Synthesis and TNF-α inducing activities of mycoloyl-arabinan motif of mycobacterial cell wall components

Article abstract of DOI:10.1016/j.bmc.2005.12.037

The extract of the cell wall skeleton of Bacillus Calmette-Guerin (BCG-CWS) from Mycobacterium bovis is known to be an activator of innate immunity. Synthesis of pentaarabinofuranoside as part of the arabinan moiety of BCG-CWS was achieved by double α-ara

Full text of DOI:10.1016/j.bmc.2005.12.037

Bioorganic & Medicinal Chemistry 14 (2006) 3049–3061
Synthesis and TNF-a inducing activities of mycoloyl-arabinan
motif of mycobacterial cell wall components
Akihiro Ishiwata,a Hiroko Akao,a Yukishige Ito,a,*
Makoto Sunagawa,b Naoto Kusunoseb and Yasuo Kashiwazakib
aRIKEN (The Institute of Physical and Chemical Research), 2-1 Hirosawa, Wako-shi, Saitama 351-0198, Japan
bResearch Division, Sumitomo Pharmaceuticals Co., Ltd, 3-1-98 Kasugadenaka, Konohana-ku, Osaka 554-0022, Japan
Received 6 September 2005; revised 8 December 2005; accepted 13 December 2005
Available online 19 January 2006
´
Abstract—The extract of the cell wall skeleton of Bacillus Calmette–Guerin (BCG-CWS) from Mycobacterium bovis is known to be
an activator of innate immunity. Synthesis of pentaarabinofuranoside as part of the arabinan moiety of BCG-CWS was achieved by
double a-arabinofuranosylation followed by double b-arabinofuranosylation with orthogonally protected donors. Mycolic esters of
the arabinan in the terminal lipo-arabinan motif of BCG-CWS were synthesized through alkylation of unprotected mycolic acid
with bis- and tetra-tosylates of pentaarabinofuranoside. A series of compounds were subjected to a tumor necrosis factor alpha
(TNF-a) secretion-inducing assay, disclosing aspects of the structure–activity relationship which should be useful in finding the site
of the activity.
ꢀ 2005 Elsevier Ltd. All rights reserved.
1. Introduction
D-arabinose (Araf), which are potentially immunogenic
to mammals. At non-reducing terminal, it has branched
pentasaccharides, which are decorated as esters of MA.
MAs are a group of long-chain fatty acids, which con-
tain b-hydroxycarboxylic acid moiety and two hydro-
phobic chains; a linear saturated alkyl (C22 or C24)
chain and a longer functionalized (e.g., cis-cyclopropyl,
cis-olefin, methyl, and oxo or methoxy) chain called
mero group.7 Although the biological roles of MA are
largely undefined, it resides at the outer region of the cell
wall and therefore is likely to be important for initial
contact with receptors.8 In addition, correlation of
MA composition and virulence has been reported.9
Mycobacterium tuberculosis, the causative agent of
tuberculosis, is rampant across the world and poses a
growing threat due to its multi-drug resistance
(MDR).1 For mycobacteria, as well as other bacteria,
one of the most important drug targets is cell wall bio-
synthesis, which is critical for bacterial survival. Clinical
use of a variety of anti-mycobacterials has resulted in
resistance, although its precise mechanism is yet to be
defined.2 An adjuvant consisting of Bacillus Calmette–
´
Guerin cell wall skeleton (BCG-CWS) from Mycobacte-
rium bovis is known to be an activator of innate
immunity.3,4 It has been proposed that macrophages
are activated via Toll-like receptors.5 However, the
structural basis of BCG-CWS-initiated immunity is
not clear, because of the complexity of BCG-CWS.
The aim of this study was to synthesize a series of mono-
(1), di- (2, 3, 4), and tetramycolated (5) arabinans, which
constitute the terminal region of BCG-CWS. In addi-
tion, their activities to induce tumor necrosis factor
(TNF)-a were evaluated.
BCG-CWS is a supramolecular architecture, composed
of a peptidoglycan (PG) linked to mycolic acids (MAs)
and arabinogalactan, through linker disaccharide
(b-Rhap-(1,3)-a-GlcNAc) (Fig. 1).6 Arabinogalactan
consists of furanose forms of D-galactose (Galf) and
2. Results and discussion
2.1. b-Arabinofuranosylation for the synthesis of myco-
loyl-arabinan
Keywords: BCG-CWS; Mycoloyl-arabinan; TNF-a secretion-inducer;
Stereoselective arabinofuranosylation.
The terminal pentaarabinofuranoside of BCG-CWS is
composed of two b-arabinofuranosides (b-Araf) linked
*
Corresponding author. Tel.: +81 48 467 9430; fax: +81 48 462
4680; e-mail: yukito@riken.jp
0968-0896/$ - see front matter ꢀ 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.037

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A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
Figure 1. Structure of monomer of BCG-CWS and targets of mycoloyl-arabinans.
to the penultimate a-arabinofuranosides (a-Araf). To
approach the branched arabinan portion of BCG-
CWS,10 formation of both the a- (B, C, D) and b-config-
ured (E, F) Araf is required. Basic strategy for the
stereoselective synthesis of furanosidic linkage is less ex-
plored, compared to that of hexopyranoside. Due to
their conformational elasticity, stereoelectronic factors
that control the stereochemistry of O-furanosides are
difficult to be generalized. Formation of b-Araf has been
difficult, because of its 1,2-cis orientation.11 To achieve
this, various approaches have been reported.10a,12–14
We looked into the effect of O-5 protecting group, be-
cause this position was going to be selectively deprotec-
ted later on, in order to be esterified with MA.
lated product 6f, which was then converted to 6a–e. It
was revealed that the nature of the protecting group
introduced to this position gave a significant effect on
the stereochemical outcome (Scheme 1); a:b = 1.3:1
(CAc, 6a), 9.4:1 (TBPS, 6b), 1.9:1 (2-NBn, 6c), 1:1.5
(NAP, 6d), and 1:2.8 (PMB, 6e).14 With these results,
we decided to use 6e as the donor for b-Araf synthesis.
As an orthogonally protected donor, 5-O-TBPS protect-
ed donor 9b was prepared from 9a and used for the
a-arabinofuranosylation.
2.2. Synthesis of arabinan moieties
Syntheses of tri (B/C/D) and pentasaccharide (B/C/E/D/
F) were conducted as shown in Scheme 2. Thus, diol
1015 was first subjected to glycosylation with 2-O-benzo-
yl protected donor 9b, which uneventfully provided bis-
a-glycosylated product 11. After deacylation, diol 12
was then glycosylated with 5-O-PMB protected donor
6e. It gave a mixture of pentasaccharides, from which
We screened glycosylation of thioglycosides 6a–e with a
model acceptor 7. These glycosyl donors were prepared
from known 9a.12 Namely, treatment under standard O-
benzylation conditions (NaH, PhCH2Cl, and DMF)
caused benzoyl migration and provided 2,3-di-O-benzy-

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3051
Scheme 1. Examination of b-arabinofuranosylation of donors 6a–e.
the desired pentasaccharide 13 was isolated in 57% yield,
together with 35% yield of a mixture of stereoisomers.
Its structure was clarified by H NMR, revealing 3 b-
Araf [dH 5.01 (d, J = 4.4 Hz), 5.12 (d, J = 4.4 Hz), 5.84
(d, J = 4.0 Hz)] and 2 a-Araf [dH 5.02 (s), 5.09 (d,
J = 0.6 Hz)] residues. It was then converted to regioiso-
meric diols 14 and 15 for subsequent esterification.
THF–DMF or toluene; in the latter case, 18-crown-6
was included in the reaction mixture. Subsequently, pen-
tasaccharides 14 and 15 were converted to bis- (21, 22)
and tetrakis-(23) tosylates, and then to corresponding
mycolates 25, 26, and 27. The progress of these reactions
was monitored most conveniently with MALDI-TOF
mass spectrometer. Because of its heterogeneity, MA
and its derivatives share a characteristic peak distribu-
tion. After coupling with arabinans, this pattern migrat-
ed to the higher MW region, indicating the successful
conversion (Fig. 2). Esterificated products were finally
deprotected under hydrogenolytic conditions to provide
Myc2Araf3 2, Myc2cdAraf5 3, Myc2efAraf5 4, and
Myc4Araf5 5.
1
2.3. Synthesis of mycoloyl-arabinans
To introduce MA to arabinans,16 direct condensation
was initially attempted. However, not unexpectedly, this
option turned out to be challenging, due to the propen-
sity of hydroxy acid to cause self-condensation.
For instance, attempted condensation between mono-
saccharide 16 and mycolic acid under Yamaguchi’s
conditions17 gave only 30% yield of the product 18. Sev-
eral attempts to protect the hydroxy group proved to be
difficult. We therefore switched to the alkylative esterifi-
cation strategy,18 which was examined with monosac-
charide tosylate 17 prepared from 16. When treated
with CsHCO3 and mycolic acid, the product 18 was ob-
tained in 79% yield (Scheme 3). It was then subjected to
hydrogenolysis to afford Myc1Araf1 1.
2.4. TNF-a secretion-inducing assay of mycoloyl-
arabinans
To evaluate the activities of mycolated arabinans as im-
mune potentiators, tumor necrosis factor-a (TNF-a)
secretion-inducing assay was conducted,9,19,5a in com-
parison with BCG-CWS (Tokyo strain),20 trehalose
dimycolate (TDM)20 as the positive control, and mycol-
ic acid (MA). Namely, RAW 264.7 mouse macrophage
was used for this assay in vitro. All of the synthesized
mycolates (Myc1Araf1 1, Myc2Araf3 2, Myc2cdAraf5 3,
Myc2efAraf5 4, and Myc4Araf5 5) induced TNF-a in vi-
tro, with activities similar to that of BCG-CWS,
In a similar manner, trisaccharide bis-tosylate (20) was
prepared from 12 via diol 19 and mycolated to give 24
(Scheme 4). This reaction was conducted either in

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A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
Scheme 2. Synthesis of tri- and pentaarabinfuranosides.
Scheme 3. Examination of esterification of arabinofuranoside with mycolic acid.
although mycolic acid and arabinan21 by themselves in-
duced TNF-a secretion to a minimum extent (Fig. 3). To
induce TNF-a, both hydrophilic arabinan and lipophilic
mycolic acid were required.
a-arabinofuranosylation followed by double b-arabino-
furanosylation with orthogonal protections. Alkylative
esterification via an arabinan tosylate is effective for the
synthesis of mycolate without protection of the b-hydrox-
yl group in mycolic acid moiety. A series of compounds
were subjected to a TNF-a secretion-inducing assay and
it was found that all the synthesized mycolates of arab-
inan showed potent TNF-a inducing activities in vitro
in almost the same order as BCG-CWS itself. A detailed
mechanism of the action of these compounds is still not
clear, although the lipo-arabinan moieties are thought
to play an important role in this activity.
3. Conclusion
We have demonstrated the first synthesis of mycobacteri-
al lipo-pentaarabinofuranoside as a terminal section of
BCG-CWS from M. bovis. Synthesis of the non-reducing
terminal arabinan of BCG-CWS was achieved via double

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3053
Scheme 4. Completion of synthesis of mycoloyl-arabinans. Reagents and conditions: (a) BnBr, NaH, DMF, 99%; (b) TBAF, THF, 82%; (c) TsCl,
pyridine, DMAP, 67%; (d) method A; mycolic acid, CsHCO3, DMF–THF, 70 ꢁC, or method B; mycolic acid, CsHCO3, 18-crown-6, toluene, 70 ꢁC;
(e) TsCl, pyridine, 86%; (f) DDQ, 68%; (g) TsCl, pyr, 91%; (h) TsCl, pyridine, 86%.
4. Experimental
4.1. General procedures
was performed on Sephadex LH-20 (Pharmacia).
Melting point was determined with a Buchi 510 melt-
¨
ing point apparatus. Optical rotations were measured
with a JASCO DIP 370 polarimeter. H NMR spectra
1
All reactions sensitive to air and/or moisture were
carried out under nitrogen or argon atmosphere with
anhydrous solvents. Column chromatography was
performed on silica gel 60 N, 100–210 mesh (Kanto
Kagaku Co., Ltd). Preparative TLC was performed
on silica gel 60 F254, 0.5 mm (E. Merck). Gel filtration
were recorded at 400 MHz on a JEOL JNM-AL
400 spectrometer and chemical shifts are referred to
internal CDCl3 (7.24 ppm). 13C NMR spectra were
recorded at 100 MHz on the same instrument and
chemical shifts are referred to internal CDCl3
(77 ppm). MALDI-TOF mass spectra were recorded

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A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
Figure 2. Mass spectra for monitoring of the synthesis of mycolate. (1) Monitoring of the synthesis of Myc1Araf1 (1): (1a) MA; (1b) 18; (1c) 1. (2)
Monitoring of the synthesis of Myc2Araf3 (2): (2a) MA; (2b) 24; (2c) 2. (3) Monitoring of the synthesis of Myc2Araf5 (3, 4): (3a) 25; (3b) 3; (3c) 26;
(3d) desilylated product of 26; (3e) 4. (4) Monitoring of the synthesis of Myc4Araf5 (5): (4a) 27; (4b) 5.
2500
formed with a Fisons EA1108 instrument. Mass units
of the mycoloyl esters were referred to those of C84
ketomycolic acid for convenience sake.
2000
1500
4.1.1. 3-O-Benzyl-5-O-t-butyldiphenylsilyl-a-D-arabino-
furanosyl-(1 ! 3)-[3-O-benzyl-5-O-t-butyldiphenylsilyl-
1000
a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-isopropylidene-b-D-
arabinofuranose (12).
5.26 mmol), 9b (7.91 g, 12.6 mmol), 2,6-di-t-butyl-4-
A
mixture of 10 (1.00 g,
500
0
methylpyridine (5.29 g, 25.2 mmol), and dried pow-
˚
dered molecular sieves 4 A (5 g) in dry CH2Cl2
a
b
c
d
e
f
g
h
i
(75 mL) was stirred at room temperature for 15 min un-
der N2 and cooled to 0 ꢁC. To the mixture was added
dropwise methyl triflate (2.86 mL, 25.3 mmol) and the
temperature of the reaction mixture was allowed to rise
to room temperature. After stirring overnight, triethyl-
amine (2.5 mL) was added before being filtered. The fil-
trate was washed successively with satd aq NaHCO3,
satd aq NH4Cl, and brine, dried over MgSO4, and con-
centrated in vacuo. The resulting residue was purified
by silica gel column chromatography (hexane–EtOAc,
8:1–3:1) to give the trisaccharide 11 (6.29 g, 4.77 mmol,
91%) as a viscous oil.
Figure 3. TNF-a secretion-inducing assay; (a) BCG-CWS, (b) TDM,
(c) MA, (d) 1 (Myc1Araf1), (e) 2 (Myc2Araf3), (f) 3 (Myc2cdAraf5), (g) 4
(Myc2efAraf5), (h) 5 (Myc4Araf5), (i) solvent only (control). , 0.01 lg/
well; h, 0.1 lg/well; , 1 lg/well.
on a SHIMADZU Kompact MALDI AXIMA-CFR
spectrometer with 2,5-dihydroxybenzoic acid as the
matrix. ESI-TOF mass spectra were recorded on a
JEOL AccuTOF JMS-T700LCK with CF3CO2Na as
the internal standard. Elemental analyses were per-

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3055
24
D
27
D
1
½aꢀ +61.5 (c 0.52, CHCl3); 1H NMR (CDCl3): d 0.94 (s,
½aꢀ +153.0 (c 0.34, CHCl3); m.p. 35–36 ꢁC; H NMR
9H), 0.97 (s, 9H), 1.30 (s, 3H), 1.50 (s, 3H), 3.71 (dd,
J = 5.2, 10.8 Hz, 1H), 3.72–3.89 (m, 4H), 3.97 (dd,
J = 6.4, J = 10.8 Hz, 1H), 4.18 (br d, J =6.0 Hz, 1H),
4.23–4.30 (m, 4H), 4.38 (br d, J = 3.2 Hz, 1H), 4.51 (d,
J = 12.4 Hz, 1H), 4.55 (d, J = 12.0 Hz, 1H), 4.68 (br d,
J = 4.2 Hz, 1H), 4.70 (d, J = 12.4 Hz, 1H), 4.72 (d,
J = 12.0 Hz, 1H), 5.21 (br s, 1H), 5.32 (br s, 1H), 5.36
(br d, J = 0.8 Hz, 1H), 5.40 (br d, J = 12.0 Hz, 1H),
5.91 (d, J = 4.2 Hz, 1H), 7.15–7.40 (m, 26H), 7.52–7.63
(m, 10H), 7.91–7.96 (m, 4H); 13C NMR (CDCl3): d
19.3, 19.3, 26.5, 26.8, 26.8, 27.2, 62.6, 62.6, 66.6, 72.0,
72.2, 80.0, 82.2, 82.4, 82.5, 82.6, 83.2, 83.4, 83.7, 85.1,
105.1, 105.5, 106.1, 113.0, 127.4, 127.5, 127.6, 127.7,
127.8, 128.2, 128.3, 129.2, 129.4, 129.5, 129.7, 133.0,
133.2, 133.3, 135.4, 135.5, 137.5, 137.7, 165.1, 165.4.
MALDI-TOF MS: [M+Na]+ calcd for C78H86O15Si2Na,
1341.5. Found 1341.8. Anal. Calcd for C78H86O15Si2: C,
70.99; H, 6.57. Found: C, 70.81; H, 6.55.
(CDCl3): d 1.29 (t, J = 7.4 Hz, 3H), 2.07 (br s, 1H),
2.57–2.75 (m, 2H), 3.67 (dd, J = 4.0, 12.0 Hz, 1H), 3.83
(dd, J = 2.8, 12.0 Hz, 1H), 3.98–4.02 (m, 2H), 4.23 (m,
1H), 4.48 (d, J = 12.0 Hz, 1H), 4.51 (d, J = 12.0 Hz,
1H), 4.57 (d, J = 12.0 Hz, 1H), 4.59 (d, J = 12.0 Hz,
1H), 5.35 (d, J = 2.0 Hz, 1H), 7.25–7.37 (m, 10H); 13C
NMR (CDCl3): d 14.8, 25.3, 61.7, 71.9, 72.3, 81.3, 82.8,
87.4, 88.5, 127.6, 127.7, 127.8, 128.3, 128.3, 137.1,
137.5. MALDI-TOF MS: [M+Na]+ calcd for
C21H26O4SNa, 397.1. Found 396.8. Anal. Calcd for
C21H26O4S: C, 67.35; H, 7.00. Found: C, 67.35; H, 6.86.
To the alcohol 6f (3.15 g, 8.41 mmol) in dry DMF
(25 mL) was added sodium hydride (60% dispersion in
oil, 640 mg, 16.0 mmol) at 0 ꢁC and the suspension
was stirred for 10 min. Then p-methoxybenzyl chloride
(1.28 mL, 9.25 mmol) was added dropwise. After the
temperature of the solution was allowed to rise to room
temperature overnight, methanol (3.5 mL) was added at
0 ꢁC. The reaction mixture was diluted with CH2Cl2.
The organic layer was washed successively with H2O,
satd aq NH4Cl, and brine, dried over MgSO4, and con-
centrated in vacuo. The resulting residue was purified by
silica gel column chromatography (hexane–EtOAc,
20:1–15:1) to give 6e (3.89 g, 7.86 mmol, 94%) as a col-
orless oil.
The dibenzoate 11 (6.39 g, 4.84 mmol) was dissolved in
CH2Cl2 (20 mL) and NaOMe (390 mg, 7.3 mmol) in
MeOH (20 mL) was added at 0 ꢁC. After stirring at
0 ꢁC for 2 h, the reaction mixture was neutralized with
dil HCl and extracted with EtOAc. The organic layer
was washed successively with satd aq NaHCO3 and
brine, dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by silica gel column
chromatography (hexane–EtOAc, 5:1–2:1) to give 12
(5.03 g, 4.53 mmol, 94%) as a viscous oil.
28
D
1
½aꢀ +107.8 (c 1.40, CHCl3); H NMR (CDCl3): d 1.30
(t, J = 7.6 Hz, 3H), 2.68 (m, 2H), 3.59 (dd, J = 4.8,
10.8 Hz, 1H), 3.64 (dd, J = 3.6, 10.8 Hz, 1H), 3.79 (s,
3H), 3.95–3.97 (m, 2H), 4.28 (m, 1H), 4.46–4.62 (m,
6H), 5.37 (d, J = 2.0 Hz, 1H), 6.84 (br d, J = 8.8 Hz,
2H), 7.21–7.37 (m, 12H); 13C NMR (CDCl3): d 14.9,
25.3, 55.3, 68.7, 72.0, 72.2, 73.0, 79.9, 83.6, 87.1, 88.8,
113.6, 127.7, 127.8, 127.9, 128.3, 129.3, 130.1, 137.4,
137.7, 159.0. MALDI-TOF MS: [M+Na]+ calcd for
C29H34O5SNa, 517.2. Found 517.4. Anal. Calcd for
C29H34O5S: C, 70.42; H, 6.93. Found: C, 70.57; H, 6.88.
26
D
½aꢀ +74.6 (c 0.54, CHCl3); 1H NMR (CDCl3): d 0.96 (s,
18H), 1.31 (s, 3H), 1.51 (s, 3H), 3.20 (d, J = 10.0 Hz,
1H), 3.40 (d, J = 10.8 Hz, 1H), 3.41–3.44 (m, 2H),
3.62–3.70 (m, 3H), 3.90–3.93 (m, 3H), 4.10–4.16 (m,
5H), 4.39 (br d, J = 4.0 Hz, 1H), 4.41 (d, J = 12.0 Hz,
1H), 4.43 (d, J = 12.0 Hz, 1H), 4.57 (d, J = 12.0 Hz,
1H), 4.59 (d, J = 12.0 Hz, 1H), 4.62 (br d, J = 4.0 Hz,
1H), 5.05 (br s, 1H), 5.18 (br s, 1H), 5.82 (d,
J = 4.0 Hz, 1H), 7.18–7.39 (m, 22H), 7.51–7.60 (m,
8H); 13C NMR (CDCl3): d 19.0, 19.1, 26.7, 26.7, 26.8,
27.3, 63.8, 63.9, 66.4, 71.7, 71.8, 77.2, 77.9, 79.3, 83.0,
83.8, 84.1, 84.5, 84.7, 85.7, 105.1, 107.5, 108.8, 113.1,
127.5, 127.6, 127.7, 127.8, 128.3, 129.7, 129.8, 129.9,
131.9, 132.1, 132.2, 132.4, 135.4, 135.5, 137.6, 137.7.
MALDI-TOF MS: [M+Na]+ calcd for C64H78O13Si2Na,
1133.5. Found 1133.4. Anal. Calcd for C64H78O13Si2: C,
69.16; H, 7.07. Found: C, 69.14; H, 7.05.
4.1.3. 2,3-Di-O-benzyl-5-O-p-methoxybenzyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-t-butyldiphenylsilyl-
a-D-arabinofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-meth-
oxybenzyl-b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-O-
t-butyldiphenylsilyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-
isopropylidene-b-D-arabinofuranose (13). A mixture of 12
(31.9 mg, 0.0287 mmol), 6e (45.0 mg, 0.0906 mmol), N-iodo-
succinimide (26 mg, 0.12 mmol), and dried powdered
˚
molecular sieves 4 A (5 g) in dry CH2Cl2 (1.5 mL) was
4.1.2. Ethyl 2,3-di-O-benzyl-5-O-p-methoxybenzyl-1-
thio-a-D-arabinofuranoside (6e). To compound 9a13
(5.08 g, 13.1 mmol) in dry DMF (40 mL) was added sodi-
um hydride (60% dispersion in oil, 1.10 g, 27.5 mmol) at
0 ꢁC. After stirring at 0 ꢁC for 20 min, benzyl chloride
(1.58 mL, 13.7 mmol) was added dropwise. The reaction
mixture was stirred at 0 ꢁC for additional 2 h and then
methanol (25 mL) was added before being diluted with
CH2Cl2. The organic layer was washed successively with
H2O, satd aq NH4Cl, and brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was purified
by silica gel column chromatography (hexane–EtOAc,
6:1–3:1) to give dibenzylated primary alcohol 6f (3.43 g,
9.17 mmol, 70%) as a colorless solid.
cooled to ꢁ78 ꢁC and stirred for 15 min under N2. To
this mixture was dropwise added silver triflate (7.4 mg,
0.029 mmol) in dry toluene (0.08 mL) and the suspen-
sion was stirred for 10 min. Then the temperature of
the solution was allowed to rise to ꢁ40 ꢁC. After stirring
for additional 1 h, triethylamine (1 mL) was added be-
fore being filtered. The filtrate was washed successively
with 20% aq sodium thiosulfate, satd aq NH4Cl, and
brine, dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by LH-20 column
chromatography (CHCl3–MeOH, 1:1) and preparative
TLC (developed with hexane–EtOAc, 2:1 and eluted
with CHCl3–MeOH, 4:1) to give 13 (32.2 mg,
0.0163 mmol, 57%) as a colorless foam and also a mix-

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A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
ture of diastereomers (19.8 mg, 0.0100 mmol, 35%) as a
colorless foam.
CH2Cl2–H2O (18:1, 1.3 mL) was added DDQ (29 mg,
0.13 mL) and stirred at room temperature for 1 h. The reac-
tion mixture was diluted with EtOAc. The organic layer was
washed successively with ascorbic acid/citric acid buffer*
and brine, dried over MgSO4, and concentrated in vacuo.
The resulting residue was purified by silica gel column chro-
matography (hexane–EtOAc, 1:1) and LH-20 column chro-
matography (CHCl3–MeOH, 1:1) to give 15 (51 mg,
0.030 mmol, 72%) as a colorless foam. *L-Ascorbic acid
(0.7 g), citric acid monohydrate (1.2 g) and NaOH (0.92 g)
in H2O (100 mL).
23
For desired bb diastereomer of 13: ½aꢀ ꢁ6.14 (c 0.96,
D
CHCl3); 1H NMR (CDCl3): d 1.04 (s, 9H), 1.04 (s, 9H),
1.32 (s, 3H), 1.52 (s, 3H), 3.48–3.67 (m, 4H), 3.60 (dd,
J = 4.0, 10.4 Hz, 1H), 3.75 (s, 3H), 3.75 (s, 3H), 3.73–
3.83 (m, 4H), 3.96 (dd, J = 6.8, 10.4 Hz, 1H), 4.02–4.38
(m, 18H), 4.56 (br d, J = 4.0 Hz, 1H), 4.45–4.69 (m,
12H), 5.01 (d, J = 4.4 Hz, 1H), 5.02 (br s, 1H), 5.09 (d,
J = 4.4 Hz, 1H), 5.12 (d, J = 0.6 Hz, 1H), 5.84 (d,
J = 4.0 Hz, 1H), 6.78 (d, J = 8.8 Hz, 1H), 6.79 (d,
J = 8.8 Hz, 1H), 7.11 (d, J = 8.8 Hz, 1H), 7.13 (d,
J = 8.8 Hz, 1H), 7.22–7.38 (m, 42H), 7.64–7.69 (m, 8H);
13C NMR (CDCl3): d 19.3, 26.6, 26.9, 27.3, 55.2, 63.5,
66.8, 71.9, 72.0, 72.1, 72.2, 72.3, 72.5, 72.6, 72.7, 77.2,
79.9, 80.0, 82.1, 82.9, 83.0, 83.2, 83.3, 83.5, 83.8, 83.9,
85.0, 85.7, 85.9, 100.0, 100.1, 104.3, 105.4, 105.8, 112.9,
113.5, 113.6, 127.2, 127.4, 127.5, 127.6, 127.8, 128.1,
128.2, 128.3, 129.0, 129.2, 129.5, 129.9, 130.0, 133.1,
133.2, 133.4, 135.5, 137.6, 137.9, 138.0, 138.1, 158.9,
27
D
1
½aꢀ +104.0 (c 0.05, CHCl3); H NMR (CDCl3): d 1.03
(s, 18H), 1.30 (s, 3H), 1.49 (s, 3H), 3.48–3.81 (m, 9H),
3.90–4.16 (m, 7H), 4.20–4.26 (m, 6H), 4.32 (m, 1H),
4.37 (m, 1H), 4.47–4.73 (m, 13H), 5.00 (br s, 1H), 5.04
(d, J = 4.4 Hz, 1H), 5.10 (d, J = 4.4 Hz, 1H), 5.14 (br
s, 1H), 5.82 (d, J = 4.0 Hz, 1H), 7.24–7.39 (m, 42H),
7.62–7.66 (m, 8H); 13C NMR (CDCl3): d 19.3, 26.5,
26.9, 27.2, 63.4, 63.4, 63.5, 63.5, 67.2, 72.0, 72.2, 72.2,
72.4, 72.5, 72.6, 79.9, 80.6, 80.7, 81.5, 81.8, 81.9, 82.4,
82.5, 82.6, 83.3, 83.8, 84.0, 85.0, 85.4, 85.5, 99.6, 99.8,
104.3, 105.4, 105.5, 112.9, 127.4, 127.6, 127.7, 127.8,
127.9, 128.0, 128.2, 128.3, 128.4, 129.5, 129.6, 133.0,
133.1, 133.2, 135.5, 137.4, 137.5, 137.6, 137.7, 137.9,
138.0. MALDI-TOF MS: [M+Na]+ calcd for
C102H118O21Si2Na, 1757.8. Found 1757.8. Anal. Calcd
for C102H118O21Si2: C, 70.56; H, 6.85. Found: C,
69.99; H, 6.74.
158.9. MALDI-TOF MS: [M+Na]+ calcd for C118H134
-
O23Si2Na, 1997.9. Found 1997.4. Anal. Calcd for
C118H134O23Si2: C, 71.71; H, 6.83. Found: C, 71.35; H, 6.84.
4.1.4. 2,3-Di-O-benzyl-5-O-p-methoxybenzyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-a-D-arabinofuranosyl-
(1 ! 3)-[2,3-di-O-benzyl-5-O-p-methoxybenzyl-b-D-ara-
binofuranosyl-(1 ! 2)-3-O-benzyl-a-D-arabinofuranosyl-
(1 ! 5)]-1,2-O-isopropylidene-b-D-arabinofuranose (14).
To compound 13 (29 mg, 0.015 mmol) in THF
(0.1 mL) was added 1 M THF solution of TBAF
(0.03 mL, 0.03 mmol) at 0 ꢁC. After stirring for 2 h at
ambient temperature, the reaction mixture was diluted
with EtOAc. The organic layer was washed successively
with satd aq NH4Cl and brine, dried over MgSO4, and
concentrated in vacuo. The resulting residue was puri-
fied by preparative TLC (developed with hexane–
EtOAc, 1:1 and eluted with CHCl3–MeOH, 5:1) to give
14 (22 mg, 0.015 mmol, 100%) as a colorless oil.
4.1.6. Methyl 2,3-di-O-benzyl-5-O-p-toluenesulfonyl-a-D-
arabinofuranoside (17). A mixture of 16 (118 mg,
0.343 mmol), pyridine (230 mg, 2.9 mmol) and 4-dim-
ethylaminopyridine (catalytic amount) in dry CH2Cl2
(1 mL) was cooled to 0 ꢁC and p-toluenesulfonyl chlo-
ride (140 mg, 0.734 mmol) was added. After the temper-
ature of the solution was allowed to rise to room
temperature overnight, the reaction mixture was diluted
with EtOAc. The organic layer was washed successively
with dil HCl, satd aq NaHCO3 and brine, dried over
MgSO4 and concentrated in vacuo. The resulting residue
was purified by silica gel column chromatography (hex-
ane–EtOAc, 4:1) to give 17 (155 mg, 0.311 mmol, 91%)
as a colorless oil.
24
D
½aꢀ ꢁ8.30 (c 0.13, CHCl3); 1H NMR (CDCl3): d 1.25 (s,
3H), 1.43 (s, 3H), 3.42–3.75 (m, 9H), 3.71 (s, 6H), 3.85–
4.07 (m, 11H), 4.15–4.63 (m, 21H), 4.92 (d, J = 4.4 Hz,
1H), 4.95 (br s, 1H), 4.98 (d, J = 3.2 Hz, 1H), 5.03 (br
s, 1H), 5.76 (d, J = 4.0 Hz, 1H), 6.76 (d, J = 8.4 Hz,
4H), 7.11 (d, J = 8.4 Hz, 4H), 7.20–7.30 (m, 30H); 13C
NMR (CDCl3): d 27.0, 27.5, 55.2, 62.8, 62.8, 65.2,
71.5, 72.1, 72.3, 72.4, 72.4, 72.6, 72.6, 72.7, 72.7, 79.9,
80.0, 82.2, 82.6, 82.7, 83.3, 83.3, 83.4, 83.7, 83.8, 83.9,
85.4, 85.5, 86.3, 100.0, 100.5, 104.8, 105.2, 105.7,
113.5, 113.6, 127.5, 127.6, 128.0, 128.2, 128.4, 129.2,
129.8, 129.9, 137.4, 137.4, 137.7, 137.8, 137.9, 138.0,
159.0, 159.0. MALDI-TOF MS: [M+Na]+ calcd for
C86H98O23Na, 1521.6. Found 1521.9. Anal. Calcd for
C86H98O23: C, 68.88; H, 6.59. Found: C, 68.64; H, 6.57.
27
D
½aꢀ +57.0 (c 0.40, CHCl3); 1H NMR (CDCl3): d 2.37 (s,
3H), 3.30 (s, 3H), 3.78 (dd, J = 2.8, 5.6 Hz, 1H), 3.92 (br
d, J = 2.8 Hz, 1H), 4.10 (br d, J = 5.2 Hz, 2H), 4.15 (m,
1H), 4.37–4.52 (m, 4H), 4.82 (s, 1H), 7.22–7.33 (m,
12H), 7.73 (d, J = 8.0 Hz, 2H); 13C NMR (CDCl3): d
21.6, 55.0, 68.8, 71.8, 72.2, 79.1, 82.7, 87.4, 107.3,
127.7, 127.8, 127.9, 128.3, 129.6, 132.6, 137.1, 137.2,
144.6. MALDI-TOF MS: [M+Na]+ calcd for
C27H30O7SNa, 521.2. Found 521.4. HRMS ESI-TOF:
[M+Na]+ calcd for C27H30O7SNa, 521.1610. Found
521.1617. Anal. Calcd for C27H30O7S: C, 65.04; H,
6.06. Found: C, 65.03; H, 5.92.
4.1.5. 2,3-Di-O-benzyl-b-D-arabinofuranosyl-(1 ! 2)-3-
O-benzyl-5-O-t-butyldiphenylsilyl-a-D-arabinofuranosyl-
(1 ! 3)-[2,3-di-O-benzyl-b-D-arabinofuranosyl-(1 ! 2)-
3-O-benzyl-5-O-t-butyldiphenylsilyl-a-D-arabinofurano-
syl-(1 ! 5)]-1,2-O-isopropylidene-b-D-arabinofuranose (15).
To a solution of compound 13 (82 mg, 0.041 mmol) in
4.1.7. Preparation of mycolic acid. Mycolic acid was pre-
pared from the extract of BCG-CWS following a Patent
Application No. 2005-135840. BCG-CWS,20 shown in
Figure 1, was hydrolyzed with KOH in ethanol–tolu-
ene–H2O at 65 ꢁC for 3 h. After addition of 2 M HCl

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3057
aq hydrophobic residues were extracted with hexane.
Combined organic phase was filtered, washed with
water and concentrated.
enzylated compound (101 mg, 0.0778 mmol, 99%) as a
colorless syrup.
24
D
½aꢀ +47.1 (c 1.46, CHCl3); 1H NMR (CDCl3): d 0.99 (s,
MALDI-TOF MS: [M+Na]+ calcd for C84H164O4Na,
1260.3. Found 1260.4.
9H), 1.00 (s, 9H), 1.29 (s, 3H), 1.47 (s, 3H), 3.56 (dd,
J = 4.8, 10.8 Hz, 1H), 3.70–3.78 (m, 4H), 3.92 (dd,
J = 6.4, 10.8 Hz, 1H), 4.02–4.22 (m, 8H), 4.39–4.52 (m,
9H), 5.02 (s, 1H), 5.14 (s, 1H), 5.82 (d, J = 4.0 Hz,
1H), 7.20–7.35 (m, 32H), 7.59–7.63 (m, 8H); 13C NMR
(CDCl3): d 19.3, 26.6, 26.9, 27.2, 63.3, 67.0, 71.8, 71.9,
72.1, 72.1, 80.1, 81.9, 82.7, 82.8, 83.0, 83.7, 85.0, 88.5,
105.2, 105.5, 106.1, 112.9, 127.4, 127.5, 127.6, 127.7,
127.8, 128.2, 128.3, 128.4, 129.4, 129.5, 129.6, 133.2,
133.3, 135.5, 135.6, 137.5, 137.7, 137.8. MALDI-TOF
MS: [M+Na]+ calcd for C78H90O13Si2Na, 1313.6.
Found 1313.6. Anal. Calcd for C78H90O13Si2: C, 72.53;
H, 7.02. Found: C, 72.67; H, 7.07.
4.1.8. Methyl 2,3-di-O-benzyl-5-O-mycoloyl-a-D-arabino-
furanoside (18). [General procedure for the synthesis of
mycolate: Method A]. To a mixture of 17 (6.0 mg,
0.012 mmol) and MA (12 mg, 0.0097 mmol) in DMF–
THF (1:5, 1.2 mL) was added CsHCO3 (10 mg,
0.052 mmol). After stirring at 70 ꢁC for 2 days, the sus-
pension was diluted with EtOAc. The organic layer was
washed successively with H2O and brine, dried over
MgSO4 and concentrated in vacuo. The resulting residue
was purified by LH-20 column chromatography
(CHCl3–MeOH, 1:1) and silica gel column chromatog-
raphy (hexane–EtOAc, 4:1) to give 18 (11.9 mg,
0.00761 mmol, 79%) as a colorless oil.
To this tetrabenzylated compound (70 mg, 0.054 mmol)
in THF (0.6 mL) was added 1 M THF solution of
TBAF (0.36 mL, 0.36 mmol) at 0 ꢁC. After stirring for
4 h at ambient temperature, the reaction mixture was
diluted with EtOAc. The organic layer was washed suc-
cessively with satd aq NH4Cl and brine, dried over
MgSO4, and concentrated in vacuo. The resulting resi-
due was purified by silica gel column chromatography
(hexane–EtOAc, 2:1–1:2) to give 19 (36 mg, 0.044 mmol,
82%) as a colorless oil.
1H NMR (sugar moiety) (CDCl3): d 3.35 (s, 3H), 3.81
(dd, J = 2.8, 6.4 Hz, 1H), 3.96 (br d, J = 2.8 Hz, 1H),
4.19 (m, 1H), 4.27–4,28 (m, 2H), 4.45 (d, J = 12.0 Hz,
1H), 4.48 (d, J = 12.0 Hz, 1H), 4.54 (d, J = 12.0 Hz,
1H), 4.56 (d, J = 12.0 Hz, 1H), 7.24–7.33 (m, 10H);
13C NMR (sugar moiety) (CDCl3): d 54.9, 63.5, 72.2,
72.4, 79.4, 83.7, 87.8, 107.1, 126.2, 127.8, 127.9, 128.4,
133.0, 137.2. MALDI-TOF MS: [M+Na]+ calcd for
C104H186O8Na, 1586.4. Found 1586.2.
25
D
½aꢀ +78.6 (c 1.85, CHCl3); 1H NMR (CDCl3): d 1.30 (s,
3H), 1.47 (s, 3H), 3.50 (dd, J = 6.1, 12.2 Hz, 1H), 3.59
(dd, J = 5.2, 11.8 Hz, 1H), 3.64–3.68 (m, 3H), 3.73 (dd,
J = 3.2, 11.8 Hz, 1H), 3.80 (dd, J = 3.2, 7.2 Hz, 1H),
3.82–3.86 (m, 2H), 3.95–4.04 (m, 4H), 4.17 (m, 1H),
4.28 (dd, J = 1.2, 4.8 Hz, 1H), 4.40–4.51 (m, 9 H), 5.02
(s, 1H), 5.09 (s, 1H), 5.76 (d, J = 4.0 Hz, 1H), 7.19–
7.31 (m, 20 H); 13C NMR (CDCl3): d 27.0, 27.5, 62.6,
62.7, 65.3, 72.0, 72.2, 72.2, 72.3, 80.2, 82.3, 83.0, 83.2,
86.3, 87.7, 88.3, 104.8, 105.7, 105.8, 113.5, 127.6,
127.7, 127.8, 127.9, 128.3, 128.4, 128.5, 137.1, 137.2,
137.5, 137.5. MALDI-TOF MS: [M+Na]+ calcd for
C46H54O13Na, 837.4. Found 837.4. HRMS ESI-TOF:
[M+Na]+ calcd for C46H54O13Na, 837.3462. Found
837.3480. Anal. Calcd for C46H54O13: C, 67.80; H,
6.68. Found: C, 67.65; H, 6.70.
4.1.9. Methyl 5-O-mycoloyl-a-D-arabinofuranoside (1).
Compound 18 (4.0 mg, 0.0026 mmol) was dissolved in
hexane–EtOAc (1:1, 0.5 mL) and 20% Pd(OH)2/C
(2.5 mg) was added. After the reaction mixture was
stirred under hydrogen atmosphere at room tempera-
ture for 5 days, it was filtered through Celite and
washed with EtOAc and the combined filtrate was con-
centrated in vacuo. The resulting residue was purified
by LH-20 column chromatography (CHCl3–MeOH,
1:1) to give 1 (2.9 mg, 0.0021 mmol, 82%) as a colorless
powder.
1H NMR (sugar moiety) (CDCl3): d 3.38 (s, 3H), 3.96
(m, 1H), 4.05 (br s, 1H), 4.16 (m, 1H), 4.30 (dd,
J = 4.0, 12.0 Hz, 1H), 4.48 (dd, J = 4.4, 12.0 Hz, 1H),
4.87 (s, 1H); 13C NMR (sugar moiety) (CDCl3): d
55.0, 63.2, 78.4, 80.4, 83.8, 108.7. MALDI-TOF MS:
[M+Na]+ calcd for C90H174O8Na, 1406.3. Found
1406.2.
4.1.11. 2,3-Di-O-benzyl-5-O-p-toluenesulfonyl-a-D-arabi-
nofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-toluenesulfo-
nyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-isopropylidene-b-
D-arabinofuranose (20). Compound 20 was synthesized
from 19 according to the procedure for the synthesis of
17 except that twofold equivalent of TsCl and pyridine
was used and that the mixture was stirred for 4 h (67%
as a colorless oil).
4.1.10. 2,3-Di-O-benzyl-a-D-arabinofuranosyl-(1 ! 3)-
[2,3-di-O-benzyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-
isopropylidene-b-D-arabinofuranose (19). To a mixture of
12 (87 mg, 0.078 mmol) and benzyl bromide (43 mg,
0.25 mmol) in dry DMF (0.5 mL) was added sodium hy-
dride (60% dispersion in oil, 10 mg, 0.25 mmol) at 0 ꢁC.
After stirring at 0 ꢁC for 5 h, the reaction was quenched
with satd aq NH4Cl. The reaction mixture was extracted
with CH2Cl2. The organic layer was washed with brine,
dried over MgSO4, and concentrated in vacuo. The
resulting residue was purified by silica gel column chro-
matography (hexane–EtOAc, 7:1–5:1) to give the tetrab-
26
D
½aꢀ +62.2 (c 0.24, CHCl3); 1H NMR (CDCl3): d 1.31 (s,
3H), 1.48 (s, 3H), 2.37 (s, 3H), 2.38 (s, 3H), 3.50 (dd,
J = 5.2, 10.4 Hz, 1H), 3.79 (dd, J = 5.2, 10.4 Hz, 1H),
3.81–3.84 (m, 2H), 3.94 (dd, J = 0.8, 3.2 Hz, 1H), 4.00
(dd, J = 0.8, 3.2 Hz, 1H), 4.02–4.15 (m, 7H), 4.19–4.23
(m, 1H), 4.38–4.51 (m, 9H), 4.93 (s, 1H), 5.01 (s, 1H),
5.78 (d, J = 4.0 Hz, 1H), 7.22–7.35 (m, 24H), 7.71 (d,
J = 8.8 Hz, 2H), 7.73 (d, J = 8.4 Hz, 2H); 13C NMR

3058
A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
(CDCl3): d 21.6, 26.6, 27.2, 66.5, 68.6, 68.7, 72.0, 72.1,
72.3, 78.8, 79.4, 80.0, 82.9, 82.9, 83.3, 85.0, 87.8, 87.8,
105.3, 106.1, 113.0, 127.7, 127.8, 127.9, 128.0, 128.3,
128.4, 129.7, 132.6, 132.7, 137.1, 137.2, 137.3, 137.5,
144.6, 144.7. MALDI-TOF MS: [M+Na]+ calcd for
C60H66O17S2Na, 1145.4. Found 1145.4. HRMS ESI-
TOF: [M+Na]+ calcd for C60H66O17S2Na, 1145.3639.
Found 1145.3601.
J = 4.0 Hz, 1H); 13C NMR (sugar moiety) (CDCl3): d
26.4, 27.2, 63.3, 63.5, 66.9, 78.8, 80.6, 81.0, 81.2, 83.0,
83.1, 83.4, 84.9, 85.4, 105.4, 106.9, 107.5, 113.1. MAL-
DI-TOF MS: [M+Na]+ calcd for C186H354O19Na,
2915.7. Found 2916.1.
4.1.14. 2,3-Di-O-benzyl-5-O-p-methoxybenzyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-p-toluenesulfonyl-a-
D-arabinofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-meth-
oxybenzyl-b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-O-
p-toluenesulfonyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-
isopropylidene-b-D-arabinofuranose (21). Compound 21
was synthesized from 14 according to the procedure
for the synthesis of 17 except that twofold equivalent
of TsCl and pyridine was used (86% as a colorless oil).
4.1.12.
2,3-Di-O-benzyl-5-O-mycoloyl-a-D-arabinofur-
anosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-mycoloyl-a-D-arabi-
nofuranosyl-(1 ! 5)]-1,2-O-isopropylidene-b-D-arabinof-
uranose (24). [Method A] Compound 24 was synthesized
from 20 according to a general procedure (Method A)
for the synthesis of 18 except that twofold equivalent
of MA and CsHCO3 was used and that the mixture
was stirred for 4 days (65% as a colorless foam).
23
D
½aꢀ ꢁ4.07 (c 2.80, CHCl3); 1H NMR (CDCl3): d 1.29 (s,
3H), 1.47 (s, 3H), 2.31 (s, 3H), 2.33 (s, 3H), 3.46–3.54
(m, 5H), 3.74 (s, 6H), 3.69–3.77 (m, 1H), 3.93–4.23 (m,
18H), 4.31–4.37 (m, 4H), 4.44 (br d, J = 4.0 Hz, 1H),
4.47–4.66 (m, 12H), 4.86 (br s, 1H), 4.88 (d,
J = 4.4 Hz, 1H), 4.93 (br s, 1H), 5.02 (d, J = 4.4 Hz,
1H), 5.75 (d, J = 4.0 Hz, 1H), 6.78 (d, J = 8.4 Hz, 2H),
6.79 (d, J = 8.8 Hz, 2H), 7.13–7.31 (m, 38H), 7.69 (d,
J = 8.4 Hz, 2H), 7.72 (d, J = 8.8 Hz, 2H); 13C NMR
(CDCl3): d 21.6, 26.7, 27.2, 55.2, 66.2, 68.8, 69.0, 71.4,
71.6, 72.1, 72.2, 72.4, 72.4, 72.6, 72.7, 72.7, 79.6, 79.8,
79.9, 80.0, 80.2, 82.5, 82.7, 83.0, 83.4, 83.5, 83.8, 83.9,
85.1, 85.2, 85.7, 100.1, 100.5, 104.8, 105.2, 106.1,
113.1, 113.6, 113.7, 127.5, 127.6, 127.7, 127.8, 127.9,
128.0, 128.1, 128.2, 128.3, 128.4, 128.9, 129.2, 129.6,
129.7, 129.9, 130.0, 132.6, 132.7, 137.5, 137.6, 137.7,
137.9, 138.1, 144.5, 144.6, 159.0, 159.0. MALDI-TOF
MS: [M+Na]+ calcd for C100H110O27S2Na, 1829.7.
Found 1830.0. HRMS ESI-TOF: [M+Na]+ calcd for
C100H110O27S2Na, 1829.6574. Found 1829.6600.
[General procedure for the synthesis of mycolate: Method
B] A mixture of 20 (6.5 mg, 0.0058 mmol), MA (18 mg,
0.015 mmol), CsHCO3 (7.2 mg, 0.037 mmol), and 18-
crown-6 (13 mg, 0.049 mmol) was dissolved in dry tolu-
ene (2 mL). After stirring at 70 ꢁC for 4 days, the reac-
tion mixture was diluted with EtOAc. The organic
layer was washed successively with satd aq NaHCO3,
satd aq NH4Cl, and brine, dried over MgSO4, and con-
centrated in vacuo. The resulting residue was purified by
preparative TLC (developed with hexane–EtOAc, 3:1
and eluted with CHCl3–MeOH, 5:1) to give 24
(12.4 mg, 0.0038 mmol, 66%) as a colorless foam.
1H NMR (sugar moiety) (CDCl3): d 1.31 (s, 3H), 1.49 (s,
3H), 3.56–3.60 (m, 1H), 3.85–3.90 (m, 3H), 3.97 (br d,
J = 2.8 Hz, 1H), 4.01 (br d, J = 2.8 Hz, 1H), 4.09 (m,
1H), 4.15 (m, 1H), 4.20–4.29 (m, 6H), 4.40 (br d,
J = 4.0 Hz, 1H), 4.43–4.54 (m, 8H), 5.02 (br s, 1H),
5.09 (br s, 1H), 5.79 (d, J = 4.0 Hz, 1H); 13C NMR (su-
gar moiety) (CDCl3): d 57.7, 62.7, 62.9, 72.1, 72.1, 72.3,
72.4, 79.2, 79.7, 79.9, 83.2, 83.7, 85.2, 85.4, 88.1, 88.1,
105.2, 105.2, 105.9, 113.1, 127.7, 127.8, 127.9, 128.0,
128.3, 128.4, 128.5, 137.1, 137.3, 137.4, 137.5. MAL-
DI-TOF MS: [M+Na]+ calcd for C214H379O19Na,
3275.9. Found 3275.6.
4.1.15. 2,3-Di-O-benzyl-5-O-p-toluenesulfonyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-t-butyldiphenylsilyl-
a-D-arabinofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-tol-
uenesulfonyl-b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-
O-t-butyldiphenylsilyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-
O-isopropylidene-b-D-arabinofuranose (22). Compound
22 was synthesized from 15 according to the procedure
for the synthesis of 17, except that twofold equivalent
of TsCl and pyridine was used (86% as a colorless oil).
4.1.13. 5-O-Mycoloyl-a-D-arabinofuranosyl-(1 ! 3)-[5-
O-mycoloyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-isopro-
pylidene-b-D-arabinofuranose (2). Compound 24
(15.9 mg, 0.00488 mmol) was dissolved in hexane–
EtOAc (1:1, 1 mL) and 20% Pd(OH)2/C (6 mg) was add-
ed. Hydrogen was bubbled through the suspension at
room temperature for 5 days. The solution was filtered
over Celite and the filtrate was concentrated in vacuo.
The resulting residue was purified by LH-20 column
chromatography (CHCl3–MeOH, 1:1) and preparative
TLC (developed with CHCl3–MeOH, 10:1 and eluted
27
D
½aꢀ ꢁ26.0 (c 0.06, CHCl3); 1H NMR (CDCl3): d 1.00 (s,
18H), 1.29 (s, 3H), 1.47 (s, 3H), 2.31 (s, 3H), 2.32 (s,
3H), 3.55 (dd, J = 4.4, 5.6 Hz, 1H), 3.66–3.78 (m, 4H),
3.89–4.26 (m, 19H), 4.39–4.64 (m, 13H), 4.94 (br s,
1H), 4.96 (d, J = 4.0 Hz, 1H), 5.04 (d, J = 4.4 Hz, 1H),
5.05 (br s, 1H), 5.79 (d, J = 4.0 Hz, 1H), 7.12 (br d,
J = 8.8 Hz, 2H), 7.14 (br d, J = 8.8 Hz, 2H), 7.23–7.37
(m, 42H), 7.61–7.65 (m, 12H); 13C NMR (CDCl3): d
19.3, 26.6, 26.9, 27.3, 63.5, 63.5, 66.8, 70.1, 70.2, 72.1,
72.2, 72.3, 72.4, 72.5, 78.3, 78.4, 79.9, 82.1, 82.1, 82.3,
83.1, 83.2, 83.3, 83.4, 85.0, 85.9, 86.1, 100.3, 100.4,
104.0, 105.4, 105.6, 113.0, 127.3, 127.4, 127.5, 127.6,
127.7, 127.8, 127.9, 128.1, 128.2, 128.3, 128.4, 129.5,
129.6, 129.7, 132.7 133.1, 133.2, 133.4, 135.5, 135.7,
137.3, 137.4, 137.5, 137.6, 137.9, 138.1. MALDI-TOF
MS: [M+Na]+ calcd for C116H130O25S2Si2Na, 2065.8.
with CHCl3–MeOH, 5:1) to give
0.00352 mmol, 72%) as a colorless foam.
2
(10.2 mg,
1H NMR (sugar moiety) (CDCl3): d 1.30 (s, 3H), 1.51 (s,
3H), 3.58 (dd, J = 7.6, 9.2 Hz, 1H), 3.92–3.95 (m, 2H),
4.00 (m, 1H), 4.08–4.15 (m, 4H), 4.19 (m, 1H), 4.25–
4.30 (m, 3H), 4.45 (dd, J = 3.6, 10.8 Hz, 2H), 4.58 (d,
J = 4.0 Hz, 1H), 4.99 (s, 1H), 5.11 (s, 1H), 5.83 (d,

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3059
Found 2066.1. Anal. Calcd for C116H130O25S2Si2: C,
68.14; H, 6.41. Found: C, 68.18; H, 6.66.
[M+Na]+ calcd for C98H106O29S4Na, 1897.6. Found
1898.4. HRMS ESI-TOF: [M+Na]+ calcd for
C98H106O29S4Na, 1897.5600. Found 1897.5637.
4.1.16. 2,3-Di-O-benzyl-5-O-p-toluenesulfonyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-p-toluenesulfonyl-a-
D-arabinofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-toluene-
sulfonyl-b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-O-
p-toluenesulfonyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-
isopropylidene-b-D-arabinofuranose (23). To a solution
of 21 (64 mg, 0.035 mmol) in CH2Cl2–H2O (18:1,
1.9 mL) was added DDQ (27 mg, 0.12 mmol) and stirred
at room temperature for 2 h. The reaction mixture was
diluted with EtOAc. The organic layer was washed suc-
cessively with ascorbic acid/citric acid buffer and brine,
dried over MgSO4, and concentrated in vacuo. The
resulting residue was purified by silica gel column chro-
matography (toluene–acetone, 15:1–10:1) and LH-20
column chromatography (CHCl3–MeOH, 1:1) to give
the corresponding diol (38 mg, 0.024 mmol, 68%) as a
colorless oil.
4.1.17. 2,3-Di-O-benzyl-5-O-p-methoxybenzyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-mycoloyl-a-D-arabi-
nofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-p-methoxyben-
zyl-b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-O-myco-
loyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-isopropylidene-
b-D-arabinofuranose (25). Compound 25 was synthe-
sized from 21 according to the general procedure
(Method A) for the synthesis of 18 except that two-
fold equivalent of MA and CsHCO3 was used and
that the mixture was stirred for 5 days (65% as a
colorless powder).
1H NMR (sugar moiety) (CDCl3): d 1.29 (s, 3H), 1.49 (s,
3H), 3.47–3.52 (m, 4H), 3.57–3.62 (m, 1H), 3.73 (s, 3H),
3.73 (s, 3H), 3.88 (dd, J = 5.6, 10.4 Hz, 1H), 3.88–4.11
(m, 9H), 4.17–4.36 (m, 13H), 4.42–4.65 (m, 13H), 4.90
(d, J = 4.4 Hz, 1H), 4.96 (br s, 1H), 5.02 (d,
J = 4.4 Hz, 1H), 5.03 (br s, 1H), 5.78 (d, J = 3.6 Hz,
1H), 6.76 (d, J = 8.8 Hz, 2H), 6.77 (d, J = 8.8 Hz, 2H),
7.11 (d, J = 8.8 Hz, 2H), 7.11 (d, J = 8.8 Hz, 2H),
7.21–7.32 (m, 30H); 13C NMR (sugar moiety) (CDCl3):
d 55.2, 63.1, 68.5, 71.6, 72.0, 72.1, 72.2, 72.3, 72.4, 72.5,
72.6, 72.7, 72.8, 79.9, 79.9, 80.5, 82.7, 82.9, 83.8, 83.9,
84.0, 84.2, 85.1, 85.4, 99.8, 100.2, 104.6, 104.7, 105.2,
113.2, 113.6, 113.7, 127.5, 127.6, 127.7, 127.9, 128.0,
128.2, 128.3, 128.4, 128.5, 129.2, 129.8, 129.9, 137.4,
137.7, 137.8, 138.0, 138.5, 159.0, 159.0. MALDI-TOF
MS: [M+Na]+ calcd for C254H422O29Na, 3960.1. Found
3960.3.
23
D
½aꢀ +6.17 (c 0.79, CHCl3); 1H NMR (CDCl3): d 1.29 (s,
3H), 1.47 (s, 3H), 2.35 (s, 3H), 2.36 (s, 3H), 3.50 (dd,
J = 5.6, 10.4 Hz, 1H), 3.49–3.59 (m, 2H), 3.60–3.67 (m,
2H), 3.77 (dd, J = 5.6, 10.4 Hz, 1H), 3.93–4.13 (m,
11H), 4.15–4.20 (m, 6H), 4.24 (dd, J = 0.9, 3.2 Hz, 1H),
4.45 (dd, J = 0.6, 4.0 Hz, 1H), 4.37–4.71 (m, 12H), 4.87
(d, J = 0.9 Hz, 1H), 4.91 (d, J = 4.4 Hz, 1H), 4.98 (d,
J = 0.9 Hz, 1H), 5.02 (d, J = 4.4 Hz, 1H), 5.77 (d,
J = 4.0 Hz, 1H), 7.20–7.33 (m, 34H), 7.71 (br d,
J = 8.4 Hz, 2H), 7.73 (br d, J = 8.4 Hz, 2H); 13C NMR
(CDCl3): d 21.6, 26.6, 27.2, 63.5, 63.6, 66.6, 68.6, 68.8,
72.2, 72.3, 72.5, 72.6, 72.7, 79.1, 79.9, 79.9, 80.7, 80.8,
81.8, 81.9, 82.8, 82.8, 83.2, 83.9, 84.0, 85.1, 85.7, 100.1,
100.2, 104.9, 105.3, 105.8, 113.1, 127.6, 127.7, 127.8,
127.9, 128.0, 128.3, 128.4, 128.5, 129.7, 129.8, 132.5,
137.4, 137.9, 144.7, 144.8. MALDI-TOF MS: [M+Na]+
calcd for C84H94O25S2Na, 1589.5. Found 1590.3. HRMS
ESI-TOF: [M+Na]+ calcd for C84H94O25S2Na,
1589.5423. Found 1589.5411. Anal. Calcd for
C84H94O25S2: C, 64.35; H, 6.04. Found: C, 64.20; H, 6.07.
4.1.18. 2,3-Di-O-benzyl-5-O-mycoloyl-b-D-arabinofur-
anosyl-(1 ! 2)-3-O-benzyl-5-O-t-butyldiphenylsilyl-a-D-
arabinofuranosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-mycoloyl-
b-D-arabinofuranosyl-(1 ! 2)-3-O-benzyl-5-O-t-butyldi-
phenylsilyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-isopro-
pylidene-b-D-arabinofuranose (26). Compound 26 was
synthesized from 22 according to the general procedure
(Method B) for the synthesis of 24 except that coevapo-
rated substrates and 18-crown-6 with toluene were used
and that the mixture was stirred for 7 days (42% as a col-
orless oil).
Compound 23 was synthesized from the diol according
to the procedure for the synthesis of 17 except that two-
fold equivalent of TsCl and pyridine was used (91% as a
viscous oil).
1H NMR (sugar moiety) (CDCl3): d 1.2–1.3 (s, 3H), 1.46
(s, 3H), 3.52–3.58 (m, 1H), 3.63–3.77 (m, 4H), 3.90 (dd,
J = 6.8, 10.8 Hz, 1H), 3.95–4.31 (m, 18H), 4.38–4.68 (m,
13H), 4.96 (br s, 1H), 4.96 (d, J = 3.6 Hz, 1H), 5.05 (br s,
1H), 5.06 (d, J = 4.0 Hz, 1H), 5.77 (d, J = 4.0 Hz, 1H),
7.22–7.30 (m, 42H), 7.60–7.62 (m, 8H). MALDI-TOF
MS: [M+Na]+ calcd for C270H442O27Si2Na, 4196.3.
Found 4196.2.
23
D
½aꢀ +4.18 (c 0.49, CHCl3); 1H NMR (CDCl3): d 1.23 (s,
3H), 1.39 (s, 3H), 2.26 (s, 3H), 2.27 (s, 3H), 2.29 (s, 3H),
2.30 (s, 3H), 3.39 (dd, J = 4.8, 10.4 Hz, 1H), 3.68 (dd,
J = 6.0, 10.4 Hz, 1H), 3.87–4.10 (m, 22H), 4.40 (d,
J = 4.0 Hz, 1H), 4.27–4.59 (m, 12H), 4.77 (br s, 1H),
4.81 (d, J = 4.0 Hz, 1H), 4.87 (br s, 1H), 4.97 (d,
J = 4.0 Hz, 1H), 5.68 (d, J = 4.0 Hz, 1H), 7.10–7.25
(m, 38H), 7.62–7.69 (m, 8H); 13C NMR (CDCl3): d
21.6, 26.7, 27.3, 66.1, 69.0, 69.1, 70.0, 70.3, 71.6, 72.3,
72.3, 72.5, 72.6, 72.6, 78.2, 78.3, 79.8, 79.9, 80.5, 81.5,
81.7, 83.0, 83.3, 83.4, 83.6, 83.6, 85.3, 85.5, 86.1, 100.6,
101.0, 104.7, 105.2, 106.0, 113.2, 127.5, 127.6, 127.7,
127.8, 127.9, 128.0, 128.1, 128.2, 128.3, 128.4, 128.5,
128.6, 128.7, 128.9, 129.6, 129.7, 129.8, 130.8, 130.9,
132.5, 132.6, 137.1, 137.3, 137.4, 137.5, 137.6, 137.7,
144.5, 144.7, 144.8, 144.9. MALDI-TOF MS:
4.1.19.
2,3-Di-O-benzyl-5-O-mycoloyl-b-D-arabinofur-
anosyl-(1 ! 2)-3-O-benzyl-5-O-mycoloyl-a-D-arabinofur-
anosyl-(1 ! 3)-[2,3-di-O-benzyl-5-O-mycoloyl-b-D-arabi-
nofuranosyl-(1 ! 2)-3-O-benzyl-5-O-mycoloyl-a-D-arabi-
nofuranosyl-(1 ! 5)]-1,2-O-isopropylidene-b-D-arabinof-
uranose (27). Compound 27 was synthesized from 23
according to the general procedure (Method B) for
the synthesis of 24 except that twofold equivalent of

3060
A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
MA and CsHCO3 were used. Furthermore, coevaporat-
ed substrates and 18-crown-6 with toluene were used
and the mixture was stirred for 5 days (28% as a color-
less powder).
LH-20 column chromatography (CHCl3–MeOH, 1:1) to
give 4 (4.1 mg, 0.0013 mmol, 96%) as a colorless
powder.
1H NMR (sugar moiety) (CDCl3): d 1.2–1.3 (s, 3H), 1.52
(s, 3H), 3.6–4.4 (br m, 24H), 4.61 (br s, 1H), 4.99 (br s,
1H), 5.02 (br s, 1H), 5.09 (br s, 1H), 5.15 (br s, 1H), 5.83
(br s, 1H). MALDI-TOF MS: [M+Na]+ calcd for
C196H370O27Na, 3179.8. Found 3180.4.
1H NMR (sugar moiety) (CDCl3): d 1.30 (s, 3H), 1.50 (s,
3H), 3.55–3.65 (m, 1H), 3.85–3.92 (m, 1H), 3.99–4.30
(m, 22H), 4.47–4.70 (m, 13H), 4.90 (d, J = 3.6 Hz,
1H), 4.97 (br s, 1H), 5.03 (br s, 1H), 5.03 (d,
J = 3.6 Hz, 1H), 5.77 (d, J = 3.6 Hz, 1H), 7.22–7.31
(m, 30H). MALDI-TOF MS: [M+Na]+ calcd. for
C406H730O33Na, 6158.5. Found 6159.2.
4.1.22. 5-O-Mycoloyl-b-D-arabinofuranosyl-(1 ! 2)-5-O-
mycoloyl-a-D-arabinofuranosyl-(1 ! 3)-[5-O-mycoloyl-b-
D-arabinofuranosyl-(1 ! 2)-5-O-mycoloyl-a-D-arabino-
furanosyl-(1 ! 5)]-1,2-O-isopropylidene-b-D-arabinofura-
nose (5). Compound 27 (8.8 mg, 0.0014 mmol) and 20%
Pd(OH)2/C (5.3 mg) were suspended in hexane–EtOAc
(1:1, 4 mL) and hydrogen was bubbled at room temper-
ature for 3 days. The reaction mixture was filtered over
Celite and washed with CHCl3–MeOH (4:1). The filtrate
was combined and concentrated in vacuo. The resulting
residue was purified by LH-20 column chromatography
(CHCl3–MeOH, 3:1) to give 5 (7.5 mg, 0.0013 mmol,
94%) as a colorless powder.
4.1.20. b-D-Arabinofuranosyl-(1 ! 2)-5-O-mycoloyl-a-D-
arabinofuranosyl-(1 ! 3)-[b-D-arabinofuranosyl-(1 ! 2)-
5-O-mycoloyl-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-iso-
propylidene-b-D-arabinofuranose (3). Compound 25
(4.2 mg, 0.0011 mmol) and 20% Pd(OH)2/C (3 mg)
were suspended in hexane–EtOAc (1:1, 1 mL) and
hydrogen was bubbled at room temperature. After 2
days, EtOAc–MeOH (1:1, 2 mL) was added to the sus-
pension and stirred for additional 5 h under hydrogen
atmosphere. The reaction mixture was filtered over
Celite and washed with CHCl3, MeOH, and EtOAc,
successively. The filtrate was combined and concen-
trated in vacuo. The resulting residue was purified by
LH-20 column chromatography (CHCl3–MeOH, 1:1)
to give 3 (3.0 mg, 0.00095 mmol, 89%) as a colorless
powder.
1H NMR (sugar moiety) (CDCl3): d 1.2–1.3 (s, 3H), 1.51
(s, 3H), 3.6–4.4 (br m, 24H), 4.58 (br d, J = 4.0 Hz, 1H),
5.00–5.02 (br m, 2H), 5.02 (br d, J = 4.0 Hz, 1H), 5.09
(br s, 1H), 5.83 (br d, J = 4.0 Hz, 1H). MALDI-TOF
MS: [M+Na]+ calcd for C364H694O33Na, 5618.3. Found
5618.8.
1H NMR (sugar moiety) (CDCl3): d 1.2–1.3 (s, 3H), 1.51
(s, 3H), 3.6–4.6 (br m, 25H), 5.0–5.2 (br m, 4H), 5.8–5.9
(br m, 1H). MALDI-TOF MS: [M+Na]+ calcd for
C196H370O27Na, 3179.8 Found 3179.8.
4.2. Methods and materials for TNF-a secretion-inducing
assay
4.2.1. Cells and reagents. A murine macrophage cell line
RAW264.7 was purchased from American Type Culture
Collection (Manassas, VA). The cells were cultured in
Dulbecco’s modified Eagle’s medium supplemented with
10% fetal calf serum, 2 mM L-glutamine, 50 U/mL pen-
icillin, and 50 lg/mL streptomycin. BCG-CWS, and tre-
halose-dimycolate (TDM) were prepared from M. bovis
BCG Tokyo strain.20
4.1.21. 5-O-Mycoloyl-b-D-arabinofuranosyl-(1 ! 2)-a-D-
arabinofuranosyl-(1 ! 3)-[5-O-mycoloyl-b-D-arabinofur-
anosyl-(1 ! 2)-a-D-arabinofuranosyl-(1 ! 5)]-1,2-O-iso-
propylidene-b-D-arabinofuranose (4). To compound 26
(2.4 mg, 0.00058 mmol) in THF (0.1 mL) were added
AcOH (0.47 lL, 0.0082 mmol) and TBAF (0.01 M
THF solution, 1.2 mL, 0.012 mmol) at 0 ꢁC. After stir-
ring for 8 days at ambient temperature, the solution was
concentrated in vacuo. The resulting residue was puri-
fied by LH-20 column chromatography (CHCl3–
MeOH, 1:1) to give the desilylated diol (1.5 mg,
0.00041 mmol, 71%) as a colorless powder.
4.2.2. In vitro macrophage stimulation. Arabinomyco-
lates (1–5), BCG-CWS and TDM were dissolved or sus-
pended in chloroform and diluted with hexane–EtOH
(9:1 v/v). After the samples were dispensed into 96-well
polypropylene microplates, the solvent was evaporated
in the clean bench. RAW264.7 cell suspension was add-
ed in the plates at 5 · 105 cells/well and incubated
overnight.
1H NMR (sugar moiety) (CDCl3): d 1.32 (s, 3H), 1.50 (s,
3H), 3.47–3.73 (m, 5H), 3.90 (m, 1H), 3.94–4.12 (m, 9H),
4.18–4.28 (m, 8H), 4.34 (br d, J = 4.4 Hz, 1H), 4.46–4.69
(m, 13H), 4.93 (d, J = 4.0 Hz, 1H), 4.97 (br s, 1H), 4.99
(d, J = 3.6 Hz, 1H), 5.05 (br s, 1H), 5.77 (d, J = 4.4 Hz,
1H), 7.25–7.31 (m, 30H). MALDI-TOF MS: [M+Na]+
calcd for C238H406O27Na, 3720.0. Found 3720.4.
4.2.3. ELISA. TNF-a in the culture supernatant was mea-
sured using a commercial kit (R&D Systems, Minneapo-
lis, MN) according to the manufacturer’s protocol.
This diol (5.0 mg, 0.0014 mmol) and 20% Pd(OH)2/C
(6 mg) were suspended in hexane–EtOAc (1:1, 2 mL)
and hydrogen was bubbled at room temperature over-
night. The reaction mixture was filtered over Celite
and washed with CHCl3–MeOH (4:1), CHCl3, and
EtOAc, successively. The filtrate was combined and con-
centrated in vacuo. The resulting residue was purified by
Acknowledgments
We thank Dr. M. Murata for in vitro assay, Ms. Y.
Uenishi for a preparation of mycolic acid (Sumitomo
Pharmaceuticals Co.), and Ms. A. Takahashi for her
technical assistance (RIKEN).

A. Ishiwata et al. / Bioorg. Med. Chem. 14 (2006) 3049–3061
3061
12. Callam, C. S.; Gadikota, R. R.; Krein, D. M.; Lowary, T.
L. J. Am. Chem. Soc. 2003, 125, 13112–13119.
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