 2H), 4.81 (d,
J = 12.2 Hz, 1H), 4.70–4.43 (m, 7H), 4.39–4.35 (m, 1H), 4.28–4.11
(m, 5H), 4.07–4.03 (m, 1H), 3.99–3.68 (m, 9H), 3.60–3.46 (m,
2H), 2.22–2.14 (m, 2H), 1.82–1.70 (m, 3H). 13C NMR (75 MHz,
CDCl3): d 165.5, 165.2, 165.2, 165.1, 138.2, 137.8, 137.8, 137.5,
133.4, 133.2, 129.7, 129.6, 129.5, 129.5, 129.4, 128.4, 128.3,
128.2, 127.8, 127.7, 127.6, 127.6, 127.5, 114.7, 106.2, 106.1,
106.0, 83.3, 83.2, 82.8, 82.2, 82.0, 81.8, 81.5, 72.3, 72.1, 72.1,
72.0, 66.7, 65.7, 65.6, 61.9, 30.2, 28.6. IR (CHCl3): mmax 2926,
1722, 1602, 1452, 1316, 1298, 1268, 1114, 1071, 1028 cmꢁ1. MAL-
DI-HRMS: m/z (M+Na)+ calcd for C81H82NaO21: 1413.5246, obsd
1413.5264.
3.2.5. Pent-4-enyl 5-O-(
a
-D
-arabinofuranosyl)-
a
-D
-arabinofur-
Rf = 0.15 (hexanes/EtOAc, 1:1); [a]
+79.6 (c 0.67, CHCl3). 1H NMR
D
anoside (1)
(300 MHz, CDCl3): d 5.86–5.72 (m, 1H), 5.14–5.11 (m, 6H), 5.03–
4.91 (m, 5H), 4.44 (dd, J = 2.7, 11.3 Hz, 1H), 4.28–4.13 (m, 4H),
3.95–3.87 (m, 2H), 3.74–3.63 (m, 3H), 3.43 (td, J = 6.2, 9.7 Hz,
1H), 2.15–2.06 (m, 23H), 1.72–1.63 (m, 2H). 13C NMR (75 MHz,
CDCl3): d 170.5, 170.2, 170.1, 170.1, 169.8, 169.6, 169.3, 138.0,
114.9, 105.5, 105.4, 81.9, 81.5, 81.5, 80.9, 80.9, 66.8, 65.5, 65.3,
63.2, 30.1, 28.6, 20.7, 20.6, 20.6. IR (CHCl3): mmax 3011, 2928,
1742, 1372, 1113, 1065, 973 cmꢁ1. MALDI-HRMS: m/z (M+Na)+
calcd for C34H48NaO20: 799.2637, obsd 799.2617.
The peracetylated trisaccharide (0.033 g, 0.042 mmol) was dis-
solved in anhydrous MeOH (1 mL) and 0.5 M NaOMe in MeOH
(0.084 mL, 0.042 mmol) was added. The mixture was stirred at
room temperature for 13 h and then neutralized with Amberlite
IR-120. The mixture was filtered, the resin washed with MeOH
and the solvents were evaporated. Purification by Sephadex G-10
column chromatography (H2O/EtOH, 1:1) gave 2 (0.0166 g, 81%)
Disaccharide 523,24 (0.060 g, 0.081 mmol) was dissolved in
anhydrous THF (5 mL) under argon, cooled to ꢁ78 °C and ammonia
(about 20 mL) was condensed into the flask. Small pieces of sodium
(washed with n-pentane) were then added to the solution until the
color of the reaction mixture turned dark blue. After 40 min at
ꢁ78 °C, MeOH was added until the blue color disappeared. Small
pieces of sodium were again added to the mixture until the blue
color of the solution persisted. After 50 min at ꢁ78 °C, the reaction
was quenched by addition of MeOH (3 mL), the cooling bath was
removed and the ammonia was evaporated using a stream of nitro-
gen. The solution was diluted with MeOH (5 mL), neutralized with
Amberlite IR-120 and filtered. The resin was washed with MeOH
and the solution was concentrated in vacuo. The residue was dis-
solved in pyridine (3 mL) and acetic anhydride (3 mL) and the solu-
tion was stirred at room temperature for 18 h. The solvents were
evaporated and the residue was purified by flash silica gel column
chromatography (hexanes/EtOAc, 3:1–2:1) to give the peracetylat-
ed disaccharide (0.039 g, 86% over two steps) as a colorless oil;
as a colorless solid; Rf = 0.20 (CH2Cl2/MeOH, 4:1); [
a
]
+94.2 (c
D
0.83, MeOH). 1H NMR (300 MHz, CD3OD): d 5.90–5.76 (m, 1H),
5.06–4.92 (m, 2H), 4.94–4.93 (m, 2H), 4.84 (s, 1H), 4.10–3.55 (m,
23H), 3.42 (td, J = 6.5, 9.7 Hz, 1H), 2.17–2.09 (m, 2H), 1.72–1.63
(m, 2H). 13C NMR (75 MHz, CD3OD): d 139.4, 115.3, 109.7, 109.6,
109.5, 85.9, 84.1, 83.7, 83.5, 83.2, 83.1, 79.2, 79.1, 78.8, 68.2,
68.1, 63.1, 31.4, 30.0. ESI-HRMS: m/z (M+Na)+ calcd for
Rf = 0.10 (hexanes/EtOAc, 2:1); [a]
+94.2 (c 0.33, CHCl3). 1H NMR
D
(300 MHz, CDCl3): d 5.86–5.72 (m, 1H), 5.15–5.12 (m, 3H), 5.03–
4.91 (m, 5H), 4.43 (dd, J = 3.0, 11.5 Hz, 1H), 4.29–4.13 (m, 3H),
3.90 (dd, J = 4.1, 11.5 Hz, 1H), 3.73–3.61 (m, 2H), 3.43 (td, J = 6.3,
9.6 Hz, 1H), 2.14–2.05 (m, 17H), 1.71–1.62 (m, 2H). 13C NMR
(75 MHz, CDCl3): d 170.5, 170.2, 170.1, 169.8, 169.4, 138.0, 114.9,
105.5, 105.5, 81.9, 80.9, 80.8, 66.8, 65.5, 63.3, 30.1, 28.6, 20.7,
20.6. IR (CHCl3): mmax 3026, 2923, 1739, 1364, 1113, 1062, 1048,
C20H34NaO13: 505.1897, obsd 505.1898.
3.2.7. Pent-4-enyl 5-O-(5-O-(5-O-(
a
-D
-arabinofuranosyl)-
a-D-
arabinofuranosyl)-
a-
D
-arabinofuranosyl)- -arabinofurano-
a-D
974 cmꢁ1. MALDI-HRMS: m/z (M+Na)+ calcd for C25H36NaO14
:
side (3)
583.2003, obsd 583.1987.
Tetrasaccharide 10 (0.050 g, 0.036 mmol) was dissolved in
anhydrous THF (4 mL) under argon, cooled to ꢁ78 °C and ammonia
(about 20 mL) was condensed into the flask. Small pieces of sodium
(washed with n-pentane) were then added to the solution until the
color of the reaction mixture turned dark blue. After 40 min at
ꢁ78 °C, MeOH was added until the blue color disappeared. Small
pieces of sodium were again added to the mixture until the blue col-
or of the solution persisted. After 40 min at ꢁ78 °C, the reaction was
quenched by addition of MeOH (3 mL), the cooling bath was re-
moved and the ammonia was evaporated using a stream of nitro-
gen. The solution was diluted with MeOH (5 mL), neutralized
with Amberlite IR-120 and filtered. The resin was washed with
MeOH and the solution was concentrated in vacuo. The residue
was dissolved in pyridine (3 mL) and acetic anhydride (2 mL) and
The peracetylated disaccharide (0.036 g, 0.064 mmol) was dis-
solved in anhydrous MeOH (1.3 mL) and 0.5 M NaOMe in MeOH
(0.13 mL, 0.065 mmol) was added. The mixture was stirred at room
temperature for 24 h and then neutralized with Amberlite IR-120.
The mixture was filtered, the resin washed with MeOH and the sol-
vents were evaporated. Purification by Sephadex G-10 column
chromatography (H2O/EtOH, 1:1) gave 1 (0.020 g, 89%) as a color-
less solid; Rf = 0.60 (MeOH/EtOAc, 1:4); [a] +103.2 (c 1.0, MeOH).
D
1H NMR (300 MHz, CD3OD): d 5.90–5.77 (m, 1H), 5.06–4.93 (m,
2H), 4.95 (d, J = 1.4 Hz, 1H), 4.84 (d, J = 1.5 Hz, 1H), 4.03–3.61 (m,
16H), 3.43 (td, J = 6.5, 9.5 Hz, 1H), 2.18–2.10 (m, 2H), 1.72–1.64
(m, 2H). 13C NMR (75 MHz, CD3OD): d 139.4, 115.2, 109.6, 109.5,
85.9, 83.6, 83.5, 83.1, 79.1, 78.8, 68.2, 68.1, 63.1, 31.4, 30.0. ESI-

J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
7129
the solution was stirred at room temperature for 16 h. The solvents
were evaporated and the residue was purified by flash silica gel col-
umn chromatography (hexanes/EtOAc, 1:1–1:2) to give the per-
acetylated tetrasaccharide (0.028 g, 78% over two steps) as a
165.6, 138.3, 133.8, 130.1, 130.0, 129.9, 129.4, 129.3, 128.8,
128.7, 115.2, 105.7, 83.8, 82.0, 78.0, 67.0, 62.6, 30.5, 28.9.
3.3.3. 5-[2-[(t-Butoxylcarbonyl)amino]-1-thioethyl]-pentyl 2,3-
colorless oil; Rf = 0.30 (hexanes/EtOAc, 1:2); [
a
]
+111.7 (c 0.23,
di-O-benzyl-
A solution of compound 13 (140 mg, 0.33 mmol), AIBN (30 mg,
0.15 mmol), 2-[(t-butoxycarbonyl)amino]-1-ethanethiol (900 L,
a-D-arabinofuranoside (14)
D
CHCl3). 1H NMR (300 MHz, CDCl3): d 5.86–5.73 (m, 1H), 5.14–4.91
(m, 14H), 4.44 (dd, J = 2.7, 11.4 Hz, 1H), 4.28–4.14 (m, 5H), 3.95–
3.87 (m, 3H), 3.75–3.62 (m, 4H), 3.43 (td, J = 6.2, 9.7 Hz, 1H),
2.17–2.00 (m, 29H), 1.72–1.63 (m, 2H). 13C NMR (75 MHz, CDCl3):
d 170.6, 170.3, 170.2, 170.2, 170.2, 169.9, 169.7, 169.4, 138.0,
114.9, 105.4, 105.4, 105.3, 105.2, 81.9, 81.8, 81.6, 81.5, 81.4, 80.9,
80.9, 80.8, 66.7, 65.2, 65.1, 63.2, 30.1, 28.5, 20.8, 20.7, 20.6, 20.6.
IR (CHCl3): mmax 3026, 2933, 1739, 1369, 1108, 1062, 1041,
l
5.0 mmol) in dry dioxane (5 mL) was thoroughly degassed by N2
before being reacted at 75 °C (preheated oilbath). TLC detected
the processing of the reaction. After the consumption of the start-
ing material (8 h), the mixture was concentrated and loaded to sil-
ica gel column (hexane/EtOAc, 2:1) to give compound 14 (160 mg,
80.8%). 1H NMR (400 MHz, CDCl3): d 8.08–8.00 (m, 4H), 7.60–7.55
(m, 2H), 7.46–7.41 (m, 4H), 5.49 (m, 1H), 5.39 (m, 1H), 5.20 (m,
1H), 4.29 (dd, 1H, J = 4.0, 8.0 Hz), 3.99–3.93 (m, 2H), 3.74 (m,
1H), 3.53–3.49 (m, 1H), 3.25 (t, 2H, J = 6.4 Hz), 2.58 (m, 2H), 2.44
(m, 2H), 1.64–1.46 (m, 6H), 1.42 (m, 9H). 13C NMR (100 MHz,
CDCl3): d 166.4, 165.6, 133.8, 130.1, 130.0, 129.4, 129.3, 128.8,
128.7, 105.7, 83.8, 82.0, 78.1, 67.4, 62.6, 40.1, 32.4, 31.9, 29.6,
29.3, 28.6, 28.5, 25.6. ESI-MS m/z 504.13 [MꢁBoc+2H]+, 603.93
[M+H]+, 620.00 [M+NH4]+, 626.27 [M+Na]+, 642.27 [M+K]+.
969 cmꢁ1. MALDI-HRMS: m/z (M+Na)+ calcd for C43H60NaO26
1015.3271, obsd 1015.3244.
:
The peracetylated tetrasaccharide (0.025 g, 0.025 mmol) was
dissolved in anhydrous MeOH (0.5 mL) and 0.5 M NaOMe in MeOH
(0.050 mL, 0.025 mmol) was added. The mixture was stirred at
room temperature for 48 h and then neutralized with Amberlite
IR-120. The mixture was filtered, the resin washed with MeOH
and the solvents were evaporated. Purification by Sephadex G-10
column chromatography (H2O/EtOH, 1:1) gave 3 (0.0125 g, 80%)
as a colorless solid; Rf = 0.15 (MeOH/EtOAc, 1:5); [
a
]
+114.3 (c
3.3.4. 5-[2-[(t-Butoxylcarbonyl)amino]-1-thioethyl]-pentyl 5-O-
D
0.63, MeOH). 1H NMR (300 MHz, CDCl3): d 5.90–5.77 (m, 1H),
5.06–4.80 (m, 6H), 4.11–3.61 (m, 21H), 3.43 (td, J = 6.5, 9.8 Hz,
1H), 2.17–2.10 (m, 2H), 1.72–1.63 (m, 2H). 13C NMR (75 MHz,
CDCl3): d 139.4, 115.3, 109.7, 109.5, 85.9, 84.1, 83.7, 83.5, 83.2,
83.1, 79.1, 78.8, 68.2, 63.1, 31.4, 30.0. ESI-HRMS: m/z (M+Na)+ calcd
for C25H42NaO17: 637.2320, obsd 637.2314.
(2,3,5-tri-O-benzyl-a-D-arabinofuranosyl)-2,3-di-O-benzyl-a-D-
arabinofuranoside (16)
To a solution of compound 14 (120 mg, 0.20 mmol) and com-
pound 1528 (145 mg, 0.24 mmol) in dry CH2Cl2 (5 mL) was added
Me3SiOTf (5.4
lL, 0.030 mmol) under N2 at ꢁ20 °C. The reaction
mixture was stirred for 1 h allowing the temperature to gradually
rise to 0 °C, then neutralized with Et3N and concentrated. The res-
idue was purified by silica gel column (hexane/EtOAc, 4:1?2:1) to
give compound 16 (180 mg, 86.5%). 1H NMR (400 MHz, CDCl3): d
8.07–7.90 (m, 10H), 7.55–7.20 (m, 15H), 5.60 (m, 2H), 5.55–5.26
(m, 2H), 5.48 (s, 1H), 5.43 (s, 1H), 5.18 (s, 1H), 4.82–4.77 (m, 1H),
4.72 (dd, 1H, J = 4.4, 8.4 Hz), 4.66–4.61 (m, 1H), 4.41 (m, 1H),
4.21 (m, 1H), 3.94 (m, 1H), 3.75–3.69 (m, 1H), 3.49–3.44 (m, 1H),
3.25 (m, 2H), 2.56 (m, 2H), 2.44 (m, 2H), 1.60–1.53 (m, 4H), 1.57
(m, 2H), 1.41 (m, 9H). 13C NMR (100 MHz, CDCl3): d 166.5, 166.0,
165.9, 165.7, 165.5, 156.0, 133.9, 133.7, 133.5, 133.2, 130.2,
130.1, 130.0, 129.9, 129.5, 129.2, 128.8, 128.7, 128.6, 128.5,
106.1, 105.8, 82.7, 82.1, 82.0, 81.7, 81.5, 78.2, 78.1, 67.3, 66.4,
63.9. ESI-MS m/z 1047.93 [M+H]+, 1065.07 [M+NH4]+, 1070.33
[M+Na]+.
3.3. Chemical synthesis of biotinylated acceptors (4)
3.3.1. Pent-4-enyl 2,3-di-O-benzyl-5-O-t-butyldiphenylsilyl-a-D-
arabinofuranoside (12)
To a solution of compound 1125 (1.98 g, 2.83 mmol) and n-pen-
tenol (576
BF3ꢂEt2O (716
l
L, 5.66 mmol) in dry CH2Cl2 (20 mL) at 0 °C was added
L, 1.43 mmol) dropwise. The reaction mixture was
l
stirred for 2 h at 0 °C, and then at room temperature for 1 h, before
Et3N (1.2 mL) was added. The mixture was diluted with CH2Cl2,
washed with a saturated solution of NaHCO3, dried over Na2SO4,
concentrated, and purified by chromatography on silica gel (hex-
ane/EtOAc, 10:1?8:1) to give compound 12 (1.20 g, 63.8%). 1H
NMR (400 MHz, CDCl3): d 8.12 (d, 2H, J = 7.2 Hz), 8.04 (d, 2H,
J = 6.9 Hz), 7.78–7.30 (m, 16H), 5.97–5.84 (m, 1H), 5.67 (dd, 1H,
J = 1.2, 5.1 Hz), 5.51 (d, 1H, J = 1.5 Hz), 5.13–5.01 (m, 2H), 4.46–
4.41 (m, 1H), 4.09–4.06 (m, 2H), 3.89–3.81 (m, 1H), 3.64–3.57
(m, 1H), 2.29–2.24 (m, 2H), 1.87–1.80 (m, 2H), 1.17–1.09 (m,
9H). 13C NMR (100 MHz, CDCl3): d 165.8, 165.7, 138.4, 135.9,
134.6, 133.5, 133.4, 130.5, 130.1, 129.9, 129.7, 129.5, 128.6,
128.1, 127.9, 115.1, 105.9, 83.1, 82.5, 77.7, 66.9, 63.9, 30.5, 29.0,
27.0, 26.2, 19.5.
3.3.5. 5-[2-[(t-Butoxylcarbonyl)amino]-1-thioethyl]-pentyl 5-O-
(
a
-
D
-arabinofuranosyl)-
To a solution of compound 16 (130 mg, 0.12 mmol) in dry
CH2Cl2 (3 mL) and dry MeOH (9 mL) was added MeONa (1 M in
MeOH, 360 L). After 2 h of stirring at room temperature, the reac-
a-D-arabinofuranoside (17)
l
tion mixture was neutralized with Amberlite IR-120 (H+) resin, fil-
tered, and the residue was purified by silica gel column (CHCl3/
MeOH, 8:1?6:1) to give compound 17 (53 mg, 81.5%). NMR
(400 MHz, CDCl3): d 5.12 (br, 1H), 5.06 (s, 1H), 4.96 (s, 1H), 4.12–
3.96 (m, 7H), 3.83–3.70 (m, 4H), 3.43 (m, 1H), 3.30 (m, 2H), 2.64
(m, 2H), 2.54 (m, 2H), 2.22 (br, 2H), 1.60 (m, 4H), 1.45 (m, 9H),
1.26 (m, 2H). 13C NMR (100 MHz, CDCl3): d 156.2, 108.2, 108.0,
85.8, 83.8, 81.1, 80.8, 79.8, 78.1, 77.4, 67.8, 66.6, 62.1, 40.0, 32.4,
31.8, 29.9, 29.5, 29.2, 28.6, 25.5. ESI-MS m/z 528.00 [M+H]+,
550.27 [M+Na]+.
3.3.2. Pent-4-enyl 2,3-di-O-benzyl-a-D-arabinofuranoside (13)
To a solution of compound 12 (1.10 g, 1.66 mmol) in dry THF
(10 mL) at room temperature was added dropwise the solution of
TBAF (866 mg, 3.3 mmol) in THF (5 mL). After stirring at room tem-
perature for 1 h, the reaction mixture was diluted with CH2Cl2,
washed with a saturated solution of (NH4)2SO4 and brine. The or-
ganic phase was dried over Na2SO4, and concentrated. The residue
was purified by chromatography on silica gel (hexane/EtOAc,
4:1?2:1) to give compound 13 (442 mg, 62.7%). 1H NMR
(400 MHz, CDCl3): d 8.10–8.05 (m, 4H), 7.63–7.58 (m, 2H), 7.49–
7.45 (m, 4H), 5.86–5.79 (m, 1H), 5.54 (d, 1H, J = 1.6 Hz), 5.45 (d,
1H, J = 4.4 Hz), 5.24 (s, 1H), 5.04–4.96 (m, 2H), 4.35–4.31 (m, 1H),
4.05–3.96 (m, 2H), 3.83–3.77 (m, 1H), 3.58–3.52 (m, 1H), 2.18
(m, 2H), 1.80–1.73 (m, 2H). 13C NMR (100 MHz, CDCl3): d 166.4,
3.3.6. 5-(2-Amino-1-thioethyl)-pentyl 5-O-(
syl)- -arabinofuranoside (18)
a-D-arabinofurano-
a-D
Compound 17 (50 mg, 0.017 mmol) was dissolved in CH2Cl2
(2 mL) and then treated with TFA (1 mL) at 0 °C. After stirring for
0.5 h, the mixture was concentrated and purified by silica gel col-
umn (CHCl3/MeOH, 1:1) to give compound 18 (32 mg, 79.0%). 1H

7130
J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
NMR (400 MHz, D2O): d 5.05 (d, 1H, J = 0.8 Hz), 4.99 (d, 1H,
J = 2.0 Hz), 4.15–4.12 (m, 1H), 4.10 (m, 1H), 4.08–4.02 (m, 2H),
3.99–3.96 (m, 1H), 3.94–3.91 (m, 1H), 3.87–3.77 (m, 3H), 3.75–
3.66 (m, 2H), 3.58–3.52 (m, 1H), 3.19 (t, 2H, J = 6.4 Hz), 2.82 (t,
2H, J = 6.4 Hz), 2.58 (t, 2H, J = 7.2 Hz), 1.64–1.57 (m, 4H), 1.47–
1.41 (m, 2H). 13C NMR (100 MHz, D2O): d 107.5, 107.3, 84.0, 81.9,
81.0, 80.9, 76.6, 76.5, 68.4, 66.8, 61.2, 38.4, 30.6, 28.3, 28.2, 28.1,
24.5. ESI-MS m/z 428.13 [M+H]+, 450.27 [M+Na]+.
the residue was reconstituted in Milli-Q water (10 lL) for analysis
by silica gel TLC. The plates were developed in CHCl3/MeOH/
NH4OH/H2O (65:25:0.4:3.6) or CHCl3/MeOH/1 M NH4OAc/
NH4OH/H2O (180:140:9:9:23), followed by autoradiography at
ꢁ70 °C using Biomax MR film (Kodak) and acceptors were visual-
ized using a-naphthol spray reagent. For analysis of the sugar com-
position of the enzymatic products, the water layer after 1-butanol
extraction was purified by Biogel P-2 column, eluted with Milli-Q
water, and approximately 2000 dpm of the purified product was
3.3.7. S-
arabinofuranosyl)-
D
-Biotinoyl-5-(2-amino-1-thioethyl)-pentyl 5-O-(
-arabinofuranoside (4)
a
-
D
-
hydrolyzed in 200 ll of 2 M trifluoroacetic acid (TFA) at 120 °C
a-
D
for 2 h. TFA was removed under a stream of air and the hydrolysate
was analyzed on silica gel TLC plate developed in pyridine/ethyl
acetate/acetic acid/water (5:5:1:3) followed by autoradiography
as described above. Radioactive spots were identified by co-chro-
matography with standard radioactive arabinose and galactose
from radioactive [14C] labeled AG.
Compound 18 (8 mg, 0.047 mmol) was dissolved in DMF
(0.8 mL), and the pH was adjusted to 9.0 using Et3N. NHS-biotin
(16 mg, 0.047 mmol) was added and the reaction was stirred at
room temperature for 12 h. The solvent was removed by Savant
and the residue was purified by silica gel column (CHCl3/MeOH,
1:1) to give biotinylated acceptor 4 (12 mg, 96.0%). 1H NMR
(400 MHz, D2O): d 5.05 (s, 1H), 4.99 (s, 1H), 4.58 (m, 1H), 4.41
(m, 1H), 4.13–4.03 (m, 3H), 3.97 (m, 1H), 3.91 (m, 1H), 3.87–3.82
(m, 1H), 3.78 (m, 1H), 3.75–3.66 (m, 2H), 3.54 (m, 1H), 3.39 (m,
2H), 3.32 (m, 1H), 2.99 (m, 1H), 2.96 (d, 1H, J = 4.8 Hz), 2.92 (s,
1H), 2.76 (m, 4H), 2.69 (t, 2H, J = 6.4 Hz), 2.58 (t, 2H, J = 7.2 Hz),
2.25 (t, 2H, J = 6.8 Hz), 1.71–1.55 (m, 8H), 1.44–1.39 (m, 4H). ESI-
MS m/z 654.13 [M+H]+, 676.47 [M+Na]+.
3.7. Preparation of DP[3H]A
The reaction containing 1 mg of wild-type M. smegmatis mem-
branes, 62.5
and buffer A in final volume of 160
l
M ATP, 10 mM NADH, 500,000 dpm of p[3H]Rpp,
lL was incubated at 37 °C for
1 h, then stopped by the addition of 3 mL of CHCl3/MeOH (2:1),
and the mixture was centrifuged. The supernatant was removed
from the pellet, and 340 lL of buffer A was added to form two
3.4. Preparation of mycobacterial membranes and cell wall
enriched fraction (P60)
phases. After thorough mixing and a brief centrifugation, the aque-
ous phase was discarded. The organic phase was backwashed with
CHCl3/MeOH/H2O (3:47:48). The sample was then dried under air
at room temperature. The radiolabeled material was quantified
and stored in CHCl3/MeOH/H2O/NH4OH (65:25:3.6:0.5) at
ꢁ20 °C.
Wild-type M. smegmatis mc2155 cells were grown in 7H9 con-
taining oleic acid-albumin-dextrose-catalase (OADC) to mid-log
phase (O.D. ꢃ0.7). Cells were harvested by centrifugation, washed
and re-suspended in ice cold buffer A containing 50 mM MOPS (pH
7.9), 5 mM 2-mercaptoethanol and 10 mM MgCl2. Cells were dis-
rupted by three passages through a French Press at 1500 psi. The
suspension was centrifuged at 27,000g for 60 min at 4 °C. The cell
wall pellet was removed and the supernatant re-centrifuged at
100,000g for 2 h at 4 °C. The supernatant was discarded and the
pellet of enzymatically active membranes was gently re-suspended
3.8. SPA beads stock solution
SPA beads were suspended in PBS with 20% glycerol to a con-
centration of 10 mg/mL. This stock solution was stored at 4 °C as
long as one month. A typical assay used 100
lL of this solution
which contains 1 mg of SPA beads.
in 500 ll of buffer A; the protein concentrations of the membrane
fractions were typically between 15 and 20 mg/ml. The 27,000g
pellet was suspended in 10 ml of buffer A and Percoll was added
to achieve a 60% suspension. The suspension was mixed and centri-
fuged at 27,000g for 60 min at 4 °C. The resulting flocculent, white
layer was collected and washed three times with buffer A at
12,000g to yield P60. This fraction was re-suspended in 1 ml of buf-
fer A to yield a protein concentration of 5–6 mg/ml.
3.9. AftB enzyme assay using SPA technology
The typical reaction mixture contained the following compo-
nents in a final volume of 50 L: buffer A, 62.5 M ATP, 20
p[3H]Rpp (200,000 dpm), biotinylated acceptor 4 (100
M), and
l
l
lM
l
indicated amount of membranes and P60. In the cases when using
DP[3H]A (10,000 dpm), Igepal CA-630 (sigma) was used in the as-
say at the final concentration of 0.1%. Reactions were incubated
at 37 °C for 1 h, terminated by adding indicated amount of SPA
beads suspension. The mixture was then incubated overnight at
room temperature with gentle shaking. All incubation and shaking
were performed in 96-well plate (Wallac 1450-401) with sealing
tape (Wallac 1450-461). The [3H]Araf incorporation was measured
by a Wallac MicroBeta scintillation count plate reader (Model
1450) using a parameter in SPA mode. Because the efficiency of
capture by the SPA beads was not known, results were generally
expressed as CPM.
3.5. Arabinosyltransferase assays
Typical reaction mixtures contained buffer A, 62.5
M p[14C]Rpp (500,000 dpm), acceptors 1, 2, 3, or 4 (300
membranes (0.5 mg) and P60 (0.3 mg) in a total volume of
200 L. The reaction mixtures were incubated at 37 °C for 1 h
and then terminated by adding 200 L of ethanol. The resulting
l
M ATP,
3.8
l
lM),
l
l
mixture was centrifuged at 14,000 rpm and the supernatants were
loaded onto prepacked strong anion exchange (SAX) columns (Bur-
dick and Jackson). The columns were eluted with 2 ml of 50% eth-
anol. The eluate was evaporated to dryness and partitioned
between the two phases (1:1) of water saturated 1-butanol and
water. The 1-butanol fractions were measured for radioactive
incorporation by liquid scintillation counting.
Acknowledgements
Research was supported by NIH NIAID AI37139 (D.C., J.Z. and
A.A.); by the ETH Zürich (ETH Grant No. TH-37/05-1) and the Deut-
sche Forschungsgemeinschaft (Emmy Noether postdoctoral fellow-
ship for A.H.). We thank Mr. Shiva Angala for developing the CS35
based ELISA assay with biotinylated acceptor.
3.6. Analytical procedures
For TLC analysis of the enzymatic products, radiolabeled prod-
uct, an aliquot of the 1-butanol extract, was dried under air and

J. Zhang et al. / Bioorg. Med. Chem. 18 (2010) 7121–7131
7131
16. Mikusova, K.; Huang, H.; Yagi, T.; Holsters, M.; Vereecke, D.; D’Haeze, W.;
Scherman, M. S.; Brennan, P. J.; McNeil, M. R.; Crick, D. C. J. Bacteriol. 2005, 187,
8020.
17. Escuyer, V. E.; Lety, M. A.; Torrelles, J. B.; Khoo, K. H.; Tang, J. B.; Rithner, C. D.;
Frehel, C.; McNeil, M. R.; Brennan, P. J.; Chatterjee, D. J. Biol. Chem. 2001, 276,
48854.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2010.07.040.
18. Zhang, N.; Torrelles, J. B.; McNeil, M. R.; Escuyer, V. E.; Khoo, K. H.; Brennan, P.
J.; Chatterjee, D. Mol. Microbiol. 2003, 50, 69.
19. Shi, L.; Berg, S.; Lee, A.; Spencer, J. S.; Zhang, J.; Vissa, V.; McNeil, M. R.; Khoo, K.
H.; Chatterjee, D. J. Biol. Chem. 2006, 281, 19512.
References and notes
1. Migliori, G. B.; Loddenkemper, R.; Blasi, F.; Raviglione, M. C. Eur. Respir. J. 2007,
29, 423.
2. Raviglione, M. C.; Smith, I. M. N. Eng. J. Med. 2007, 356, 656.
3. Gandhi, N. R.; Moll, A.; Sturm, A. W.; Pawinski, R.; Govender, T.; Lalloo, U.;
Zeller, K.; Andrews, J.; Friedland, G. Lancet 2006, 368, 1575.
4. Blanchard, J. S. Annu. Rev. Biochem. 1996, 65, 215.
5. McNeil, M.; Daffe, M.; Brennan, P. J. J. Biol. Chem. 1990, 265, 18200.
6. Besra, G. S.; Khoo, K.-H.; McNeil, M. R.; Dell, A.; Morris, H. R.; Brennan, P. J.
Biochemistry 1995, 34, 4257.
7. McNeil, M.; Daffe, M.; Brennan, P. J. J. Biol. Chem. 1991, 266, 13217.
8. Chatterjee, D.; Bozic, C. M.; McNeil, M.; Brennan, P. J. J. Biol. Chem. 1991, 266,
9652.
9. Daffe, M.; Brennan, P. J.; McNeil, M. J. Biol. Chem. 1990, 265, 6734.
10. Alderwick, L. J.; Seidel, M.; Sahm, H.; Besra, G. S.; Eggeling, L. J. Biol. Chem. 2006,
281, 15653.
11. Seidel, M.; Alderwick, L. J.; Birch, H. L.; Sahm, H.; Eggeling, L.; Besra, G. S. J. Biol.
Chem. 2007, 282, 14729.
20. Makarov, V.; Manina, G.; Mikusova, K.; Mollmann, U.; Ryabova, O.; Saint-
Joanis, B.; Dhar, N.; Pasca, M. R.; Buroni, S.; Lucarelli, A. P.; Milano, A.; De Rossi,
E.; Belanova, M.; Bobovska, A.; Dianiskova, P.; Kordulakova, J.; Sala, C.; Fullam,
E.; Schneider, P.; McKinney, J. D.; Brodin, P.; Christophe, T.; Waddell, S.;
Butcher, P.; Albrethsen, J.; Rosenkrands, I.; Brosch, R.; Nandi, V.; Bharath, S.;
Gaonkar, S.; Shandil, R. K.; Balasubramanian, V.; Balganesh, T.; Tyagi, S.;
Grosset, J.; Riccardi, G.; Cole, S. T. Science 2009, 324, 801.
21. He, X.; Mueller, J. P.; Reynolds, K. A. Anal. Biochem. 2000, 282, 107.
22. Schaeffer, M. L.; Carson, J. D.; Kallender, H.; Lonsdale, J. T. Tuberculosis (Edinb)
2004, 84, 353.
23. Holemann, A.; Stocker, B. L.; Seeberger, P. H. J. Org. Chem. 2006, 71, 8071.
24. Lu, J.; Fraser-Reid, B. Org. Lett. 2004, 6, 3051.
25. Zhang, J.; Khoo, K. H.; Wu, S. W.; Chatterjee, D. J. Am. Chem. Soc. 2007, 129,
9650.
26. Gurjar, M. K.; Reddy, L. K.; Hotha, S. J. Org. Chem. 2001, 66, 4657.
27. Buskas, T.; Söderberg, E.; Konradsson, P.; Fraser-Reid, B. J. Org. Chem. 2000, 65,
958.
12. Birch, H. L.; Alderwick, L. J.; Bhatt, A.; Rittmann, D.; Krumbach, K.; Singh, A.;
Bai, Y.; Lowary, T. L.; Eggeling, L.; Besra, G. S. Mol. Microbiol. 2008, 69, 1191.
13. Skovierova, H.; Larrouy-Maumus, G.; Zhang, J.; Kaur, D.; Barilone, N.;
Kordulakova, J.; Gilleron, M.; Guadagnini, S.; Belanova, M.; Prevost, M. C.;
Gicquel, B.; Puzo, G.; Chatterjee, D.; Brennan, P. J.; Nigou, J.; Jackson, M.
Glycobiology 2009, 19, 1235.
14. Scherman, M.; Weston, A.; Duncan, K.; Whittington, A.; Upton, R.; Deng, L.;
Comber, R.; Friedrich, J. D.; McNeil, M. J. Bacteriol. 1995, 177, 7125.
15. Scherman, M. S.; Kalbe-Bournonville, L.; Bush, D.; Xin, Y.; Deng, L.; McNeil, M. J.
Biol. Chem. 1996, 271, 29652.
28. Du, Y.; Pan, Q.; Kong, F. Synlett 1999, 1648.
29. Ma, Z.; Zhang, J.; Kong, F. Carbohydr. Res. 2004, 339, 1761.
30. Oscarson, S.; Alpe, M.; Svahnberg, P.; Nakouzi, A.; Casadevall, A. Vaccine 2005,
23, 3961.
31. Kaur, D.; Lowary, T. L.; Vissa, V. D.; Crick, D. C.; Brennan, P. J. Microbiology 2002,
148, 3049.
32. Murase, T.; Zheng, R. B.; Joe, M.; Bai, Y.; Marcus, S. L.; Lowary, T. L.; Ng, K. K. J.
Mol. Biol. 2009, 392, 381.
33. Joe, M.; Bai, Y.; Nacario, R. C.; Lowary, T. L. J. Am. Chem. Soc. 2007, 129, 9885.
34. Keppler, B. R.; Jarstfer, M. B. Anal. Biochem. 2006, 353, 75.