The synthesis and biological evaluation of mycobacterial p-hydroxybenzoic acid derivatives (p-HBADs)

Article abstract of DOI:10.1039/c3ob42277a

Mycobacterium tuberculosis establishes chronic infection and causes disease through manipulation of the host's innate and adaptive immune response. The bacterial cell wall is highly complex and contains a rich variety of glycosylated compounds that are se

Full text of DOI:10.1039/c3ob42277a

Organic &
Biomolecular Chemistry
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The synthesis and biological evaluation of
mycobacterial p-hydroxybenzoic acid derivatives
(p-HBADs)†
Cite this: Org. Biomol. Chem., 2014,
12, 1114
Jean Bourke,a Corinna F. Brereton,b Stephen V. Gordon,c Ed C. Lavelle*b and
Eoin M. Scanlan*a
Mycobacterium tuberculosis establishes chronic infection and causes disease through manipulation of
the host’s innate and adaptive immune response. The bacterial cell wall is highly complex and contains a
rich variety of glycosylated compounds that are secreted during infection and have been proposed as
immunomodulatory molecules. Amongst the most important of these are the p-hydroxybenzoic acid
derivatives (p-HBADs). Here we report the synthesis of this important class of biomolecules and the first
in vitro study of the immunomodulatory effects of these compounds in isolation from the host bacterium.
The compounds do not have stimulatory properties but, in contrast, can inhibit the production of inflam-
matory cytokines, particularly interferon-γ (IFN-γ), by T-cells. This study offers a fundamental insight into
the effect of these glycans on the immune response.
Received 14th November 2013,
Accepted 20th December 2013
DOI: 10.1039/c3ob42277a
www.rsc.org/obc
Introduction
Mycobacterium tuberculosis (Mtb), the causative agent of tuber-
culosis is one of the world’s most deadly pathogens, with
1.4 million deaths in 2011 alone. The pathogen is character-
ized by a sophisticated and complex cell wall architecture.1–4
Many of the lipids and glycolipids that make up the Mtb cell
wall are believed to be virulence factors that play a key role in
host-pathogen interactions and disease pathogenesis.5–10
Glycosyl processing enzymes are essential for bacterial cell
wall biosynthesis and many of the key components of the cell
wall display complex carbohydrates.11,12 The critical and
diverse biological functions of these glycans in the life-cycle of
the mycobacterium are only beginning to be understood. Phe-
nolic glycolipids (PGLs) are a family of bacterial glycolipids Fig. 1 Structures of p-HBAD-I, p-HBAD-II and the related PGL-tb1
from M. tuberculosis. The lipid core of PGL is composed of
phenolphthiocerol esterified by mycocerosic acids.
with a lipid core similar to dimycocerosates of phthiocerol
(DIMs).13–17 PGLs contain an aromatic nucleus that is glycosy-
lated by a strain-specific, mono, di, tri or tetrasaccharide
(Fig. 1). The carbohydrate region is composed of the 6-deoxy
a very specific methylation pattern. These glycosylated lipids
sugars, L-rhamnose and L-fucose and is usually decorated with
are important for mycobacterial disease pathogenesis and
have been associated with an inhibition of the release of key
inflammatory effector molecules by cells of the host’s innate
immune response.18,19 PGL-1 has been isolated from strains of
the related mycobacterium, M. leprae, the causative agent of
leprosy. The carbohydrate region of PGL-1 binds to the α2LG1,
α2LG4 and α2LG5 modules of the peripheral nerve laminin α2
chain and promotes phagocytosis of M. leprae by macrophages
and Schwann cells.20 The carbohydrate region of PGLs is criti-
cal to their activity. Despite the acute biological effects of these
aSchool of Chemistry, Trinity Biomedical Sciences Institute, 152-160 Pearse Street,
Trinity College, Dublin 2, Ireland. E-mail: eoin.scanlan@tcd.ie;
Fax: +353 (1) 671 2896; Tel: +353 (1) 896 2514
bAdjuvant Research Group, School of Biochemistry, Trinity Biomedical Sciences
Institute, 152-160 Pearse Street, Trinity College Dublin, Dublin 2, Ireland
cConway Institute of Biomolecular & Biomedical Research, University College Dublin,
Belfield, Dublin 4, Ireland
†Electronic supplementary information (ESI) available. See DOI:
10.1039/c3ob42277a
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molecules, it has been found that most clinical isolates of these compounds were tested in vitro using mouse splenocytes
M. tuberculosis do not synthesize PGLs.19 A subclass of carbo- and bone marrow derived macrophages.
hydrate molecules related to PGLs are the para-hydroxybenzoic
Synthesis of the trisaccharide unit of p-HBAD-II has been
acid derivatives (p-HBADs).21,22 These small glycoconjugates described previously.27,28 However, our reported strategy for
contain an identical glycosylated phenolic moiety to PGLs and regioselective protection of hydroxyl groups offers an alterna-
are formed through a related biosynthetic pathway.23–25 The tive synthetic route with comparable yields to these reported
p-HBADs are a component of the bacterial cell wall and unlike syntheses. Recently, the total synthesis of PGL-tb1 has been
the related PGLs, all strains of M. tuberculosis appear to syn- reported but this approach also differs considerably from our
thesize p-HBADs.21 The fact that all strains of M. tuberculosis reported strategy.29 The synthesis of the PGL mycoside-B and
have retained the ability to produce such complex molecules p-HBAD-I have also recently been reported, highlighting the
suggests that these carbohydrates are extremely important for interest in accessing synthetically pure samples of these com-
bacterial pathogenesis. Indeed it has been demonstrated that pounds for biological study.30 The overall synthetic route to
M. tuberculosis mutants defective in the synthesis of p-HBADs p-HBAD-I is outlined in Scheme 1. The regioselective methyl-
promote a stronger inflammatory response than wild type ation of the 2-OH position of L-rhamnose required a number
strains.22 The fact that these compounds do not have a lipid of protecting group manipulations and a general protecting
component strongly suggests that it is the carbohydrate region, group strategy was developed that allowed regioselective access
with its defined methylation pattern, that is the most impor- to each hydroxyl groups on rhamnose moiety. Glycosylation
tant factor in modulating biological activity. The p-HBADs are with methyl p-hydroxybenzoate was carried out at a late stage
secreted during host infection but despite their prominent in the synthesis. Starting from per-acetylated rhamnose 1, a
role in disease infection and pathogenesis, little is known Lewis acid catalysed glycosylation reaction with ethane thiol
about the biological targets of these molecules.
furnished thioglycoside donor 2. Thioglycosides are widely
Since publication of the Mtb genome,26 a number of genes used as glycosyl donors in carbohydrate synthesis due to their
associated with glycosylation and subsequent methylation of relative stability and ability to tolerate functional group modifi-
the p-hydroxybenzoic acid aromatic nucleus have been identi- cations on the sugar.31 Removal of the acetate protecting
fied.23,24 The structural similarities between PGLs and groups under Zemplén conditions followed by regioselective
p-HBADs suggest that they share
a similar biosynthetic protection of the 2- and 3-OH groups with a cyclic acetal furn-
pathway originating from p-hydroxybenzoic acid. A chorismate ished partially protected donor 4. Benzylation of the free 4-OH
pyruvate-lyase has been identified as the sole enzymatic source followed by hydrolysis of the acetal protecting group furnished
of p-hydroxybenzoic acid in M. tuberculosis.21 Methylation of the
2-hydroxyl group located on C-2 of the rhamnosyl residue linked
directly to the phenolic core appears to be of paramount impor-
tance for biological activity. In order to directly address the role
of p-HBADs in Mtb pathogenesis, we set out to prepare fully syn-
thetic p-HBADs with their native methylation pattern intact. A
synthetic variant possessing an unnatural methylation pattern
was also prepared and studied in vitro.
Results and discussion
The p-HBADs play a key role in M. tuberculosis disease patho-
genesis and recent studies have suggested that these glycans
have potent immunomodulatory activity. Data from Mtb
strains defective in the synthesis of p-hydroxybenzoic acid
derivatives suggest that p-HBADs influence the release of key
inflammatory cytokines by macrophages.22 It is difficult to derive
definitive conclusions regarding the biological roles of specific
p-HBADs from studies using Mtb since the bacterium secretes
heterogeneous mixtures of glycoconjugates with variable ratios
of p-HBADs. It is also challenging to probe the biological role of
the unusual methylation pattern displayed on the p-HBADs. In
order to directly study the immunomodulatory effects of the
p-HBADs in the absence of the parent bacterium, we set out to
prepare synthetic samples of p-HBAD-I (both methylated and
unmethylated at the 2-OH position) and p-HBAD-II with its
Scheme 1 Synthetic strategy for the preparation of p-HBAD-I 14 from
native methylation pattern. The immunomodulatory effects of peracetylated rhamnose 1.
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the diol 6.32,33 A regioselective hydrolysis of an orthoester was
used to access 7.34 The resulting free 3-OH was protected with
a levulinoyl protecting group which was selected to be ortho-
gonal to both the acetyl and benzyl protecting groups. Following
deacetylation of the 2-OH to give 9 (this compound was
employed as a starting point for the synthesis of p-HBAD-II), the
methyl group was introduced in good yield using methyl iodide
as the alkylating agent. It was found that using an excess
sodium hydride could remove the levulinoyl protecting group
quantitatively in a one-pot system to give 10. The free 3-OH was
acetylated to give thioglycoside 11 which was employed as a gly-
cosyl donor for the glycosylation reaction with methyl p-hydroxy-
benzoate to furnish the protected p-HBAD-I 12. Deprotection of
12 in two steps, furnished the fully synthetic p-HBAD-I 14 in a
good yield. The synthetic strategy is advantageous in that all of
the synthetic steps are high yielding and the key intermediates
9 and 13 could be used directly for the preparation of the more
complex oligosaccharide, p-HBAD-II.
Scheme 3 Synthesis of unmethylated p-HBAD-I 23 from per-OAc-
rhamnose 1.
previously isolated from a mutant strain of M. tuberculosis
taken from a transposon mutant library, and may be an inter-
mediate in the biosynthesis of p-HBADs.21 The per-acetylated
rhamnosyl donor 1 was glycosylated with the methyl p-hydroxy-
benzoate to furnish 22 which was subsequently deprotected to
furnish the fully unmethylated UM-p-HBAD-I 23 (Scheme 3).
Cell studies
The synthesis of the p-HBAD-II trisaccharide was based on Mouse splenocytes and Bone Marrow Derived Macrophages
the strategy developed for p-HBAD-I. A sequential glycosylation (BMM) were employed for the in vitro studies. Splenocytes
strategy employing two thioglycoside donors was used. The comprise a mixed population of white blood cells originating
overall synthetic scheme is outlined in Scheme 2. Starting in the spleen that play a key role in immune responses. Here,
from the levulinoyl protected compound 9, treatment with splenocytes were incubated with the T lymphocyte activating
benzyl bromide and an excess of sodium hydride gave the par- stimulus, anti-CD3e, to promote lymphocyte proliferation and
tially protected glycosyl donor 15. This compound was acetyl- cytokine production. Cells were also incubated with each of
ated at the 3-OH position and then employed in
a
the p-HBADs at various concentrations alone or together with
glycosylation reaction with the acceptor 13 that was prepared anti-CD3e antibody to assess if the molecules could induce,
previously as part of the p-HBAD-I synthesis. The disaccharide inhibit or modulate cytokine secretion. Cytokine production
17 was formed in good yield with complete alpha selectivity by splenocytes and macrophages was quantified by enzyme-
1
(as determined by H-NMR analysis), despite the absence of a linked immunosorbent assay (ELISA). Flow cytometry was used
participating group at the 2-OH position. The 3-OH position of to examine intracellular cytokines produced by CD4 and CD8
the terminal rhamnose residue was deprotected and glycosyla- cells and to assess cell proliferation by intracellular fluorescent
tion with the per-O-methylated fucosyl thioglycoside donor label carboxyfluorescein diacetate succinimidyl ester (CFSE)
1935 furnished the protected trisaccharide 20 in good yield. incorporation (Fig. 2).
Removal of the benzyl protecting groups under palladium-cata-
lysed hydrogenation conditions furnished the fully synthetic and 500 μM for their ability to induce cytokines or to modulate
p-HBAD-II with the native methylation pattern.
anti-CD3e (0.4 μg mL−1) induced cytokine secretion by spleno-
p-HBADs were tested at concentrations of 20 μM, 100 μM
In order to probe the role of the methyl group on the 2-OH cytes. p-HBAD-I, p-HBAD-II and UM-p-HBAD-I inhibited the
position of p-HBAD-I, the unmethylated derivative (UM-p- secretion of IFN-γ, IL-17 and IL-10 by anti-CD3e-stimulated
HBAD-I) was also prepared. This unmethylated p-HBAD was splenocytes, particularly at a concentration of 500 μM. This
reduction was significant at the higher concentration of 500 μM
but was still observed at the lower concentrations of the
p-HBADs. In the absence of anti-CD3e there was no enhance-
ment in cytokine secretion indicating that the p-HBADs them-
selves do not have immunostimulatory properties. The effect of
p-HBADs on intracellular IFN-y was examined by flow cytometry
(Fig. 3). p-HBAD-I, p-HBAD-II or UM-p-HBAD-I alone did not
induce CD4+ T cell proliferation or intracellular IFN-γ pro-
duction. However, p-HBAD-I, p-HBAD-II and UM-p-HBAD-I sup-
pressed intracellular IFN-γ production by activated CD4+ T cells.
From the above data we can conclude that in the presence
of anti-CD3, the three p-HBADs inhibit IFN-γ production but
not the proliferation of Th cells. Following from this we can
conclude that the p-HBADs examined antagonize the effect of
the anti-CD3 stimulus on IFN-γ production but enhance its
Scheme 2 Synthetic strategy for the preparation of p-HBAD-II.
1116 | Org. Biomol. Chem., 2014, 12, 1114–1123
effect on Th cell proliferation. Thus we can put forth the
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Fig. 2 p-HBADs inhibit the secretion of IFN-γ by splenocytes. Cells
were incubated with medium, p-HBADs alone, anti-CD3e or p-HBADs
with anti-CD3e. Supernatants were collected after 72 h and IFN-γ, IL-17
and IL-10 concentrations were determined by ELISA.
Fig. 4 p-HBAD-I and UM-p-HBAD-I suppress pro-inflammatory cyto-
kine production by BMDMs stimulated with irradiated M. tuberculosis
H37Rv. BMDMs were incubated with medium or p-HBADs alone (100 or
500 μM) in the presence or absence of irradiated H37Rv (10 : 1). Super-
natants were collected after 24 h and TNF-α, IL-12p40, IL-6 and IL-10
concentrations were determined by ELISA.
BMM were incubated with irradiated H37Rv in the presence or
absence of the p-HBADs, with the compounds added directly
to the cell supernatant. The results of the macrophage experi-
ments are presented in Fig. 4. Cells stimulated with irradiated
H37Rv produced the pro-inflammatory cytokines TNF-α,
IL-12p40 and IL-6, as well as the anti-inflammatory cytokine
Fig. 3 Flow cytometry data. p-HBADs suppress IFN-γ production by
CD4+ T cells. Splenocytes were incubated with medium, p-HBADs alone
or in the presence of anti-CD3e. Cells were incubated for 72 h and T cell IL-10. Stimulation in the presence of p-HBAD-I significantly
proliferation and IFN-γ production was assessed.
suppressed the production of TNF-α and IL-12p40 in response
to irradiated M. Tuberculosis H37RV, but had a reduced effect
on IL-6 production and no effect on IL-10 production. Simi-
postulate that p-HBADs inhibit the production of proinflam-
larly, co-incubation of Bone-marrow derived macrophages
matory cytokines (due to the knock-on effect of decreased
(BMM) with UM-p-HBAD-I significantly reduced the pro-
IFN-γ production) but do not adversely affect Th cell prolifer-
duction of TNF-α compared to cells stimulated with H37Rv
ation. The p-HBADs appear to have similar effects on both
alone, and also had no effect on IL-10 production induced by
CD3 + CD4+ and CD3 + CD4− cells. Given the central impor-
the irradiated bacteria. In contrast to the results obtained from
tance of IFN-γ production in protective immunity to Mtb36,37
the splenocyte studies, p-HBAD-II did not appear to affect pro-
these results indicate that p-HBADS may help the bacterium
inflammatory cytokine production by BMM co-stimulated with
evade the antibacterial effects of this key cytokine.
irradiated H37Rv. This data shows that p-HBAD-I and UM-p-
Macrophages play a vital role in Mtb pathogenesis and are
HBAD-I suppress TNF-α production but exert no effect on the
often the first line of defence in the host response to Mtb
production of the cytokines IL-10 or IL-6.
infection. Macrophages are also key antigen presenting cells
that can induce antigen specific CD4+ T cell responses which
have been implicated in protective immunity against TB.38 In
addition, recent studies with Mtb have determined that some
Conclusions
cell wall glycolipids can directly inhibit CD4+
T cell
activation.39–42 The effects of the p-HBADs on macrophages For the first time a number of synthetic p-HBADs have been
therefore provide a valid in vitro measure of the immunomodu- prepared and their immunomodulatory effects have
latory effects of p-HBADs in the context of Mtb pathogenesis. been studied in isolation from the parent bacterium. It has
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been determined that these small molecules have the ability to (m/z): [M + Na]+ calculated for C14H22O7SNa 357.0984; found
suppress host immune response in vitro but that they are not 357.0973.
immunostimulatory in themselves. In particular, an inhibition
of key immunomodulatory cytokines, IFN-γ and IL-17 by anti-
CD3e-stimulated splenocytes was observed, albeit at a high
Compound 343
concentration of the corresponding p-HBAD. This research has Thioglycoside 2 (650 mg, 1.95 mmol) was dissolved in metha-
important implications for developing an understanding of nol with catalytic sodium methoxide, quenched with DOWEX
Mtb pathogenesis and for developing new treatments for TB after 3 hours, filtered and concentrated to give 3 in 94% yield
1
infection.
(410 mg). H-NMR (400 MHz, CDCl3, δ): 5.29 (1H, s, H1), 4.07
(1H, m, H2), 4.05 (1H, m, H5), 3.77 (1H, dd, J3,2 = 2.9 Hz, J3,4
=
9.4 Hz, H3), 3.53 (1H, app t, H4), 2.65 (2H, m, CH2), 1.36 (3H,
d, J6,5 = 6.2 Hz, H6), 1.32 (3H, t, J = 7.5 Hz, CH3). 13C-NMR
(CDCl3, δ): 84.2 (C1), 73.5 (C4), 72.6 (C2), 72.3 (C3), 68.5 (C5),
25.2 (CH2) 17.5 (C6), 14.9 (CH3). HRMS-ESI (m/z): [M + Na]+ cal-
culated for C8H16O4NaS 231.0667; found 231.0676.
Experimental
General experimental methods
1
For NMR spectra, 400 MHz spectrometer was employed for H
(400.13 MHz) and 13C (100.61 MHz) spectra, 600 MHz spectro-
meter was employed for 1H (600.13 MHz) and 13C
(150.90 MHz) spectra. Resonances δ, are in ppm units down-
Compound 443
3 (1.56 g, 7.50 mmol) was dissolved in anhydrous acetone
(20 mL), 2,2-dimethoxy propane (3.67 mL, 30 mmol) was
added along with catalytic para-toluenesulfonic acid. After 3 h
the reaction was quenched by the addition of sat. aq. NaHCO3
(ca. 15 mL). The mixture was filtered and concentrated and
extracted with DCM. The organic layer was dried with MgSO4,
filtered and concentrated to give 4 in 96% yield (1.85 g).
1H-NMR (400 MHz, CDCl3, δ): 5.19 (1H, s, H1), 4.17 (1H, d,
J2,3 = 5.5 Hz, H2), 4.04 (1H, dd, J3,2 = 5.5 Hz, J3,4 = 7.6 Hz, H3),
3.44 (1H, m, H4), 2.61 (2H, m, CH2), 1.53, 1.34 (3H, s, CH3),
1.30 (3H, app t, CH2CH3), 1.29 (3H, d, J6,5 = 6.3 Hz, H6).
13C-NMR (CDCl3, δ): 109.0 (Cq), 79.4 (C1), 78.3 (C3), 76.8 (C2),
75.4 (C4), 66.0 (C5), 28.2, 26.4(CH3), 24.4 (CH2), 17.2 (C6), 14.6
(CH3). HRMS-ESI (m/z): [M + Na]+ calculated for C11H20O4NaS
271.0980; found 271.0991.
field from an internal reference in CDCl3 (δH = 7.26 ppm, δC
=
77.0 ppm), MeOH (δH = 3.31 ppm, δC = 49.0 ppm). Optical
rotations are quoted in deg cm3 g−1 dm−1. For oligosacchar-
ides the notation a, b, c…. refers to the monosaccharide from
the reducing end. Mass spectrometry analysis was performed
with Maldi-quadrupole time-of-flight (Q-Tof) mass spectro-
meter equipped with Z-spray electrospray ionization (ESI).
Silica gel (200 mesh) was used for column chromatography.
Analytical thin-layer chromatography was performed using
silica gel (pre-coated sheets, 0.2 mm thick, 20 cm × 20 cm)
and visualized by UV irradiation or molybdenum staining.
DCM, MeOH, THF and toluene were dried over flame dried 3 Å
or 4 Å sieves. Dimethylformamide (DMF), triethylamine (Et3N)
and trifluoroacetic acid (TFA) were used dry from sure/seal
bottles. Other reagents were purchased from an industrial
supplier.
Compound 527
Compound 243
4 (14.46 g, 58.3 mmol) was dissolved in DMF (5 mL) under N2;
Peracetylated rhamnose 1 (4.8 g, 14.7 mmol) was dissolved in NaH (2.80 g, 70.0 mmol) and BnBr (9.0 mL, 75.8 mmol) were
anhydrous dichloromethane (40 mL) at 0 °C under N2. Ethane added. After 18 h the reaction was quenched with ca. 2 mL
thiol (1.2 mL, 19.0 mmol) was added followed by BF3·OEt2 MeOH, concentrated, redissolved in diethyl ether (50 mL),
(9.1 mL, 73.5 mmol) in increments. The stirred solution was washed with water (2 × 40 mL) and saturated NaHCO3 (2 ×
allowed to warm to room temperature and stirred for 18 h to 40 mL), dried with MgSO4, and concentrated. Purification by
give a red solution which was quenched by treatment with column chromatography (hexane–EtOAc 80 : 20) furnished 5 as
saturated sodium bicarbonate solution (ca. 20 mL) and solid a colourless solid in 97% yield (19.0 g). 1H-NMR (400 MHz,
NaHCO3. The organic layer was filtered and concentrated. Puri- CDCl3, δ): 7.36 (5H, m, Ar-H), 5.50 (1H, s, H1), 4.91 (1H, d, J =
fication by column chromatography (hexane–EtOAc 80 : 20) 11.6 Hz, CH2), 4.74 (2H, dd, J = 11.4 Hz, J = 21.4 Hz, CH2), 4.63
furnished 2 as a colourless oil with an overall yield of 82% (1H, d, J = 11.6 Hz, CH2), 4.24 (1H, dd, J3,2 = 5.4 Hz, J3,4 = 7.2
(4.01 g) and 61% for the α anomer (3.01 g). Data for α anomer: Hz, H3), 4.18 (1H, app d, J = 5.4 Hz, H2), 4.03 (1H, m, H5),
1H-NMR (400 MHz, CDCl3, δ): 5.36 (1H, dd, J2,1 = 1.6 Hz, J2,3
3.4 Hz, H2), 5.26 (1H, dd, J3,2 = 3.4 Hz, J3,4 = 10.1 Hz, H3), 5.22 CH2CH3), 1.52, 1.36 (3H, s, CH3), 1.29 (3H, t, J = 7.3 Hz,
(1H, d, J = 1.5 Hz, H1), 5.12 (1H, app t, H4), 4.26 (1H, dq, J5,6
CH2CH3), 1.28 (3H, d, J6,5 = 6.1 Hz, H6). 13C-NMR (CDCl3, δ):
6.2 Hz, J5,4 = 9.7 Hz, H5), 2.67 (2H, m, CH2), 2.13, 2.08, 2.01 138.3 (Cq), 128.7, 128.3, 128.1, 127.9, 127.6 (Ar–C), 109.4 (Cq),
(3H, s, CH3), 1.32 (3H, t, J = 7.5 Hz, CH2CH3), 1.26 (3H, d, J6,5 82.0 (C4), 79.5 (C1), 78.4 (C3), 77.0 (C2), 75.0, 73.1 (CH2), 65.3
6.1 Hz, H6); 13C-NMR (CDCl3, δ): 170.1, 170.0, 169.9 (CvO), (C5), 28.1, 26.5 (CH3), 24.4 (CH2), 17.9 (C6), 17.7 (CH2CH3).
82.0 (C1), 71.5 (C2), 71.3 (C4), 69.5 (C3), 66.7 (C5), 25.4 (CH2), HRMS-ESI (m/z): [M
Na]+ calculated for C18H26O4NaS
21.0, 20.8, 20.7 (CH3), 17.4 (C6), 14.9 (CH2CH3); HRMS-ESI 361.1450; found 361.1436. IR: 2895, 1369, 1248, 1093 cm−1
=
3.30 (1H, J4.3 = 7.2 Hz, J4.5 = 9.8 Hz, H4), 2.60 (2H, m,
=
=
+
.
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Compound 627,44
(C4), 74.8 (CH2), 72.3 (C3), 71.8 (C2), 68.1 (C5), 37.7 (CH2),
29.6 (CH3), 27.7, 25.2 (CH2), 20.8 (CH3), 17.7 (C6), 14.7
(CH2CH3). HRMS-ESI (m/z): [M
5 (585 mg, 1.73 mmol) was dissolved in 9 : 1 acetic acid–H2O
(5 mL) and reacted for 18 h at 50 °C then concentrated. Purifi-
cation by column chromatography (hexane–EtOAc 60 : 40) gave
6 as a colourless solid in 98% yield (515 mg). [α]2D0 −181.8
(c 0.11, CHCl3). 1H-NMR (400 MHz, CDCl3, δ): 7.40 (5H, m,
Ar-H), 5.27 (1H, d, J1,2 = 1.3 Hz, H1), 4.77 (2H, dd, J = 11.4 Hz,
J = 19.8 Hz, CH2), 4.13 (1H, m, H5), 4.06 (1H, dd, J2,3 = 3.4 Hz,
J2,1 = 1.4 Hz, H2), 3.91 (1H, dd, J3,2 = 3.4 Hz, J3,4 = 9.1 Hz, H3),
+
Na]+ calculated for
C22H30O7NaS 461.1610; found 461.1625. IR: 2931, 1743, 1368,
1231, 1096 cm−1
.
Compound 9
8 (16.00 g, 36.49 mmol) was dissolved in MeOH (50 mL) and a
catalytic amount of NaOMe was added. After stirring at rt for
18 h the reaction was quenched with a catalytic amount of
DOWEX, filtered and concentrated to give 9 as a white solid in
99% yield (14.33 g). [α]D20 −156.8 (c 0.0051, CHCl3). 1H-NMR
3.41 (1H, app t, H4), 2.65 (2H, m, CH2CH3), 1.39 (3H, J6,5
=
6.3 Hz, H6), 1.32 (3H, t, J = 7.4 Hz, CH2CH3). 13C-NMR (CDCl3,
δ): 138.2 (Cq), 128.7, 128.1, 127.9 (Ar–C), 83.6 (C1), 82.0 (C4),
75.0 (CH2), 72.6 (C2), 71.9 (C3), 67.7 (C5), 25.1 (CH2CH3), 18.0
(C6), 14.9 (CH2CH3). HRMS-ESI (m/z): [M − H]− calculated for
C15H21O4S 297.1161; found 297.1151. IR: 3289, 2929, 1749,
(400 MHz, CDCl3, δ): 7.39 (5H, m, Ar–H), 5.27 (1H, d, J1,2
=
1.4 Hz, H1), 4.77 (2H, dd, J = 11.4 Hz, J = 15.6 Hz, CH2), 4.13
(1H, m, H5), 4.05 (1H, dd, J2,1 = 1.4 Hz, J2,3 = 3.4 Hz, H2), 3.91
(1H, dd, J3,2 = 3.4 Hz, J3,4 = 9.1 Hz, H3), 3.41 (1H, app t, J =
9.3 Hz, H4), 2.79 (2H, t, J = 6.6 Hz, CH2), 2.63 (2H, m,
CH2CH3), 2.61 (2H, t, J = 6.6 Hz, CH2), 2.22 (3H, s, CH3), 1.38
(3H, d, J6,5 = 6.3 Hz, H6), 1.31 (3H, t, J = 7.4 Hz, CH2CH3).
13C-NMR (CDCl3, δ): 206.7, 173.3 (CvO), 138.2 (Cq), 128.7,
128.0, 127.9 (Ar–C), 83.6 (C1), 82.0 (C4), 75.0 (CH2Bn), 72.6
(C2), 71.9 (C3), 67.7 (C5), 38.0 (CH2), 29.9 (CH3), 27.7 (CH2),
25.1 (CH2), 18.0 (C6), 14.9 (CH2CH3). HRMS-ESI (m/z): [M +
Na]+ calculate for C20H28O6NaS 419.1499; found 419.1490. IR:
1453, 1082 cm−1
.
Compound 744,45
6 (420 mg, 1.41 mmol) was dissolved in acetonitrile (15 mL)
with trimethylorthoacetate (0.36 mL, 2.82 mmol) and cam-
phorsulfonic acid (catalytic) under N2. The mixture was
allowed to react for 45 min then 5 mL 4 : 1 acetic acid–H2O was
added. After 15 min it was diluted with DCM, washed with
H2O and aq. saturated NaHCO3 solution, dried with MgSO4
and concentrated. Purification by column chromatography
3290, 2923, 1453, 1081 cm−1
.
(hexane–EtOAc 60 : 40) gave 7 as a colourless oil in 89% yield Compound 1044
(427 mg). 1H-NMR (400 MHz, CDCl3, δ): 7.36 (5H, m, Ar–H)
9 (250 mg, 0.63 mmol) was dissolved in DMF (4 mL) under N2,
NaH (35 mg, 0.88 mmol) and MeI (0.047 mL, 0.76 mmol) were
added. After 18 h the reaction was quenched with ca. 2 mL
MeOH, concentrated, redissolved in diethyl ether (20 mL),
washed with water (2 × 15 mL) and sat. NaHCO3 (2 × 15 mL),
dried with MgSO4, and concentrated. Purification by column
chromatography (hexane–EtOAc 80 : 20) gave 10 as a yellow oil
in 74% yield (146 mg) with the dimethylated compound, also a
yellow oil, as a side product. [α]2D0 −220.5 (c 0.0069, CHCl3).
1H-NMR (400 MHz, CDCl3, δ): 7.38 (5H, m, Ar-H), 5.41 (1H, d,
J1,2 = 1.2 Hz, H1), 4.93, 4.70 (1H, d, J = 11.2 Hz, CH2), 4.07 (1H,
m, H5), 3.91 (1H, dd, J3,2 = 3.7 Hz, J3,4 = 9.3 Hz, H3), 3.61 (1H,
dd, J2,1 = 1.2 Hz, J2,3 = 3.7 Hz, H2), 3.51 (3H, s, CH3), 3.33 (1H,
5.23 (1H, s, H1), 5.23 (1H, m, H2), 4.85, 4.75 (1H, d, J = 11.0
Hz, CH2), 4.14 (1H, m, H5), 4.09 (1H, m, H3), 3.42 (1H, app t,
H4), 2.65 (2H, m, CH2CH3), 2.19 (3H, s, CH3), 1.39 (3H, d,
J6.5 = 6.2 Hz, H6), 1.31(3H, t, J = 7.4 Hz, CH2CH3). 13C-NMR
(CDCl3, δ): 170.8 (CvO) 138.1 (Cq), 128.6, 128.0, 127.9 (Bn),
82.1 (C1), 82.0 (C4), 75.2 (CH2), 74.6 (C2), 70.8 (C3) 68.0 (C5),
25.6 (CH2CH3), 21.2 (CH3), 18.0 (C6), 15.0 (CH2CH3).
HRMS-ESI (m/z): [M
363.1242; found 363.1241. IR: 3457, 2931, 1740, 1373,
1231 cm−1
+
Na]+ calculated for C17H24O5SNa
.
Compound 8
7 (12.5 g, 36.7 mmol) was dissolved in DCM (30 mL), and levu- app t, H4), 2.65 (2H, m, CH2CH3), 1.34 (3H, d, J6,5 = 6.3 Hz,
linic acid (17.25 g, 146.24 mmol), DCC (30.3 g, 146.24 mmol), H6), 1.32 (3H, t, J = 7.4 Hz, CH2CH3).13C-NMR (CDCl3, δ):
TEA (catalytic 2 mL) and DMAP (catalytic) were added. After 138.5 (Cq), 128.4, 128.0, 127.7 (Ar–C), 82.5 (C4), 82.3 (C2), 80.1
18 h the mixture was filtered through cotton wool, washed (C1), 75.1 (CH2Bn), 72.1 (C3), 67.6 (C5), 58.1 (CH3), 25.2
with aq. sat. NaHCO3 (25 mL), aq. sat. NaCl (25 mL), H2O (CH2CH3), 17.9 (C6), 15.0 (CH2CH3). HRMS-ESI (m/z): [M +
(25 mL), dried with MgSO4, filtered and concentrated. Purifi- Na]+ calculated for C16H24O4NaS 335.1293; found 335.1292. IR:
cation by column chromatography (hexane–EtOAc 70 : 30) gave 3469, 2930, 1454, 1090 cm−1
the product 8 as a colourless oil in 99% yield (16.0 g).
.
Compound 1144
[α]2D0 −163 (c 0.136, CHCl3). 1H-NMR (400 MHz, CDCl3, δ): 7.34
(5H, m, Ar–H), 5.36 (1H, dd, J2,1 = 1.6 Hz, J2.3 = 3.2 Hz, H2), 10 (580 mg, 1.86 mmol) was dissolved in pyridine (5 mL) and
5.27 (1H, dd, J3.2 = 3.2 Hz, J3.4 = 9.7 Hz, H3), 5.18 (1H, app s, Ac2O (5 mL) under N2. After 18 h the mixture was quenched
H1), 4.76, 4.66 (1H, d, J = 11.3 Hz, CH2), 4.20 (1H, m, H5), 3.57 with iced water, diluted with EtOAc, washed with water
(1H, app t, H4), 2.68 (4H, m, CH2), 2.51 (2H, m, CH2), 2.19, (20 mL), dilute aq. CuSO4 (6 × 20 mL), washed with water
2.18 (3H, s, CH3), 1.37 (3H, d, J6.5 = 6.3 Hz, H6), 1.30 (3H, t, J = (20 mL), dried with MgSO4, filtered and concentrated. Purifi-
7.3 Hz, CH2CH3). 13C-NMR (CDCl3, δ): 206.1, 171.5, 169.9 cation by column chromatography (hexane–EtOAc 80 : 20) gave
(CvO), 137.9 (Cq), 128.3, 127.62, 127.59 (Ar–C), 81.1 (C1), 78.8 11 as a colourless oil 77% yield (518 mg). [α]2D0 −143.9
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(c 0.0532, CHCl3). 1H-NMR (400 MHz, CDCl3, δ): δ 7.34 (5H, m, 425.1576; found 425.1565. IR: 2931, 1716, 1605, 1278,
Ar–H), 5.34 (1H, d, J1,2 = 1.8 Hz, H1), 5.19 (1H, dd, J3,2
1098 cm−1
=
.
3.3 Hz, J3,4 = 9.5 Hz, H3), 4.70 (2H, dd, J = 11.4 Hz, J = 33.7 Hz,
CH2), 4.15 (1H, m, H5), 3.78 (1H, dd, J2,1 = 1.8 Hz, J2,3 = 3.3 Hz,
Compound 14
H2), 3.60 (1H, app t, H4), 3.47 (3H, s, OCH3), 2.65 (2H, m, 13 (60.8 mg, 0.151 mmol) was dissolved in THF and a catalytic
CH2CH3), 2.08 (3H, s, CH3), 1.35 (3H, d, J6,5 = 6.2 Hz, H6), 1.32 amount of palladium on carbon added. The mixture was first
(3H, t, J = 7.5 Hz, CH2CH3). 13C-NMR (CDCl3, δ): 170.2 (CvO), degassed by bubbling N2 through the solution for 1 hour then
138.3 (Cq), 128.4, 127.7, 127.6 (Ar–C), 80.9 (C1), 80.2 (C2), 79.4 saturated with H2 by bubbling H2 through the mixture for
(C4), 75.0 (CH2), 74.1 (C3), 68.1 (C5), 58.6 (OCH3), 25.3 (CH2), 30 min. This saturation was repeated periodically over 2 days
21.1 (CH3), 17.9 (C6), 14.9 (CH2CH3). HRMS-ESI (m/z): [M + and the reaction kept under H2 throughout. When TLC analy-
Na]+ calculated for C18H26O5NaS 377.1399; found 377.1399. IR: sis indicated the consumption of the starting material N2 was
2932, 1737, 1369, 1233, 1084 cm−1
.
again bubbled through before exposing the mixture to air. The
palladium was filtered off and the resulting solution concen-
trated. Purification by column chromatography (acetone–
Compound 12
Trifluoromethanesulfonic anhydride (0.328 mL, 1.49 mmol) toluene 20 : 80) furnished 14 as a colourless solid in 83% yield
was added to a solution of dimethyl disulfide (0.195 mL, (39.4 mg). [α]2D2 −86.9 (c 0.0184, CHCl3).1H-NMR (400 MHz,
2.3 mmol) in anhydrous DCM (1 mL) at 0 °C and reacted for CDCl3, δ): 8.01, 7.11 (2H, d, J = 8.7 Hz, Ar–H), 5.67 (1H, d, J1,2
=
30 min at 0 °C to give a dark yellow solution. 11 (520 mg, 1.4 Hz, H1), 3.94 (1H, dd, J3,2 = 3.7 Hz, J3,4 = 9.6 Hz, H3), 3.90
1.29 mmol) was dissolved in anhydrous DCM (7 mL) under N2 (3H, s, CO2CH3), 3.70 (1H, dd, J2,1 = 1.4 Hz, J2,3 = 3.7 Hz, H2),
at 0 °C along with methyl 4-hydroxybenzoate (296 mg, 3.67 (1H, m, H5), 3.56 (3H, s, OCH3−), 3.47 (1H, app t, J = 9.6
1.94 mmol). 1.5 eq. of the activating Me2S2–Tf2O solution was Hz, H4), 1.27 (3H, d, J6,5 = 6.5 Hz, H6). 13C-NMR (CDCl3, δ):
added. The mixture was reacted at 0 °C for 1 h, quenched with 166.7 (CvO), 159.9 (Cq), 131.6 (Ar–C), 124.2 (Cq), 115.8 (Ar–C),
ca. 1 mL TEA, diluted with DCM (15 mL), washed with 1 M 94.3 (C1), 79.9 (C2), 73.8 (C4), 71.3 (C3), 68.9 (C5), 59.3, 52.0
HCl (20 mL), sat. aq. NaHCO3 (20 mL), H2O (20 mL), dried (OCH3), 17.6 (C6). HRMS-ESI (m/z): [M − H]− calculated for
with MgSO4, filtered and concentrated. Purification by column C15H19O7 311.1131; found 311.1134. IR: 3426, 2933, 1718,
chromatography (hexane–EtOAc 70 : 30) gave 12 as a pale 1281, 1106 cm−1
yellow oil in 58% yield (330 mg). [α]1D8 −92.8 (c 0.0070, CHCl3).
.
Compound 1546
1H-NMR (400 MHz, CDCl3, δ): 8.02 (2H, m, Ar–H), 7.34 (5H, m,
Ar-H), 7.14 (2H, m, Ar), 5.61 (1H, d, J1,2 = 1.9 Hz, H1), 5.44 (1H, 9 (1.00 g, 2.52 mmol) was dissolved in DMF (10 mL) with NaH
dd, J3,2 = 3.4 Hz, J3,4 = 9.4 Hz, H3), 4.72 (2H, dd, J = 11.2 Hz, J = (121 mg, 3.03 mmol) and BnBr (0.375 mL, 3.03 mmol) under
33.2 Hz, CH2), 3.91 (3H, s, OCH3), 3.90 (1H, m, H2), 3.83 (1H, N2. After 18 h the reaction was quenched with ca. 2 mL MeOH,
m, H5), 3.65 (1H, app t, H3), 3.55 (3H, s, OCH3), 2.13 (3H, s, concentrated, diluted with diethyl ether (75 mL), washed with
CH3), 1.30 (3H, d, J6,5 = 6.2 Hz, H6). 13C-NMR (CDCl3, δ): 170.4 water (2 × 50 mL) and sat. NaHCO3 (2 × 50 mL), dried with
(CvO), 169.7, 159.8, 138.0 (Cq), 131.6, 128.5, 127.8, 127.6 (Ar– MgSO4, filtered, and concentrated. Purification by column
C), 124.1 (Cq), 115.9 (Ar–C), 95.2 (C1), 78.9 (C4), 78.3 (C2), 75.1 chromatography (hexane–EtOAc 80 : 20) gave the product as a
(CH2), 73.5 (C3), 68.8 (C5), 59.6, 52.0 (OCH3), 21.2 (CH3), 17.0 colourless oil in 87% yield (849 mg). [α]2D0 −139 (c 0.1, CHCl3).
(C6). HRMS-ESI (m/z): [M + Na]+ calculated for C24H28O8Na 1H-NMR (400 MHz, CDCl3, δ): 7.39 (10H, m, Ar-H), 5.40 (1H,
467.1682; found 467.1689. IR: 2936, 1718, 1606, 1279, app s, H1), 4.95 (1H, d, J = 11.2 Hz, CH2), 4.81 (1H, d, J = 11.7
1233 cm−1
.
Hz, CH2), 4.70 (1H, d, J = 11.2 Hz, CH2), 4.60 (1H, d, J = 11.7
Hz, CH2), 4.10 (1H, m, H5), 3.97 (1H, dd, J3,2 = 3.7 Hz, J3,4 = 9.1
Hz, H3), 3.88 (1H, dd, J2,1 = 1.4 Hz, J2,3 = 3.7 Hz, H2), 3.41 (1H,
Compound 13
12 (97.3 mg, 0.219 mmol) was dissolved in 7 mL MeOH along app t, H4), 2.63 (2H, m, CH2CH3), 1.38 (3H, d, J6,5 = 6.3 Hz,
with a catalytic amount of NaOMe and allowed to react for H6), 1.30 (3H, t, J = 7.4 Hz, CH3). 13C-NMR (CDCl3, δ): 138.6,
18 h. The reaction was quenched with a catalytic amount of 137.6 (Cq), 128.7, 128.4, 128.2, 128.1, 128.0, 127.7 (Ar–C), 82.6
DOWEX, filtered and concentrated to give 13 as a pale yellow (C4), 81.0 (C1), 80.3 (C2), 75.1, 72.5 (CH2), 72.2 (C3), 67.7 (C5),
oil in 81% yield (71.3 mg). [α]1D8.6 −79.3 (c 0.097, CHCl3). 25.2 (CH2CH3), 18. (C6), 15.0 (CH2CH3). HRMS-ESI (m/z): [M +
1H-NMR (400 MHz, CDCl3, δ): 8.01 (2H, m, Ar), 7.34 (5H, m, Na]+ calculated for C22H28O4NaS 411.1606; found 411.1599. IR:
Ar–H), 7.10 (2H, m, Ar–H), 5.65 (1H, d, J1,2 = 1.6 Hz, H1), 4.94, 3472, 2929, 1716, 1454, 1091 cm−1
.
4.64 (1H, d, J = 10.9 Hz, CH2), 3.90 (3H, s, CH3), 3.82(1H, dd,
Compound 1646
J2,1 = 1.6 Hz, J2,3 = 3.3 Hz, H2), 3.80 (1H, dd, J3,2 = 3.3 Hz, J3,4
=
9.1 Hz, H3), 3.69 (1H, m, H5), 3.61 (3H, s, CH3), 3.53 (1H, app 15 (803 mg, 2.07 mmol) was dissolved in pyridine (5 mL) and
t, H4), 1.27 (3H, d, J6,5 = 6.1 Hz, H6). 13C-NMR (CDCl3, δ): Ac2O (5 mL) under N2. After 18 h the mixture was quenched
210.8, 166.6 (CvO), 159.8 (Cq), 131.5, 128.3, 127.9, 127.6 (Ar– with iced water, diluted with EtOAc, washed with water
C), 123.9 (Cq), 115.7 (Ar), 94.9 (C1), 81.2 (C3), 80.1 (C4), 77.0 (20 mL), dil. aq. CuSO4 (6 × 20 mL), washed with water
(C2), 75.3 (CH2), 68.8 (C5), 59.4, 57.9, 51.8 (CH3), 17.8 (C6). (20 mL), dried with MgSO4, filtered and concentrated. Purifi-
HRMS-ESI (m/z): [M
+
Na]+ calculated for C22H26O7Na cation by column chromatography (hexane–EtOAc 90 : 10) gave
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16 as pale yellow oil 96% yield (859 mg). [α]D20 −73.3 (c 0.105, CH2), 4.29 (1H, dd, J3,2 = 3.0 Hz, J3,4 = 9.5 Hz, H3b), 4.24 (1H,
1
CHCl3). H-NMR (400 MHz, CDCl3, δ): 7.36 (10H, m, Bn), 5.30 d, J = 11.9 Hz, CH2), 4.06 (1H, dd, J3,2 = 3.6 Hz, J3,4 = 9.3 Hz,
(1H, d, J1,2 = 1.7 Hz, H1), 5.18 (1H, dd, J3,2 = 3.4 Hz, J3,4 = 9.5 H3a), 3.95 (1H, m, H5a), 3.93 (3H, s, OCH3), 3.78 (3H, m, H2a,
Hz, H3), 4.72 (2H, dd, J = 11.7 Hz, J = 30.1 Hz, CH2), 4.71 (1H, H2b, H5b), 3.61 (1H, app t, H4b), 3.59 (3H, s, OCH3) 3.40 (1H,
d, J = 12.6 Hz, CH2), 4.57 (1H, d, J = 12.2 Hz, CH2), 4.17 (1H, app t, H4a), 1.42 (3H, d, J6,5 = 6.3 Hz, H6a), 1.29 (3H, d, J6,5
=
m, H5), 4.00 (1H, dd, J2,1 = 1.7 Hz, J2,3 = 3.4 Hz, H2), 3.70 (1H, 5.8 Hz, H6b). 13C-NMR (CDCl3, δ): 166.7(CvO), 159.9, 138.5,
app t, H4), 2.62 (2H, m CH2), 1.99 (3H, s, CH3), 1.38 (3H, d, 138.3, 137.7 (Cq), 131.7, 128.5, 128.46, 128.4, 128.0, 127.9,
J6,5 = 6.2 Hz, H6), 1.29 (3H, t, J = 7.4 Hz, CH2CH3). 13C-NMR 127.8, 127.7, 127.6, 127.0 (Ar–C), 124.1 (Cq), 115.9 (Ar–C), 99.4
(CDCl3, δ): 170.1 (CvO), 138.3, 137.8 (Cq), 128.5, 128.4, (C1a). 94.9 (C1b), 82.1 (C4a), 80.3 (C4b), 80.1(C2b), 79.2 (C2a),
127.96, 127.95, 127.7, 127.6 (Ar–C), 81.7 (C1), 79.4 (C4), 77.7 78.4 (C3b), 75.1, 75.0, 72.7 (CH2), 71.6 (C3a), 69.3 (C5b), 68.0
(C2), 74.9 (CH2), 74.0 (C3), 72.6 (CH2), 68.2 (C5), 25.3 (CH2), (C5a), 59.2, 52.0 (OCH3), 18.2 (C6b), 18.0 (C6a). HRMS-ESI
21.1 (CH3), 18.0 (C6), 15.0 (CH2CH3). HRMS-ESI (m/z): [M + (m/z): [M + Na]+ calculated for C43H52O10Na 751.3458; found
Na]+ calculated for C24H30O5NaS 453.1712; found 435.1714. IR: 751.3466. IR: 3376, 2926, 1717, 1606, 1279 cm−1
.
2930, 1737, 1454, 1292, 1080 cm−1
.
Compound 20
19 (91.7 mg, 0.1258) was dissolved in anhydrous DCM (5 mL)
Compound 17
13 (118 mg, 0.293 mmol) was dissolved in anhydrous DCM under N2. 12 (28 mg, 0.112 mmol) was added followed by NIS
(5 mL) under N2. 16 (152 mg, 0.352 mmol) was added followed (40.0 mg, 0.1792 mmol) and a catalytic amount (ca. 10 μL) of
by NIS (106 mg, 0.352 mmol) and a catalytic amount (ca. TMS·OTf at 0 °C. After 18 h at rt the mixture was quenched
10 μL) of TMS·OTf at 0 °C. After 18 h at rt the mixture was with ca. 1 mL of TEA, washed with sat. NaHCO3 solution
quenched with ca. 1 mL of TEA, washed with sat. NaHCO3 (15 mL), dried with MgSO4, filtered and concentrated. Purifi-
solution (15 mL), dried with MgSO4, filtered and concentrated. cation by column chromatography (hexane–EtOAc 80 : 20) furn-
Purification by column chromatography (hexane–EtOAc ished 20 as a pale yellow oil in 67% yield (69.0 mg). [α]2D0 −82.0
1
80 : 20) gave 17 as a pale yellow oil in 95% yield (214 mg). [α]2D0 (c 0.1, CHCl3). H-NMR (600 MHz, CDCl3, δ): 8.02 (2H, m, Ar),
−34.9 (c 0.103, CHCl3). 1H-NMR (600 MHz, CDCl3, δ): 8.03 (2H, 7.34 (15H, m, Ar–H), 7.12 (2H, m, Ar), 5.63 (1H, d, J1,2 = 1.4 Hz,
m, Ar–H), 7.33 (15H, m, Ar–H), 7.12 (2H, m, Ar–H), 5.62 (1H, H1c), 5.26 (1H, d, J1,2 = 1.3 Hz, H1b), 5.24 (1H, d, J1,2 = 1.5 Hz,
d, J1,2 = 1.6 Hz, H1b), 5.32 (1H, dd, J3,2 = 3.1 Hz, J3,4 = 9.5 Hz, H1a), 5.21 (1H, d, J = 11.3 Hz, CH2), 4.89, 4.70 (1H, d, J = 11.6
H3a), 5.17 (1H, d, J1,2 = 1.5 Hz, H1a), 4.87 (1H, d, J = 11.5 Hz, Hz, CH2), 4.64 (1H, d, J = 11.3 Hz, CH2), 4.59, 4.28 (1H, d, J =
CH2), 4.77 (1H, d, J = 11.3 Hz, CH2), 4.69 (2H, d, J = 11.7 Hz, 12.2 Hz, CH2), 4.27 (1H, dd, J2,3 = 3.2 Hz, J3,4 = 9.7 Hz, H3c),
CH2), 4.47, 4.32 (1H, d, J = 12.1 Hz, CH2), 4.25 (1H, dd, J3,2
=
4.12 (1H, dd, J2,3 = 3.2 Hz, J3,4 = 9.5 Hz, H3b), 3.98 (1H, m,
3.1 Hz, J3,4 = 9.5 Hz, H3b), 4.06 (1H, m, H5b), 3.94 (1H, dd, H5b), 3.91 (3H, s, OCH3), 3.85 (1H, dd, J2,1 = 1.8 Hz, J2,3 = 3.2
J2,1 = 1.9 Hz, J2,3 = 1.5 Hz, H2a), 3.92 (3H, s, CO2CH3), 3.80 Hz, H2b), 3.81 (1H, dd, J2,1 = 1.8 Hz, J2,3 = 3.2 Hz, H2c), 3.76
(1H, dd, J2,1 = 1.9 Hz, J2,3 = 3.0 Hz, H2b), 3.75 (1H, m, H5b), (1H, m, H5c), 3.71 (1H, m, H5a), 3.66 (1H, app t, J = 9.4 Hz,
3.71 (1H, app t, H4a), 3.62 (1H, app t, H4b), 3.60 (3H, s, H4b), 3.60 (1H, app t, H4c), 3.59 (3H, s, OCH3), 3.58 (2H, m,
OCH3), 1.98 (3H, s, CH3), 1.41 (3H, J6.5 = 6.3 Hz, H6a), 1.27 H2a, H3a), 3.56, 3.53, 3.36 (3H, s, OCH3), 3.24 (1H, app s,
(3H, d, J6,5 = 6.2 Hz, H6b). 13C-NMR (CDCl3, δ): 170, 166.5 H4a), 1.37 (3H, d, J6,5 = 6.3 Hz, H6b), 1.25 (3H, d, J6,5 = 6.3 Hz,
(CvO), 159.8 (Cq), 138.3, 138.1, 137.8 (Ar–C), 137.1 (Cq), H6c), 1.01 (3H, d, J6,5 = 6.3 Hz, H6a). 13C-NMR (CDCl3, δ):
128.3, 128.24, 128.23, 128.12, 128.08, 128.0, 127.9, 127.85, 166.7 (CvO), 156.0, 139.1, 138.4, 138.3 (Cq), 131.6, 128.4,
127.82, 127.6, 127.59, 127.55, 127.54, 127.51, 127.4, 115.8 (Ar– 128.3, 128.2, 127.7, 127.5, 127.4, 127.3, 227.2, 127.1 (Ar–C),
C), 100.0 (C1b), 94.7 (C1a), 79.9 (C2b), 79.7(C4b), 79.0 (C3b), 124.1 (Cq), 115.8 (Ar–C), 99.6 (H1c), 99.4 (H1a), 94.7 (H1), 80.4
78.9 (C4a), 76.9 (C2a), 74.9, 74.8 (CH2), 73.4 (C3a), 72.8 (CH2), (H2c), 80.1 (H2), 79.77 (H4), 79.75 (H4b), 79.4 (H3, H3b),79.2
69.1 (C5b), 68.4(C5a), 59.0 (OCH3), 51.8 (CO2CH3), 20.9 (CH3), (H4c), 78.9 (H2b), 77.9 (H3c), 75.0, 74.7, 71.5 (CH2), 69.2 (H5),
18.1 (C6a), 17.8 (C6b). HRMS-ESI (m/z): [M + Na]+ calculated 68.9 (H5b), 67.4 (H5c), 61.7, 59.2, 59.0, 58.0, 52.0 (OCH3), 18.2
for C44H50O12Na 793.3200; found 793.3180. IR 2932, 1718, (H6b), 17.9 (H6), 16.7 (H6c). HRMS-ESI (m/z): [M + Na]+ calcu-
1605, 1234, 1098 cm−1
.
lated for C51H64O15Na 939.4143; found 939.4139. IR: 2917,
1750, 1606, 1509, 1220 cm−1
.
Compound 18
Compound 21
17 (91.7 mg, 0.126 mmol) was dissolved in 10 mL MeOH, a cat-
alytic amount of NaOMe was added and the mixture stirred at 20 (118 mg, 0.129 mmol) was dissolved in THF and a catalytic
rt for 48 h. The reaction was quenched with a catalytic amount amount of palladium on carbon added. The mixture was
of DOWEX, filtered and concentrated to give the product 18 as degassed under vacuum and then stirred vigorously under a
a pale yellow oil in 95% yield (69.0 mg). [α]2D0 −50.9 (c 0.159, hydrogen atmosphere until TLC analysis indicated the con-
CHCl3). 1H-NMR (400 MHz, CDCl3, δ): 8.04 (2H, m, Ar), 7.30 sumption of the starting material. A N2 atmosphere was intro-
(15H, m, Ar–H), 7.14 (2H, m, Ar–H), 5.63 (1H, app s, H1b), 5.26 duced before exposing the mixture to air. The palladium was
(1H, app s, H1a), 4.95(1H, d, J = 10.6 Hz, CH2), 4.83 (1H, J = filtered off and the resulting solution concentrated. Purifi-
11.1 Hz, CH2), 4.73 (2H, m, CH2), 4.45 (1H, d, J = 11.9 Hz, cation by column chromatography (40 : 60 acetone–toluene)
This journal is © The Royal Society of Chemistry 2014
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Organic & Biomolecular Chemistry
furnished 21 as a pale yellow, amorphous solid in 70% yield Cl]− calculated for C14H18O7Cl 333.0741; found 333.0750. IR:
(58.4 mg). [α]2D0 −89.3 (c 0.103, CHCl3). 1H-NMR (600 MHz, 3365, 2937, 1700, 106, 1436, 1238 cm−1
.
CDCl3, δ): 8.03, 7.14 (2H, m, Ar–H), 5.65 (1H, d, J1,2 = 1.6 Hz,
Mice. C3H/HeJ mice were purchased from Harlan UK. All
H1c), 5.165 (1H, d, J1,2 = 3.3 Hz, H1a), 5.162 (1H, d, J1,2
=
mice were maintained according to EU regulations and exper-
1.4 Hz, H1b), 4.13 (1H, dd, J2,1 = 1.4 Hz, H2b), 4.08 (1H, m, iments were performed under licence from the Department of
H4a), 4.05 (1H, dd, J3,2 = 3.3 Hz, J3,4 = 9.0 Hz, H3c), 3.96 (1H, Health and Children and with approval from the Trinity
m, H5b), 3.92 (3H, s, CO2Me), 3.82 (1H, dd, J3,2 = 3.3 Hz, J3,4
=
College Dublin Bioresources Ethics Committee.
9.6 Hz, H3b), 3.80 (1H, dd, J2,1 = 1.7 Hz, J2,3 = 3.3 Hz, H2c),
Spleen cells. Cells were isolated from the spleens of C3H/
3.73 (1H, m, H5c), 3.72 (1H, m, H4c), 3.69 (1H, dd, J2,1 = 3.3 HeJ mice. 1.5 × 106 cells per mL were activated on 96-well flat-
Hz, J2,3 = 9.9 Hz, H2a), 3.68 (1H, m, H4b), 3.66 (1H, m, H4a), bottomed tissue-culture plates (Greiner) that had been coated
3.63, 3.61, 3.55, 3.54 (3H, s, OCH3), 3.50 (1H, app d, J = 2.5 Hz, with 0.4 μg anti-CD3e (BD Pharmingen) for 3 h at 37 °C, and
H3a), 1.40 (3H, d, J6,5 = 6.4 Hz, H6b), 1.30 (3H, d, J6,5 = 5.6 Hz, then washed 3 times with sterile PBS. Spleen cells were cul-
H6c), 1.30 (3H, d, J6,5 = H6a). 13C-NMR (CDCl3, δ): 166.8 tured in medium alone or with p-HBad I, p-HBad II or UM-p-
(CvO), 160.0 (Cq), 131.6 (Ar–C), 124.2 (Cq), 115.9 (Ar–C), 102.2 HBad (100 μM). Supernatants were collected after 4 d and IFNγ
(C1b), 100.9 (C1a), 94.5 (C1c), 83.2 (C3b), 81.0 (C4a), 79.8 (BD Pharmingen), IL-17 and IL-10 (R&D Systems) concen-
(C3c), 79.0 (C3a), 78.8 (C2 s), 71.6 (C4b), 71.5 (C5c), 71.2 (C2b), trations were determined by ELISA. Spleen cells were stimu-
69.5 (C4c), 68.7 (C5b), 67.6 (C4a), 61.9, 60.3, 58.7, 57.7 (OCH3), lated for 5 h with PMA (0.1 μg mL−1) and ionomycin (0.5 μg
52.0 (CO2CH3), 17.9 (C6b), 17.7 (C6c), 16.6 (C6a). HRMS-ESI mL−1) and for the final 4 h with brefeldin A (10 μg mL−1). Cells
(m/z): [M + Na]+ calculated for C30H46O15Na 669.2734 found were washed, and blocked with Fcγ blocker (BD Pharmingen
669.2763. νmax(L) 3443, 2928, 1717, 1283, 1091 cm−1
.
1 μg mL−1) before staining with aqua Live/Dead stain (Invitro-
gen). Extracellular staining for surface CD3, CD4 and CD8
(eBioscience) was carried out and then spleen cells were fixed
Compound 22
1 (2.00 g, 6.04 mmol) was dissolved in anhydrous DCM and permeabilized (Intracellular Fixation and Permeabilization
(20 mL) at 0 °C under N2. Methyl-4-hydroxybenzoate (1.20 g, kit (eBioscience)) and stained intracellularly for IFNγ
7.85 mmol) was added followed by BF3·OEt2 (3.73 mL, (eBioscience). Flow cytometric analysis was performed using a
30.2 mmol) in increments. After 18 h the mixture was BD LSRFortessa cell analyser (BD Biosciences).
quenched by treatment with sat. NaHCO3 solution (ca. 50 mL)
Bone-marrow
derived
macrophages
(BMDMs). Bone
and solid NaHCO3. The organic layer was filtered, dried with marrow-derived macrophages (BMDMs) were prepared by cul-
MgSO4, filtered and concentrated. Purification by column turing bone marrow cells from C3H/HeJ mice in medium sup-
chromatography (hexane–EtOAc 80 : 20) gave 22 as a colourless plemented with 15% macrophage colony-stimulating factor
solid in 96% yield (2.459 g). [α]2D0 −88.8 (c 0.072, CHCl3). (M-CSF). M-CSF was collected in the supernatant of L929 cells
1H-NMR (400 MHz, CDCl3, δ): 8.02, 7.13 (2H, m, Ar–H), 5.55 in culture. On day 4, fresh medium containing 20% MCSF was
(1H, d, J1,2 = 1.8 Hz, H1), 5.52 (1H, dd, J3,2 = 3.5 Hz, J3,4 = 10.1 added to the cell cultures. On day 7 cells were removed from
Hz, H3), 5.45 (1H, dd, J2,1 = 1.8 Hz, J2,3 = 3.5 Hz, H2), 5.18 (1H, the culture flasks and re-suspended in fresh medium contain-
app t, H4), 3.95 (1H, m, H5), 3.91(3H, s, OCH3), 2.22, 2.07, ing 15% M-CSF. BMDMs were cultured with medium alone or
2.05 (3H, s, CH3), 1.21 (3H, d, J6,5 = 6.2 Hz, H6). 13C-NMR irradiated H37Rv (10 : 1), with or without p-HBad I, p-HBad II
(CDCl3, δ): 170.0, 166.6, 159.3 (CvO), 131.6 (Ar), 124.5 (Cq), or UM-p-HBad (1, 10 or 100 μM). Supernatants were recovered
115.8 (Ar), 95.3 (C1), 90.7 (Cq), 70.8 (H4), 69.5 (C2), 68.8 (C3), after 24 h and IL-12p40 (BD Pharmingen) and TNF-α (R&D
67.5 (C5), 52.0 (OCH3), 20.9, 20.8, 20.7 (CH3), 17.4 (C6). Systems) concentrations were determined by ELISA.
HRMS-ESI (m/z): [M
447.1267; found 447.1259. IR: 2986, 1746, 1606, 1369,
1215 cm−1
+
Na]+ calculated for C20H24O10Na
.
Acknowledgements
Compound 23
This work was funded by Trinity College Dublin. We would
22 (1.07 g, 2.51 mmol) was dissolved in MeOH (15 mL) with a like to thank Dr John O’Brien and Dr Manuel Ruether for
catalytic amount of NaOMe. After 18 h the reaction was assistance with NMR studies.
quenched with a catalytic amount of DOWEX, filtered and con-
centrated to give 23 as a colourless solid in 98% yield
(734 mg). [α]2D0 −117.6 (c 0.142, MeOH). 1H-NMR (400 MHz,
CD3OD, δ): 7.96, 7.13 (2H, m, Ar–H), 5.52 (1H, d, J1,2 = 1.2 Hz,
Notes and references
H1), 4.04 (1H, dd, J2,1 = 1.2 Hz, J2,3 = 3.3 Hz, H2), 3.86 (3H, s,
CH3), 3.83 (1H, dd, J3,2 = 3.3 Hz, J3,4 = 9.5 Hz, H3), 3.57 (1H, m,
H5), 3.46 (1H, app t, H4), 1.21 (3H, d, J6,5 = 6.1 Hz, H6).
13C-NMR (CD3OD, δ): 167.2 (CvO), 160.9 (Cq), 131.6 (Ar–C),
124.0 (Cq), 116.1 (Ar–C), 98.7 (C1), 72.7 (C4), 71.1 (C3), 70.8
(C2), 70.0 (C5), 51.5 (OCH3), 17.0 (C6). HRMS-ESI (m/z): [M +
1 M. Daffe and P. Draper, Adv. Microb. Physiol., 1998, 39, 131–
203.
2 P. J. Brennan and D. C. Crick, Curr. Top. Med. Chem., 2007,
7, 475–488.
3 C. E. Barry, D. C. Crick and M. R. McNeil, Infect. Disord.:
Drug Targets, 2007, 7, 182–202.
1122 | Org. Biomol. Chem., 2014, 12, 1114–1123
This journal is © The Royal Society of Chemistry 2014

View Article Online
Organic & Biomolecular Chemistry
Paper
4 D. C. Crick, L. Quadri and P. J. Brennan, Handbook of Tuber-
culosis: Molecular Biology and Biochemistry, 2008, pp. 1–19.
5 V. Rao, N. Fujiwara, S. A. Porcelli and M. S. Glickman,
J. Exp. Med., 2005, 201, 535–543.
6 M. S. Glickman and W. R. Jacobs, Jr., Cell, 2001, 104, 477–485.
7 K. Sakamoto, J. Kim Mi, R. Rhoades Elizabeth, E. Allavena
Rachel, S. Ehrt, C. Wainwright Helen, G. Russell David and
H. Rohde Kyle, Infect. Immun., 2013, 81, 764–776.
III, F. Tekaia, K. Badcock, D. Basham, D. Brown,
T. Chillingworth, R. Connor, R. Davies, K. Devlin,
T. Feltwell, S. Gentles, N. Hamlin, S. Holroyd, T. Hornsby,
K. Jagels, A. Krogh, J. McLean, S. Moule, L. Murphy,
K. Oliver, J. Osborne, M. A. Quail, M. A. Rajandream,
J. Rogers, S. Rutter, K. Seeger, J. Skelton, R. Squares,
S. Squares, J. E. Sulston, K. Taylor, S. Whitehead and
B. G. Barrell, Nature, 1998, 393, 537–544.
8 A. M. Cooper and E. Torrado, Curr. Opin. Immunol., 2012, 27 G. H. Veeneman, S. H. Van Leeuwen, H. Zuurmond
24, 431–437.
and J. H. Van Boom, J. Carbohydr. Chem., 1990, 9,
783–796.
28 T. Fujiwara, Agric. Biol. Chem., 1991, 55, 2123–2128.
9 E. Torrado, R. T. Robinson and A. M. Cooper, Trends
Immunol., 2011, 32, 66–72.
10 A. Forrellad Marina, I. Klepp Laura, A. Gioffre, J. Sabio y 29 S. Barroso, R. Castelli, M. P. Baggelaar, D. Geerdink, B. ter
Garcia, R. Morbidoni Hector, M. de la Paz Santangelo,
A. Cataldi Angel and F. Bigi, Virulence, 2013, 4, 3–66.
11 S. Berg, D. Kaur, M. Jackson and P. J. Brennan, Glycobio-
logy, 2007, 17, 35R–56R.
12 A. K. Mishra, N. N. Driessen, B. J. Appelmelk and
G. S. Besra, FEMS Microbiol. Rev., 2011, 35, 1126–1157.
13 J. Yu, V. Tran, M. Li, X. Huang, C. Niu, D. Wang, J. Zhu,
J. Wang, Q. Gao and J. Liu, Infect. Immun., 2012, 80, 1381–
1389.
14 C. Guilhot, C. Chalut and M. Daffe, Mycobact. Cell Envelope,
2008, 273–289.
15 J. A. Ferreras, K. L. Stirrett, X. Lu, J.-S. Ryu, C. E. Soll,
D. S. Tan and L. E. N. Quadri, Chem. Biol., 2008, 15, 51–61.
16 G. J. Blaauw and B. J. Appelmelk, Protein-Carbohydr. Inter-
act. Infect. Dis., 2006, 6–29.
Horst, E. Casas-Arce, H. S. Overkleeft, G. A. van der Marel,
J. D. C. Codee and A. J. Minnaard, Angew. Chem., Int. Ed.,
2012, 51, 11774–11777.
30 S. Barroso, D. Geerdink, B. ter Horst, E. Casas-Arce and
A. J. Minnaard, Eur. J. Org. Chem., 2013, 21, 4642–4654.
31 J. D. C. Codee, R. E. J. N. Litjens, L. J. Van den Bos,
H. S. Overkleeft and G. A. Van der Marel, Chem. Soc. Rev.,
2005, 34, 769–782.
32 E. M. Scanlan, M. M. Mackeen, M. R. Wormald and
B. G. Davis, J. Am. Chem. Soc., 2010, 132, 7238–7239.
33 B. Mukhopadhyay and R. A. Field, Carbohydr. Res., 2003,
338, 2149–2152.
34 J. F. King and A. D. Allbutt, Can. J. Chem., 1970, 48, 1754–
1769.
35 L. Wei, G. Wei, H. Zhang, P. G. Wang and Y. Du, Carbohydr.
Res., 2005, 340, 1583–1590.
17 I. Vergne and M. Daffe, Front. Biosci., 1998, 3, D865–D876.
18 M. B. Reed, P. Domenech, C. Manca, H. Su, A. K. Barczak, 36 A. M. Cooper, D. K. Dalton, T. A. Stewart, J. P. Griffin,
B. N. Kreiswirth, G. Kaplan and C. E. Barry, Nature, 2004,
431, 84–87.
D. G. Russell and I. M. Orme, J. Exp. Med, 1993, 178, 2243–
2247.
19 D. Sinsimer, G. Huet, C. Manca, L. Tsenova, M.-S. Koo, 37 J. L. Flynn, J. Chan, K. J. Triebold, D. K. Dalton,
N. Kurepina, B. Kana, B. Mathema, S. A. E. Marras,
B. N. Kreiswirth, C. Guilhot and G. Kaplan, Infect. Immun.,
2008, 76, 3027–3036.
T. A. Stewart and B. R. Bloom, J. Exp. Med., 1993, 178,
2249–2254.
38 H. McShane, S. Behboudi, N. Goonetilleke, R. Brookes and
A. V. S. Hill, Infect. Immun., 2002, 70, 1623–1626.
39 R. N. Mahon, R. E. Rojas, S. A. Fulton, J. L. Franko,
C. V. Harding and W. H. Boom, Infect. Immun., 2009, 77,
4574–4583.
20 V. Ng, G. Zanazzi, R. Timpl, J. F. Talts, J. L. Salzer,
P. J. Brennan and A. Rambukkana, Cell, 2000, 103, 511–524.
21 G. Stadthagen, J. Kordulakova, R. Griffin, P. Constant,
I. Bottova, N. Barilone, B. Gicquel, M. Daffe and
M. Jackson, J. Biol. Chem., 2005, 280, 40699–40706.
22 G. Stadthagen, M. Jackson, P. Charles, F. Boudou,
40 A. J. Gehring, K. M. Dobos, J. T. Belisle, C. V. Harding and
W. H. Boom, J. Immunol., 2004, 173, 2660–2668.
N. Barilone, M. Huerre, P. Constant, A. Liav, I. Bottova, 41 A. J. Gehring, R. E. Rojas, D. H. Canaday, D. L. Lakey,
J. Nigou, T. Brando, G. Puzo, M. Daffe, P. Benjamin,
S. Coade, R. S. Buxton, R. E. Tascon, A. Rae,
C. V. Harding and W. H. Boom, Infect. Immun., 2003, 71,
4487–4497.
B. D. Robertson, D. B. Lowrie, D. B. Young, B. Gicquel and 42 N. D. Pecora, A. J. Gehring, D. H. Canaday, W. H. Boom
R. Griffin, Microbes Infect., 2006, 8, 2245–2253. and C. V. Harding, J. Immunol., 2006, 177, 422–429.
23 E. Perez, P. Constant, A. Lemassu, F. Laval, M. Daffe and 43 G. H. Veeneman, L. J. F. Gomes and J. H. van Boom, Tetra-
C. Guilhot, J. Biol. Chem., 2004, 279, 42574–42583. hedron, 1989, 45, 7433–7448.
24 E. Perez, P. Constant, F. Laval, A. Lemassu, M.-A. Laneelle, 44 K. Zegelaar-Jaarsveld, G. A. van der Marel and J. H. van
M. Daffe and C. Guilhot, J. Biol. Chem., 2004, 279, 42584–
42592.
25 P. Constant, E. Perez, W. Malaga, M.-A. Laneelle, O. Saurel,
M. Daffe and C. Guilhot, J. Biol. Chem., 2002, 277, 38148–38158.
Boom, Tetrahedron, 1992, 48, 10133–10148.
45 M.-J. L. Thijssen, K. M. Halkes, J. P. Kamerling and
J. F. G. Vliegenthart, Bioorg. Med. Chem., 1994, 2, 1309–
1317.
26 S. T. Cole, R. Brosch, J. Parkhill, T. Garnier, C. Churcher, 46 I. Bajza, J. Kerekgyarto, J. Hajko, L. Szilagyi and A. Liptak,
D. Harris, S. V. Gordon, K. Eiglmeier, S. Gas, C. E. Barry,
Carbohydr. Res., 1994, 253, 111–120.
This journal is © The Royal Society of Chemistry 2014
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