Design, synthesis, and x-ray analysis of a glycoconjugate bound to mycobacterium tuberculosis antigen 85C

Article abstract of DOI:10.1021/bc3004342

Tuberculosis (TB) is a global health threat with nearly 500 000 new cases of multidrug-resistant TB estimated to occur every year, so new drugs are desperately needed. A number of current antimycobacterial drugs work by interfering with the biosynthesis of key components of the mycolylarabinogalactan (mAG). In light of this observation, other enzymes involved in the synthesis of the mAG should also serve as targets for antimycobacterial drug development. One potential target is the Antigen 85 (Ag85) complex, a family of mycolyltransferases that are responsible for the transfer of mycolic acids from trehalose monomycolate (TMM) to the arabinogalactan. Virtual thiophenyl-arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5) using Glide. Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides. The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-methylumbelliferyl butyrate fluorescence assay. The conjugates showed K _i values ranging from 18.2 to 71.0 μM. The most potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined. The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.

Full text of DOI:10.1021/bc3004342

Article
pubs.acs.org/bc
Design, Synthesis, and X‑ray Analysis of a Glycoconjugate Bound to
Mycobacterium tuberculosis Antigen 85C
Diaa A. Ibrahim,† Julie Boucau,‡ Daniel H. Lajiness,‡ Sri Kumar Veleti,‡ Kevin R. Trabbic,‡
Samuel S. Adams,‡ Donald R. Ronning,*,‡ and Steven J. Sucheck*,‡
†National Organization for Drug Control & Research, Cairo, Gizaa, Egypt
‡Department of Chemistry, The University of Toledo, 2801 W. Bancroft Street, Toledo, Ohio 43606, United States
S
* Supporting Information
ABSTRACT: Tuberculosis (TB) is a global health threat with
nearly 500 000 new cases of multidrug-resistant TB estimated
to occur every year, so new drugs are desperately needed. A
number of current antimycobacterial drugs work by interfering
with the biosynthesis of key components of the mycolylar-
abinogalactan (mAG). In light of this observation, other
enzymes involved in the synthesis of the mAG should also
serve as targets for antimycobacterial drug development. One
potential target is the Antigen 85 (Ag85) complex, a family of
mycolyltransferases that are responsible for the transfer of
mycolic acids from trehalose monomycolate (TMM) to the
arabinogalactan. Virtual thiophenyl−arabinoside conjugates were docked to antigen Ag85C (PDB code: 1va5) using Glide.
Compounds with good docking scores were synthesized by a Gewald synthesis followed by linking to 5-thioarabinofuranosides.
The resulting thiophenyl-thioarabinofuranosides were assayed for inhibition of mycoyltransferase activity using a 4-
methylumbelliferyl butyrate fluorescence assay. The conjugates showed Ki values ranging from 18.2 to 71.0 μM. The most
potent inhibitor was soaked into crystals of Mycobacterium tuberculosis antigen 85C and the structure of the complex determined.
The X-ray structure shows the compound bound within the active site of the enzyme with the thiophene moiety positioned in
the putative α-chain binding site of TMM and the arabinofuranoside moiety within the known carbohydrate-binding site as
exhibited for the Ag85B-trehalose crystal structure. Unexpectedly, no specific hydrogen bonding interactions are being formed
between the arabinofuranoside and the carbohydrate-binding site of the active site suggesting that the binding of the arabinoside
within this structure is driven by shape complementarily between the arabinosyl moiety and the carbohydrate binding site.
attention is the Antigen 85 complex (Ag85). Ag85 represents a
family (Ag85A, Ag85B, and Ag85C) of homologous mycolyl-
transferases that are responsible for the synthesis of trehalose-
6,6′-dimycolate (TDM) from trehalose-6-monomycolate
(TMM)9−12 and for transfer of mycolic acids from TMM to
the arabinogalactan (AG).13,14 Results from inhibitor studies
suggest that Ag85 is essential for bacterial viability. For
example, 6-azido-6-deoxytrehalose, a known inhibitor of
Ag85s, has been shown to completely inhibit the growth of
Mycobacterium aurum a surrogate for Mycobacterium tuber-
culosis.15 Other studies which support the essentiality of Ag85
include the use of Ag85-specific antisense oligonucleotides,
which were shown to reduce Mycobacterium tuberculosis growth
as well significantly enhance bacterial sensitivity to isonia-
zid.16,17 Similarly, an Ag85A knockout strain of Mycobacterium
smegmatis exhibits increased sensitivity to drugs that target
peptidoglycan biosynthesis, i.e., vancomycin and imipenem.18
INTRODUCTION
■
Tuberculosis (TB) is a global health threat with nearly 500 000
new cases of multidrug-resistant TB (MDF-TB) estimated to
occur every year. More concerning is that, since 2006, strains of
extensively drug-resistant TB (XDR-TB), estimated to be 4% of
MDR-TB, have been reported in over 50 countries. It is also
estimated that 30−40% of XDR-TB is untreatable with the
current antitubercular drug repertoire.1 On this basis, it is clear
that new approaches to treating the disease are needed. Several
of the current drugs used to treat TB work by interfering with
mycobacterial cell wall synthesis.2 Contained within the
structure of the cell is a unique macromolecular structure
called the mycolylarabinogalactan (mAG), which is composed
of the arabinogalactan (AG) and mycolic acids.3 The structure
serves as a protective barrier for the organism and limits the
diffusion of hydrophobic and hydrophilic drugs.4
A number of antimycobacterial drugs, e.g., ethambutol,5,6
isoniazid,7 and ethionamide,8 work by interfering with the
biosynthesis of key components of the mAG. In light of this
observation, other enzymes involved in the synthesis of the
mAG may also serve as targets for antimycobacterial drug
development. One potential target that has attracted some
Received: August 7, 2012
Revised: November 7, 2012
Published: November 29, 2012
© 2012 American Chemical Society
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Figure 1. Proposed mechanism for Ag85-mediated mycolyl transfer resulting in the formation of mAG and TDM.
A variety of putative Ag85 inhibitors have been described.
For example, alkyl phosphonates19 expected to mimic the
tetrahedral intermediate of Ag85s were shown to be active
against Mycobacterium avium and inhibited mycolyltransferase
activity.20 Other examples include trehalose-based substrate
analogues that contain aliphatic chains which showed activity
against Mycobacterium smegmatis,21 and related compounds
consisting of 6,6′-bis(sulfonamido) and N,N′-dialkylamino
derivatives active against Mycobacterium tuberculosis and
Mycobacterium avium.22 Further, a fluorophosphonate deriva-
tive of trehalose was shown to form a covalent adduct with the
active site serine of M. tuberculosis Ag85C.23 More recently, we
have found arabinofuranoside-based substrate analogues
containing S-alkyl chains (Figure 2) that inhibited the growth
of Mycobacterium smegmatis;24−26 however, the latter com-
pounds did not inhibit acyltransferase activity using our
recently developed colorimetric acyltransferase assay.27 On
the other hand, α-ketoester derivatives of arabinofuranosides
showed weak (mM) inhibitory activity against Ag85C but were
inactive in cell-based assays.28 In light of this data, we thought it
was necessary to identify more potent Ag85 inhibitors.
mycolyl moiety. However, it seems more likely that the β-
branch of the mycolyl moiety will remain embedded in the
mycobacterial outer membrane during the mycolyltransfer
reaction. According to the proposed mechanism of the catalytic
mycolyl transfer reaction, Ser124 attacks the carboxyl carbon of
TMM to give a mycolyl-enzyme intermediate and free
trehalose. In the next step, the 6-OH group of a second
TMM molecule attacks the carboxylate carbon of the acyl-
enzyme intermediate to produce TDM. Both the acylation and
deacylation steps proceed via a high-energy tetrahedral
transition state.33 In the case of the formation of the mAG,
the terminal and penultimate primary hydroxyl groups of
arabinan serve as acyl acceptors (Figure 1). Recombinant
Ag85C has been shown to catalyze this reaction in vitro.34 On
the basis of this model and the available crystallographic data,
we envisioned conjugates that would contain both a rigid, drug-
like moiety, that could mimic the affinity of the mycolyl moiety
and could also be conjugated to a fragment of the arabinan for
selectivity (Figure 2). We reasoned that the carbohydrate
component of compounds could occupy the carbohydrate
binding site on Ag85s and provide specificity, while a
hydrophobic component could occupy the pocket leading
into the tunnel proposed to accommodate the α-branch of the
mycolyl moiety. The main difficulty in this endeavor would be
to select an appropriate motif that could accommodate at least
a portion of the putative mycolyl moiety binding site. Our
interests were drawn toward 2-amino-4,5,6,7-tetrahydrobenzo-
[b]thiopenes due to their structural similarity with ebselen
(Figure 2). We have identified the latter compound as a
nanomolar inhibitor of Ag85s via the screening of the NIH
Clinical Collection.35 The ability of this motif to accommodate
the putative binding pocket of mycolyl moiety was confirmed
The crystal structures of secreted forms of Ag85A,29
Ag85B,30,31 and Ag85C29,32,33 from Mycobacterium tuberculosis
have all been determined. The structures reveal an α/β-
hydrolase fold, with a catalytic triad formed by Ser124, Glu228,
and His260 (Ag85C numbering). The carbohydrate binding
site is highly conserved between the three acyltransferases. Near
the catalytic triad is a binding site for the carbohydrate moiety
with a negative electrostatic potential, as well as a hydrophobic
tunnel, well-suited to accommodate the shorter α-branch of the
mycolyl moiety. Additionally, a nearby shallow cleft possessing
hydrophobic character may bind the longer β-branch of the
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growth of M. smegmatis with an MIC of 50−100 μg/mL.39
More recently, 2-amino-6-propyl-4,5,6,7-tetrahydro-1-benzo-
thiophene-3-carbonitrile (I3-AG85, Figure 2) was identified as
a second-generation inhibiter with MICs of 100 μg/mL against
M. tuberculosis. The I3-AG85 reduced survival of M. tuberculosis
inside macrophages and at a 100 μM concentration. The
compound inhibited the growth of 7 MDR and 3 XDR M.
tuberculosis strains at 200 μM or lower. Finally, I3-AG85 was
shown to inhibit the synthesis of TDM. The generalized
structures, which we explored in docking studies, are shown in
Figure 2.
EXPERIMENTAL PROCEDURES
■
Biological Methods. KM Determination for 4-MUB.
Ag85C was purified as described previously.27 All assays were
performed under atmospheric pressure at 37 °C in a 384-well
format on a Synergy H4 Hybrid Multi-Mode Microplate
Reader (Biotek). All 4-methylumbelliferyl butyrate (4-MUB)
stock solutions were made and stored in 100% DMSO at −20
°C. Excitation and emission wavelengths, 365 and 450 nm,
respectively, were used to monitor the production of the 4-
methylumbelliferone (4-MU) at 20 s intervals for 30 min. For
each reaction, a master mix was made consisting of 600 nM
Ag85C, 10 mM trehalose, and an appropriate volume of 20 mM
bis(2-hydroxyethyl)amino-tris(hydroxymethyl)methane (Acros
Organics) for an overall reaction volume of 50 μL. 0.5 μL of 4-
MUB was added from each corresponding stock solution to
reach the desired substrate concentration (25−400 μM). The
data were normalized using negative controls where no Ag85C
Figure 2. Design of arabinose−thiophene conjugates designed to act
as inhibitors of Ag85s. Structures of an S-alkyl arabinofuranoside,
ebselen, and I3-AG85.
by a docking strategy. Once identified, we noted that the 2-
amino-4,5,6,7-tetrahydrobenzo[b]thiophene had been used
extensively as a scaffold for enzyme inhibitor development.36−40
We have previously disclosed the structure of 1a (Scheme
1).41,42 We also note, 2-amino-4,5,6,7-tetrahydrobenzo[b]-
thiophene-3-carbonitrile (1b, Scheme 1) has been independ-
ently identified as a binder of Ag85C using an 15N-HSQC
NMR spectroscopy-based fragment screening protocol.43,44
The resulting thiophene was found to selectively inhibit the
a
Scheme 1. Synthesis of Tetrahydrobenzo[b]thiophenes
a
Reagent and conditions: (i) sulfur, N-ethylmorpholine, EtOH, 1a (27%), 1b (33%); (ii) Et3N, CH2Cl2, rt, 2a (11%), 2b (9%), 2c (15%), 2d (29%);
(iii) KSCOCH3, DMF, rt, 3a (62%), 3b (66%), 3c (96%), 3d (78%); (iv) toluene, 112 °C, 3 h (53%); (v) CH2Cl2, rt, 4a (39%), 4b (67%).
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was added. The background fluorescence from the negative
control was subtracted from the intensities measured from the
enzyme-catalyzed reaction. To correlate relative fluorescence
intensity with the amount of product, the fluorescence of 4-MU
(1−1000 nM) was determined using reaction conditions. RFU/
min rates from the enzyme-catalyzed reaction were directly
correlated to the amount of 4-MU produced per minute using
the equation for the line of best fit, using Microsoft Excel.
GraphPad Prism 5 software was used to determine Michelis-
Menten parameters via nonlinear regression analysis.
Inhibition Assays. All stock solutions for each compound
were made and stored in 100% DMSO. Excitation and emission
scans of each compound to be tested were conducted to avoid
regions of overlap with 4-methylumbelliferone. The excitation
and emission wavelengths used for each compound were as
follows: SA-1, λex = 320 nm, λem = 500 nm; KT-1-189, λex = 380
nm, λem = 500 nm; DI-1, λex = 380 nm, λem = 500 nm; DI-2, λex
= 320 nm, λem = 450 nm. All fluorescence intensities were
determined at 20 s intervals over 30 min. The identities and
concentrations of the reaction components used for determin-
ing the KM were employed in these reactions as well; however,
the 4-MUB concentration was held constant at 100 μM. 0.5 μL
of each tested inhibitor was added from its corresponding stock
solution to give final concentrations ranging from 20 μM to 320
μM. Inhibition was determined by comparing the relative rate
of the reaction performed with inhibitor against a reaction that
contained no inhibitor (vi′/vi, where vi′ and vi are steady-state
rates with and without inhibitor, respectively). The equilibrium
dissociation constant (Ki) for each inhibitor was obtained by
fitting the data into the equation below using Prism:
in DMSO (20 mg/mL) and INH (500 μg/mL) were applied to
6-mm-diameter sterile paper disks. The plates were incubated at
37 °C for 24 h and then analyzed.
Synthetic Methods. Ethyl 2-Amino-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate (1a). Cyclohex-
anone (30.0 mL, 290 mmol), sulfur (9.30 g, 290 mmol), N-
ethylmorpholine (25.2 mL, 200 mmol), ethyl cyanoacetate
(51.5 mL, 290 mmol) and ethanol (320 mL) were combined in
a round-bottom flask and stirred at room temperature. After 3 h
of continuous stirring, the reaction was complete and filtered to
remove sulfur. The solvent was evaporated under reduced
pressure. The product was extracted with 200 mL of methanol
and crystallized by adding 100 mL of water to the methanol
solution. The crystals were filtered and rinsed with cold 1:1
methanol−water: yield = 17.6 g (27%); mp = 117−119 °C
(Lit:51113−117 °C); Rf = 0.55 (4:1 hexanes−ethyl acetate);
mass spectrum (ESIMS), m/z = 226 (M+H)+, C11H106NO2S
requires 226.
2-Amino-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboni-
trile (1b). Cyclohexanone (30.0 mL, 290 mmol), malonitrile
(19.8 g, 290 mmol), sulfur (9.30 g, 290 mmol), and 25.2 mL of
N-ethylmorpholine (25.2 mL, 200 mmol) were combined in a
round-bottom flask and stirred at room temperature. After 3 h
of continuous stirring, the reaction was complete and filtered to
remove sulfur. The solvent was evaporated under reduced
pressure, and the product was extracted by 200 mL of
methanol. The product was crystallized by adding 100 mL of
water to the methanol solution. The crystals were filtered and
rinsed with a solution of cold 4:1 methanol−water: yield = 17.0
g (33%); mp = 147−149 °C (Lit:52 147 °C); Rf = 0.35 (9:1
hexane−ethyl acetate); mass spectrum (ESIMS), m/z = 179
(M+H)+, C9H11N2S requires 179.
v′
KM + [S]
KM + [S] +
i
=
KM[I]
Ki
v
Ethyl 2-[(2-Chloroacetyl)amino]-4,5,6,7-tetrahydrobenzo-
[b]thiophene-3-carboxylate (2a).53 Compound (1a) (0.500
mg, 2.2 mmol) was treated with chloroacetyl chloride (0.35
mL, 4.4 mmol) in chloroform (5 mL) and reaction was refluxed
for 3 h. The reaction was analyzed for completion by TLC with
9:1 hexanes−ethyl acetate. The reaction solution was
concentrated under reduced pressure and the residue crystal-
lized from chloroform−hexanes: yield 0.231 g (34%); mp =
118−121 °C (Lit:54 115−117 °C); Rf = 0.49 (9:1 hexanes−
i
KM is the Michelis-Menten constant for 4-MUB; [S] and [I] are
the concentrations of 4-MUB and inhibitor, respectively.
Protein Crystallization, Diffraction, and Model Building.
The Ag85C crystals were obtained using the hanging drop
method where 1 μL of purified Ag85C was combined with 1 μL
of the well solution containing 0.3 M ammonium sulfate and
0.1 M sodium acetate. These crystals were prepared for X-ray
diffraction studies by adding 1 μL of a 10.7 mM solution of 13a
dissolved in an 80% v/v solution of DMSO to the 2 μL
crystallization drop. These crystals were incubated at 16 °C for
2 days prior to data collection. Crystals were cryoprotected by
adding glycerol directly to the drop containing the Ag85C
crystal and 13a. Diffraction data were collected at the LS-CAT
beamline at the Advanced Photon Source using a wavelength of
0.978 56 Å. Data were reduced using HKL2000.45 Molecular
replacement was performed using EPMR.46 Manual model
building was performed using Coot.47 Refinement, building
inhibitor 13a, and addition of glycerol and water molecules was
performed using Phenix.48,49 Glycerol molecules were manually
fit to Fo − Fc density using Coot.
Kirby-Bauer Disk Diffusion Assay. Disk diffusion assays were
adapted from methods outlined by the NCCLS and Wang et
al.21,50 Mycobacterium smegmatis ATCC 14468 was inoculated
into Middlebrook 7H9 containing 0.2% glycerol and ADC
enrichment, respectively. The inocula were incubated for
approximately 48 h at 37.5 °C at 160 rpm. The bacteria were
plated on agar (Lennox or Middlebrook 7H10 containing 0.5%
glycerol and OADC Enrichment) using sterile cotton-tipped
applicators. Aliquots (10 μL) of arabinose derivatives dissolved
1
ethyl acetate); H NMR (CDCl3, 400 MHz): δ1.39 (t, J = 7.2
Hz, 3H), 1.79−1.81 (m, 4H), 2.66 (t, J = 4.4 Hz, 2H), 2.79 (t, J
= 5.2 Hz, 2H), 4.26 (s, 2H), 4.36 (q, J = 7.2 Hz, 2H), 12.14 (br.
s, 1H, NH); mass spectrum (ESIMS), m/z = 324 (M+Na)+,
C13H16ClNO3S requires 324.
Ethyl 2-[(4-Chlorobutanoyl)amino]-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (2b). Com-
pound 1a (1.13 g, 5.00 mmol), triethyl amine (2.10 mL, 15.0
mmol), and dry dichloromethane (20.0 mL) were combined in
a round-bottom flask. 4-Chlorobutyryl chloride (1.20 mL, 15.0
mmol) was added and the reaction stirred at room temperature
for 6 h. The solution was washed with 30.0 mL of 1 M HCl
followed by 30.0 mL of saturated sodium bicarbonate solution.
The organic layer was dried using anhydrous sodium sulfate,
filtered, and rinsed with dichloromethane. The filtrate was
evaporated to dryness, and the product was purified by flash
column chromatography on silica gel (30 mm × 150 mm). The
column was eluted with 3:1 chloroform−hexanes and
concentrated to dryness under reduced pressure. The residue
was crystallized from ethyl acetate−hexanes. After cooling to
room temperature, the crystals were filtered and rinsed with
20.0 mL of cold 10:1 hexanes−ethyl acetate: yield = 0.47 g
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(9.4%); mp = 70−72 °C; Rf = 0.63 (3:1 chloroform−hexanes);
1H NMR (DMSO-d6, 600 MHz): δ 1.39 (t, J = 6.8 Hz, 3H),
1.54−1.57 (m, 2H), 1.79−1.80 (m, 4H), 2.23 (p, J = 7.2 Hz,
2H), 2.65−2.69 (m, 2H), 2.78 (t, J = 6 Hz, 2H), 3.65 (t, J = 6
Hz, 2H), 4.33 (q, J = 7.2 Hz, 2H), 11.35 (s, 1H); 13C NMR
(DMSO-d6, 150 MHz) δ: 14.5, 23.0, 23.1, 24.5, 26.5, 27.9, 33.6,
44.3, 60.7, 111.7, 126.9, 130.9, 147.6, 166.8, 168.8; mass
spectrum (HRMS), m/z = 330.0910 (M+H)+, C15H21ClNO3S
requires 330.0931.
2-Chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thien-2-
yl)acetamide (2c). Compound (1b) (0.500 g, 2.8 mmol) was
treated with chloroacetyl chloride (0.44 mL, 5.61 mmol) in
chloroform (5 mL). The reaction was refluxed for 3 h. The
reaction was analyzed for completion by TLC with 9:1
hexanes−ethyl acetate. The reaction solution was concentrated
under reduced pressure. The residue is crystallized from
chloroform−hexanes: yield 0.418 g (59%); mp = 180−183 °C
(Lit:24 171−173 °C); Rf = 0.3 (9:1 hexanes−ethyl acetate); 1H
NMR (CDCl3, 400 MHz); δ 1.85 (m, 4H), 2.62 (t, J = 5.6 Hz,
2H), 2.66 (t, J = 4.4 Hz, 2H), 4.28 (s, 2H), 9.38 (s, 1H); mass
spectrum (ESIMS), m/z = 277 (M+Na)+, C11H11ClN2OS
requires 277.
derivative 2a (100 mg, 0.332 mmol) in anhydrous DMF (5.0
mL) under nitrogen. The reaction mixture was stirred at room
temperature for 20 h and diluted with ethyl acetate (10.0 mL).
The resulting solution was washed with water and the
combined organic extracts were dried with anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography on
silica gel (30 mm × 150 mm) eluting with 1:4 ethyl acetate−
hexanes. The solid could be crystallized from ethyl acetate−
hexanes to generate yellow microcrystals: yield = 68.9 mg
(62%); mp = 115−116 °C; Rf = 0.48 (1:4 ethyl acetate−
1
hexanes); H NMR (CDCl3, 400 MHz): δ 1.38 (t, J = 7.2 Hz,
3H) 1.77−1.78 (m, 4H), 2.46 (s, 3H), 2.63−2.64 (m, 2H),
2.76−2.77 (m, 2H), 3.82 (s, 2H), 4.36 (q, J = 6.8 Hz, 2H),
11.68 (s, 1H); 13C NMR (CDCl3, 150 MHz): δ 14.3, 22.8,
22.9, 24.3, 26.3, 30.2, 33.0, 60.5, 112.3, 127.1, 131.0, 146.6,
164.8, 166.2, 194.0; mass spectrum (HRMS), m/z = 364.0630
(M+Na)+, C15H19NNaO4S2 requires 364.0653.
Ethyl 2-(4-(Acetylthio)butanamido)-4,5,6,7-
tetrahydrobenzo[b]thiophene-3-carboxylate (3b). The solid
could be crystallized from ethyl acetate−hexanes to generate
1
yellow needles: yield 0.78g (66%); mp = 74.0−74.5 °C; H
NMR (CDCl3, 600 MHz): δ 1.38 (t, J = 7.2 Hz, 3H), 1.78 (m,
4H), 2.03 (m, 2H), 2.33 (s, 3H), 2.54 (m, 2H), 2.63 (m, 2H),
2.76 (m, 2H), 2.97 (m, 2H), 4.37 (q, J = 6.8 Hz, 2H), 11.28 (s,
1H); 13C NMR (CDCl3, 600 MHz): δ 14.3, 22.8, 22.9, 24.3,
25.0, 26.3, 28.3, 30.6, 35.2, 60.5, 111.4, 126.6, 130.6, 147.3,
166.6, 195.5; mass spectrum (HRMS), m/z = 392.0938 (M
+Na)+, C17H23NNaO4S2 requires 392.0966.
4-Chloro-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]thien-2-
yl)butanamide (2d). Compound 1b (1.07 g, 6.00 mmol),
triethyl amine (2.50 mL, 18.0 mmol), and dry dichloromethane
(20 mL) were combined in a round-bottom flask. The reaction
occurred under dry nitrogen and 4-chlorobutyryl chloride (2.03
mL, 18.0 mmol) was added. The reaction was analyzed for
completion by TLC using 3:1 hexanes−ethyl acetate. The
solution was washed with 30 mL of 1 M HCl in a separatory
funnel; the organic layer was then washed with 30 mL saturated
sodium bicarbonate solution. The organic layer was dried using
anhydrous sodium sulfate, filtered, and rinsed with dichloro-
methane. The product was concentrated to dryness under
reduced pressure. The product was decolorized using carbon
decolorizing charcoal in ethyl acetate solution. The solution
was filtered, concentrated, and crystallized by chloroform and
hexanes. Crystals were rinsed and filtered with cold 1:1
chloroform−hexanes: yield = 1.48 g (29%); mp = 158−161 °C
(Lit:54 mp = 186−187 °C, Lit:55 mp = 143−146 °C); Rf = 0.5
4-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylami-
no)-4-oxoethyl ethanethioate (3c). The solid could be
crystallized from ethyl acetate−hexanes to generate yellow
1
needles: yield 0.89g (96%); mp = 170.0−170.5 °C; H NMR
(CDCl3, 600 MHz): δ 1.83−1.79 (m, 4H), 2.47 (s, 3H), 2.58−
2.57 (m, 2H), 2.63−2.59 (m, 2H), 3.78 (s, 2H), 9.48 (s, 1H);
13C NMR (CDCl3, 600 MHz): δ 22.0, 23.0, 23.9, 23.9, 30.2,
32.8, 94.1, 113.9, 128.6, 131.1, 146.1, 165.1, 196.5; mass
spectrum (ESIMS), m/z = 295 (M+H)+, C13H15N2O2S2
requires 295.
4-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-ylami-
1
1
(3:1 hexanes−ethyl acetate); H NMR (CDCl3, 400 MHz): δ
no)-4-oxobutyl ethanethioate (3d). Yield 0.79g (78%); H
1.92−1.87 (m, 2H), 2.15 (p, J = 6.4 Hz, 2H), 2.70 (t, J = 5.6
Hz, 1H), 2.77 (t, J = 4.4 Hz, 1H), 2.89 (t, J = 7.2 Hz, 2H), 3.63
(t, J = 6.0 Hz, 2H) 9.08 (s, 1H); 13C NMR (CDCl3, 100 MHz):
δ 22.3, 23.3, 24.1(2), 32.8, 27.9, 44.4, 92.5, 115.0, 128.2, 130.9,
147.7, 169.6; mass spectrum (ESIMS), m/z = 283 (M+H)+,
C13H16ClN2OS requires 283.
2-(Acetylthio)alkanamido-4,5,6,7-tetrahydrobenzo[b]-
thiophene derivatives 3a−d. Potassium thioacetate (0.423
mg, 3.78 mmol) was added to a solution of 2-(chloroalkyl)
aminothiophene derivatives 2a−d (3.15 mmol) in anhydrous
DMF (25 mL) under nitrogen. The reaction mixture was
stirred overnight, and the solvent was removed under reduced
pressure. The residue was dissolved in ethyl acetate (15 mL),
and the resulting solution was washed with water. The
combined organic extracts were dried over anhydrous
Na2SO4, filtered, and the solvent was removed under reduced
pressure. The residue was purified by flash chromatography to
give the acetylthio derivatives 3a−d.
NMR (CDCl3, 600 MHz): δ 1.85 (m, 4H), 2.03 (m, 2H), 2.33
(m, 2H), 2.34 (s, 3H), 2.55 (m, 4H), 2.95 (m, 2H), 9.40 (s,
1H); 13C NMR (CDCl3, 600 MHz): δ 22.8, 22.9, 24.3, 25.0,
26.3, 29.3, 32.6, 34.2, 91.5, 114.4, 128.6, 132.6, 148.3, 170.8,
196.5; mass spectrum (HRMS), m/z = 322.0527 (M+Na)+,
requires 322.0548.
Ethyl 2-[(4-Bromobutanoyl)amino]-4, 5, 6, 7-
tetrahydrobenzo[b]thiophene-3-carboxylate (4a). Com-
pound 1a (0.600 g, 2.66 mmol) was dissolved in dichloro-
methane (15 mL) and cooled to 0 °C. 4-Bromobutyryl chloride
(0.366 mL, 3.16 mmol) was added dropwise under nitrogen
atmosphere. The reaction was stirred continuously for 1.5 h
and analyzed for completion by TLC using 3:1 (hexanes−ethyl
acetate). Upon completion, the reaction mixture was washed
with brine (3 × 20 mL), satd NaHCO3 (3 × 20 mL), and 1 N
HCl (3 × 20 mL). The organic layer was dried using anhydrous
sodium sulfate, filtered, and rinsed with dichloromethane. The
crude product was purified by flash column chromatography on
silica gel (30 mm × 150 mm) using 1:3 hexanes−ethyl acetate
as the eluent. The product was concentrated to dryness and
recrystallized from hot hexanes: yield = 0.392 g (39%); mp =
E t h y l 2 - ( 2 - ( A c e t yl t hi o ) a c e ta m i d o ) - 4 , 5 , 6 , 7 -
tetrahydrobenzo[b]thiophene-3-carboxylate (3a). Potassium
thioacetate (43.9 mg, 0.385 mmol) was added to a conical vial
containing a solution of 2-(chloroalkyl) aminothiophene
1
80−81 °C; Rf = 0.5 (3:1 hexanes−ethyl acetate); H NMR
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(CDCl3, 400 MHz): δ 1.39 (t, J = 6.8 Hz, 2H, 3H), 1.77−1.79
(m, 4H), 2.31 (p, J = 6.4 Hz, 2H), 2.62−2.67 (m, 4H), 2.69−
2.77 (m, 2H), 3.52 (t, J = 6.4 Hz, 2H), 4.33 (p, J = 7.2 Hz, 2
H), 11.34 (s,1H); 13C NMR (CDCl3, 400 MHz) δ 14.5, 23.0,
23.1, 24.5, 26.5, 28.0, 33.0, 34.9, 60.7, 111.6, 126.9, 131.0,
147.4, 166.3, 168.7; mass spectrum (HRMS), m/z = 396.0253
(M+H)+ C15H2oBrNO3SNa requires 396.0245; Anal. Calcd for
C15H20BrNO3S: C, 48.13; H, 5.39; N, 3.74. Found: C, 48.78;
H, 5.41; N, 3.77.
mL). The organic layers were combined and back extracted
with water and brine. Anhydrous sodium sulfate was used to
dry the solution, which was then filtered and concentrated. The
crude product was purified by flash column chromatography on
silica gel (50 mm × 150 mm) eluting with 3:3:14 acetone−
chloroform−hexanes (500 mL), followed by 1:1:3 acetone−
chloroform−hexanes (250 mL), and finally followed by 1:1:2
acetone−chloroform−hexanes (200 mL). The product frac-
tions were pooled and concentrated: yield 633 mg (65%); Rf =
0.45 (1:9 methanol−chloroform).
Methyl 5-O-p-Toluenesulfonyl-2,3-di-O-acetyl-α-D-arabi-
nofuranoside (7)..57,58 Compound 7 (1.28 g, 3.88 mmol)
was dissolved in 30 mL of 1:1 pyridine−acetic anhydride in a
round-bottom flask. The solution was allowed to stir at room
temperature under dry nitrogen for 24 h. The reaction was
quenched with ice (30.0 g) and extracted using dichloro-
methane and back-extracted with brine. The solution was dried
with anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The crude product was purified by flash
column chromatography on silica gel (30 mm × 150 mm). The
product was eluted with 1:3 ethyl acetate−hexanes, followed by
1:1 ethyl acetate−hexanes (100 mL). The product fractions
were pooled and concentrated under reduced pressure: yield
1.15 g (73%); Rf = 0.16 (1:3 ethyl acetate−hexanes); 1H NMR
(CDCl3, 600 MHz): δ 2.08 (s, 6H), 2.45 (s, 3H), 3.36 (s, 3H),
4.16−4.18 (m, 1H), 4.26 (dd, 1H, J = 5.4, 10.8 Hz), 4.32 (dd,
1H, J = 3, 10.8 Hz), 4.87 (m, 1H), 4.89 (s, 1H), 5.02 (s, 1H),
7.35 (d, 2H, 6.6 Hz), 7.81 (d, 1H, J = 6.6 Hz); 13C NMR (600
MHz, CDCl3): δ 20.9, 21.9, 55.6, 68.9, 71.4, 75.9, 78.8, 80.7,
81.1, 81.1, 107.0, 128.1, 128.2, 128.3, 130.1, 130.1, 133.0, 145.2,
170.1, 170.5, 170.7.
4-Bromo-N-(3-cyano-4,5,6,7-tetrahydrobenzo[b]-
thiophen-2-yl)butanamide (4b). Compound 1b (1.00 g, 5.61
mmol) was dissolved in dichloromethane (25 mL) and cooled
to 0 °C. 4-Bromobutyryl chloride (0.773 mL, 6.68 mmol) was
added dropwise under nitrogen atmosphere and the reaction
was stirred continuously for 1.5 h. The reaction was analyzed
for completion by TLC using 3:1 hexanes−ethyl acetate. Upon
completion, the reaction mixture was washed with brine (3 ×
20 mL), satd NaHCO3 (3 × 20 mL), and 1 N HCl (3 × 20
mL). The organic layer was dried using anhydrous sodium
sulfate, filtered, and concentrated under reduced pressure. The
crude product was purified by flash column chromatography on
silica gel (30 mm × 150 mm) using 1:3 hexanes−ethyl acetate
as the eluent. The product was concentrated to dryness and
recrystallized using dichloromethane−hexanes: yield = 1.23 g
(67%); mp = 173−174 °C; Rf = 0.5 (3:1 hexanes−ethyl
1
acetate); H NMR (CDCl3, 600 MHz): δ 1.53−1.59 (m, 1H),
1.83−1.84 (m, 4H), 2.31 (p, J = 6.6 Hz, 2H), 2.60−2.64 (m,
4H), 2.69 (t, J = 7.2 Hz, 2H), 3.53 (t, J = 6.0 Hz, 2H), 8.57
(m,1H, NH); 13C NMR (CDCl3, 600 MHz): δ 22.3, 23.3, 24.1,
24.2, 27.9, 33.1, 34.1, 92.8, 114.9, 128.4, 131.0, 147.3, 169.2;
mass spectrum (ESIMS), m/z = 349 (M+Na)+
C13H15BrN2NaOS requires 349; Anal. Calcd for
C13H15BrN2OS: C, 47.71; H, 4.62; N, 8.56. Found: C, 47.82;
H, 4.44; N, 8.53.
Methyl 2,3-Tri-O-acetyl-5-S-acetyl-5-thio-α-D-arabinofura-
noside (8).21 Compound 7 (1.15 g, 2.86 mmol) was dissolved
in 10 mL of anhydrous DMF in a round-bottom flask. An
additional 10 mL of anhydrous DMF was used to dissolve
KSCOCH3 (0.40 g, 3.50 mmol). The KSCOCH3 solution was
then added dropwise to the starting material and allowed to stir
at room temperature under nitrogen for 24 h. The solution was
diluted with 50 mL of water and extracted with three 30 mL
portions of ether. The ether layers were combined and washed
with saturated sodium chloride solution. The ether was dried
with anhydrous sodium sulfate, filtered, and concentrated under
reduced pressure. The crude product was purified by flash
column chromatography on silica gel (30 mm × 150 mm). The
product was eluted with 1:1:5 acetone−chloroform−hexanes
and concentrated to dryness under reduced pressure: yield =
Ethyl 2-(3-(2-Chloroethyl)ureido)-4,5,6,7-tetrahydrobenzo-
[b]thiophene-3-carboxylate (5). A solution of ethyl 2-amino-
4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxylate 1a (1.125 g,
5.00 mmol) in toluene (5 mL) was treated with 2-chloroethyl
isocyanate (0.636 mL, 7.50 mmol). The resulting solution was
refluxed at 112 °C for 3 h under dry nitrogen. The solution was
allowed to cool to room temperature and was then
concentrated under reduced pressure. The crude product was
purified by flash column chromatography on silica gel (75 mm
× 50 mm) eluting with 1:9 ethyl acetate−hexanes. The product
was concentrated to dryness and crystallized from 2:2:6
dichloromethane−diethyl ether−hexanes): yield 867 mg
(53%); Rf = 0.56 (3:7 ethyl acetate−hexanes); mp = 119−
1
525 mg (60%); Rf = 0.39 (2:3 ethyl acetate−hexanes); H
123 °C (Lit:56 123−125 °C); H NMR (600 MHz, CDCl3): δ
NMR (CDCl3, 600 MHz): δ 2.03 (s, 6H), 2.33 (s, 3H), 3.12−
3.17 (m, 1H), 3.32−3.37 (m, 4H), 4.14 (dd, 1H, J = 5.2, 12
Hz), 4.84 (s, 1H), 4.86 (d, 1H, J = 6.0), 4.98 (s, 1H). 13C NMR
(600 MHz, CDCl3): 21.0, 30.7, 31.3, 55.2, 78.2, 79.7, 81.9,
106.8, 170.0, 170.3, 194.9; mass spectrum (HRMS), m/z =
329.0646 (M+Na)+, C12H18NaO7S requires 329.0671.
1
1.37−1.35 (t, J = 7.2 Hz, 3H), 1.78−1.77 (m, 4H), 2.60−2.58
(d, J = 6 Hz, 2H), 2.74−2.73 (d, J = 6 Hz, 2H), 3.68−3.65 (m,
4H), 4.31−4.27 (q, J = 7.2, Hz, 2H), 5.52 (s, 1H); 13C NMR
(600 MHz, CDCl3): δ 14.4, 23.0, 24.4, 26.5, 42.5, 44.2, 60.4,
77.2, 109.4, 125.1, 130.6, 150.8, 153.7, 167.1; mass spectrum
(ESIMS), m/z = 332 (M+H)+, C14H20ClN2O3S requires 332.
Methyl 5-O-p-Toluenesulfonyl-α-D-arabinofuranoside
(6).24 Methyl α-D-arabinofuranoside24 (3.05 mmol) was
dissolved in 20 mL of pyridine. Tosyl chloride was then
added in slight excess (3.25 mmol) and the solution stirred at
37 °C for 24 h under dry nitrogen. The heat was removed and
the reaction was allowed to progress at room temperature for
an additional 5 days. Ice was used to quench the reaction and
an additional 60 mL of water was added along with 15 mL of
brine. The solution was extracted with ethyl acetate (3 × 35
Methyl α-D-Arabinofuranoside-5-thiol (9). A solution of 8
(3.37 g, 11.0 mmol) in 0.1 M methanolic NaOMe (40 mL) was
stirred for 4 h at 50 °C under nitrogen and neutralized with
Amberlite IR120 (H+ form). The resin was filtered off, and the
filtrate was concentrated under reduced pressure. The residue
was extracted with ethyl acetate to provide thiol 9 after
1
concentration: yield 1.21 g (61%); H NMR (CDCl3, 600
1
MHz): δ 1.68−1.65 (t, H, J = 6.0 Hz), 2.81−2.77 (m, 1H),
2.89−2.81 (m, 1H), 3.39 (s, 3H), 3.92−3.91 (m, 2H), 4.09−
4.06 (m, 3H), 4.85 (s, 1H); 13C NMR (CDCl3, 600 MHz): δ
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26.9, 55.8, 79.9, 81.7, 84.4, 108.3; mass spectrum (HRMS), m/z
= 203.0337 (M+Na)+, C6H12NaO4S requires 203.0354.
5-Iodo-α-D-arabinofuranoside (11). Methyl α-D-arabinofur-
anoside (10) (1.00 g, 6.66 mmol) was dissolved in dry THF
(10 mL) and placed on ice. The reaction was cool to 0 °C in an
ice bath and imidazole (1.07 g, 15.8 mmol), iodine (2.01 g, 7.92
mmol), and triphenylphosphine (2.00 g, 7.92 mmol) were
added. The solution was warmed to 65 °C and allowed to stir
for 1.5 h. The solution was diluted with 50 mL of water and
extracted with dichloromethane (3 × 30 mL). The organic
layers were combined, dried with anhydrous sodium sulfate,
filtered, and concentrated under reduced pressure. The crude
product was purified by flash column chromatography on silica
gel (30 mm × 150 mm). The product was eluted with 8:1
chloroform−acetone and concentrated to dryness under
reduced pressure: yield = 1.13 g (65%); Rf = 0.50 (3:1
chloroform−acetone); 1H NMR (DMSO, 400 MHz) δ 3.25 (s,
2H), 3.35 (s, 3H), 3.46−3.59 (m, 2H), 3.83 (s, 1H), 4.64 (s,
1H), 5.39−5.40 (m,1H), 5.49−5.50 (m,1H); 13C NMR
(DMSO, 100 MHz) δ 09.1, 54.5, 80.6, 81.2, 82.0, 108.8;
mass spectrum (ESIMS), m/z = 297 (M+Na)+. Anal. Calcd for
C6H11IO4: C, 26.30; H, 4.05; N, 0.00; Found: C, 26.04; H,
4.02; N, 0.03.
concentrated to dryness under reduced pressure. Compound
12b was obtained as brown color residue: yield 0.216 g (83%);
mass spectrum (ESIMS), m/z = 295 (M)+, C12H15N4OS2
+
requires 295. Step 2: Compound 12b (0.118 g, 0.36 mmol) was
combined with Compound 11 (0.05 mg, 0.18 mmol) and
cesium carbonate (0.234 g, 0.72 mmol) in dry DMF (2 mL).
The solution was stirred for 16 h at 85 °C under nitrogen
atmosphere. The reaction was monitored in thin layer
chromatography using (2:1 toluene−acetone). On completion,
the crude reaction mixture was loaded onto silica gel and the
residual solvent evaporated under reduced pressure. The
sample was loaded on silica gel column (30 mm × 150 mm)
and eluted with hexanes followed by 4:1 toluene−acetone to
afford the product. The product was orange amorphous solid:
yield 0.040 g (56%); Rf = 0.39 (2:1 toulene−acetone); mp =
1
160−163 °C. H NMR (CD3OD, 600 MHz): δ 1.8 (m, 4H),
2.51 (m, 2H), 2.59 (m, 2H), 2.85 (dd, J = 24.6 Hz, 7.2 Hz, 1H),
2.93 (dd, J = 18.6 Hz, 4.2 Hz, 1H), 3.26 (s, OCH3), 3.52 (d, J =
15 Hz, 1H), 3.54 (d, J = 15 Hz, 1H), 3.76 (m, 1H), 3.86 (m,
1H), 3.98 (m, 1H), 4.68 (d, J = 1.8 Hz, 1H). 13C NMR
(CD3OD, 400 MHz): δ 23.4, 24.3, 24.9, 25.1, 35.9, 36.5, 55.4,
81.7, 83.6, 84.5, 110.5, 114.9, 129.9, 132.6, 169.9; mass
spectrum (HRMS), m/z
=
421.0873 (M+Na)+ ,
Ethyl 2-(Methyl 5-thio-α-D-arabinofuranoside-5-S-yl)-
acetamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxy-
late (13a). Step 1: Compound (2a) (0.100 g, 0.33 mmol) and
thiourea (0.504 g, 0.66 mmol) were suspended in 2 mL of
absolute ethanol and stirred for 3 h at 65 °C. The reaction was
analyzed for completion by TLC 3:1 hexanes−ethyl acetate.
The reaction was cooled and filtered. The filtrate was rinsed
with absolute ethanol and concentrated to dryness under
reduced pressure. Compound 12a was obtained as yellow color
residue: yield 0.100 g (80%); mass spectrum (ESIMS), m/z =
C17H22N2O5S2 requires 421.0868.
Ethyl 2-(Methyl 5-thio-α-D-arabinofuranoside-5-S-yl)-
butamido-4,5,6,7-tetrahydrobenzo[b]thiophene-3-carboxy-
late (13c). Step 1: Compound 4a (0.275 g, 0.725 mmol) and
thiourea (0.110 g, 1.45 mmol) were suspended in 2 mL of
absolute ethanol and stirred at reflux for 4 h. The reaction was
analyzed for completion by TLC using 3:1 hexanes−ethyl
acetate. The reaction was cooled on ice and filtered. The filtrate
was rinsed with a minimal amount of absolute ethanol and
concentrated to dryness under reduced pressure. Compound
12c was obtained as colorless crystals. yield = 0.170 g (52%);
mp = 212−213 °C; 1H NMR (DMSO, 400 MHz) δ 1.25 (t, J =
7.2 Hz, 3H), 1.63 (br. s, 4H), 1.85−1.92 (m, 2H), 2.59−2.62
(m, 4H), 3.18 (t, J = 7.2 Hz, 2H), 3.37−3.41 (m, 1H), 4.22 (p,
J = 6.8 Hz, 2H), 9.00−9.11 (m, 4H), 10.95 (s, 1H); 13C NMR
(DMSO, 400 MHz) δ 14.1, 22.3, 22.5, 23.7, 24.3, 25.8, 29.4,
34.1, 60.3, 111.2, 125.9, 130.3, 145.9, 165.0, 168.9, 169.7; Anal.
Calcd for C16H24BrN3O3S2: C, 42.67; H, 5.37; N, 9.33; Found:
C, 42.46; H, 5.17; N, 9.34. Step 2: Compound 12c was
combined with compound 11 (0.110 g, 0.244 mmol) and
cesium carbonate (0.268 g, 0.825 mmol) in dry DMF (4 mL).
The solution was stirred for 16 h at 85 °C under nitrogen
atmosphere. The reaction was monitored by TLC using (2:1
toluene−acetone). Upon completion, the crude reaction
mixture was loaded onto silica gel and DMF was evaporated
under reduced pressure. The dry slurry was purified by flash
column chromatography on silica gel (30 mm × 150 mm).
Elution with 4:1 toluene−acetone affords a partially purified
product. The compound was further purified on a second silica
gel column to remove residual DMF, eluting with (4:1 ethyl
acetate−hexanes). The product fractions were combined,
concentrated, and dried in vacuum where the product
crystallized upon standing: yield = 0.080 g (77%); Rf = 0.53
342 (M)+, C14H20N3O3S2 requires 342. Step 2: Compound
+
(12a) (0.100 g, 0.29 mmol) was combined with compound 11
(0.040 g, 0.14 mmol), cesium carbonate (0.19 g, 0.584 mmol)
in dry DMF (2 mL). The solution was stirred for 16 h at 85 °C
under nitrogen atmosphere. The reaction was monitored by
using TLC (2:1 toluene−acetone). On completion, the crude
reaction mixture was loaded onto silica gel and the residual
solvent evaporated under reduced pressure. The sample was
loaded on silica gel column (30 mm × 150 mm) and eluted
with hexanes followed by 4:1 toluene−acetone to afford the
product. The product fractions were combined, concentrated
under reduced pressure. The product was pink amorphous
solid: yield 0.018 g (28%); Rf = 0.37 (2:1 toluene−acetone); 1H
NMR (CDCl3, 600 MHz); δ 1.39 (t, J = 7.2 Hz, 3H), 1.79 (m,
4H), 2.65 (m, 2H), 2.76 (m, 2H), 2.98 (m, 2H), 3.36 (s,
OCH3), 3.53 (d, J = 16.2 Hz, 1H), 3.64 (d, J = 16.2 Hz, 1H),
3.98 (m, 1H), 4.0 (m, 1H), 4.23 (m, 1H), 4.34 (q, J = 7.2 Hz,
2H), 4.89 (s, 1H). 13C NMR (CD3OD, 400 MHz); δ 12.4,
20.2, 20.4, 20.6, 30.4, 30.6, 50.4, 60.0, 80.0, 80.2, 80.3, 100.9,
112.0, 126.0, 129.0, 146.0, 165.0,167.0; mass spectrum
(HRMS), m/z = 468.1089 (M+Na)+, C19H27NNaO7S2 requires
468.1127.
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-
(methyl 5-thio-α-D-arabinofurano-side-5-S-yl)acetamide
(13b). Step 1: Compound (2c) (0.200 g, 0.78 mmol) and
thiourea (0.119 g, 1.57 mmol) were suspended in 4 mL
absolute ethanol and stirred for 3 h at 65 °C. The reaction was
analyzed for completion by TLC using 3:1 hexanes−ethyl
acetate. The reaction was cooled and filtered. The filtrate was
rinsed with a minimal amount of absolute ethanol and
1
(2:1 toulene−acetone); mp = 92−93 °C; H NMR (CDCl3,
400 MHz) δ 1.39 (t, J = 6.8 Hz, 3H), 1.78−1.79 (m, 4H), 2.04
(t, J = 7.2 Hz, 3H), 2.59−2.64 (m, 2H), 2.70−2.77 (m, 3H),
2.83−2.90 (m, 2H), 3.38 (s,3H), 3.90−3.93 (m,1H), 4.06 (d, J
= 6.8 Hz, 1H), 4.20−4.22 (m, 1H), 4.34 (q, J = 7.2 Hz, 2H),
4.90 (s, 1H), 5.39 (d, J = 6.0 Hz, 1H), 11.32 (s, 1H) ppm; 13
C
NMR (CDCl3, 400 MHz) δ 14.5, 23.0, 23.2, 24.5, 25.0, 26.6,
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32.8, 34.5, 55.1, 60.8, 80.5, 80.7, 85.7, 109.1, 111.7, 127.0,
131.0, 147.5, 167.0, 169.4 ppm; mass spectrum (ESIMS), m/z
= 496.2 (M+Na)+ Anal. Calcd for C21H31NO7S2: C, 53.26; H,
6.60; N, 2.96; O, 23.65; S, 13.54. Found: C, 53.32; H, 6.51; N,
2.93.
23.2, 24.7, 25.5, 30.3, 34.3, 39.8, 55.4, 80.0, 81.5, 83.9, 108.8,
114.0, 127.3, 132.2, 148.7, 152.2, 159.1; mass spectrum
(HRMS), m/z = 451.0969 (M+Na)+, C18H25N2O6S2 requires
429.1154.
N-(3-Cyano-4,5,6,7-tetrahydrobenzo[b]thiophen-2-yl)-2-
(methyl-5-thio-α-D-arabinofuranoside-5-S-yl)butamide
(13d). Step 1: Compound 4b (0.300 g, 0.916 mmol) and
thiourea (0.139g, 1.83 mmol) were suspended in 2 mL of
absolute EtOH and stirred at reflux for 4 h. The reaction was
analyzed for completion by using TLC 3:1 (hexanes−ethyl
acetate). The reaction mixture was cooled on ice and filtered
with minimal amount of abs EtOH and dried in vacuo.
Compound 12d was obtained as colorless crystals: yield =
RESULTS AND DISCUSSION
■
Molecular Modeling. The substrate analogues shown in
Figure 2 all contain the 2-amino-4,5,6,7-tetrahydrobenzo[b]-
thiophene scaffold. Different linker lengths and the arabinose
moiety were converted to three-dimensional structures (MDL
mol file format) using Chem3D (CambridgeSoft). The
compound data sets were then imported into Maestro v 8.0
(Schrodinger, Inc.) and were prepared for docking using
MacroModel (Schrodinger, Inc.) by using conformational
analysis and minimization methods to obtain the lowest energy
conformer. The crystallographic coordinates for the Ag85C-
octylthioglucoside complex (PDB code: 1va5)29 was obtained
from the Protein Data Bank (www.rcsb.org) and prepared by
removing all the solvent and adding hydrogen atoms and
minimal minimization in the presence of bound ligand using
protein preparation. Grids for molecular docking with Glide v
4.5 (Schrodinger, Inc.) were calculated without any constraint
to the protein.59 Compounds were docked using Glide in extra-
precision mode, with up to five poses saved per molecule. The
docked poses were then minimized using the local optimization
feature in MacroModel,60 and the energies were calculated using
the OPLS force field61 and the GBSA continuum model62 in
Maestro. The binding free energy ΔGbind is estimated as
1
0.262 g (71%); mp = 205−207 °C. H NMR (DMSO, 600
MHz): δ 1.67 (m, 4H), 1.83−1.85 (m, 3H), 2.01 (s, 2H),
2.40−2.44 (m, 2H), 2.55−2.58 (m, 2H), 3.12−3.132 (m, 2H),
8.98 (m, 1H) ppm; 13C NMR (DMSO, 600 MHz): δ 21.7,
22.6, 23.3, 23.5, 24.0, 29.5, 30.7, 33.1, 114.3, 127.1, 130.6,
146.4, 169.6, 170.1 ppm; Anal. Calcd for C14H19BrN4OS2: C,
41.69; H, 4.75; Br, 19.81; N, 13.89; O, 3.97; S, 15.90. Found: C,
40.88; H, 4.57; N, 14.20. Step 2: Compound 12d was
combined to compound 11 (0.210 g, 0.520 mmol), cesium
carbonate (0.268 g, 0.825 mmol) were dissolved in dry DMF (5
mL). The solution was stirred for 24 h at 85 °C under nitrogen
atmosphere. The reaction was monitored by thin layer
chromatography using (1:1 acetone−hexanes). Upon comple-
tion, the crude reaction mixture was loaded onto silica gel and
the DMF was evaporated under reduced pressure. The dry
slurry was purified by flash column chromatography on silica
gel (30 mm × 150 mm). Elution was with 2:1 acetone−
hexanes. The product fractions were combined and concen-
trated to dryness. The crude product was crystallized using
ethanol and filtered with cold ethanol: yield = 0.110 g (49%); Rf
ΔGbind = ΔEMM + ΔGsolv + ΔGSA
where ΔEMM is the difference in energy between the complex
structure and the sum of the energies of the ligand and the
unliganded protein, using the OPLS force field, ΔGsolv is the
difference in the GBSA solvation energy of the complex and the
sum of the solvation energies for the ligand and unliganded
protein, and ΔGSA is the difference in the surface area energy
for the complex and the sum of the surface area energies for the
ligand and uncomplexed protein. Corrections for entropic
changes were not applied. Finally, according to the docking
score and binding energy we selected the candidate structures
for preparing and testing as inhibitors for Ag85C. Structures
with good docking scores included compounds containing a 2-
amino-4,5,6,7-tetrahydro-1-benzothiophene-3-carbonitrile or an
ethyl 2-amino-4,5,6,7-tetrahydro-3-carboxylate linked through
the 2-amino moiety to a methyl 5-thio-α-D-arabinofuranoside.
The docking scores are shown in Table 1 and include 13a−d
and compound 14. Of these compounds, 13a showed the
highest binding energy of −62 kJ/mol.
1
= 0.3 (1:1 acetone−hexanes); mp = 183−184 °C; H NMR
(DMSO, 600 MHz) δ 1.69 (s, 4H), 1.80 (p, J = 6.0 Hz, 4H),
2.42 (br. s, 2H), 2.51−2.62 (m, 4H), 2.73 (s, 1H), 3.18 (s, 3H),
3.56−3.58 (m, 1H), 3.71−3.72 (m, 1H), 3.74−3.76 (m, 1H),
4.55 (s, 1H), 5.25 (d, J = 6, 1H), 5.39 (d, J = 6.0 Hz, 1H), 11.51
(br. s, 1H) ppm; 13C NMR (DMSO, 400 MHz) δ 21.8, 22.7,
23.3, 23.5, 24.8, 31.6, 33.8, 33.9, 54.4, 79.9, 80.1, 81.9, 82.6,
92.2, 109.0, 114.4, 127.04, 130.6, 146.67, 170.66 ppm; mass
spectrum (HRMS), m/z = 449.1199 (M+Na)+
C19H26N2O5S2Na requires 449.1181; Anal. Calcd for
C19H26N2O5S2: C, 53.50; H, 6.14; N, 6.57; O, 18.75; S,
15.03. Found: C, 48.78; H, 5.41; N, 3.71.
Methyl 5-S-[2-(2,4-Dioxo-1,4,5,6,7,8-hexahydro[1]-
benzothieno[2,3-d]-pyrimidin-3(2H)-yl)ethyl]-5-thioarabino-
furanoside (14). Compound 10 (0.194 g, 0.634 mmol) and
compound 5 (0.289 g, 0.868 mmol) were combined in 3 mL
NaOMe (3.06 mmol Na metal in 3 mL NaOH). The solution
was stirred at room temperature for 24 h then quenched using
20 mL 1 N HCl. The solution was diluted with water and
extracted with chloroform (3 × 20 mL). The chloroform was
dried with anhydrous sodium sulfate, filtered, and dried under
reduced pressure. The product was purified by flash column
chromatography on silica gel (30 mm × 150 mm) eluting with
50:50 hexanes−acetone. The product fractions were concen-
trated to afford 14, a colorless waxy solid: yield = 36 mg (13%);
Rf = 0.46 (1:1 hexanes−acetone); 1H NMR (CDCl3, 600
MHz): δ 1.78 (m, 4H), 2.60 (s, 2H), 2.83−3.04 (m, 6H), 3.37
(s, 3H), 4.03 (br. s, 2H), 4.13 (br. s, 1H), 4.19 (m, 4H), 4.88 (s,
1H), 10.91 (br. s, 1H); 13C NMR (CDCl3, 600 MHz): δ 22.2,
Potential intermolecular hydrogen-bonded interactions
between compound 13a and the Ag85C protein showed four
Table 1. Docking Scores, Binding Energies, and Measured Ki
of Compounds 13a−d and Compound 14
a
compound
docking score
binding energy
Ki (μM)
native ligand
−11.3
−12
−12.88
−11.4
−10.9
−11.85
−53
−62
−19
−6
−29
−15
N.D.
13a
13b
13c
13d
14
18.2 1.8
34.8 3.8
71.0 5.3
N.D.
47.1 3.9
a
N.D. = no data.
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Alternatively, 1a was reacted with 2-chloroethyl isocyanate to
form urea 556 in 53% yield. With key intermediates in hand, we
explored different approaches to form a thioether linkage
between the thiophenes and the C-5 position of a methyl α-D-
arabinofuranoside. In the first approach, the chloro substituent
of thiophenes 2a−d was displaced with potassium thioacetate
in anhydrous DMF to afford the thioesters 3a−d in yields of
62−92%. The resulting thiophenes were treated with NaOEt,
concentrated, and directly reacted with the known methyl 5-O-
p-toluenesulfonyl-α-D-arabinofuranoside (6) (Scheme 3).24−26
Unfortunately, we failed to isolate an identifiable product using
this protocol. We then chose to reverse the polarity of the
electrophile by converting the tosylate to a sulfhydryl. This
required peracetylation of arabinofuranoside 6 with acetic
anhydride to afford the diacetylated compound 7 in 73% yield
(Scheme 2). Acetylated sulfonate 7 was then treated with
potassium thioacetate to afford the thioester 8 in 60% yield.
Arabinofuranoside 8 was then treated with NaOMe at 50 °C
for 2 h. The reaction was neutralized with Amberlite IR120 (H
+ form), filtered and dried. The resulting methyl 5-deoxy-5-
thio-α-D-arabinofuranoside 9 was resuspended in THF and
combined with an equivalent of alkyl chlorides 2a−d and
triethyl amine to afford glycoconjugates 13a−d (Scheme 3). In
our hands, only the α-chloroamides 2a and 2c reacted with the
mercaptan 9 to produce thioethers 13a and 13b, respectively, in
unacceptable yield. Modification of bases and solvents and
addition of disulfide reducing agents failed to generate the
longer-chain thioethers.
Table 2. X-ray Diffraction and Crystallographic Refinement
Data
diffraction statistics
resolution range (å) (highest
shell)
35−2.15 (2.20−2.15)
P212121
space group
unit cell parameters
a = 67.5,b = 80.2, c = 136.6, α = β = γ =
90°
total reflections (unique
reflections)
246 157 (40 674)
completeness (%) (highest shell) 99.0 (99.9)
average I/σI
7.0
Rsym (%) (highest shell)
8.8 (40.4)
refinement statistics
protein molecules/atoms (per A.S.U.)
work/Rfree (%)
2/4695
R
15.86/19.66
25.66/43.58/36.17
100
average B-factor (Å2) (protein/ligand/solvent)
ligand occupancy (%)
r.m.s.d. bonds/angles (Å/°)
Ramachanran (favored/disallowed) (%)
0.007/1.041
96.2/0.0
hydrogen bonds (OH-3 of the arabinose with NH of Arg41 and
HOH 2037, OH-4 of the arabinose with NH of Trp262, and S
of the linker with HOH 2242) (Figure 3). In addition, the
Unsatisfied with failure of the reaction between halides 2b
and 2d and mercaptan 9, as well as poor coupling yields to this
point, we began to explore alternate options for synthesizing
the desired thioether linkages. There are numerous examples of
alkyl halides reacting with isothiourionium salts to form
thioethers.63 Therefore, we converted methyl α-D-arabinofur-
anoside (10)24−26 to the known methyl 5-deoxy-5-iodo-α-D-
arabinofuranoside (11) using I2-P(Ph)3-imidizole in 65% yield
(Scheme 2).64 Compounds 1a and 1b were then acylated with
4-chlorobutyryl chloride to provide amides 4a and 4b,
respectively (Scheme 1). Amides 2a, 2c, 4a, and 4b were
then treated with thiourea in refluxing ethanol to afford the
respective isothiourionium salts 12a−d, in good yield (Scheme
3). The isothiourionium salts reacted cleanly with iodide 11 in
the presence of cesium carbonate in N,N-dimethylformamide to
form thioethers 13a−d, respectively.
We also found it noteworthy that chloride 5 and methyl 5-
thio-α-D-arabinofuranoside 8 in methanol-NaOMe, afforded the
3-substituted pyrimidinedione 14 linked to a methyl 5-thio-α-D-
arabinofuranoside moiety (Scheme 4). The resulting materials
were then used to evaluate the structural class as inhibitors of
the Ag85C acyltransferase activity.
Enzyme Inhibition Studies. To determine if any
compounds from this library inhibit the Ag85C enzyme, assays
measuring the enzymatic activity were performed. These assays
employed 4-methylumbelliferyl butyrate (4MUB) as the acyl
donor and trehalose as the acyl acceptor. Binding of 4MUB
within the enzyme active site allows nucleophilic attack on the
substrate to produce an acyl-Ag85C intermediate and release of
the fluorescent molecule 4-methylumbelliferone (4-MU).
Subsequent binding of trehalose in the active site allows
nucleophilic attack by trehalose on the acyl-Ag85C inter-
mediate to form trehalose-6-butyrate. Measuring the fluo-
rescence emission of 4-MU at 500 nm affords an accurate
determination of the initial rate of the enzymatic reaction.
Figure 3. Hypothetical binding mode of inhibitor 13a in the Ag85C
active site predicted using Glide 4.5. The arabinofuranoside substituent
is located in the trehalose binding pocket, and the ester moiety is
oriented in the vicinity of Ser124. The tetrahydrobenzo[b]thiophene is
accommodated in the mycolate α-chain binding channel extending
through the core of Ag85C.
calculated distance between the OH of Ser124 and the carbon
of carbonyl carbon of the ester was found to be 6.876 Å. Finally,
we calculated the electrostatic interactions between compound
13a and the Ag85C protein (Figure 4). According to the
hydrogen bond and electrostatic interactions, compound 13a
was expected to be a good template for occupying the active
site of the enzyme.
Synthesis of Glycoconjugates. Both the 2-amino-4,5,6,7-
tetrahydro-1-benzothiophene-3-carbonitrile and ethyl 2-amino-
4,5,6,7-tetrahydro-1-benzothiophene-3-carboxylate classes of
thiophenes could be accessed through a classic Gewald
synthesis52 followed by amine functionalization with an
appropriate linker. The Gewald synthesis was performed by
treating cyclohexanone with malononitrile or ethyl cyanoace-
tate and sulfur in the presence of N-ethylmorpholine to yield 3-
carboxylate thiophene 1a51 and 3-carbonitrile thiophene 1b,
respectively, in a modest yield. Thiophenes 1a and 1b were
then reacted with either chloroacetyl chloride or 4-chlorobutyrl
chloride to prepare amides 2a−d (Scheme 1) in modest yield.
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Figure 4. Electrostatic potential and hydrogen bonding interaction between compound 13a and Ag85C. The distances between the OH of Ser124
and the ester and amide CO moieties of 13a is indicated by green lines.
a
Scheme 2. Synthesis of Arabinofuranosides
Scheme 3. Isothiourionium Salt-Based Synthesis of
Thioethers
a
a
Reagent and conditions: (i) Ac2O, pyridine, 24 h, rt (73%); (ii)
KSCOCH3, DMF, 24 h, rt (60%); (iii) 0.1 M methanolic NaOMe 2 h,
50 °C then Amberlite IR120; (iv) imidazole, I2, PPh3, THF, 0 to 65
°C, (65%).
To determine the Ki values for the library of inhibitors, the
initial reaction rate using this fluorescence-based assay was
performed in the presence of varying concentrations of each
inhibitor. The ratio of the rates of the inhibited reaction versus
the uninhibited reaction plotted as a function of inhibitor
concentration exhibits a clear dose-dependent decrease in
enzymatic activity. Fitting these data allowed the calculation of
Ki values for each of the inhibitors, which ranged from 18.2 to
71.0 μM (Table 1 and Figure 5A).
a
Reagent and conditions: (i) NaOEt, MeOH, rt, no reaction; (ii)
THF, TEA, 6 h, rt, no reaction; (iii) CS(NH2)2, EtOH, 4 h, reflux, 12a
(83%), 12b (80%), 12c (52%), 12d (71%); (iv) 12a−12d, Cs2CO3,
DMF, 16 h, 85 °C, 13a (28%), 13b (56%), 13c (77%), 13d (49%).
a
Scheme 4. Synthesis of Pyrimidinedione 14
While the Ki values of the inhibitors do not vary significantly,
a general trend is observed that offers insight for the design of
second-generation compounds with improved affinity for the
Ag85C enzyme and better inhibitory activity. Because of the
choices of arabinofuranosides tested, the importance of two
structural variables can be related to inhibitory activity. First, of
the thiophene containing compounds, those elaborated with
the ethyl ester moiety on the thiophene, have slightly better
inhibitory activity than compounds with a corresponding nitrile
moiety as seen when comparing the Ki values of 13a and 13b.
a
Reagents and conditions: (i) NaOMe, MeOH, 24 h, rt (13%).
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single three-ringed compound tested is difficult to analyze in
isolation. The Ki is near the middle of the range observed in the
inhibition studies. Synthesis of similar compounds and further
testing will be required to determine if there is a benefit to
having the third ring. The compounds were screened for
growth inhibition activity against M. smegmatis; however, no
growth inhibition was observed.
Crystal Structure of the Ag85C-Inhibitor Complex. To
gain insight into the mechanism of inhibition and information
necessary to develop better inhibitors, specific structural
information is needed. To this end, crystals of recombinant
Ag85C were soaked with compound 13a. Compound 13a was
chosen because it possesses the lowest Ki value. The difference
map calculated with these data show four strong regions of
electron density within the Ag85C active site. These are
consistent with one compound of 13a, a single water molecule,
and two molecules of glycerol. The binding of 13a within the
active site indicates that it functions as a competitive inhibitor
of Ag85C (Figure 5B).
The orientation of 13a within the active site of the crystal
structure (Figure 6) is different with respect to the predicted
structure from the inhibitor modeling data. The thiophene
moiety is positioned in roughly the same location, but is flipped
180° along the major axis of the two-ringed system to position
the ethyl ester pointing away from the enzyme rather than into
the hydrophobic tunnel. The arabinofuranoside moiety binds
within the known carbohydrate-binding site as exhibited for the
Ag85B-trehalose crystal structure.30 However, the arabinosyl
moiety of 13a overlaps with the glucosyl moiety of trehalose
that is distal to Ser124 of Ag85B rather than binding in the
proximal carbohydrate binding pocket that accommodates the
sugar to be acylated by the enzyme.
Unexpectedly, no specific hydrogen bonding interactions are
being formed between the arabinofuranoside and the
carbohydrate-binding site of the active site. This would suggest
that the binding is driven by hydrophobic interactions between
the thiophene moiety and the mycolyl binding site, and that
linker length as well as shape complementarity between the
arabinosyl moiety and the carbohydrate binding site ultimately
Figure 5. Inhibition of Ag85C. (A) vi′ represents the initial velocity of
the inhibited enzymatic reaction at the respective inhibitor
concentration. vi represents the initial velocity of the uninhibited
enzymatic reaction. (B) The enzyme active site is shown (with green
carbon atoms) with one molecule of 13a, one water molecule, and two
molecules of glycerol (molecules with gray carbon atoms). The
placement of these compounds was based on the Fo−Fc omit maps
(light blue difference density) shown contoured at 3σ. Colors for the
atoms other than carbon are blue, red, and yellow for nitrogen, oxygen,
and sulfur, respectively.
Second, the length of the linker between the thioether and the
aromatic fused ring system affects inhibitory activity. Com-
pound 13a, which contains a methylene linker, exhibits stronger
inhibition than 13c, which possesses a propylene linker. The
Figure 6. Interactions between 13a and the Ag85C active site. Two views of the Ag85C active site are shown from different orientations. (A)
Nonspecific interactions. The Ag85C protein backbone is shown as the green ribbon. The carbon atoms are in green, nitrogen atoms are in blue,
oxygen in red, and sulfur in yellow. The location of the serine nucleophile is shown as a reference. All of the hydrophobic residues within 4 Å of the
thiophene moiety are shown, as well as the R41 side chain that interacts with the thioether linker. (B) The superposition of the trehalose-bound
Ag85B and the 13a-bound Ag85C structures shows overlap of the trehalose and 13a binding sites. The trehalose from the Ag85B structure is shown
with gray carbon atoms. All other atom types are colored as in panel A. The distal glucosyl moiety of trehalose and the methylarabinoside of 13a are
clearly occupying the same region within the active site. The proximal glucosyl and an ordered glycerol molecule also superimpose well and form
similar hydrogen bonding interactions with Ag85. The ordered water molecule near O6 of the Ag85b-bound trehalose is shown. The dashed lines
represent hydrogen bonds between this water molecule and the 13a amide carbonyl, the side chain of Ser124, and the backbone amide of Leu40,
which forms part of the oxyanion hole. The length of these hydrogen bonds range from 2.6 to 3.0 Å.
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determine the location of the arabinosyl binding. The
interaction between Ag85C and the arabinofuranosyl moiety
is rather dissimilar to the interactions observed in the Ag85B-
trehalose crystal structure, where O2 and O3 of the distal
glucosyl moiety of trehalose are hydrogen-bonded to the side
chain of Arg41 and O6 is hydrogen-bonded to the backbone
carbonyl of His260.30
limited set of structures we identified showed no activity against
M. smegmatis, a model organism, in a disk diffusion assay;
however, the structures we identified are very similar to
thiophenes which show activity in macrophages infected with
drug resistant strains M. tuberculosis. On the basis of this
observation, we plan to evaluate these compounds and others
in a macrophage infection model. The crystal structure
highlights interactions between both the thiophene and
arabinofuranosides moieties and will be leveraged to design
improved inhibitors of Ag85s.
The fused, two-ring system of 13a is positioned in the
proposed mycolic acid-binding pocket and forms van der Waals
interactions with the side chains of Phe150, Trp158, Leu161,
Ala165, and Leu227.29,33 In addition, the linker connecting the
thiophene to the arabinofuranosides moiety forms van der
Waals interactions with the side chain of Arg41. The carbonyl
oxygen of the amide moiety appears to be poised to form a
hydrogen bond with the backbone amide of Leu40, although
the 4.5 Å distance between these is too long for a bonafide
hydrogen bond. The approach of 13a to the oxyanion hole is
blocked by an ordered water molecule that forms hydrogen
bonds with both the oxyanion hole and the amide carbonyl of
13a. One key to improving binding and inhibitory activity of a
second-generation inhibitor would be to displace this water
molecule with a hydrogen bond acceptor to promote a direct
interaction between the inhibitor and the oxyanion hole.
Another important piece of information from this structure
can be used for future design efforts to improve both strength
and specificity of binding. The pocket initially assumed to be
important for binding the trehalose still has an undefined role.33
To date, no structure has revealed binding of any substrate or
product analogues within this site even though the residues
forming this site (Asp38, Asp45, and Trp262) are conserved in
all Ag85 complex mycolyltransferases. The only compounds
observed in any structures within this site are glycerol and
water, which implies that it may accommodate additional
carbohydrate moieties exposed on the surface of the
mycobacterial outer membrane.28,29,33 One potential substrate
accommodated by this conserved site would be a neighboring
subunit of the arabinan. As the conserved sequence and
structure in this region implies an important role, the Ag85C-
13a complex structure gives very specific information regarding
the future design of inhibitors that could bind within this site.
Specifically, the methyl group on the methyl-arabinofuranoside
can be replaced with a much bulkier moiety such as another
arabinofuranosyl moiety, capable of forming a complex
hydrogen-bonded network with the conserved residues in this
site. Alternatives other than carbohydrates could also be used.
Ideally, these groups would possess a large number of both
hydrogen bond donors and acceptors to maximize the
interaction potential within this site.
ASSOCIATED CONTENT
■
S
* Supporting Information
1H and 13C spectra for compounds 2a−2d, 3a−3d, 4a, 4b, 5,
7−9, 11, 12c, 12d, 13a−d, 14, HRMS for compound 14. The
material is available free of charge via Internet at http://pubs.
acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*S.J.S.: phone, 419-530-1054; fax, 419-530-1190; e-mail, steve.
sucheck@utoledo.edu. D.R.R.: phone, 419-530-1585; e-mail,
donald.ronning@utoledo.edu.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Use of the Advanced Photon Source was supported by the U.S.
Department of Energy, Office of Science, Office of Basic Energy
Sciences, under Contract No. DE-AC02-06CH11357. Use of
the LS-CAT Sector 21 was supported by the Michigan
Economic Development Corporation and the Michigan
Technology Tri-Corridor for the support of this research
program (Grant 085P1000817). This was supported by an NIH
grants to D. R. Ronning (AI089653) and S. J. Sucheck
(GM094734). We also thank students Shen Zhang and
Shuangqianzi Li for assistance with the resynthesis and
characterization of key intermediates.
REFERENCES
■
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■
Virtual thiophene−arabinoside conjugates were docked to
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NOTE ADDED AFTER ASAP PUBLICATION
■
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