Chemical synthesis and immunosuppressive activity of dipalmitoyl phosphatidylinositol hexamannoside

Article abstract of DOI:10.1021/jo200588u

Phosphatidylinositol mannosides (PIMs) isolated from mycobacteria have been identified as an important class of phosphoglycolipids with significant immune-modulating properties. We present here the synthesis of dipalmitoyl phosphatidylinositol hexamannosi

Full text of DOI:10.1021/jo200588u

ARTICLE
pubs.acs.org/joc
Chemical Synthesis and Immunosuppressive Activity of Dipalmitoyl
Phosphatidylinositol Hexamannoside
Gary D. Ainge,†,‡ Benjamin J. Compton,† Colin M. Hayman,† William John Martin,§ Steven M. Toms,†
David S. Larsen,‡ Jacquie L. Harper,*,§ and Gavin F. Painter*,†
†Carbohydrate Chemistry Team, Industrial Research Limited, PO Box 31-310, Lower Hutt, New Zealand
§The Malaghan Institute of Medical Research, PO Box 7060, Wellington, New Zealand
‡University of Otago, PO Box 56, Dunedin, New Zealand
S Supporting Information
b
ABSTRACT: Phosphatidylinositol mannosides (PIMs) isolated from myco-
bacteria have been identified as an important class of phosphoglycolipids with
significant immune-modulating properties. We present here the synthesis of
dipalmitoyl phosphatidylinositol hexamannoside (PIM6) 1 and the first
reported functional biology of a synthetic PIM6. Key steps in the synthetic
protocol included the selective glycosylation of an inositol 2,6-diol with a
suitably protected mannosyl donor and construction of the glycan core utilizing
a [3 þ 4] thio-glycosylation strategy. The target 1 was purified by reverse phase
chromatography and characterized by standard spectroscopic methods, HPLC,
and chemical modification by deacylation to dPIM6. The 1H NMR spectrum of
synthetic dPIM6 obtained from 1 matched that of dPIM6 obtained from nature.
PIM6 (1) exhibited dendritic cell-dependent suppression of CD8þ T cell
expansion in a human mixed lymphocyte reaction consistent with the well
established immunosuppressive activity of whole mycobacteria.
’ INTRODUCTION
was purified by reverse phase chromatography and evaluated for
its effect on DC and antibody-mediated human T cell expansion.
Mycobacteria and their soluble products have strong inherent
immunomodulatory properties. The bulk of this activity is
associated with the components of the mycobacterial cell wall1
that include lipoglycans such as lipoarabinomannan and phos-
phatidylinositol mannosides (PIMs).
PIMs are phosphoglycolipids embedded in the cell walls of
mycobacteria that anchor an array of more complex glycoli-
pids to the cell membrane2 and exhibit both immunosuppres-
sive and adjuvant activities. Previous studies have shown that
PIMs can modulate T cell-driven immune responses,3,4 by
altering T cell function and expansion.5ꢀ8 PIMs or their
corresponding glycans are reported to have vaccine adjuvant
activity in mouse models,9ꢀ11 and there is evidence that PIMs
also have the ability to interfere with LPS-induced immune
responses.12
’ RESULTS AND DISCUSSION
In previous work we demonstrated that dipalmitoyl phos-
phatidylinositol tetramannoside (PIM4) could be prepared
from synthetic protocols that utilized a glycosylation of a
suitably protected pseudotrisaccharide similar to 2 with a
dimannosyl trichloroacetimidate donor in a [3 þ 2] glycosyla-
tion strategy. Subsequent introduction of the phosphatidic acid
moiety using 3, followed by global deprotection afforded the
desired target PIM4.14 We envisaged that dipalmitoyl PIM6 (1)
could be prepared in an analogous manner by utilizing the
acceptor 2 along with the thio-tetramannosyl donor 4 as the
coupling partner to effect a convergent [3 þ 4] glycosylation
approach (Scheme 1). Subsequent phosphitylation with a
phosphoramidite and deprotection would afford PIM6 (1). In
related work a similar approach to the synthesis of higher order
PIMs has been reported by Seeberger et al. for Ac1PIM615 and
PIM glycans.10
Native PIM6 has been reported to expand T cell subsets;8
however, to our knowledge, other than DC-SIGN binding
experiments,13 currently there is no functional data published
on the biological activities of the higher glycosylated synthetic
PIMs (i.e., PIM6). Therefore we decided to embark on the
synthesis of a PIM6, utilizing a thioglycoside as the tetrasacchar-
ide donor in a convergent [3 þ 4] glycosylation strategy. A key
aspect of the synthesis was the use of the anchimeric directing
group 2-(azidomethyl)benzoate (AZMB) as a temporary pro-
tecting group orthoganol to benzoate. The target compound
Synthesis of Thio-tetramannosyl Donor 4. Tetramannoside
donor 4 was prepared from monomannoside building blocks 5, 6
and 7. The incorporation of the directing benzoyl and AZMB
Received: March 19, 2011
Published: May 16, 2011
r
2011 American Chemical Society
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Scheme 1. Retrosynthetic Analysis of PIM6
Scheme 2. Synthesis of Thiomannosyl Donors 7 and 10
synthesized donors 6 and 7 using the approach shown in
Schemes 2 and 3. The diol 818 was a convenient starting material
for the synthesis of 7 and was readily prepared from D-mannose
in a modified, four step procedure19 without the need for column
chromatography. The C-3 hydroxyl group of 8 was selectively
benzylated via prior stannylene acetal formation and the C-2
hydroxyl was subsequently benzoylated to afford 9 in good yield.
Traces of tin residues were removed by chromatography on silica
gel impregnated with KF.20 The benzylidene acetal was regiose-
lectively reduced with borane-THF complex in the presence of
dibutylboron triflate or scandium triflate to give primary alcohol
7, the initial acceptor for the synthesis of tetrasaccharide 4.
Furthermore, thiomannoside 7 was silylated with triisopropylsi-
lyl chloride (TIPSCl) to afford 10, a glycosyl donor for the
synthesis of pseudotrisaccharide 2.
Scheme 3. Syntheis of 2-O-AZMB Protected Donor 6
Trichloroacetimidate (TCA) donor 6 was synthesized from
thioglycoside 1121 by acylation with 2-(azidomethyl)benzoyl
chloride (AZMBCl) to give 12 followed by hydrolysis of the
thioglycoside moiety with aqueous N-iodosuccinimide and reac-
tion with trichloroacetonitrile (Scheme 3).
Glycosylation of 7 with 6 promoted by TMSOTf afforded
disaccharide 13 in 94% yield (Scheme 4). Selective hydrolysis of
the AZMB group was achieved using a Staudinger reduction of
the azide moiety with tributylphosphine22 to give the disacchar-
ide acceptor 14 in 83% yield. Reiteration of glycosylation with 6
to give 15 and removal of the AZMB protecting group afforded
trisaccharide 16 in 68% yield for the two steps. In the final step,
16 was mannosylated with donor 5 to give the key tetrasacchar-
ide building block 4 in 56% yield.
Synthesis of Pseudotrisaccharide Building Block. With
tetrasaccharide 4 in hand it was used in the [3 þ 4] glycosylation
of pseudotrisaccharide 2. We have previously reported the
synthesis of an analogue of 223 for the synthesis of PIM4 from
an intermediate where the primary hydroxyl group of the
mannosyl residue at O-6 of the inositol was protected as a tert-
butyldiphenylsilyl (TBDPS) ether.14 However, difficulties in
removing TBDPS protecting group in that instance led us to
groups16 at the O-2 positions of these donors would ensure high
R-selectivity in manosylation reactions. Furthermore, the AZMB
group can be selectively removed in the presence of benzoyl
groups and would allow subsequent construction of the two
(1f2) mannosyl linkages at the nonreducing end of 4.
Often 2-O-acyl-R-D-mannopyranosyl donors are generated
using orthoester chemistry and we used this approach to
synthesize mannosyl donor 5 using standard protocols.17 Due
to the accessibility of thiophenyl R-D-mannopyranosides we
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Scheme 4. Synthesis of Tetrasaccharide Donor 4
Scheme 5. Synthesis of Pseudotrisaccharide 2
design the mannosyl donor 10 in which the more acid-labile
TIPS group provides the temporary protection required en
route to 2 (Scheme 5). Selective O-6 glycosylation of inositol
diol 17 was achieved with thiophenyl-mannosyl donor 10 using
conditions reported by Codꢀee et al.24 to give pseudodisacchar-
ide 18 in 57% yield. The corresponding O-6 β-glycoside was
not detected under these conditions consistent with previous
work in this area.4,14 The regioselectivity of the glycosylation
was evident from NMR data and confirmed by benzoylation
that gave the O-2-benzoate 19. The 1H NMR chemical shift of
21, benzylation to 22 and deallylation afforded alcohol 23. It
was pleasing to note that the analytical data for the intermediate
compounds 22 and 23 matched that previously reported.10
Phosphitylation of 23 with 3 and subsequent mCPBA oxidation
gave protected PIM6 24 as a mixture of diastereoisomers in 82%
yield. Our experience with this and similarly prepared lipidated
late-stage, PIM intermediates is that they are difficult to isolate
in high purity from putative phospholipid reaction byproducts.
Standard petroleum ether-based silica chromatography is nor-
mally complemented well with a second CHCl3-based purifica-
tion on silica and this was indeed the case here. However, in this
instance a reverse-phase purification step (CHCl3/MeOH) was
also introduced to remove minor impurities (RP-HPLC).
Global deprotection of 24 under hydrogenolytic conditions
and reverse-phase chromatography afforded dipalmitoyl PIM6
(1) as its ammonium salt in good yield and high purity (96%
by HPLC).
The negative ion high resolution mass spectrum of 1 was
consistent with the molecular ion C77H138O43P. The choice of
solvent was critical for obtaining informative NMR spectra and a
30:60:25 mixture of deuterated chloroform, methanol and water
provided the best result. In the 13C NMR spectrum four of the six
signals for the anomeric carbons were well resolved with the
remaining two overlapping at 102.9 ppm.
the characteristic inositol H-2 proton signal of 19 (dd, J1,2, J2,3
2.6 Hz) resonated at 6.00 ppm whereas that for 18 (dd, J1,2
=
=
2.6 Hz, J2,3 = 2.7 Hz) was at 4.18 ppm. Glycosylation of 18 with
TCA donor 5 afforded an inseparable mixture of R- and β-
glycosides. Methanolic HCl-promoted removal of the TIPS
protecting group revealed the primary hydroxyl functionality
which fortunately facilitated the separation of anomers and the
key pseudotrisaccharide building block 2 was obtained in 75%
yield from 18 along with the corresponding β-glycoside in 11%
yield. The R-stereochemistry of the two glycosidic bonds in 2
were each confirmed as R from the two anomeric 1JC,H coupling
constants of 173 and 176 Hz, whereas the R,β-pseudotrisac-
charide isomer was observed to have 1JC,H coupling constants of
161 and 176 Hz.
We have confirmed that the nonlipid core of 1 is identical to
Synthesis of PIM6. The [3 þ 4] glycosylation of 2 and
thioglycoside 4 was effected by treatment with diphenylsulf-
oxide (DPS) and triflic anhydride affording the pseudohepta-
saccharide 20 in good yield (Scheme 6). All six signals for the
anomeric carbons were well resolved in the 125 MHz 13C NMR
spectrum. The newly formed glycosidic bond was established
confirmed to have an R-configuration as all six of the JC,H
coupling constants ranged from 170 to 174 Hz. Routine
protecting group manipulations, namely debenzoylation to
that found in nature. To support this 1 was deacylated with
1
hydrazine to afford deacyl PIM6 (dPIM6). The H NMR data
recorded in d6-acetone of the synthetic dPIM6 was consistent
with that obtained by deacylation of a natural PIM sample as
previously reported by Severn et al. (Figure 1).25
Further support for the structural relationship of 1 to natural
PIM6 is provided by the comparison of the negative ion ESI-
MS2 spectrum of 1 with that reported by Hsu et al.26 for a
1
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Scheme 6. Synthesis of 1
Figure 1. Partial 1H NMR spectra of synthetic dPIM6 (bottom) and dPIM6 from MTb (top)24 recorded in D2O.
natural PIM6 present in Mycobacterium bovis. The MS2 spec-
trum of 1 (Supporting Information) selecting for the MS [M ꢀ
H]ꢀ ion m/z 1781 displays prominent signals at m/z 1525 and
1213 corresponding to loss of the palmitic acid and diacyl
glycerol substituents respectively. Taking into account the
different acylation state of the natural 19:0/16:0-PIM6 for the
spectrum reported by Hsu, a similar fragmentation pattern in
the MS2 spectrum for the MS [M ꢀ H]ꢀ ion at m/z = 1823 is
observed.
Biological Activity of Synthetic PIM6 in a Human Mixed
Lymphocyte Reaction Assay. We have shown previously that
natural and synthetic PIM molecules can suppress T cell-driven
inflammatory immune responses in vivo.3,4 However, lipid anti-
gen presentation via CD1 molecules is also known to induce
expansion of CD1-restricted T cell subsets.6ꢀ8,27,28 Therefore
PIM6 has the potential to either suppress or enhance T cell
immune responses, or both. Five CD1 molecules, CD1aꢀe, have
been identified in humans,27 and there is some evidence that both
CD1b and CD1d can present PIM molecules.6,8,7 Furthermore, it
appears that efficient presentation of PIM6 to CD1b involves
CD1e-dependent intracellular processing.29ꢀ31 As murine sys-
tems only express CD1d, we decided to investigate the effect of
PIM6 (1) on human T cell expansion using a human mixed
lymphocyte reaction (MLR), thus ensuring that all relevant
CD1 molecules were present for glycolipid processing and
The positive ion MS2 spectrum of 1 selecting the MS [M þ
NH4]þ ion m/z 1800 shows a very clear sequential loss of all six
mannose pyranosyl residues after initial loss of ammonia {[M-
NH3-n(162)]þ} (Supporting Information) and the correspond-
ing signal for the resulting protonated phosphatidyl inositol ion
(m/z 811) and the diacylglyceryl ion (m/z 551). This data is
consistent with the structure of 1.
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’ EXPERIMENTAL SECTION
General Experimental. Specific optical rotations, given in
10ꢀ1 deg cm2 gꢀ1, were measured at ambient temperature using either
a Perkin-Elmer 241 or a Rudolph Autopol IV polarimeter with a cell of
path length 1.0 dm. 1H NMR spectra were obtained at 300 or 500 MHz
and referenced to tetramethylsilane (TMS) (0.0 ppm) or the residual
solvent peak (CHCl3 7.26 ppm). Chemical shifts are reported as parts
per million (ppm) using the δ scale. Water suppression NMR spectra
were collected with the standard Bruker supplied ZGCPPR pulse
sequence which employs presaturation with an approximately 7 Hz rf
field for a period of 2 s prior to excitation with an on resonance
composite excitation pulse (Bax, A. J. Magn. Reson. 1985, 65,
142ꢀ145). 13C NMR spectra were recorded at 75 or 125 MHz and
referenced either to TMS (0.0 ppm) or internal solvent (CDCl3 77.0
ppm). Chemical shifts are reported to 1 decimal place with the chemical
shifts for signals that are similar reported to 2 decimal places. 31P NMR
spectra were recorded at 202 MHz and are reported to 1 decimal place
with H3PO4 (0.0 ppm) as the external reference. Electro-spray ioniza-
tion (ESI) mass spectra were recorded either on a PerSpective Biosys-
tems Mariner time-of-flight mass spectrometer or a Q-TOF Premier
mass spectrometer. Thin layer chromatography (TLC) was performed
on Merck silica gel DC Alurolle Kieselgel 60F254 plates and were
visualized under an UV lamp and/or with a spray consisting of 5% w/v
dodecamolybdophosphoric acid in ethanol with subsequent heating.
Flash column chromatography was carried out using Merck Kieselgel 60
(230ꢀ400 mesh) unless otherwise stated. All chromatography solvents
were AR-grade. Petroleum ether used was bp 60ꢀ80° range. Anhydrous
solvents were sourced from Aldrich and unless stated were used without
further treatment. Powdered molecular sieves were flame-dried under
vacuum immediately prior to use. All compounds were isolated after
silica-gel column chromatography and fractions collected were one spot
by TLC. Analytical HPLC analyses were performed on one of two
systems. HPLC System 1 Column: Phenomenex Luna 5 μm C18 4.6 ꢁ
250 mm. Detection: UV at 220 nm. Solvents; A water þ0.1% TFA; B 1:1
CH3CN/THF þ0.1% TFA. Gradient Program; 0ꢀ20 min 10ꢀ0% A,
90ꢀ100% B. Column temperature; 40 °C. HPLC System 2 HPLC:
Column: Phenomenex Synergi 4 μm Fusion-RP (80 Å, 3.0 ꢁ 250 mm).
Detection: ESA Corona charged aerosol detector (Filter = none).
Solvents; A H2O; B Water containing 50 mM NH4OAc; C Methanol.
Gradient Program; 0ꢀ10 min 10ꢀ0% A, 10% B, 80ꢀ90%C, 10ꢀ28 min
hold (0% A, 10% B, 90% C), 28ꢀ30 min return to starting conditions.
Column temperature; 40 °C. Chromatograms were corrected by sub-
traction of a blank injection trace prior to integrating signals after a 3.5
min cutoff.
Phenyl 2-O-Benzoyl-3-O-benzyl-4,6-O-benzylidene-1-thio-
R-D-mannopyranoside (9). A mixture of diol 818 (8.40 g, 23.3 mmol)
and Bu2SnO (6.10 g, 24.5 mmol) were refluxed in dry toluene (100 mL)
under an atmosphere of argon. After 2 h, the resulting clear solution was
allowed to cool to rt and the solvent removed. The residue was dried
under high-vacuum then placed under argon. Cesium fluoride (3.72 g,
24.5 mmol) was added, then dry DMF (100 mL) was cannulated onto the
mixture. Benzyl bromide (3.42 mL, 25.6 mmol) was added and the
reaction stirred at rt overnight. The reaction mixture was diluted with
Et2O (200 mL) and washed with H2O (2 ꢁ 200 mL) and saturated NaCl
solution (200 mL), then dried (MgSO4), filtered and concentrated to a
residue that was dissolved in CH2Cl2 and adsorbed on silica gel. This was
purified by chromatography on silica gel containing 10% w/w potassium
fluoride20 (Et2O/pet. ether =1:9 to 2:8) to afford phenyl 3-O-benzyl-4,6-
O-benzylidene-1-thio-R-D-mannopyranoside (9.43 g, 90%) as a solid.
[R]D20 = þ206 (c 1.58, CHCl3) [Lit.16 [R]D = þ228 (c 1.07, CHCl3)]. 1H
NMR (500 MHz, CDCl3) δ 2.88 (br s, 1H), 3.86 (dd, J = 10.3, 10.3 Hz,
1H), 3.97 (dd, J = 9.6, 3.3Hz, 1H), 4.19 (dd, J = 9.5, 9.5 Hz, 1H), 4.21 (dd,
J = 1.3, 4.9 Hz, 1H), 4.28ꢀ4.30 (m, 1H), 4.31ꢀ4.37 (m, 1H), 4.75 (d, J =
Figure 2. Effect of PIM6 (1) on T cell proliferation in (A) mixed
lymphocyte reaction assay and (B) antibody-induced T cell expansion
assay. (C) Flow cytometry plot of CD8þ T cell expansion in MLR.
(D) PIM6-induced suppression of CD8þ T cell expansion in MLR (Dex-
Dexamethasone).
presentation. This assay system was used to determine whether
PIM6 treatment suppressed or enhanced human T cell expansion
in vitro.
As shown in Figure 2A, incubation of dendritic cells (DC) and
peripheral blood monocytes (PBMC) from different donors
induced an allogeneic-driven T cell expansion that was inhibited
by PIM6 (1) in a dose-dependent manner (IC50 4.9 μM).
Previous work in murine systems showed that direct interaction
of PIM molecules with T cells can suppress antibody-induced T
cell proliferation in vitro.32 Interestingly, 1 did not directly
suppress human T cell expansion induced by anti-CD3/anti-
CD28 (Figure 2B) indicating that PIM6-dependent suppression
of T cell expansion in the MLR was DC mediated. Further
analysis of carboxyfluorescein succinimidyl ester-labeled T cells
by flow cytometry showed that the CD8þ T cells were the
predominant population expanding in the MLR at the time of
analysis (Figure 2C). PIM6 (1) treatment suppressed CD8þ
T
cell expansion in a dose-dependent manner and PIM6-dependent
inhibition was comparable to the control anti-inflammatory
steroid dexamethasone (Figure 2D). Control CD4þ T cell
expansion was not observed (data not shown). It has been shown
that in mice the PIM6 glycan has adjuvant activity when
conjugated to the carrier protein KLH.10 However, our results
clearly show the synthetic phosphoglycolipid 1 compound has
suppressive activity in the human cell-based assay. These results
are potentially representative of the known immunosuppressive
effects of Mtb when resident in human host cells.
’ CONCLUSION
In this study, we report the first synthesis of dipalmitoyl PIM6
1 utilizing a thio-glycoside donor in the key [3 þ 4] glycosylation
step. Importantly, common purification difficulties associated
with lipoglycans were overcome by employing a combination of
reverse phase chromatography and analytical LCMS techniques
to deliver target compound 1 in greater than 95% purity. We also
report for the first time that a synthetic PIM6, found in nature,33
exhibits DC-mediated suppression of human CD8þ T cell
proliferation.
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11.8 Hz, 1H), 4.90 (d, J = 11.8 Hz, 1H), 5.60 (br s, 1H), 5.63 (s, 1H),
7.25ꢀ7.53 (m, 15H). 13C NMR (75 MHz, CDCl3) δ 64.7, 68.6, 71.5,
73.7, 75.8, 79.1, 87.9, 101.7, 126.1, 127.7, 127.9, 128.1, 128.3, 128.6, 129.0,
129.2, 131.8, 133.4, 137.5, 137.8. HRMS-ESI [M þ Na]þ calculated for
C26H26O5SNa: 473.1399. Found 473.1418. Benzoyl chloride (7.23 mL,
62.1 mmol) and DMAP (0.253 g, 2.07 mmol) were added to a solution of
the intermediate thiomannoside (9.35 g, 20.7 mmol) prepared above in
dry pyridine (75 mL) at rt and stirred overnight. H2O was then added and
the reaction stirred for 1 h to ensure that no benzoyl chloride remained.
The reaction was diluted into CH2Cl2/Et2O (1:9, 150 mL) and washed
with 0.5 M HCl (3 ꢁ 200 mL), saturated NaHCO3 (100 mL), saturated
NaCl (100 mL) then dried (MgSO4), filtered and the solvent removed.
The residue was purified on silica gel (pet. ether to EtOAc/pet. ether =
CDCl3) δ 8.12ꢀ8.07 (m, 2H), 7.59ꢀ7.52 (m, 1H), 7.50ꢀ7.38 (m, 4H),
7.35ꢀ7.19 (m, 13H), 5.84 (dd, J = 3.1, 1.5 Hz, 1H), 5.61 (d, J = 1.5 Hz,
1H), 4.92 (d, J = 10.7 Hz, 1H), 4.79 (d, J = 11.3 Hz, 1H), 4.74 (d, J =
10.7 Hz, 1H), 4.61 (d, J = 11.3 Hz, 1H), 4.24ꢀ4.07 (m, 3H), 4.05 (dd,
J = 8.7, 3.1 Hz, 1H), 3.95 (dd, J = 11.1, 1.2 Hz, 1H), 1.12ꢀ1.03
(m, 21H). 13C NMR (75 MHz, CDCl3) δ 165.7, 138.6, 137.9, 134.3,
133.2, 131.5, 130.1, 130.0, 129.0, 128.41, 128.36, 128.2, 128.1, 127.8,
127.7, 127.5, 86.3, 78.8, 75.5, 74.4, 74.1, 71.9, 71.1, 62.6, 18.1, 12.1.
HRMS-ESI [M þ Na]þ calculated for C42H52O6SSiNa: 735.3152.
Found 735.3144.
Phenyl 2-O-(2-Azidomethylbenzoyl)-3,4,6-tri-O-benzyl-1-
thio-R-D-mannopyranoside (12). A solution of alcohol 11 (498
mg, 0.918 mmol) in dry pyridine (5 mL) was canulated onto freshly
prepared 2-(azidomethyl)benzoyl chloride16 (2.98 mmol) and left to stir
at rt for 2 h. The reaction was quenched by the addition of H2O (10 mL),
and then extracted into Et2O (50 mL). The combined organic phases
were washed with 0.5 M HCl (2 ꢁ 50 mL), saturated NaHCO3 (50 mL),
and saturated NaCl (50 mL), then dried (MgSO4), filtered and the
solvent removed to give a crude material which was purified on silica gel
(EtOAc/pet. ether = 1:4) to yield the title compound 12 (507 mg, 79%).
[R]2D0 = þ63 (c 0.72, CHCl3). 1H NMR (300 MHz, CDCl3) δ
8.06ꢀ8.02 (m, 1H), 7.57ꢀ7.44 (m, 4H), 7.36ꢀ7.20 (m, 19H), 5.83
(dd, J = 2.7, 1.9 Hz, 1H), 5.65 (d, J = 1.7 Hz, 1H), 4.90 (d, J = 10.8 Hz,
1H), 4.79 (d, J = 10.8 Hz, 1H), 4.74 (s, 2H), 4.66 (d, J = 11.9 Hz, 1H),
4.65 (d, J = 11.4 Hz, 1H), 4.48 (d, J = 11.9 Hz, 1H), 4.42 (m, 1H), 4.12
(dd, J = 9.3, 9.3 Hz, 1H), 4.06 (dd, J = 9.2, 2.9 Hz, 1H), 3.91 (dd, J =
10.9, 4.8 Hz, 1H), 3.76 (dd, J = 10.9, 1.9 Hz, 1H). 13C NMR (75 MHz,
CDCl3) δ 165.8, 138.3, 13737, 137.6, 133.6, 133.0, 132.0, 131.7, 129.6,
129.2, 128.6ꢀ127.5, 86.3, 78.6, 75.4, 74.7, 73.5, 72.6, 71.9, 71.2, 69.0, 53.1.
HRMS-ESI [M þ Na]þ calculated for C41H39N3O6SNa: 724.2457.
Found 724.2477.
1:9) to afford the title compound 9 (9.29 g, 81%) as a white solid. [R]D20
=
þ64 (c 1.94, CHCl3). 1H NMR (300 MHz, CDCl3) δ 8.12ꢀ8.06
(m, 2H), 7.60ꢀ7.21 (m, 18H), 5.83 (dd, J = 3.4, 1.4 Hz, 1H), 5.69 (s,
1H), 5.61 (d, J = 1.4, 1.4 Hz, 1H), 4.77 (d, J = 12.4 Hz, 1H), 4.72 (d, J =
12.4 Hz, 1H), 4.48ꢀ4.38 (m, 1H), 4.31ꢀ4.23 (m, 2H), 4.12 (dd, J =
9.8, 3.4 HZ, 1H), 3.91 (dd, J = 10.3, 10.3 Hz, 1H). 13C NMR (75 MHz,
CDCl3) δ 165.7, 137.8, 137.5, 133.4, 133.4, 132.2, 130.0, 129.7, 129.3,
129.0, 128.5, 128.4, 128.3, 128.1, 127.7, 126.2, 101.8, 87.3, 79.0, 74.3, 72.3,
72.1, 68.6, 65.3. HRMS-ESI [M þ Na]þ calculated for C33H30O6SNa:
577.1661. Found 577.1664. Analysis calculated for C33H30O6S: C 71.46,
H 5.45. Found C 71.52, H 5.46.
Phenyl 2-O-Benzoyl-3,4-di-O-benzyl-1-thio-R-D-mannopyr-
anoside (7). Method A. A solution of BH3 THF (67.5 mL, 1 M in
3
THF) followed by a solution of Bu2BOTf (13.5 mL, 1 M in CH2Cl2) was
added to a stirred solution of benzylidene acetal 9 (7.57 g, 13.5 mmol) in
dry THF (50 mL) at 0 °C. After 1 h the reaction was quenched by the
careful addition of H2O. Once the evolution of H2 ceased, 0.5 M HCl
(50 mL) was then added and stirred for a further 15 min. The resultant
mixture was extracted into Et2O and the organic extract washed with
0.5 M HCl (100 mL), saturated NaHCO3 (2 ꢁ 100 mL), saturated NaCl
(100 mL) then dried (MgSO4), filtered and the solvent removed. The
residue was purified on silica gel (EtOAc/pet. ether =1:9 to 1:4) to
afford the title compound 7 (6.72 g, 90%). [R]2D0 = þ82 (c 1.72, CHCl3).
1H NMR (300 MHz, CDCl3) δ 8.08ꢀ8.04 (m, 2H), 7.61ꢀ7.55
(m, 1H), 7.49ꢀ7.42 (m, 4H), 7.37ꢀ7.23 (m, 13H), 5.84ꢀ5.82 (m,
1H), 5.58 (d, J = 1.6 Hz, 1H), 4.94 (d, J = 10.9 Hz, 1H), 4.79 (d, J =
11.5 Hz, 1H), 4.68 (d, J = 10.9 Hz, 1H), 4.62 (d, J = 11.5 Hz, 1H),
4.28ꢀ4.19 (m, 1H), 4.11ꢀ4.02 (m, 2H), 3.89ꢀ3.79 (m, 2H), 1.79 (t, J =
6.8 Hz, 1H). 13C NMR (75 MHz, CDCl3) δ 165.6, 138.2, 137.7, 133.42,
133.37, 132.3, 130.0, 129.8, 129.3, 128.6, 128.52, 128.49, 128.24, 128.17,
128.1, 127.94, 127.89, 86.5, 78.5, 75.4, 74.2, 73.1, 71.8, 70.8, 62.1.
HRMS-ESI [M þ Na]þ calculated for C33H32O6SNa: 579.1817. Found
579.1842. Analysis calculated for C33H30O6S: C 71.20, H 5.79. Found C
71.41, H 5.86.
2-O-(2-Azidomethylbenzoyl)-3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl Trichloroacetimidate (6). N-Iodosuccinamide
(379 mg, 1.68 mmol), then triflic acid (5 μL, 0.056 mmol) were
added to a solution of thioglycoside 12 (394 mg, 0.561 mmol) in
H2O/acetone (1:9, 20 mL). After stirring at rt for 2 h the reaction was
extracted with CH2Cl2 (50 mL) and washed with 10% aqueous
Na2S2O3, saturated NaCl (50 mL), dried (MgSO4), and the solvent
removed. The crude residue was dissolved in dry CH2Cl2 (20 mL)
and cooled to 0 °C. Trichloroacetonitrile (563 μL, 5.61 mmol) and
DBU (8 μL, 0.056 mmol) were added and the reaction stirred for
30 min at this temperature. The solvent was then removed and the
crude residue was purified on silica gel (EtOAc/pet. ether = 1:9 to
1
1:4) to afford the title compound 6 (250 mg, 59%). H NMR (300
MHz, CDCl3) δ 8.71 (s, 1H), 8.08ꢀ8.05 (m, 1H), 7.58ꢀ7.46 (m,
2H), 7.35ꢀ7.20 (m, 17H), 6.43 (d, J = 1.9 Hz, 1H), 5.71 (dd, J = 2.5,
2.5 Hz, 1H), 4.90 (d, J = 10.8 Hz, 1H), 4.80 (d, J = 11.4 Hz, 1H), 4.76
(s, 2H), 4.67 d, J = 11.9 Hz, 1H), 4.66 (d, J = 11.4 Hz, 1H), 4.60 (d, J =
10.8 Hz, 1H), 4.51 (d, J = 11.9 Hz, 1H), 4.22 (dd, J = 9.4, 9.3, Hz, 1H),
4.16 (dd, J = 9.3, 2.9 Hz, 1H), 4.07ꢀ4.01 (m, 1H), 3.90 (dd, J =
11.1, 3.4 Hz, 1H), 3.76 (dd, J = 11.1, 1.7 Hz, 1H). 13C NMR (75 MHz,
CDCl3) δ 165.5, 160.0, 138.3, 138.1, 137.8, 137.6, 133.1, 131.7,
129.6, 128.6ꢀ127.5, 95.3, 90.8, 77.3, 75.6, 74.5, 73.8, 73.5, 72.1, 68.5,
68.1, 53.1. HRMS-ESI [M þ Na]þ calculated for C37H35Cl3N4O7Na:
775.1469. Found 775.1500.
Phenyl (2-O-(2-Azidomethylbenzoyl)-3,4,6-tri-O-benzyl-
R-D-mannopyranosyl)-(1f6)-2-O-benzoyl-3,4-di-O-benzyl-
1-thio-R-D-mannopyranoside (13). TMSOTf (24 μL, 0.131
mmol) was added to a mixture of alcohol 7 (0.713 g, 1.28 mmol),
trichloroacetimidate 6 (0.991 g, 1.31 mmol) and 4 Å molecular sieves in
dry Et2O at ꢀ30 °C. After stirring for 30 min the reaction was quenched
with Et3N, filtered through Celite and evaporated to dryness. The crude
residue was purified on silica gel (EtOAc/pet. ether =1:9 to 1:4 to 3:7)
to afford the title compound 13 (1.374 g, 94%) as an oil. [R]2D0= þ41
Method B. A solution of BH3 THF (1.00 mL, 1 M in THF) was
3
added to a solution of benzylidene acetal 9 (111 mg, 0.200 mmol) in dry
CH2Cl2 at rt. After stirring for 5 min, Sc(OTf)3 (15 mg, 0.030 mmol)
was added and the reaction stirred overnight. Et3N was added to quench
the reaction, which was then diluted and coevaporated with MeOH (3 ꢁ
30 mL). The residue was purified on silica gel (EtOAc/pet. ether = 1:9 to
1:4) to afford the title compound 7 (111 mg, 100%).
Phenyl 2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopropylsi-
lyl-1-thio-R-D-mannopyranoside (10). Triisopropylsilyl chloride
(1.68 mL, 7.84 mmol) was added to a solution of alcohol 7 (3.62 g,
6.53 mmol) and imidazole (667 mg, 9.80 mmol) in dry DMF (50 mL)
and stirred at rt overnight. H2O was added and then the reaction was
diluted in Et2O. The mixture was washed with 0.2 M HCl (100 mL),
saturated NaHCO3 (2 ꢁ 100 mL), saturated NaCl (100 mL) then dried
(MgSO4), filtered and the solvent removed. The residue was purified on
silica gel (EtOAc/pet. ether =1:9) to afford the title compound 10
(4.25 g, 91%). [R]2D0 = þ61 (c 1.74, CHCl3). H NMR (300 MHz,
1
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ARTICLE
(c 1.42, CHCl3). 1H NMR (500 MHz, CDCl3) δ 8.11ꢀ8.08 (m, 2H),
8.05ꢀ8.02 (m, 1H), 7.54ꢀ7.10 (m, 36H), 5.88 (dd, J = 2.9, 1.7 Hz, 1H),
5.68 (m, 1H), 5.60 (d, J = 1.6 Hz, 1H), 5.08 (d, J = 1.5 Hz, 1H), 4.90
(d, J = 11.1 Hz, 1H), 4.90 (d, J = 10.9 Hz, 1H), 4.86 (d, J = 10.9 Hz, 1H),
4.82 (d, J = 11.2 Hz, 1H), 4.74ꢀ4.67 (m, 3H), 4.62 (d, J = 11.1 Hz, 1H),
4.59 (d, J = 11.1 Hz, 1H), 4.53ꢀ4.38 (m, 5H), 4.08ꢀ4.00 (m, 4H), 3.94
(dd, J = 9.7, 9.5 Hz, 1H), 3.85ꢀ3.72 (m, 3H), 3.63 (dd, J = 10.6, 1.5 Hz,
1H). 13C NMR (125 MHz, CDCl3) δ 165.68, 165.67, 138.5, 138.3,
138.2, 137.8, 137.57, 137.56, 133.8, 133.4, 132.9, 131.7, 131.5, 129.9,
solvent was removed with the aid of toluene to azeotrope all the remaining
H2O. The crude residue was purified on silica gel (EtOAc/pet. ether = 1:9 to
1:4 to 3:7) to afford the title compound 16 (0.809 g, 68%) as a foam. [R]D20
=
þ58 (c 0.64, CHCl3). 1H NMR (500 MHz, CDCl3) δ 8.06ꢀ8.03 (m, 2H),
7.47ꢀ7.07 (m, 48H), 5.82 (dd, J= 2.9, 1.8 Hz, 1H), 5.52 (d, J= 1.4 Hz, 1H),
5.13 (d, J = 1.3 Hz, 1H), 4.98 (d, J = 1.5 Hz, 1H), 4.87ꢀ4.77 (m, 4H), 4.61
(d, J= 12.2 Hzm 1H), 4.58ꢀ4.44 (m, 9H), 4.42, d, J= 12.2 Hz, 1H), 4.37 (d,
J = 12.2 Hz, 1H), 4.32 (m, 1H), 4.12ꢀ4.07 (m, 2H), 4.00 (dd, J = 9.1,
3.0 Hz, 1H), 3.97ꢀ3.82 (m, 7H), 3.76ꢀ3.67 (m, 3H), 3.64ꢀ3.55 (m, 3H).
13C NMR (125 MHz, CDCl3) δ 165.7, 138.7, 138.6, 138.5, 138.27, 138.26,
138.11, 138.10, 137.6, 134.0, 133.4, 131.5, 129.91, 129.88, 129.3, 128.6ꢀ
127.4, 101.1, 99.2, 88.5, 80.0, 79.6, 78.9, 75.19, 75.16, 75.1, 74.7 (2 ꢁ C),
74.5, 74.4, 73.4, 73.2, 72.2, 72.14, 72.11, 72.0, 71.8, 71.6, 70.7, 69.1, 68.9, 68.8,
66.7. HRMS-ESI [M þ Na]þ calculated for C87H88O16SNa: 1443.5691.
Found 1443.5649
129.5, 129.3, 128.6, 128.5, 128.4ꢀ127.7, 127.6, 127.5, 97.9 (1JC,H
=
171.6 Hz), 86.5 (1JC,H = 167.6 Hz), 78.8, 78.0, 75.29, 75.25, 74.4, 74.3,
73.4, 71.9, 71.8, 71.7, 71.6, 70.7, 69.1, 68.7, 66.6, 53.1. HRMS-ESI [M þ
Na]þ calculated for C68H65N3O12SNa: 1170.4187. Found 1170.4226.
Phenyl (3,4,6-Tri-O-benzyl-R-D-mannopyranosyl)-(1f6)-
2-O-benzoyl-3,4-di-O-benzyl-1-thio-R-D-mannopyranoside
(14). Bu3P (1.50 mL, 6.0 mmol) was added to a solution of AZMB ester
13 (1.374 g, 1.20 mmol) in H2O/THF (1:9, 40 mL). After stirring at rt
for 1 h the solvent was removed with the aid of toluene to azeotrope all
the remaining H2O. The crude residue was purified on silica gel
(EtOAc/pet. ether =1:4 to 3:7) to afford the title compound 14
(0.975 g, 83%) as an oil. [R]2D0= þ80 (c 1.44, CHCl3). 1H NMR
(500 MHz, CDCl3) δ 8.07ꢀ8.04 (m, 2H), 7.52ꢀ7.13 (m, 33H), 5.86
(dd, J = 2.8, 1.7 Hz, 1H), 5.59 (d, J = 1.5 Hz, 1H), 5.06 (d, J = 1.3 Hz, 1H),
4.91 (d, J = 11.0 Hz, 1H), 4.84ꢀ4.81 (m 2H), 4.63ꢀ4.57 (m, 3H), 4.55
(d, J = 11.1 Hz, 1H), 4.49 (d, J = 10.8 Hz, 1H), 4.45 (d, J = 12.2 Hz, 1H),
4.40ꢀ4.36 (m, 1H), 4.09ꢀ4.03 (m, 3H), 3.99 (dd, J = 11.2, 5.1 Hz, 1H),
3.94ꢀ3.78 (m, 5H), 3.69 (dd, J = 10.8, 4.1 Hz), 3.61 (dd, J = 10.6, 1.5
Hz, 1H), 2.44 (d, J = 2.4 Hz, 1H). 13C NMR (125 MHz, CDCl3) δ 165.6,
138.5, 138.19, 138.17, 137.8, 137.6, 133.9, 133.3, 131.5, 129.88,
129.86,129.2,128.54, 128.52, 128.46, 128.4, 128.33, 128.30, 128.27,
127.94, 127.93, 127.89, 127.81, 127.77, 127.7, 127.61, 127.58, 99.4, 86.4,
80.0, 78.7, 75.3, 75.1, 74.5, 74.2, 73.4, 72.2, 71.9, 71.7, 71.2, 70.7, 68.7, 68.3,
66.2. HRMS-ESI [M þ Na]þ calculated for C60H60O11SNa: 1011.3754.
Found 1011.3795.
Phenyl (2-O-Benzoyl-3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-
(1f2)-(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-
tri-O-benzyl-R-D-mannopyranosyl)-(1f6)-2-O-benzoyl-3,4-
di-O-benzyl-1-thio-R-D-mannopyranoside (4). TMSOTf (12 μL,
0.068 mmol) was added to a mixture of alcohol 16 (0.846 g, 0.595 mmol),
trichloroacetimidate 5 (0.467 g, 0.668 mmol) and 4 Å molecular sieves in
dry Et2O at ꢀ30 °C. After stirring for 30 min the reaction mixture was
quenched with Et3N, filtered through Celite and evaporated to dryness.
The crude residue was purified on silica gel (EtOAc/pet. ether = 1:4) to
give a product containing fraction that was further purified on silica gel
(Et2O/toluene =1:49 to 1:24) to afford the title compound 4 (0.656 g,
1
56%) as a foam. [R]2D0 = þ26 (c 1.46, CHCl3). H NMR (500 MHz,
CDCl3) δ 8.08ꢀ8.02 (m, 4H), 7.57ꢀ6.89 (m, 66H), 5.83 (dd, J = 3.1,
1.7 Hz, 1H), 5.75 (m, 1H), 5.51 (d, J = 1.6 Hz, 1H), 5.23 (d, J = 1.9 Hz,
1H), 5.11 (d, J = 1.9 Hz, 1H), 4.97 (d, J = 1.9 Hz, 1H), 4.86ꢀ4.78
(m, 5H), 4.73 (d, J = 11.1 Hz, 1H), 4.62ꢀ4.29 (m, 17H), 4.13ꢀ4.04 (m,
4H), 4.00 (dd, J = 9.0, 2.9 Hz, 1H), 3.97ꢀ3.78 (m, 8H), 3.77ꢀ3.53
(m, 8H). 13C NMR (125 MHz, CDCl3) δ 165.6, 165.4, 138.7, 138.64,
138.59, 138.53, 138.49, 138.4, 138.22, 138.16, 138.1, 137.6, 134.0, 133.4,
133.1, 131.5, 130.1, 130.0, 129.9, 129.3, 128.6ꢀ127.3, 100.7 (1JC,H
=
=
Phenyl (2-O-(2-Azidomethylbenzoyl)-3,4,6-tri-O-benzyl-
R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl)-(1f6)-2-O-benzoyl-3,4-di-O-benzyl-1-thio-
R-D-mannopyranoside (15). TMSOTf (19 μL, 0.102 mmol) was
added to a mixture of alcohol 14 (0.975 g, 0.986 mmol), trichloroace-
timidate 6 (0.772 g, 1.02 mmol) and 4 Å molecular sieves in dry Et2O
at ꢀ30 °C. After stirring for 30 min the reaction mixture was quenched
with Et3N, filtered through Celite and evaporated to dryness. The
residue was purified on silica gel (EtOAc/pet. ether = 1:4) to afford the
title compound 15 (1.317 g, 94%) as a foam. [R]2D0= þ29 (c 1.30,
CHCl3); 1H NMR (500 MHz, CDCl3) δ 8.06ꢀ8.03 (m, 2H),
8.02ꢀ7.11 (m, 1H), 7.56ꢀ7.07 (m, 51H), 5.83 (dd, J = 2.9, 1.7 Hz,
1H), 5.72 (dd, J = 2.7, 1.8 Hz, 1H), 5.57 (d, J = 1.5 Hz, 1H), 5.16 (d, J =
1.5 Hz, 1H), 4.99 (d, J = 1.6 Hz, 1H), 4.89 (d, J = 11.3 Hz, 1H),
4.88ꢀ4.83 (m, 2H), 4.80 (d, J = 11.2 Hz, 1H), 4.72ꢀ4.68 (m, 3H),
4.63ꢀ4.33 (m, 12H), 4.11ꢀ3.96 (m, 6H), 3.94ꢀ3.87 (m, 3H),
3.82ꢀ3.76 (m, 2H), 3.71ꢀ3.58 (m, 4H). 13C NMR (125 MHz, CDCl3)
δ 165.8, 165.6, 138.6, 138.53, 138.47, 138.4, 138.19, 138.16, 138.1,
137.6, 137.5, 134.0, 133.3, 132.8, 131.7, 131.5, 129.8, 129.4, 129.3,
128.7ꢀ127.4, 99.4, 99.0, 86.4, 79.6, 78.8, 78.0, 75.23, 75.19 (2 ꢁ
C), 75.1, 74.6, 74.44, 74.39, 73.4, 73.2, 72.1, 72.04 (2 ꢁ C), 72.02,
71.8, 70.7, 69.5, 69.0, 68.9, 66.6, 53.1. Gated decoupled 13C NMR
173 Hz), 99.5 (1JC,H = 173 Hz), 99.1 (1JC,H = 172 Hz), 86.4 (1JC,H
169 Hz), 79.6, 79.4, 78.9, 78.2, 75.5, 75.3, 75.17, 75.16, 75.1, 74.8, 74.7,
74.5, 74.3, 73.4, 73.23, 73.20, 72.3, 72.2, 72.1, 72.0, 71.73, 71.65, 70.7, 69.3,
69.1, 69.0, 66.8. HRMS-ESI [M þ Na]þ calculated for C121H120O22SNa:
1979.7890. Found 1979.7808. Further elution gave the corresponding
tetrasaccharide sulfone (0.079 g, 7%). HRMS-ESI [MþNa]þ calculated
for C121H120O24SNa: 2011.7788. Found 2011.7797. This was followed by
the corresponding tetrasaccharide sulfoxide (0.185 g, 16%). HRMS-ESI
[Mþ Na]þ calculated for C121H120O23SNa: 1995.7839. Found 1995.7793.
1-O-Allyl-6-O-(2-O-benzoyl-3,4-di-O-benzyl-6-O-triisopro-
pylsilyl-R-D-mannopyranosyl)-3,4,5-tri-O-benzyl-D-myo-ino-
sitol (18). Tf2O (144 μL, 0.854 mmol) was added to a solution of
thioglycoside donor 10 (435 mg, 0.610 mmol), diphenyl sulfoxide
(345 mg, 1.71 mmol), and DTBMP (376 mg, 1.83 mmol) in CH2Cl2
(15 mL) at ꢀ60 °C. After stirring for 5 min at this temperature, a
precooled (ꢀ60 °C) solution of diol 17 (360 mg, 0.734 mmol) in
CH2Cl2 (5 mL) was added. The reaction mixture was stirred for a further
30 min then warmed to rt. Saturated NaHCO3 was added to quench the
reaction. The organic layer was separated, washed with saturated NaCl
(30 mL), then dried (MgSO4), filtered and the solvent removed. The
crude residue was purified on silica gel (EtOAc/pet. ether = 1:9 to 1:4)
to afford the title compound 18 (383 mg, 57%) as a white foam. 1H NMR
(300MHz, CDCl3) δ8.11ꢀ8.05(m, 2H), 7.57ꢀ7.07 (m, 28H), 6.03ꢀ5.89
(m, 1H), 5.65(dd, J= 3.1, 1.9 Hz, 1H), 5.42 (d, J= 1.8 Hz, 1H), 5.25 (dd, J=
17.1, 1.3 Hz, 1H), 5.16 (dd, J = 10.2, 1.3 Hz, 1H), 4.94ꢀ4.58 (m, 10H), 4.18
(dd, J = 2.7, 2.6 Hz, 1H), 4.15ꢀ3.98 (m, 6H), 3.88ꢀ3.82 (m, 1H),
3.54ꢀ3.51 (m, 2H), 3.41 (dd, J = 9.7, 2.8 Hz, 1H), 3.35ꢀ3.26 (m, 2H), 2.40
(s, 1H), 1.05ꢀ1.01 (m, 21H). 13C NMR (75 MHz, CDCl3) δ 165.8, 139.4,
138.7, 138.5, 138.3, 136.0, 134.4, 133.0, 130.3, 130.1, 128.7ꢀ127.2, 118.4,
(75 MHz, CDCl3) selected data δ 99.4 1JC,H = 172.3 Hz, 99.0 1JC,H
=
170.7 Hz, 86.4 1JC,H = 169.3 Hz. HRMS-ESI [M þ Na]þ calculated for
C95H93N3O17SNa: 1602.6123. Found 1602.6140.
Phenyl (3,4,6-Tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-
(3,4,6-tri-O-benzyl-R-D-manno-pyranosyl)-(1f6)-2-O-ben-
zoyl-3,4-di-O-benzyl-1-thio-R-D-mannopyranoside (16). Bu3P
(1.04 mL, 4.17 mmol) was added toa solution ofAZMB ester 15 (1.317 g,
0.833 mmol) in H2O/THF (1:9, 40 mL). After stirring at rt for 1 h the
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98.6, 81.5, 81.3, 80.6, 78.8, 78.4, 76.0, 75.9, 75.6, 75.0, 74.1, 73.8, 72.5, 71.6,
71.4, 69.5, 66.9, 62.1, 18.1, 12.1. HRMS-ESI [M þ Na]þ calculated for
C66H80O12SiNa: 1115.5317. Found 1115.5292.
9.4 Hz), 3.15 (dd, J = 9.7, 2.4 Hz, 1H), 1.94 (br s, 1H). 13C NMR
(75 MHz, CDCl3) δ 166.8, 165.3, 138.9, 138.8, 138.4, 138.3, 138.2,
138.0, 137.8, 135.0, 133.2, 132.4, 130.6, 130.3, 130.0, 128.7ꢀ
127.2, 117.4, 97.8 (1JC,H = 161 Hz), 97.6 (1JC,H = 176 Hz), 81.7, 81.5,
81.4, 80.3, 78.9, 77.8, 75.9, 75.4, 75.2, 75.0, 74.8, 74.4, 74.3, 73.6, 73.3,
71.3, 71.2, 70.1, 69.9, 69.0, 68.7, 61.6. HRMS-ESI [M þNa]þ calculated
for C91H92O18Na: 1495.6181. Found 1495.6125.
1-O-Allyl-2-O-benzoyl-6-O-(2-O-benzoyl-3,4-di-O-benzyl-
6-O-triisopropylsilyl-R-D-mannopyranosyl)-3,4,5-tri-O-ben-
zyl-D-myo-inositol (19). Benzoyl chloride (54 μL, 0.470 mmol) was
added to a solution of alcohol 18 (51 mg, 0.047 mmol) and DMAP
(6 mg, 0.049 mmol) in dry pyridine (10 mL) at rt, and the mixture was
stirred for 48 h. H2O was added and the reaction stirred for a further
30 min then diluted with Et2O (75 mL). The mixture was washed with
0.5 M HCl (3 ꢁ 50 mL), saturated NaHCO3 (100 mL), saturated NaCl
(100 mL) then dried (MgSO4), and the solvent removed. The crude
residue was purified on silica gel (EtOAc/pet. ether =1:9 to 1:4) to
1-O-Allyl-2-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl)-6-O-[(2-O-benzoyl-3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-
(1f2)-(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-(1f6)-(2-
O-benzoyl-3,4-di-O-benzyl-R-D-mannopyranosyl)-(1f6)-
(2-O-benzoyl-3,4-di-O-benzyl-R-D-mannopyranosyl)]-3,4,5-tri-
O-benzyl-D-myo-inositol (20). Tf2O (43 μL, 0.258 mmol) was
added to a solution of thioglycoside donor 4 (361 mg, 0.184 mmol),
diphenyl sulfoxide (104 mg, 0.515 mmol), and DTBMP (113 mg,
0.552 mmol) in dry CH2Cl2 (15 mL) at ꢀ60 °C. After stirring for 5 min
at this temperature, a precooled (ꢀ60 °C) solution of alcohol 2 (343 mg,
0.233 mmol) in dry CH2Cl2 (5 mL) was added. The reaction was stirred
for a further 30 min then allowed to warm to rt. Saturated NaHCO3 was
added to quench the reaction. The organic layer was separated, washed
with saturated NaCl (30 mL), then dried (MgSO4), filtered and the
solvent removed. The crude residue was purified on silica gel (EtOAc/
toluene = 1:49 to 1:24) to afford the title compound 20 (466 mg, 76%)
as a foam. [R]2D0 = þ16 (c 1.36, CHCl3). 1H NMR (500 MHz, CDCl3) δ
8.15ꢀ8.13 (m, 2H), 8.09ꢀ8.05 (m, 4H), 7.99ꢀ7.96 (m, 2H),
7.56ꢀ6.79 (m, 111H), 5.92ꢀ5.83 (m, 1H), 5.80ꢀ5.77 (m, 2H), 5.76
(dd, J = 2.8, 1.8 Hz, 1H), 5.68 (dd, J = 2.2, 2.2 Hz, 1H), 5.57 (d, J =
1.7 Hz, 1H), 5.30 (d, J = 1.6 Hz, 1H), 5.25 (d, J = 1.8 Hz, 1H), 5.20 (dd,
J = 17.2, 1.6 Hz, 1H), 5.16 (d, J = 1.8 Hz, 1H), 5.06ꢀ5.01 (m, 2H),
4.94ꢀ3.72 (m, 65H), 3.65ꢀ3.54 (m, 4H), 3.48ꢀ3.28 (m, 8H), 3.22ꢀ3.17
(m, 2H). 13C NMR (125 MHz, CDCl3) δ 165.7, 165.4, 165.3, 135.1,
138.9, 138.8, 138.7, 138.54, 138.48, 138.47, 138.4, 138.2, 138.12, 138.08,
138.0, 137.9, 137.8, 137.6, 133.7, 133.2, 133.0, 132.8, 130.2ꢀ129.8,
1
afford the title compound 19 (42 mg, 74%). H NMR (300 MHz,
CDCl3) δ 8.09ꢀ8.00 (m, 4H), 7.60ꢀ7.06 (m, 31H), 6.00 (dd, J = 2.6,
2.6 Hz, 1H), 6.00ꢀ5.86 (m, 1H), 5.68 (dd, J = 3.1, 1.9 Hz, 1H), 5.45 (d,
J = 1.7 Hz, 1H), 5.23 (dd, J = 17.1, 1.5 Hz, 1H), 5.13 (dd, J = 10.2, 1.5 Hz,
1H), 4.99ꢀ4.77 (m, 6H), 4.69ꢀ4.54 (m, 4H), 4.23ꢀ3.90 (m, 7H),
3.70ꢀ3.56 (m, 3H), 3.49 (dd, J = 9.7, 2.7 Hz, 1H), 3.43 (dd, J = 9.7,
9.5 Hz, 1H), 1.06ꢀ1.02 (m, 21H). 13C NMR (75 MHz, CDCl3) δ
165.9, 165.7, 139.4, 138.6, 138.4, 138.0, 137.7, 134.3, 133.1, 132.9,
130.3ꢀ129.9, 128.5, 127.2, 118.4, 98.6, 81.9, 81.3, 78.7, 78.44, 78.36,
76.3, 76.1, 75.9, 75.1, 74.1, 72.5, 72.2, 71.6, 71.1, 69.3, 66.9, 62.1,
18.2ꢀ18.0, 12.1. HRMS-ESI [M þ Na]þ calculated for C73H84O13SiNa:
1219.5579. Found 1219.5536.
1-O-Allyl-2-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-R-D-man-
nopyranosyl)-6-O-(2-O-benzoyl-3,4-di-O-benzyl-R-D-man-
nopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (2). TMSOTf
(10 μL, 0.058 mmol) was added to a mixture of alcohol 18 (384 mg,
0.351 mmol), trichloroacetimidate 5 (405 mg, 0.579 mmol) and 4 Å
molecular sieves in dry Et2O at ꢀ30 °C. After stirring for 30 min the
reaction was quenched with Et3N, filtered through Celite and evapo-
rated to dryness. The crude residue was purified on silica gel (acetone/
toluene =1:49 to 1:24) to afford the pseudotrisaccharide as an insepar-
able anomeric mixture. This was dissolved in CH2Cl2/MeOH (3:7,
20 mL) and cooled to 0 °C. Acetyl chloride (501 μL, 7.02 mmol) was
added and the reaction was allowed to warm to rt and stirred overnight
after which it was quenched with Et3N (986 μL, 7.02 mmol) and
evaporated to dryness. The crude residue was purified on silica gel
(EtOAc/pet. ether =1:9 to 3:7) to give a product containing fraction that
was further purified on silica gel (Et2O/CH2Cl2 = 1:49 to 1:24) to afford
the title compound 2 (386 mg, 75%). [R]2D0 = ꢀ1.3 (c 1.52, CHCl3). 1H
NMR (500 MHz, CDCl3) δ 8.09ꢀ7.99 (m, 4H), 7.58ꢀ7.08 (m, 46H),
5.95ꢀ5.81 (m, 1H), 5.72 (dd, J = 2.4, 2.3 Hz, 1H), 5.68 (dd, J = 3.0,
1.9 Hz, 1H), 5.58 (d, J = 1.8 Hz, 1H), 5.26 (d, J = 1.9 Hz, 1H), 5.19 (dd,
J = 17.3, 1.4 Hz, 1H), 5.01 (dd, J = 10.4, 1.3 Hz, 1H), 4.95ꢀ4.57
(m, 14H), 4.46 (d, J = 10.9 Hz, 1H), 4.39ꢀ4.33 (m, 2H), 4.26ꢀ3.97
(m, 8H), 3.87 (dd, J = 9.6, 9.6, Hz, 1H), 3.60 (dd, J = 10.7, 3.1 Hz, 1H),
3.55ꢀ3.28 (m, 6H), 1.86 (m, 1H). 13C NMR (125 MHz, CDCl3)
δ 165.6, 163.3, 138.8, 138.7, 138.47, 138.47, 138.46, 138.45, 138.2,
138.0, 137.94, 137.93, 133.8, 133.1, 133.0, 130.1ꢀ130.0, 128.5ꢀ127.4,
118.1, 99.0 (1JC,H = 173 Hz), 98.3 (1JC,H = 176 Hz), 81.4, 81.0, 78.8,
78.0, 76.2, 75.8, 75.2, 75.1, 74.14, 74.10, 73.54, 72.3, 72.0, 71.9, 71.7,
71.4, 71.3, 69.0, 68.82, 68.79, 61.5. HRMS-ESI [M þ Na]þ calculated
for C91H92O18Na: 1495.6181. Found 1495.6161. Further elution pro-
vided a mostly β-linked mixed fraction (53 mg, 10%) and then the
β-linked compound, 1-O-allyl-2-O-(2-O-benzoyl-3,4,6-tri-O-benzyl-
β-D-mannopyranosyl)-6-O-(2-O-benzoyl-3,4-di-O-benzyl-R-D-man-
128.6ꢀ127.3, 127.0, 126.8, 118.1, 100.5 (1JC,H = 170 Hz), 99.5 (1JC,H
=
=
173 Hz), 99.2 (1JC,H = 171 Hz), 99.1 (1JC,H = 172 Hz), 98.9 (1JC,H
174 Hz), 98.4 (1JC,H = 172 Hz), 81.5, 81.3, 80.7, 79.58, 79.56, 79.4, 78.9,
78.8, 78.4, 78.3, 75.9, 75.87, 75.34, 75.32, 75.11, 75.05, 74.8, 74.63, 74.58,
74.3, 74.14, 74.11, 74.0, 73.43, 73.39, 73.38, 73.31, 73.27, 72.5, 72.4, 72.3,
72.1, 71.9, 71.71, 71.67, 71.5, 71.39, 71.37, 71.1, 70.7, 70.5, 69.1, 69.00,
68.97, 68.84, 68.78, 68.6, 68.3, 66.1, 65.6. HRMS-ESI [M þ Na]þ
calculated for C206H206O40Na: 3342.3983. Found 3342.4109.
1-O-Allyl-2-O-(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-
6-O-[(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,
6-tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-
benzyl-R-D-mannopyranosyl)-(1f6)-(3,4-di-O-benzyl-R-D-
mannopyranosyl)-(1f6)-(3,4-di-O-benzyl-R-D-mannopyra-
nosyl)]-3,4,5-tri-O-benzyl-D-myo-inositol (21). Sodium methox-
ide in MeOH (30% solution) was added dropwise to a stirred solution of
tetra-benzoate 20 (559 mg, 0.168 mmol) in CH2Cl2/MeOH (1:9,
50 mL) until the pH of the solution was 11. After being stirred for
4 days, the reaction mixture was diluted with saturated NH4Cl (50 mL).
The aqueous phase was extracted with CHCl3 (3 ꢁ 50 mL) and the
combined organic extracts were washed with H2O (100 mL). After
drying (MgSO4) and filtration the solvent was removed and the residue
purified by column chromatography on silica gel (EtOAc/pet.ether =1:4
to 1:1) to afford thetitle compound 21(294 mg, 60%) as anoil. [R]2D0 = þ
63(c 2.16, CHCl3). 1H NMR (300 MHz, CDCl3) δ 7.40ꢀ6.98 (m, 95H),
5.94ꢀ5.80 (m, 1H), 5.37 (br s, 1H), 5.30ꢀ5.16 (m, 4H), 5.14
(br s, 1H), 5.01ꢀ4.27 (m, 40H), 4.19ꢀ3.62 (m, 30H), 3.57ꢀ3.15
(m, 13H), 3.10ꢀ3.03 (m, 1H), 2.40ꢀ2.26 (m, 4H). 13C NMR (75 MHz,
CDCl3) δ 138.9ꢀ137.9, 133.8, 128.7ꢀ126.9, 117.9, 101.0, 111.7, 100.2,
100.1, 100.0, 99.1, 81.6, 81.5, 81.3, 80.7, 80.1, 79.9, 79.6, 79.4, 79.0, 78.9, 77.3,
75.9, 75.8, 75.0, 74.8, 74.6, 74.3, 73.8, 73.5, 73.3, 72.4, 72.1, 91.94, 71.89, 71.6,
nopyranosyl)-3,4,5-tri-O-benzyl-D-myo-inositol (54 mg, 11%). [R]2D0
=
ꢀ24 (c 1.08, CHCl3). 1H NMR (300 MHz, CDCl3) δ 8.08ꢀ8.02
(m, 4H), 7.67ꢀ6.90 (m, 46H), 5.95ꢀ5.81 (m, 2H), 5.47 (dd, J = 2.8,
1.9 Hz, 1H), 5.12 (dd, J = 17.3, 1.5 Hz, 1H), 5.08 (br s, 1H), 5.04ꢀ4.99
(m, 1H), 4.93 (d, J = 1.7 Hz, 1H), 4.92ꢀ4.47 (m, 16H), 4.15ꢀ3.67
(m, 12H), 3.52ꢀ3.45 (m, 1H), 3.38ꢀ3.31 (m, 3H), 3.23 (dd, J = 9.5,
4948
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The Journal of Organic Chemistry
ARTICLE
71.4, 71.3, 71.1, 70.6, 70.5, 69.3, 69.0, 68.8, 68.7, 68.4, 67.6, 66.2, 65.3.
HRMS-ESI [M þ Na]þ calculated for C178H190O36Na: 2926.2935. Found
2926.2996.
mmol) was coevaporated from dry acetonitrile (2 ꢁ 30 mL) then
placed under high vacuum for 30 min. The reagents were dissolved in
dry acetonitrile (15 mL) before the addition of 1H-tetrazole (24 mg,
0.347 mmol) at rt. After 1 h the reaction mixture was diluted with dry
CH2Cl2 (10 mL), stirred for 10 min, then cooled to ꢀ30 °C and a dried
(MgSO4) solution of mCPBA (∼60%, 100 mg, 0.347 mmol) in
CH2Cl2 (3 mL) was added to the reaction. After warming to rt over
1 h the reaction was diluted with CH2Cl2 quenched by the addition of
solid NaHCO3, water (50 mL) and Na2SO3 (10%, 50 mL) and the
mixture was extracted with CH2Cl2 (2 ꢁ 50 mL). The aqueous phase
was extracted with EtOAc (50 mL) and the combined organics dried
(MgSO4), filtered and the solvent removed. The crude residue was
purified on silica gel (EtOAc/pet. ether = 1:9 to 1:4) followed by a
further purification on silica gel (EtOAc/CHCl3 = 1:49 to 1:24) to
afford a product containing fraction (180 mg) that included an
impurity (5% by HPLC system 1). This fraction was further purified
on LiChroprep RP-18 (MeOH/CHCl3 = 4:1 to 1:1) to afford to the
title compound 24 (170 mg, 0.043 mmol, 82%) (>98% pure by HPLC
1-O-Allyl-2-O-(2,3,4,6-tetra-O-benzyl-R-D-mannopyranosyl)-
6-O-[(2,3,4,6-tetra-O-benzyl-R-D-mannopyranosyl)-(1f2)-
(3,4,6-tri-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-
benzyl-R-D-mannopyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-
D-mannopyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-D-man-
nopyranosyl)]-3,4,5-tri-O-benzyl-D-myo-inositol (22)10. NaH
(32 mg, 60% in mineral oil, 0.800 mmol) was added to a stirred solution
of tetraol 21 (291 mg, 0.100 mmol) in dry DMF (10 mL) at 0 °C. After
10 min BnBr (71 μL, 0.600 mmol) was added and the resultant
suspension left to slowly warm to rt and stirred overnight. Saturated
NH4Cl was added to quench the reaction until the evolution of H2 ceased.
The reaction was extracted into CH2Cl2/Et2O (1:4, 50 mL) then washed
with H2O (2 ꢁ 500 mL), saturated NaCl (500 mL) then dried (MgSO4),
filtered and the solvent removed. The crude residue was purified on silica
gel (EtOAc/pet. ether = 1:4 to 3:7) to afford the title compound 22 (303
mg, 93%) as a pale-yellow oil. [R]2D0 = þ39 (c 1.28, CHCl3) [Lit10 þ37.5
(c = 1, CHCl3)]. 1H NMR (300 MHz, CDCl3) δ 7.37ꢀ6.96 (m, 115H),
5.79ꢀ5.63 (m, 1H), 5.43 (br s, 1H), 5.23ꢀ5.15, (m, 4H), 5.10ꢀ5.05
(m, 1H), 5.02ꢀ4.73 (m, 12H), 4.68ꢀ4ꢀ22 (m, 37H), 4.18ꢀ3.77 (m,
27H), 3.72ꢀ3.06 (m, 16H). 13C NMR (75 MHz, CDCl3) δ 139.2ꢀ137.9,
134.0, 128.9ꢀ126.7, 117.7, 100.4, 99.4, 99.3, 99.1, 98.8, 98.4, 81.6, 81.5,
81.3, 80.8, 80.03, 79.96, 79.1, 78.9, 78.8, 75.9, 75.8, 75.4, 75.0, 74.9, 74.8,
74.6, 74.3, 73.7, 73.6, 73.4, 73.3, 72.7, 72.6, 72.5, 72.3, 72.2, 72.0, 71.3, 71.2,
71.0, 70.9, 70.5, 69.2, 69.07, 68.95, 68.9, 66.1, 65.9. HRMS-ESI [M þ Na]þ
calculated for C206H214O36Na: 3286.4813. Found 3286.4849.
1
system 1) as a clear oil. H NMR (500 MHz, CDCl3) mixture of
isomers δ 7.37ꢀ6.94 (m, 120H), 5.43ꢀ5.39 (br s, 1H), 5.31ꢀ5.24 (m,
1H), 5.22ꢀ5.17 (m, 2H), 5.16ꢀ5.06 (m, 1H), 5.05ꢀ3.75 (m, 80H),
3.72ꢀ3.23 (m, 12H), 3.10ꢀ3.03 (m, 2H), 2.24ꢀ2.11 (m, 4H),
1.56ꢀ1.43 (m, 4H), 1.34ꢀ1.14 (m, 48H), 0.91ꢀ0.84 (m, 6H). 13C
NMR (125 MHz, CDCl3) δ 173.0, 172.6, 139.1ꢀ137.8, 129.1ꢀ126.7,
100.4, 99.6, 99.4, 99.2, 99.0 (br), 98.3, 81.0, 80.3, 80.73, 70.70, 80.6,
80.04, 79.97, 79.9, 79.1, 79.0, 78.9, 78.7, 78.5, 77.3, 76.0, 75.81, 75.76,
75.6, 75.5, 75.1, 75.01, 74.95, 74.92, 74.89, 74.8, 74.63, 74.60, 74.5,
74.3, 74.1, 73.7, 73.6, 73.5, 73.41, 73.36, 73.2, 72.8, 72.6, 72.5, 72.4,
72.3, 72.2, 72.0, 71.9, 71.7, 71.3, 71.2, 70.9, 70.2, 70.1, 69.5, 69.4, 69.2,
68.9, 68.8, 66.2, 66.0, 65.8, 61.5, 34.1, 34.0, 33.9, 32.6, 29.8ꢀ29.2, 24.8,
22.7, 14.2. 31P NMR (202 MHz, CDCl3) δ ꢀ0.11, ꢀ0.43. HRMS-ESI
[M þ Na]þ calculated for C245H283O43PNa: 3966.9593. Found
3967.0671.
1-O-(1,2-Di-O-hexadecanoyl-sn-glycero-3-benzylphosphoryl)-
2-O-(R-D-mannopyranosyl)-6-O-[(R-D-mannopyranosyl)-
(1f2)-(R-D-mannopyranosyl)-(1f2)-(R-D-mannopyranosyl)-
(1f6)-(R-D-mannopyranosyl)-(1f6)-(R-D-mannopyranosyl)]-
D-myo-inositol (1). Pd(OH)2/C (20%, 35 mg) was added to a
solution of per-benzylated PIM6 24 (130 mg, 0.033 mmol) in THF/
MeOH (2:3, 10 mL). The mixture was stirred under an H2 atmosphere
for 3 h at rt, then filtered through Celite and the solvent removed. The
residue was lyophilized to afford the title compound 1 (58 mg, 0.033
mmol, 99%) as a white powder. A portion (10 mg) was purified on
LiChroprep RP-18 (MeOH/water containing 35 mmol ammonium
acetate/CHCl3 = 85:15:0 to 45:15:40) to afford to the title compound 1
(8 mg, 96% pure by HPLC system 2) as a white powder. [R]2D0= þ48.9
(c 0.5, H2O). 1H NMR (500 MHz, CDCl3/CD3OD/D2O 30:60:25) δ
5.30 (m, 1H), 5.28 (s, 1H), 5.16 (s, 1H), 5.13 (s, 1H), 5.11 (s, 1H), 5.02
(s, 1H), 4.90 (s, 1H), 4.47 (dd, J = 2.5, 12.5 Hz, 1H), 4.31 (s, 1H),
4.27ꢀ4.161 (m, 2H), 4.12ꢀ3.57 (m, 39H) 3.51 (dd, J = 2.5, 10.0 Hz,
1H), 2.39 (ddm, J = 7.5, 7.5 Hz, 2H), 2.34 (ddd, J = 7.5, 7.5, 1.8 Hz 2H),
1.61 (m, 4H), 1.36ꢀ1.26 (m, 48H), 0.91ꢀ0.87 (m, 6H). 13C NMR
(125 MHz, CD3OD/D2O 30:60:25) δ 175.9, 175.7, 103.7, 102.9 (2 ꢁ
C), 102.3, 100.9, 99.6, 80.4, 79.9, 78.8, 78.2, 74.8, 74.4, 74.3, 72.9, 72.5,
72.2, 72.1, 71.9, 71.8, 71.7, 68.9, 68.6, 68.4, 67.5, 67.1, 64.9, 64.4, 62.9,
62.8, 62.7, 35.6, 35.5, 33.2, 31.0, 30.8, 30.6, 30.5, 30.45, 26.3, 26.2, 23.9,
15.1. 31P NMR (202 MHz, CDCl3) δ ꢀ0.7. HRMS-ESI [M ꢀ H]ꢀ
calculated for C77H138O43P: 1781.8349. Found 1781.8326.
2-O-(2,3,4,6-Tetra-O-benzyl-R-D-mannopyranosyl)-6-O-[(2,3,
4,6-tetra-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-
benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-
R-D-mannopyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-D-manno-
pyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-D-mannopyranosyl)]-
3,4,5-tri-O-benzyl-D-myo-inositol (23)10. (1,5-Cyclooctadiene)bis-
(methyldiphenyl-phosphine)iridium(I) hexafluorophosphate (15 mg,
0.018 mmol) was added to a stirred solution of allyl ether 22 (295 mg,
0.090 mmol) in dry THF (10 mL) at rt under argon. The argon was
replaced with H2 for ca. 1 min followed by a gentle stream of argon being
passed over the reaction. After 1 h the solvent was removed and the
residue dissolved in MeOH/CH2Cl2 (1:2, 15 mL). Acetyl chloride
(150 μL) was added and the reaction was stirred overnight. Solid
NaHCO3 was added to quench the reaction then the resulting mixture
partitioned between CHCl3 and H2O. The aqueous layer was extracted
with CHCl3 (3 ꢁ 30 mL) and the combined organics dried (MgSO4),
filtered and the solvent removed. The crude residue was purified on silica
gel (EtOAc/pet. ether = 1:4 to 3:7) to afford the title compound 23 (265
mg, 91%) as an oil. [R]D20 = þ34.4 (c 2.3, CHCl3). [Lit10 þ35 (c = 1,
CHCl3)]. 1H NMR (300 MHz, CDCl3) δ 7.36ꢀ6.98 (m, 115H), 5.45
(br s, 1H), 5.21ꢀ5.17 (m, 3H), 4.97ꢀ4.76 (m, 10H), 4.75ꢀ4.22 (m,
40H), 4.18ꢀ3.74 (m, 26H), 3.70ꢀ3.15 (m, 14H), 2.88 (br s, 1H). 13
C
NMR (75 MHz, CDCl3) δ 138.9ꢀ138.0, 128.6ꢀ127.2, 100.6, 99.34,
99.31, 98.9, 98.6, 96.2 (br), 81.3, 80.5, 80.0, 79.9, 78.9, 78.7, 75.6, 75.3,
75.01, 74.96, 74.9, 74.8, 74.7, 74.6, 74.4, 74.0, 73.38, 73.35, 73.3, 73.2, 72.8,
72.59, 72.56, 72.4, 72.2, 72.1, 72.04, 72.01, 71.9, 71.8, 71.61, 71.57, 71.3,
71.1, 69.3, 69.1, 69.0, 68.9, 66.7, 66.2. HRMS-ESI [M þ Na]þ calculated
for C203H210O36Na: 3246.4500. Found 3246.4465.
2-O-(2,3,4,6-Tetra-O-benzyl-R-D-mannopyranosyl)-6-O-[(2,3,
4,6-tetra-O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-
O-benzyl-R-D-mannopyranosyl)-(1f2)-(3,4,6-tri-O-benzyl-
R-D-mannopyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-D-man-
nopyranosyl)-(1f6)-(2,3,4-tri-O-benzyl-R-D-mannopyranosyl)]-
3,4,5-tri-O-benzyl-1-O-(1,2-di-O-hexadecanoyl-sn-glycero-
3-benzylphosphoryl)-D-myo-inositol (24). A mixture of alcohol
23 (170 mg, 0.053 mmol) and phosphoramidite 3 (280 mg, 0.347
’ BIOLOGICAL ASSAYS
Reagents. Carboxyfluorescein succinimidyl ester (CSFE), RPMI-
1640, and Penicillin-streptomycin were from Invitrogen, Auckland, NZ.
Human CD3 and CD28 antibodies, and fluorescent antibodies for CD4
4949
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ARTICLE
(2) Cao, B.; Williams, S. J. Nat. Prod. Rep. 2010, 27, 919–947.
(3) Sayers, I.; Severn, W.; Scanga, C. B.; Hudson, J.; Le Gros, G.;
Harper, J. L. J. Allergy Clin. Immunol. 2004, 114, 302–309.
(4) Ainge, G. D.; Hudson, J.; Larsen, D. S.; Painter, G. F.; Gill, G. S.;
Harper, J. L. Bioorg. Med. Chem. 2006, 14, 5632–5642.
(5) Torrelles, J. B.; Azad, A. K.; Schlesinger, L. S. J. Immunol. 2006,
177, 1805–16.
(6) Ernst, W. A.; Maher, J.; Cho, S.; Niazi, K. R.; Chatterjee, D.;
Moody, D. B.; Besra, G. S.; Watanabe, Y.; Jensen, P. E.; Porcelli, S. A.;
Kronenberg, M.; Modlin, R. L. Immunity 1998, 8, 331–340.
(7) Fischer, K.; Scotet, E.; Niemeyer, M.; Koebernick, H.; Zerrahn,
J.; Maillet, S.; Hurwitz, R.; Kursar, M.; Bonneville, M.; Kaufmann,
S. H. E.; Schaible, U. E. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
10685–10690.
(8) Sieling, P. A.; Chatterjee, D.; Porcelli, S. A.; Prigozy, T. I.;
Mazzaccaro, R. J.; Soriano, T.; Bloom, B. R.; Brenner, M. B.; Kronenberg,
M.; Brennan, P. J.; Modlin, R. L. Science 1995, 269, 227–230.
(9) Sprott, G. D.; Dicaire, C. J.; Gurnani, K.; Sad, S.; Krishnan, L.
Infect. Immun. 2004, 72, 5235–46.
(10) Boonyarattanakalin, S.; Liu, X.; Michieletti, M.; Lepenies, B.;
Seeberger, P. H. J. Am. Chem. Soc. 2008, 130, 16791–16799.
(11) Denis, M.; Ainge, G. D.; Larsen, D. S.; Severn, W. S.; Painter,
G. F. Immunopharmacol. Immunotoxiccol. 2009, 31, 577–582.
(12) Doz, E.; Rose, S.; Court, N.; Front, S.; Vasseur, V.; Charron, S.;
Gilleron, M.; Puzo, G.; Fremaux, I.; Delneste, Y.; Erard, F.; Ryffel, B.;
Martin, O. R.; Quesniaux, V. F. J. J. Biol. Chem. 2009, 284, 23187.
(13) Driessen, N. N.; Ummels, R.; Maaskant, J. J.; Gurcha, S. S.;
Besra, G. S.; Ainge, G. D.; Larsen, D. S.; Painter, G. F.; Vandenbroucke-
Grauls, C. M. J. E.; Geurtsen, J.; Role, B. J. A. Infect. Immun. 2009, 77,
4538–4547.
and CD8 were from eBiosciences, San Diego, CA, USA. [3H]-thymidine
was from Sigma-Aldrich, Auckland, NZ. Lymphoprep was from Medica
Pacifica Ltd., Auckland, NZ.
Peripheral Blood Mononuclear Cells (PBMC) Isolation and
Generation of Human Dendritic Cells (DC). Whole blood was
collectedfromhealthyvolunteersintoheparinizedvacutainertubes. Blood
collection for this study was approved by the Central Regional Ethics
Committee (NZ) and all donors provided written, informed consent.
PBMC were isolated from whole blood using a Lymphoprep density
gradient according to manufacturer’s instructions. Autologous serum
was retained for in vitro cell cultures. DC were generated by six day
culture in RPMI-1640 supplemented with GM-CSF and IL-4 as
previously described.34
Mixed lymphocyte Reaction (MLR) Assays. [3H]-Thymidine
Assay. DC from donor one were cultured in 96 well plates (1 ꢁ 104/
well) with freshly isolated PBMC from donor two (1 ꢁ 105/well) in
RPMI-1640 (100 μL, 100 U/mL penicillin-streptomyocin, 1% heat
inactivated autologous human serum) in the presence of test com-
pounds. Cells were incubated for four days (37 °C, 5% CO2) then
incubated with 0.25 μCi [3H]-thymidine for one day. The cells were
then harvested onto filtermats using an automated cell harvester and
read on a Topcount Microplate scintillation counter.
Carboxyfluorescein Succinimidyl Ester (CFSE) Assay. Freshly isolated
PBMC from donor two were incubated with CFSE (1 μM in RPMI-
1640, 8 min) then washed three times with PBS. CFSE-labeled PBMC
were cultured with DC from donor one as described above and the cells
harvested after 72 h. Cells were fluorescently labeled with antimouse
CD8-APC and analyzed on a BD FACSCalibur flow cytometer (Becton
Dickinson, San Jose, CA). Proliferation was determined by the percen-
tage of proliferating CFSE-diluted cells compared to the undiluted
CFSE-labeled nonproliferating control population. Dexamethasone
(10 μg/mL) was included as a positive control.
(14) Ainge, G. D.; Parlane, N. A.; Denis, M.; Hayman, C. M.; Larsen,
D. S.; Painter, G. F. Bioorg. Med. Chem. 2006, 14, 7615–7624.
(15) Liu, X.; Stocker Bridget, L.; Seeberger Peter, H. J. Am. Chem.
Soc. 2006, 128, 3638–48.
Antibody-Mediated T-Cell Proliferation Assay. Freshly iso-
lated human PBMC ((1ꢀ2) ꢁ 105 per well in RPMI-1640, 100 U/mL
penicillin-streptomyocin, 1% heat inactivated autologous serum) were
plated onto 96-well plates coated with antibodies for CD3 (5 mg/mL)
and CD28 (0.5 mg/mL). Cells were incubated with the compounds
(37 °C, 5% CO2) for four days. Cells were then incubated in the
presence of 0.25 μCi [3H]-thymidine for one day and thymidine uptake
measured as above.
(16) Wada, T.; Ohkubo, A.; Mochizuki, A.; Sekine, M. Tetrahedron
Lett. 2001, 42, 1069–1072.
(17) Hoelemann, A.; Stocker, B. L.; Seeberger, P. H. J. Org. Chem.
2006, 71, 8071–8088.
(18) Oshitari, T.; Shibasaki, M.; Yoshizawa, T.; Tomita, M.; Takao,
K.-i.; Kobayashi, S. Tetrahedron 1997, 53, 10993–11006.
(19) Tennant-Eyles, R. J.; Davis, B. G.; Fairbanks, A. J. Tetrahedron:
Asymmetry 2003, 14, 1201–1210.
(20) Harrowven, D. C.; Guy, I. L. Chem. Commun. (Cambridge, U.K.)
2004, 17, 1968–1969.
(21) Zhang, Y.-M.; Brodzky, A.; Sinai, P.; Saint-Marcoux; Perly, B.
Tetrahedron: Asymmetry 1995, 6, 1195–1216.
(22) Doi, T.; Kinbara, A.; Inoue, H.; Takahashi, T. Chem. Asian J.
2007, 2, 188–198.
(23) Actetates were used instead of benzoates as the choice of
a-directing group on the mannose moieties.
(24) Codee Jeroen, D. C.; Litjens Remy, E. J. N.; den Heeten, R.;
Overkleeft Herman, S.; van Boom Jacques, H.; van der Marel Gijs, A.
Org. Lett. 2003, 5, 1519–22.
(25) Severn, W. B.; Furneaux, R. H.; Falshaw, R.; Atkinson, P. H.
Carbohydr. Res. 1998, 308, 397–408.
’ ASSOCIATED CONTENT
S
Supporting Information. General experimental details,
b
1H, 13C, and 31P NMR spectra for all new compounds reported in
the manuscript, HPLC trace for compounds 24 and 1 and the
ESI-MS2 spectrum of compound 1. This material is available free
of charge via the Internet at http://pubs.acs.org.
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: g.painter@irl.cri.nz; jharper@malaghan.org.nz
(26) Hsu, F.-F.; Turk, J.; Owens, R. M.; Rhoades, E. R.; Russell, D. G.
J. Am. Soc. Mass Spectrom. 2007, 18, 466–478.
(27) Barral, D. C.; Brenner, M. B. Nat. Rev. Immunol. 2007, 7,
929–941.
(28) Salio, M.; Silk, J. D.; Cerundolo, V. Curr. Opin. Immunol. 2010,
22, 81–88.
(29) de la Salle, H.; Mariotti, S.; Angenieux, C.; Gilleron, M.; Garcia-
Alles, L.-F.; Malm, D.; Berg, T.; Paoletti, S.; Maitre, B.; Mourey, L.;
Salamero, J.; Cazenave, J. P.; Hanau, D.; Mori, L.; Puzo, G.; De Libero,
G. Science 2005, 310, 1321–1324.
(30) Maitre, B.; Angenieux, C.; Salamero, J.; Hanau, D.; Fricker, D.;
Signorino, F.; Proamer, F.; Cazenave, J.-P.; Goud, B.; Tourne, S.; de la
Salle, H. Traffic 2008, 9, 431–445.
’ ACKNOWLEDGMENT
The authors wish to thank the New Zealand Foundation of
Research Science and Technology (Contract C08X0808) for
financial support.
’ REFERENCES
(1) Kaur, D.; Guerin, M. E.; Skovierova, H.; Brennan, P. J.; Jackson,
M. Adv. Appl. Microbiol. 2009, 69, 23–78.
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The Journal of Organic Chemistry
ARTICLE
(31) Tourne, S.; Maitre, B.; Collmann, A.; Layre, E.; Mariotti, S.;
Signorino-Gelo, F.; Loch, C.; Salamero, J.; Gilleron, M.; Angenieux, C.;
Cazenave, J.-P.; Mori, L.; Hanau, D.; Puzo, G.; De Libero, G.; de la Salle,
H. J. Immunol. 2008, 180, 3642–3646.
(32) Mahon, R. N.; Rojas, R. E.; Fulton, S. A.; Franko, J. L.; Harding,
C. V.; Boom, W. H. Infect. Immun. 2009, 77, 4574–4583.
(33) Hsu, F. F.; Turk, J.; Owens, R. M.; Rhoades, E. R.; Russell, D. G.
J. Am. Soc. Mass Spectrom. 2007, 18, 466–478.
(34) Hsieh, S.; Pan, S.; Hung, C.; Tsai, H.; Chen, M.; Lee, C.; Chang,
S. J. Immunol. 2001, 167, 6286–6291.
4951
dx.doi.org/10.1021/jo200588u |J. Org. Chem. 2011, 76, 4941–4951