Synthesis of a tristearoyl lipomannan via preactivation-based iterative one-pot glycosylation

Article abstract of DOI:10.1021/jo4021979

A convergent and efficient strategy was developed for the synthesis of lipomannan (LM), useful for vaccine development. Thioglycosides were employed as glycosyl donors to construct two key pseudotrisaccharide and tetramannose intermediates through preacti

Full text of DOI:10.1021/jo4021979

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Article
Synthesis of a Tristearoyl Lipomannan via Pre-
activation-based Iterative One-pot Glycosylation
Jian Gao, and Zhongwu Guo
J. Org. Chem., Just Accepted Manuscript • Publication Date (Web): 22 Nov 2013
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Synthesis of a Tristearoyl Lipomannan via Preꢀactivationꢀbased Iterative
Oneꢀpot Glycosylation
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Jian Gao† and Zhongwu Guo†,‡,*
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† Department of Chemistry, Wayne State University, Detroit, Michigan 48202, USA
‡ National Glycoengineering Research Center, Shandong University, Jinan 250100, China
* Corresponding author, Eꢀmail: zwguo@chem.wayne.edu
Table of Contents (TOC) graphic
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Abstract
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A convergent and efficient strategy was developed for the synthesis of lipomannan (LM), useful
for vaccine development. Thioglycosides were employed as glycosyl donors to construct two key
pseudotrisaccharide and tetramannose intermediates through preꢀactivationꢀbased glycosylation
strategy. These building blocks were then successfully coupled to form the LM core, which was
lapidated, phospholipidated, and finally globally deprotected to afford the target molecule. The
intermediate LM core involved in this synthesis contained orthogonal protections, which would
facilitate its variable modifications for the preparation of other complex LM derivatives and for
the synthesis of LM conjugates as LMꢀbased vaccines.
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Introduction
Mycobacterium tuberculosis (Mtb) is the causative pathogen of tuberculosis (TB), one of the
most detrimental diseases worldwide, which causes more than two million deaths each year. A
major virulent factor of Mtb is its cell envelope glycolipids, especially the phosphatidylinositolꢀ
anchored lipoglycans, such as lipoarabinomannans (LAMs) and lipomannans (LMs).1,2 LMs
have exhibited a variety of bioactivities, such as stimulating proinflammatory cytokine secretion
through the tollꢀlike receptor 2/CD14ꢀdependent pathway and inducing macrophage apoptosis
and ILꢀ12 expression.3ꢀ6 Due to the structural complexity and the intriguing immunoregulatory
activities, LMs and related molecules have been the targets of a number of chemical total
syntheses.7ꢀ18
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In an effort to explore LMꢀderived vaccines, we have developed a highly convergent and
efficient strategy for LM synthesis via preꢀactivationꢀbased iterative oneꢀpot glycosylation using
thioglycosides as glycosyl donors.19,20 Many syntheses have proved that this strategy can save
time and improve efficiency by abolishing multiple experimental preparation and intermediate
separation steps. Our synthetic plan (Scheme 1) was to assemble the target molecule 1 from
pseudotrisaccharide 2, tetramannose 3, and phosphoglycerolipid 4. The key intermediate 3 was
constructed from two monosaccharide building blocks 6 and 8 via iterative oneꢀpot glycosylation,
whereas the orthogonal allyl (All), tertꢀbutyldimethylsilyl (TBS) and paraꢀmethoxybenzyl (PMB)
protecting groups in 2 allowed for regioselective glycosylation, lipidation, and phospholipidation.
Furthermore, the 2ꢀOꢀpositions of 6, 7, 8, and 9 were protected as acetyl esters to safeguard
αꢀselective glycosylation resulting from neighboring group participation.
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Scheme 1. Retrosynthesis of the target molecule 1
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Results and Discussion
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Mannosyl donors and acceptors 6, 7, 8, and 9 were prepared from 1021 and 1222 according to
the procedures shown in Scheme 2. Stannylene acetalꢀdirected benzylation of 10 was selective
for the 3ꢀOꢀposition, which was followed by 2ꢀOꢀacetylation and then regioselective benzylidene
ringꢀopening in the presence of borane tetrahydrofuran complex (BH3ꢁTHF) and trimethylsilyl
trifluoromethanesulfonate (TMSOTf) to afford 6. Thereafter, the free hydroxyl group in 6 was
silylated with TBSCl under the influence of imidazole to obtain 7. Compound 8 was prepared
from orthoester 12 following perbenzylation and subsequent thioglycosylation using SnCl4 as the
promoter. En route to 9, the 6ꢀOꢀposition of 12 was regioselectively protected with TBS first,
followed by benzylation of the remaining free hydroxyl groups to produce 14. Consecutively, the
6ꢀOꢀTBS group was swapped for an allyl group to distinguish from 7, via sequential desilylation
mediated by tetrabutylammonium fluoride (TBAF) and reaction with allyl bromide and sodium
hydride. Finally, reaction of 15 with pꢀthiocresol and SnCl4 gave 9 which was derived from 12 in
five steps and a 47% overall yield, involving only two column purification operations.
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Scheme 2. Synthesis of mannose building blocks 6ꢀ9
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The assembly of pseudotrisaccharide 2 (Scheme 3), which had two mannose residues linked
to the inositol 2ꢀOꢀ and 6ꢀOꢀpositions while the 1ꢀOꢀposition was uniquely protected to facilitate
subsequently selective deprotection and phospholipidation, commenced from an optically pure
1,2,6ꢀOꢀdifferentiated myoꢀinositol derivative 5 obtained from methyl αꢀDꢀglucopyranoside
according to a reported procedure.23 Glycosylation of 5 with 7 under the influence of
pꢀtoluenesulfenyl triflate (pꢀTolSOTf) generated in situ from the reaction of pꢀtoluenesulfenyl
chloride (pꢀTolSCl) and silver triflate (AgOTf)19,20 gave αꢀlinked pseudodisaccharide 16 (81%)
stereospecifically, as a result of neighboring acetyl group participation in the reaction. The allyl
group on the inositol 6ꢀOꢀposition was thereafter removed by Iridium complexꢀcatalyzed olefin
rearrangement and then Hg(II)ꢀcatalyzed hydrolysis24 to form 17. Mannosylation of 17 with 9
was again promoted by pꢀTolSOTf to give the desired 2 in an 83% yield. The anomeric CꢀH
coupling constants (1JC,H = 175 and 177 Hz) of 2 confirmed the αꢀstereochemistry of its
glycosidic linkages.25 Cleavage of the allyl protection by the Irꢀcomplex/Hg(II) method
mentioned above finally afforded 18, which was ready for further elongation of the glycan to get
the target molecule or other LM derivatives.
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Scheme 3. Synthesis of the key pseudotrisaccharide 2
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The synthesis of tetramannose 3 via preꢀactivationꢀbased iterative oneꢀpot glycosylation is
outlined in Scheme 4. Preꢀactivation of the thioglycosyl donors was achieved at ꢀ78 ˚C with
pꢀTolSOTf as the promoter. Glycosylation reactions were furnished using a sterically hindered
base, 2,4,6ꢀtriꢀtertꢀbutylpyrimidine (TTBP), as a scavenger for trifluoromethanesulfonic acid
generated from the reactions. Each glycosylation was kept at room temperature for ca. 20 min to
endorse complete reaction as shown by TLC. It is noteworthy that 1.0 eq. of pꢀTolSCl and 0.9 eq.
of 6 (relative to the donors) were used in the reactions to further assure complete consumption of
the glycosyl acceptor in each step so as to minimize any potential interference with the reactions
followed. Clearly, iterative oneꢀpot glycosylation for oligosaccharide synthesis could improve
the synthetic efficiency by obviating some timeꢀconsuming purification processes.26ꢀ28 Thus,
after three sequential glycosylation steps, 3 was obtained in 6 h and a 39% overall yield, giving
an average of more than 73% yield for each glycosylation step. All of the reactions were proved
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αꢀspecific (anomeric JC,H values of 3 were between 169 and 176 Hz). Although 3 could be
directly utilized as a glycosyl donor for the glycosylation of 18, the reactions employing
pꢀTolSCl/AgOTf/TTBP or NIS/AgOTf/TTBP as the promoters gave relatively low yields of the
desired product with an orthoester as the main byproduct. Due to neighboring group participation,
glycosylation reactions using 2ꢀOꢀacylated donors, such as 3, usually involve orthoesters as the
reaction intermediates, which can be transformed into the desired glycosides in the presence of
strong Lewis acids. However, 3 and 18 were both complex and sterically hindered, and the
reaction condition was almost neutral, thus the reaction might have stopped at the orthoester
stage. To avoid this problem, 3 was converted to trichloroacetimidate 19 via hydrolysis of the
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thioglycoside in the presence of NIS/AgOTf/TTBP and then reaction of the resultant hemiacetal
with trichloroacetonitrile and 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU).
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Scheme 4. Preꢀactivationꢀbased iterative oneꢀpot synthesis of 3 and 19
Glycosylation of 18 with 19 in the presence of TMSOTf was αꢀspecific to afford 20 in a 77%
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yield (Scheme 5). The C NMR spectrum of 20 displayed six discrete carbon signals at δ 98.6,
98.3, 98.2, 98.1, 98.0 and 97.9 with 1JC,H values between 172 and 176 Hz. Upon Oꢀdeacetylation
and benzylation, the acetyl groups in 20 were replaced with benzyl groups to provide 22, which
was ready for the installation of lipids. The purpose for the protecting group exchange was to
avoid using bases for global deprotection later on, as the target molecule contained ester linkages
that were rather sensitive to basic conditions. Treatment of 22 with Et3Nꢁ3HF to remove TBS
was followed by acylation of the exposed hydroxyl group with stearic acid under the influence of
N,N′ꢀdicyclohexylcarbodiimide (DCC) and 4ꢀdimethylaminopyridine (DMAP) and thereafter
cleavage of the PMB ether with 10% TFA in CH2Cl2 to generate 25. Compound 25 was smoothly
phospholipidated using a twoꢀstep oneꢀpot protocol, including reaction with freshly prepared
phosphoramidite 4 in the presence of 1Hꢀtetrazole and oxidation of resultant phosphite
intermediate with metaꢀchloroperoxybenzoic acid (mꢀCPBA) to afford 26 (72%) as a 1:3
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diastereomeric mixture, originating from the stereogenic phosphorus atom. Global debenzylation
of 26 was achieved in a mixture of chloroform, methanol, and water (3:3:1) under a H2
atmosphere with 10% Pd/C as the catalyst to eventually yield the synthetic target 1, which was
characterized with 1H and 31PꢀNMR spectroscopy and MALDIꢀTOF MS.
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Scheme 5. Final assembly of the synthetic target 1
Conclusions
In summary, an efficient and convergent strategy was developed for the synthesis of LMs. It is
highlighted by the construction of tetramannose 3 through preꢀactivationꢀbased iterative oneꢀpot
glycosylation, which has markedly reduced the number of synthetic and purification steps as
compared to the reported syntheses7ꢀ18 and thus improved the synthetic efficiency. Furthermore,
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taking advantage of neighboring group participation, all of the glycosylation reactions involved
in the synthesis were stereoselective to form αꢀglycosidic linkages. The synthetic strategy reported
here can be generally applicable to the preparation of various LM derivatives and conjugates. For
example, intermediate 22, which contained orthogonal TBS and PMB protecting groups, can be
selectively deprotected to facilitate regioselective introduction of linkers for the conjugation with
proteins or other carrier molecules to formulate LMꢀbased vaccines or introduction of various
lipids or phospholipids at these positions to obtain different LM derivatives. Furthermore, using
differently protected monosaccharides from 8 as starting materials for Scheme 4, intermediates
suitable for further elongation of the carbohydrate chain of the LM skeleton can be obtained for
the synthesis of more complex LM molecules.
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Experimental Section
General Experimental Methods. Chemicals and materials were obtained from commercial
sources, and were used as received without further purification unless otherwise noted. MS 4Å
was flameꢀdried under high vacuum and used immediately after cooling under a N2 atmosphere.
Analytical TLC was carried out on Silica Gel 60Å F254 plates with detection by a UV detector
and/or by charring with 15% (v/v) H2SO4 in EtOH. NMR spectra were recorded on a 400, 500 or
600 MHz machine with chemical shifts reported in ppm (δ) downfield from tetramethylsilane
(TMS), which was used as an internal reference.
pꢀTolyl 2ꢀOꢀacetylꢀ3ꢀOꢀbenzylꢀ4,6ꢀO-benzylideneꢀ1ꢀthioꢀαꢀDꢀmannopyranoside (11):29 The
mixture of diol 10 (2.0 g, 5.35 mmol) and Bu2SnO (1.6 g, 6.42 mmol) in toluene (80 mL) was
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refluxed under a N2 atmosphere for 6 h. After the reaction was cooled to room temperature,
toluene was removed under vacuum. The residue was dissolved in 20 mL of anhydrous DMF and
mixed with CsF (2.4 g, 16.05 mmol) and BnBr (960 ꢂL, 8.03 mmol). The mixture was stirred at
room temperature for 24 h, at the end of which time TLC indicated the complete reaction. The
solution was diluted with EtOAc (300 mL) and washed with saturated aq. NaCl. The organic
layer was dried with Na2SO4 and evaporated. The residue was dried under highꢀvacuum for 1 h
and then dissolved in 15 mL of pyridine and 2 mL of acetic anhydride. After 1 h of reaction, the
mixture was coꢀevaporated with toluene and the residue was finally purified by silica gel column
chromatography with a 1:7 mixture of EtOAc and hexane as eluent to give 11 (2.2 g, 81% for
two steps) as syrup. The following NMR data agreed well with that of the reported.29 1H NMR
(600 MHz, CDCl3) δ: 7.52ꢀ7.49 (m, 2H, Ph), 7.40ꢀ7.24 (m, 10H, Ph), 7.12 (d, J = 8.4 Hz, 2H,
Ph), 5.63 (s, 1H, PhCH), 5.61 (dd, J = 3.3, 1.0 Hz, 1H, Hꢀ2), 5.37 (d, J = 1.0 Hz, 1H, Hꢀ1), 4.70
(m, 2H, Bn), 4.36 (dt, J = 10.2, 4.8 Hz, 1H, Hꢀ5), 4.22 (dd, J = 10.2, 4.8 Hz, 1H, Hꢀ6), 4.12 (t, J
= 10.2, 1H, Hꢀ4), 4.01 (dd, J = 10.2, 3.3 Hz, 1H, Hꢀ3), 3.85 (t, J = 10.2 Hz, 1H, Hꢀ6’), 2.32 (s,
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3H, Tol), 2.14 (s, 3H, Ac). C NMR (150 MHz, CDCl3) δ 170.0, 138.3, 137.7, 137.4, 132.7,
129.9, 128.9, 128.3, 128.1, 127.7, 126.1, 101.6, 87.5 (Cꢀ1), 78.5, 74.1, 72.3, 71.3, 68.4, 65.1,
21.1, 21.0. MS (ESIꢀTOF) m/z: calcd for C29H30O6SNa [M + Na]+ 529.1; found, 528.8.
pꢀTolyl 2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ1ꢀthioꢀαꢀDꢀmannopyranoside (6): To a solution of 11 (2.0
g, 3.95 mmol) and MS 4Å in 30 mL of anhydrous THF at ꢀ40 ˚C was added BH3ꢁTHF (19.75 mL,
19.75 mmol) under a N2 atmosphere. Fifteen minutes later, TMSOTf (924 ꢂL, 5.14 mmol) was
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added dropwise to this solution. The mixture was stirred under this condition for 1 h, and then
stirred at room temperature for another 24 h. The reaction was quenched with saturated aq.
NaHCO3 at 0 ˚C. The solution was diluted with CH2Cl2 (400 mL) and washed with saturated aq.
NaHCO3 and NaCl solutions. The organic layer was dried with Na2SO4 and concentrated; the
residue was purified by silica gel column chromatography with a mixture of EtOAc and hexane
(1:4) as the eluent to give 6 (76%, 1.53 g) as syrup. 1H NMR (400 MHz, CDCl3) δ 7.40ꢀ7.29 (m,
12H, Ph), 7.13 (d, J = 8.0 Hz, 2H, Ph), 5.62 (dd, J = 3.2, 1.6 Hz, 1H, Hꢀ2), 5.40 (d, J = 1.6 Hz,
1H, Hꢀ1), 4.95 (d, J = 11.2 Hz, 1H, Bn), 4.75 (d, J = 11.7 Hz, 1H, Bn), 4.66 (d, J = 10.7 Hz, 1H,
Bn), 4.59 (d, J = 11.2 Hz, 1H, Bn), 4.22 (dt, J = 9.6, 3.4 Hz, 1H, Hꢀ5), 3.98 (dd, J = 9.4, 3.2 Hz,
1H, Hꢀ3), 3.90 (t, J = 9.2 Hz, 1H, Hꢀ4), 3.80ꢀ3.85 (m, 2H, Hꢀ6,6’), 2.33 (s, 3H, Tol), 2.14 (s, 3H,
Ac). 13C NMR (100 MHz, CDCl3) δ 170.2, 138.3, 138.2, 137.6, 132.8, 129.9, 129.4, 128.5, 128.2,
128.0, 127.9, 127.8, 86.6 (Cꢀ1), 78.3, 75.3, 74.3, 72.9, 71.9, 70.3, 70.2, 62.0, 21.1. HR MS
(ESIꢀTOF) m/z: calcd for C29H32O6SNa [M + Na]+ 531.1817; found, 531.1824. MS (ESIꢀTOF):
found, 530.9.
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pꢀTolyl
2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀO-tertꢀbutydimethylsilylꢀ1ꢀthioꢀαꢀDꢀmannopyranoꢀ
side (7): To a solution of 6 (388 mg, 0.764 mmol) in anhydrous DMF (10 mL) was added
imidazole (104 mg, 1.53 mmol) and TBSCl (192 mg, 1.15 mmol) at 0 ˚C. The mixture was
stirred at room temperature for 2 h and then diluted with EtOAc (150 mL). The organic phase,
after being washed with saturated aq. NaCl solution, was dried over Na2SO4, concentrated in
vacuo and the residue was purified by silica gel column chromatography with EtOAc and hexane
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(1:14) as the eluent to give 7 (427 mg, 90%) as a white solid. H NMR (600 MHz, CDCl3) δ
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J = 10.8 Hz, 1H, Bn), 4.71 (d, J = 11.2 Hz, 1H, Bn), 4.64 (d, J = 10.8 Hz, 1H, Bn), 4.56 (d, J =
11.2 Hz, 1H, Bn), 4.10ꢀ4.12 (m, 1H, Hꢀ5), 3.95ꢀ3.91 (m, 3H, Hꢀ3,4,6), 3.81 (d, J = 11.4 Hz, 1H,
Hꢀ6’), 2.30 (s, 3H, Tol), 2.09 (s, 3H, Ac), 0.89 (s, 9H, tBu), 0.03 (s, 3H, SiMe), 0.04 (s, 3H,
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SiMe). C NMR (150 MHz, CDCl3) δ 170.2, 138.6, 137.7, 137.6, 132.1, 130.3, 129.7, 128.4,
128.3, 128.1, 127.9, 127.8, 127.6, 86.5 (Cꢀ1), 78.3, 75.2, 74.4, 73.8, 71.9, 70.6, 62.2, 60.0, 25.9,
21.0, 20.9, 18.3, ꢀ5.2, ꢀ5.4. HR MS (ESIꢀTOF) m/z: calcd for C35H46O6SSiNa [M + Na]+
645.2682; found, 645.2689. MS (ESIꢀTOF): found, 644.9.
pꢀTolyl 2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀ1ꢀthioꢀαꢀDꢀmannopyranoside (8):30 To a solution of 12
(410 mg, 1.74 mmol) in anhydrous DMF were added NaH (208 mg, 8.69 mmol) and BnBr (1.04
mL, 8.69 mmol) at 0 ˚C under N2. Half an hour later, the reaction was quenched with saturated
aq. NaCl solution, and the mixture was diluted with EtOAc (150 mL). The organic phase, after
being washed with saturated aq. NaCl, was dried over Na2SO4, concentrated in vacuo and the
residue was purified by silica gel column with EtOAc and hexane (1:8) as the eluent to give 13
as a pale yellow solid. Compound 13, pꢀthiocresol (248 mg, 2.0 mmol), and activated 4Å MS
were mixed in 20 ml of anhydrous CH2Cl2. Then, a catalytic amount of SnCl4 (300 ꢂL, 0.3 mmol,
1M in CH2Cl2) was added at 0 ˚C under N2. The mixture was stirred at room temperature for 30
min, and TLC indicated the completion of the reaction. After the reaction was quenched with
triethylamine, the solvent was evaporated in vacuum to give the crude oil, which was purified by
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silica gel column chromatography with EtOAc and hexane (1:10) as the eluent to give 8 (717 mg,
69% for 2 steps) as a white solid. The following NMR data agreed well with that of the
reported.30 1H NMR (600 MHz, CDCl3) δ 7.38ꢀ7.25 (m, 15H, Ph), 7.20 (d, J = 7.2 Hz, 2H, Ph),
7.05 (d, J = 7.2 Hz, 2H, Ph), 5.60 (d, J = 1.8 Hz, 1H, Hꢀ2), 5.46 (s, 1H, Hꢀ1), 4.89 (d, J = 10.8
Hz, 1H, Bn), 4.73 (d, J = 10.8 Hz, 1H, Bn), 4.66 (d, J = 12.0 Hz, 1H, Bn), 4.57 (d, J = 11.4 Hz,
1H, Bn), 4.51 (d, J = 10.8 Hz, 1H, Bn), 4.46 (d, J = 12.0 Hz, 1H, Bn), 4.34 (m, 1H, Hꢀ5),
3.96ꢀ3.93 (m, 2H, Hꢀ3,4), 3.85 (ddd, J = 10.8, 4.8, 1.8 Hz, 1H, Hꢀ6), 3.73 (d, J = 10.8 Hz, 1H,
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Hꢀ6’), 2.30 (s, 3H), 2.13 (s, 3H). C NMR (150 MHz, CDCl3) δ 170.3, 138.3, 138.2, 137.8,
137.6, 132.3, 129.9, 129.8, 128.4, 128.3, 128.2, 128.1, 127.8, 127.7, 127.6, 127.5, 86.5 (Cꢀ1),
78.5, 75.2, 74.6, 73.3, 72.4, 71.9, 70.3, 68.9, 21.07, 21.06. MS (ESIꢀTOF) m/z: calcd for
C36H38O6SNa [M + Na]+ 621.2; found, 620.9.
3,4ꢀDiꢀOꢀbenzylꢀ6ꢀOꢀallylꢀ1,2ꢀOꢀmethoxyethylideneꢀβꢀDꢀmannopyranose (15): To a solution
of 12 (330 mg, 1.40 mmol) in anhydrous DMF (20 mL) was added imidazole (190 mg, 2.80
mmol) and TBSCl (252 mg, 1.68 mmol) at 0 ˚C. The mixture was stirred at room temperature for
2 h and then diluted with EtOAc (200 mL). The organic phase, after being washed with saturated
aq. NaCl solution, was dried over Na2SO4 and concentrated in vacuo to afford a residue, which
was dissolved in anhydrous DMF (10 mL) and then mixed with NaH (101 mg, 4.2 mmol) and
BnBr (670 ꢂL, 5.6 mmol) at 0 ˚C under N2. Half an hour later, the reaction was quenched with
methanol, diluted with EtOAc (200 mL) and washed with saturated aq. NaCl solution. The
organic phase was dried over Na2SO4 and concentrated in vacuo to give the crude 14. To a
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solution of crude 14 in THF (8 mL) was added TBAF (4.2 mL, 4.2 mmol, 1M in THF). The
mixture was stirred at room temperature for 3 h, at which time TLC indicated the completion of
the reaction, and then diluted with EtOAc (200 mL). The organic phase, after being washed with
saturated aq. NaCl solution, was dried over Na2SO4 and concentrated in vacuo to afford a residue,
which was dissolved in anhydrous DMF (10 mL) and mixed with NaH (67 mg, 2.8 mmol) and
allyl bromide (242 ꢂL, 2.8 mmol) at 0 ˚C under N2. An hour later, the reaction was quenched
with methanol, diluted with EtOAc (100 mL) and then washed with saturated aq. NaCl solution.
The organic phase was dried over Na2SO4, concentrated in vacuo and the product was purified
by silica gel column chromatography with EtOAc and hexane (1:8) as the eluent to give 15 (408
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mg, 64% for 4 steps) as a white solid. H NMR (600 MHz, CDCl3) δ 7.39ꢀ7.28 (m, 10H, Ph),
5.87 (m, 1H, All), 5.33 (d, J = 2.4 Hz, 1H, Hꢀ1), 5.26 (dd, J = 17.2, 1.8 Hz, 1H, All), 5.14 (dd, J
= 10.2, 1.2 Hz, 1H, All), 4.92 (d, J = 10.8 Hz, 1H, Bn), 4.80ꢀ4.75 (m, 2H, Bn), 4.66 (d, J = 10.8
Hz, 1H, Bn), 4.38 (dd, J = 3.6, 2.4 Hz, 1H, Hꢀ2), 4.04 (dd, J = 13.2, 5.4 Hz, 1H, All), 3.99 (dd, J
= 13.2, 5.4 Hz, 1H, All), 3.89 (t, J = 9.6 Hz, 1H, Hꢀ4), 3.72ꢀ3.63 (m, 3H, Hꢀ3,4,6’), 3.39ꢀ3.36 (m,
13
1H, Hꢀ5), 3.27 (s, 3H, OMe), 1.73 (s, 3H, Me). C NMR (150 MHz, CDCl3) δ 138.3, 137.8,
134.6, 128.5, 128.4, 128.0, 127.9, 127.7, 123.9, 116.6, 97.5 (Cꢀ1), 79.0, 77.1, 75.2, 74.2, 74.1,
72.4, 72.3, 68.9, 49.7, 24.3. HR MS (ESIꢀTOF) m/z: calcd for C26H32O7Na [M + Na]+ 479.2046;
found, 479.2052. MS (ESIꢀTOF): found, 478.9.
pꢀTolyl 2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀO-allyꢀ1ꢀthioꢀαꢀDꢀmannopyranoside (9): To a solution
of 15 (400 mg, 0.877 mmol), pꢀthiocresol (131 mg, 1.05 mmol) and activated MS 4Å in 15 mL
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of anhydrous CH2Cl2 was added SnCl4 (176 ꢂL, 0.176 mmol, 1M in CH2Cl2) at 0 ˚C under N2.
The reaction mixture was stirred at room temperature for 30 min, at which time TLC indicated
the completion of the reaction. The reaction was quenched with triethylamine, and the solvent
was evaporated in vacuo. The residue was purified by silica gel column chromatography EtOAc
and hexane (1:8) as the eluent to give 9 (344 mg, 74%) as colorless syrup. 1H NMR (400 MHz,
CDCl3) δ 7.38ꢀ7.30 (m, 12H, Ph), 7.11 (d, J = 8.0 Hz, 2H, Ph), 5.98ꢀ5.88 (m, 1H, All), 5.61 (s,
1H, Hꢀ2), 5.47 (s, 1H, Hꢀ1), 5.30 (dd, J = 17.2, 1.2 Hz, 1H, All), 5.18 (d, J = 10.4 Hz, 1H, All),
4.95 (d, J = 10.8 Hz, 1H, Bn), 4.75 (d, J = 11.2 Hz, 1H, Bn), 4.63 (d, J = 10.8 Hz, 1H, Bn), 4.58
(d, J = 11.2 Hz, 1H, Bn), 4.32 (m, 1H, Hꢀ5), 4.11 (dd, J = 12.8, 5.2 Hz, 1H, All), 4.01ꢀ3.94 (m,
3H, Hꢀ3,4,All), 3.82 (dd, J = 10.8, 4.4 Hz, 1H, Hꢀ6), 3.69 (d, J = 10.8 Hz, 1H, Hꢀ6’), 2.33 (s, 3H,
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Tol), 2.16 (s, 3H, Ac). C NMR (100 MHz, CDCl3) δ 170.3, 138.4, 137.8, 137.7, 134.7, 132.2,
129.9, 129.8, 128.5, 128.4, 128.2, 127.9, 127.7, 116.9, 86.6 (Cꢀ1), 78.5, 75.3, 74.6, 72.36, 72.34,
71.9, 70.4, 68.9, 21.1. HR MS (ESIꢀTOF) m/z: calcd for C32H36O6SNa [M + Na]+ 571.2130;
found, 571.2137. MS (ESIꢀTOF): found, 570.9.
Pre-activation-based glycosylation (General Procedure A): After a solution of glycosyl donor
(1.1 eq.) and acceptor (1.0 eq.) in anhydrous CH2Cl2 mixed with TTBP (1.0 eq.) and MS 4Å was
stirred at room temperature for 40 min, it was cooled to ꢀ78 ˚C. Then, a solution of AgOTf (3.3
eq.) in acetonitrile was added. Fifteen minutes later, pꢀTolSCl (1.1 eq.) was added dropwise. The
reaction mixture was warmed to room temperature slowly within 1 h and stirred for another 20
min. The reaction was quenched with Et3N, and the mixture was diluted with CH2Cl2 and filtered.
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The filtrate was concentrated in vacuum, and the resultant residue was purified by flash silica gel
column chromatography with a mixture of EtOAc and toluene as eluent to afford the desired
product.
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Ir-complex/Hg(II)-catalyzed removal of the allyl group (General Procedure B): The solution of
[Ir(COD)(PMePh2)2]PF6 (0.15 eq.) in anhydrous THF was stirred under a H2 atmosphere at room
temperature until the red color turned to pale yellow (in ca. 15 min). Then, H2 was exchanged
with Argon for three times, before a solution of the allyl protected intermediate (1.0 eq.) in
anhydrous THF was added slowly. The reaction mixture was stirred at room temperature for 40
min, at which point TLC showed the complete reaction. The reaction mixture was concentrated
in vacuum, and after the residue was dissolved in acetone and water (9:1, V/V), the solution was
treated with HgCl2 (5.0 eq.) and HgO (0.15 eq.). Ten minutes later, the solution was concentrated
and the residue was purified by silica gel column chromatography to give the desired product.
(2ꢀOꢀAcetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutydimethylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ1ꢀOꢀ
(paraꢀmethoxybenzyl)ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀallylꢀDꢀmyoꢀinositol (16): General Procedure
A was used to prepare 16 (279 mg, 81%) from 7 (213 mg, 0.342 mmol) and 5 (190 mg, 0.311
1
mmol). H NMR (600 MHz, CDCl3) δ 7.35ꢀ7.19 (m, 27H), 6.86 (d, J = 8.4 Hz, 2H), 5.99ꢀ5.93
(m, 1H, All), 5.42 (s, 1H), 5.28 (dd, J = 17.4, 1.8 Hz, 1H, All), 5.20 (d, J = 1.2 Hz, 1H, Man Hꢀ1),
5.17 (dd, J = 10.8, 1.2 Hz, 1H, All), 4.86ꢀ4.53 (m, 12H), 4.38 (dd, J = 12.0, 6.0 Hz, 1H), 4.32 (dd,
J = 12.0, 6.0 Hz, 1H), 4.27 (s, 1H), 3.98ꢀ3.92 (m, 3H), 3.81ꢀ3.76 (m, 5H), 3.67 (dd, J = 12.0, 2.4
Hz, 1H), 3.44 (d, J = 10.8 Hz, 1H), 3.37 (t, J = 9.6 Hz, 1H), 3.27 (m, 2H), 2.05 (s, 3H), 0.89 (s,
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9H), 0.04 (s, 3H), 0.007 (s, 3H). C NMR (150 MHz, CDCl3) δ 169.9, 159.1, 139.1, 138.7,
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138.6, 138.1, 138.0, 135.4, 130.3, 129.0, 128.3, 128.2, 128.1, 128.0, 127.9, 127.6, 127.4, 127.3,
127.1, 116.7, 113.7, 98.5 (Man Cꢀ1, JCH = 177 Hz), 83.4, 81.1, 81.0, 80.3, 78.9, 77.6, 76.1, 75.7,
75.0, 74.6, 73.9, 72.37, 72.35, 72.0, 71.8, 68.8, 61.8, 55.2, 25.9, 20.9, 18.3, ꢀ5.1, ꢀ5.4. HR MS
(ESIꢀTOF) m/z: calcd for C66H80O13SiNa [M + Na]+ 1131.5266; found, 1131.5243. MS
(MALDIꢀTOF): found, 1132.4.
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(2ꢀOꢀAcetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutydimethylsilylꢀαꢀDꢀmannopyranosyl)ꢀ(1→2)ꢀ1ꢀOꢀ(
paraꢀmethoxybenzyl)ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀDꢀmyoꢀinositol (17): General Procedure B was used
1
to prepare 17 (197 mg, 89%) from 16 (230 mg, 0.208 mmol). H NMR (400 MHz, CDCl3) δ
7.38ꢀ7.20 (m, 27H), 6.87 (d, J = 8.4 Hz, 2H), 5.40 (t, J = 1.6 Hz, 1H), 5.16 (d, J = 1.6 Hz, 1H,
Man Hꢀ1), 4.89ꢀ4.51 (m, 12H), 4.32 (t, J = 2.0 Hz, 1H), 4.02ꢀ3.94 (m, 4H), 3.84ꢀ3.79 (m, 4H),
3.69 (dd, J = 11.6, 2.4 Hz, 1H), 3.46 (d, J = 10.8 Hz, 1H), 3.36ꢀ3.29 (m, 2H), 3.17 (dd, J = 9.6,
13
2.0 Hz, 1H), 2.07 (s, 3H), 0.89 (s, 9H, tBu), 0.04 (s, 3H, SiMe), 0.01 (s, 3H, SiMe). C NMR
(100 MHz, CDCl3) δ 170.0, 159.4, 139.0, 138.7, 138.6, 138.1, 138.0, 129.7, 129.4, 128.5, 128.4,
128.3, 128.1, 128.0, 127.9, 127.7, 127.6, 127.5, 127.2, 113.9, 98.5 (Man Cꢀ1), 83.2, 80.9, 79.7,
79.1, 77.6, 75.7, 75.6, 75.1, 73.9, 72.9, 72.48, 72.44, 72.0, 71.9, 71.3, 68.9, 61.8, 55.3, 25.9, 21.0,
18.3, ꢀ5.0, ꢀ5.3. HR MS (ESIꢀTOF) m/z: calcd for C63H76O13SiNa [M + Na]+ 1091.4953; found,
1091.4956. MS (MALDIꢀTOF): found, 1092.1.
(2ꢀOꢀAcetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀallylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀO-
(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutydimethylsilylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(paraꢀme
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thoxybenzyl)ꢀDꢀmyoꢀinositol (2): General Procedure A was used to parepare 2 (173 mg, 83 %)
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from 9 (84 mg, 0.154 mmol) and 17 (150 mg, 0.140 mmol). H NMR (500 MHz, CDCl3) δ
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7.42ꢀ7.17 (m, 35H), 7.11 (t, J = 7.5 Hz, 2H), 6.88 (d, J = 8.5 Hz, 2H), 5.80 (m, 1H, HꢀAll), 5.49
(m, 2H), 5.46 (s, 1H, ManA Hꢀ1), 5.16 (m, 2H, ManB Hꢀ1, HꢀAll), 5.05 (d, J = 10.0 Hz, 1H,
HꢀAll), 4.95ꢀ4.48 (m, 16H), 4.32 (s, 1H), 4.08 (t, J = 9.5 Hz, 1H), 4.02ꢀ3.94 (m, 7H), 3.88 (t, J =
9.5 Hz, 1H), 3.82 (s, 3H), 3.75ꢀ3.70 (m 2H), 3.52 (d, J = 11.5 Hz, 1H), 3.36ꢀ3.24 (m, 5H), 2.10
(s, 6H), 0.92 (s, 9H), 0.06 (s, 3H), 0.02 (s, 3H). 13C NMR (125 MHz, CDCl3) δ 170.3, 169.7,
159.4, 139.2, 139.0, 138.4, 138.2, 138.1, 137.9, 134.9, 129.9, 129.2, 128.6, 128.3, 128.2, 128.1,
128.0, 127.9, 127.6, 127.5, 127.3, 127.2, 116.6, 113.8, 98.6 (ManB Cꢀ1), 98.3 (ManA Cꢀ1), 81.4,
80.9, 78.9, 78.2, 77.4, 76.0, 75.7, 75.5, 75.06, 75.00, 74.1, 73.8, 72.6, 72.4, 72.3, 71.7, 71.6, 71.3,
70.6, 68.6, 68.5, 68.1, 61.9, 55.2, 25.9, 21.2, 21.0, 18.3, ꢀ5.1, ꢀ5.3. HR MS (ESIꢀTOF) m/z: calcd
for C88H104O19SiNa [M + Na]+ 1515.6839; found, 1515.6865. MS (MALDIꢀTOF): found,
1516.6.
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(2ꢀOꢀAcetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀO-(2ꢀOꢀacet
ylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutydimethylsilylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(paraꢀmethoxyben
zyl)ꢀDꢀmyoꢀinositol (18): General Procedure B was used to prepare 18 (124 mg, 91 %) from 2
1
(140 mg, 0.094 mmol). H NMR (500 MHz, CDCl3) δ 7.40ꢀ7.19 (m, 35H), 7.10 (t, J = 7.5 Hz,
2H), 6.89 (d, J = 8.5 Hz, 2H), 5.49 (dd, J = 2.5, 1.0 Hz, 1H), 5.46ꢀ5.43 (m, 2H, ManA Hꢀ1, ManB
Hꢀ2), 5.19 (d, J = 1.0 Hz, 1H, ManB Hꢀ1), 4.92ꢀ4.47 (m, 16H), 4.35 (s, 1H), 4.10 (t, J = 9.5 Hz,
1H), 4.02ꢀ3.91 (m, 5H), 3.87 (t, J = 9.5 Hz, 1H), 3.83ꢀ3.78 (m, 4H), 3.76 (dd, J = 11.0, 2.0 Hz,
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1H), 3.55 (d, J = 11.5 Hz, 1H), 3.49 (dd, J = 12.0, 2.0 Hz, 1H), 3.40 (dd, J = 12.0, 3.0 Hz, 1H),
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3.35ꢀ3.27 (m, 3H), 2.11 (s, 3H), 2.09 (s, 3H), 0.92 (s, 9H), 0.08 (s, 3H), 0.04 (s, 3H). C NMR
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(125 MHz, CDCl3) δ 170.2, 170.1, 159.4, 138.9, 138.8, 138.4, 138.1, 138.0, 137.8, 130.0, 129.1,
128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 113.8, 98.2
(ManB Cꢀ1), 98.0 (ManA Cꢀ1), 81.5, 81.4, 80.9, 78.8, 77.9, 76.2, 75.7, 75.1, 74.0, 73.9, 72.6, 72.5,
71.6, 71.5, 70.0, 68.7, 68.6, 61.9, 61.4, 55.2, 25.9, 21.1, 21.0, 18.3, ꢀ5.1, ꢀ5.3. HR MS (ESIꢀTOF)
m/z: calcd for C85H100O19SiNa [M + Na]+ 1475.6526; found, 1475.6366. MS (MALDIꢀTOF):
found, 1476.4.
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(2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4
ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranos
yl)ꢀ(1→6)ꢀ3,4ꢀdiꢀOꢀbenzylꢀ2ꢀOꢀacetylꢀαꢀDꢀmannopyranoside (3): A mixture of 8 (370 mg,
0.619 mmol) and activated MS 4Å in anhydrous CH2Cl2 (8 mL) was stirred at room temperature
for 40 min, and then cooled to ꢀ78 ˚C. A solution of AgOTf (477 mg, 1.856 mmol) in acetonitrile
(1.5 mL) was added. After 10 min of stirring, pꢀTolSCl (89 ꢂL, 0.619 mmol) was added
dropwise. Fifteen minutes later, a solution of 6 (286 mg, 0.563mmol) and TTBP (140 mg, 0.563
mmol) in anhydrous CH2Cl2 (1.5 mL) was added. The reaction mixture was warmed to room
temperature slowly in 1 h, stirred for another 20 min, and then cooled to ꢀ78 ˚C, which was
followed by the same sequence of addition of AgOTf (434 mg, 1.689 mmol) in acetonitrile (1
mL), pꢀTolSCl (81 ꢂL, 0.563 mmol), a solution of 6 (260 mg, 0.512 mmol) and TTBP (127 mg,
0.512 mmol) in anhydrous CH2Cl2 (1.5 mL). The reaction mixture was warmed to room
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temperature slowly in 1 h, stirred for another 20 min, and then cooled to ꢀ78 ˚C, and again was
followed by the same sequential addition of AgOTf (395 mg, 1.536 mmol) in acetonitrile (1 mL),
pꢀTolSCl (74 ꢂL, 0.512 mmol), a solution of 6 (236 mg, 0.465 mmol) and TTBP (115 mg, 0.465
mmol) in anhydrous CH2Cl2 (1.5 mL). The reaction mixture was warmed to room temperature
slowly in 1 h, stirred for another 20 min, and then quenched with Et3N, diluted with CH2Cl2, and
finally filtered. The filtrate was concentrated in vacuum, and the residue purified by silica gel
column chromatography with EtOAc and toluene (1:12) as the eluent to give 3 (317 mg, 39% for
three glycosylation steps) as a foamy solid.1H NMR (600 MHz, CDCl3) δ 7.35ꢀ7.08 (m, 49H),
5.61 (dd, J = 2.5, 1.2 Hz, 1H), 5.47 (m, 2H), 5.46 (dd, J = 2.5, 1.2 Hz, 1H), 5.38 (s, 1H, ManD
Cꢀ1), 4.98 (s, 1H, ManC Hꢀ1), 4.93ꢀ4.82 (m, 6H, ManA Hꢀ1, ManB Hꢀ1, 4 × BnꢀCH2ꢀ), 4.74ꢀ4.39
(m, 14H), 4.31 (dd, J = 10.2, 4.8 Hz, 1H), 3.98 (dd, J = 9.6, 3.0 Hz, 1H), 3.96ꢀ3.80 (m, 8H), 3.77
(dd, J = 11.4, 3.0 Hz, 1H), 3.74ꢀ3.68 (m, 2H), 3.68ꢀ3.58 (m, 4H), 3.57ꢀ3.50 (m, 3H), 2.19 (s, 3H),
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2.15 (s, 3H), 2.14 (s, 6H), 2.13 (s, 3H). C NMR (150 MHz, CDCl3) δ 170.3, 170.2, 170.19,
170.18, 138.5, 138.4, 138.2, 137.9, 137.7, 137.6, 137.5, 132.0, 129.9, 128.5, 128.4, 128.3, 128.2,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 98.0 (ManC Cꢀ1), 97.98 (ManA Cꢀ1), 97.97
(ManB Cꢀ1), 86.6 (ManD Cꢀ1), 78.5, 77.9, 77.7, 77.6, 75.2, 75.1, 75.0, 74.9, 74.3, 74.1, 73.8, 73.7,
73.4, 72.1, 71.8, 71.6, 71.4, 71.3, 71.15, 71.11, 70.2, 68.6, 68.2, 68.1, 68.0, 66.3, 65.5, 65.3, 29.7,
21.1, 21.04, 21.03, 21.00. HR MS (ESIꢀTOF) m/z: calcd for C102H110O24SNa [M + Na]+
1773.7005; found, 1773.7000. MS (MALDIꢀTOF): found, 1774.6.
6ꢀOꢀ[(2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenz
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ylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)]ꢀ3,4ꢀdiꢀ
Oꢀbenzylꢀ2ꢀOꢀacetylꢀαꢀDꢀmannopyranosyl trichloroacetimidate (19): To a solution of 3 (310
mg, 0.177 mmol) and TTBP (132 mg, 0.532 mmol) in wet CH2Cl2 was added Nꢀiodosuccinimide
(80 mg, 0.354 mmol) and silver triflate (91 mg, 0.354 mmol) at 0 ˚C. The reaction mixture was
stirred at room temperature for 2 h, quenched with saturated aq. Na2S2O3 solution, and diluted
with CH2Cl2 (150 mL). The organic phase, after being washed with saturated aq. NaCl solution
and H2O, was dried over Na2SO4 and concentrated in vacuo to afford a residue that was purified
with silica gel column chromatography with EtOAc and toluene (1:5) as the eluent to give a
white solid. It was dissolved in 10 mL of anhydrous CH2Cl2, and then DBU (14 ꢂL, 0.089 mmol)
and trichloroacetonitrile (91ꢂL, 0.89 mmol) were added at 0 ˚C. After 1.5 h of stirring, the
mixture was concentrated in vacuum and the residue was purified on a Et3Nꢀneutralized silica gel
column with EtOAc and toluene (1:8) as the eluent to give 19 (234 mg, 74% for two steps) as a
solid. 1H NMR (600 MHz, CDCl3) δ 8.67 (s, 1H), 7.32ꢀ7.07 (m, 45H), 6.19 (d, J = 1.8 Hz, 1H),
5.48 (dd, J = 3.0, 1.8 Hz, 1H), 5.46 (dd, J = 3.0, 1.8 Hz, 1H), 5.45 (dd, J = 3.0, 1.8 Hz, 1H), 5.41
(dd, J = 3.0, 1.8 Hz, 1H), 4.98 (d, J = 1.2 Hz, 1H, Man Hꢀ1), 4.91ꢀ4.38 (m, 20H, 3 × Man Hꢀ1,
17 × BnꢀCH2ꢀ), 4.02 (dd, J = 9.0, 3.0 Hz, 1H), 3.94ꢀ3.87 (m, 4H), 3.86ꢀ3.86 (m, 9H), 3.67 – 3.59
(m, 3H), 3.56 (d, J = 11.4 Hz, 2H), 3.51 (d, J = 10.8, 1.2 Hz, 1H), 2.19 (s, 3H), 2.15 (s, 3H), 2.13
(s, 3H), 2.12 (s, 3H). 13C NMR (150 MHz, CDCl3) δ 170.3, 170.2, 170.17, 170.15, 159.6, 138.4,
138.2, 137.9, 137.7, 137.6, 137.4, 128.4, 128.3, 128.2, 127.9, 127.8, 127.5, 127.4, 98.0 (Man
Cꢀ1), 97.9 (Man Cꢀ1), 97.6 (Man Cꢀ1), 94.9 (Man Cꢀ1), 90.7, 77.8, 77.7, 77.6, 77.5, 75.3, 75.1,
74.97, 74.95, 74.1, 73.9, 73.76, 73.72, 73.4, 73.3, 72.0, 71.5, 71.45, 71.41, 71.13, 71.11, 68.6,
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68.2, 68.1, 68.0, 67.2, 65.42, 65.40, 65.3, 21.1, 21.04, 21.03, 20.9.
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6ꢀOꢀ[(2ꢀOꢀacetylꢀ3,4,6ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenz
ylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(
2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)]ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀOꢀ(2ꢀOꢀacetylꢀ3,4ꢀdiꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutyldimeth
ylsilylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(paraꢀmethoxybenzyl)ꢀDꢀmyoꢀinositol (20) : To a stirred
mixture of 18 (60 mg, 0.041 mmol), 19 (96 mg, 0.054 mmol), and MS 4Å in anhydrous CH2Cl2
(4 mL) was added TMSOTf (1 ꢃL, 5.4 ꢃmol) under N2 protection at 0 ˚C. After the reaction
mixture was stirred for another 30 min, it was neutralized with Et3N, filtered and concentrated.
The residue was subjected to silica column chromatography with EtOAc and toluene (1:10) as
the eluent to afford 20 (97 mg, 77%) as a white solid.1H NMR (600 MHz, CDCl3) δ 7.38 (d, J =
7.8 Hz, 2H), 7.35ꢀ7.01 (m, 78H), 6.99 (d, J = 7.2 Hz, 2H), 6.84 (d, J = 7.2 Hz, 2H), 5.52 (s, 1H),
5.49 (m, 4H), 5.41 (s, 1H), 5.38 (s, 1H, ManA Hꢀ1), 5.14 (s, 1H, ManB Hꢀ1), 5.01 (d, J = 10.8 Hz,
1H), 4.97 (s, 1H, ManC Hꢀ1), 4.93ꢀ4.20 (m, 36H, 3 × Man Hꢀ1, 33 × BnꢀCH2ꢀ), 4.01ꢀ3.69 (m,
21H), 3.64ꢀ3.58 (m, 3H), 3.51ꢀ3.37 (m, 7H), 3.31ꢀ3.22 (m, 6H), 3.15 (d, J = 12.0 Hz, 1H), 3.12
(d, J = 12.0 Hz, 1H), 2.14 (s, 3H), 2.11 (s, 3H), 2.10 (s, 9H), 2.05 (s, 3H), 0.88 (s, 9H), 0.03 (s,
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3H), ꢀ0.003 (s, 3H). C NMR (150 MHz, CDCl3) δ 170.18, 170.13, 170.0, 169.9, 169.7, 159.4,
139.0, 138.9, 138.6, 138.5, 138.3, 138.2, 138.1, 138.0, 137.9, 137.8, 137.7, 137.5, 130.0, 129.0,
128.7, 128.5, 128.4, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.3, 127.2,
127.1, 126.9, 126.6, 113.8, 98.6 (ManA Cꢀ1), 98.3 (ManB Cꢀ1), 98.2 (Man Cꢀ1), 98.1 (ManC Cꢀ1),
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98.0 (Man Cꢀ1), 97.9 (Man Cꢀ1), 81.4, 81.3, 80.6, 78.8, 77.8, 77.7, 77.67, 77.61, 76.4, 75.7, 75.1,
74.9, 74.8, 74.6, 74.4, 74.0, 73.8, 73.6, 73.5, 73.4, 73.3, 72.6, 71.68, 71.65, 71.5, 71.44, 71.40,
71.3, 71.2, 72.1, 71.0, 70.8, 70.5, 69.9, 68.6, 68.5, 68.2, 67.9, 67.8, 67.7, 65.3, 65.2, 61.9, 55.2,
25.9, 21.1, 21.09, 21.03, 21.00, 18.3, ꢀ5.1, ꢀ5.4. HR MS (ESIꢀTOF) m/z: calcd for
C180H202O43SiNa2 [M + 2Na]2+ 1562.6593; found, 1562.6454. MS (MALDIꢀTOF) m/z: calcd for
C180H202O43SiNa [M + Na]+ 3102.3; found, 3103.2.
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6ꢀOꢀ[(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannop
yranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)]ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀOꢀ
(2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀtertꢀbutyldimethylsilylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(paraꢀmethoxybe
nzyl)ꢀDꢀmyoꢀinositol (22): To a solution of 20 (90 mg, 0.029 mmol) in CH2Cl2 and CH3OH (1:2,
9 mL) was added CH3ONa solution (0.5 M in CH3OH) until pH reached 11. The solution was
stirred at 35 ˚C for 24 h before the solvent was removed in vacuo. The residue was dissolved in a
mixture of CH2Cl2 and CH3OH (1:10) and filtered, and the filtrate was concentrated under
vacuum to give crude 21. To a solution of crude 21 in dry DMF was added BnBr (42 ꢂL, 0.35
mmol) and TBAI (4.0 mg, 10.8 ꢂmol). After the mixture was stirred for 10 min, NaH was added
(5.6 mg, 0.232 mmol) at 0 ˚C, which was stirred for 1.5 h. Then, MeOH was added to quench the
reaction before water was added. The aqueous phase was extracted with CH2Cl2 (3×50 mL), and
the organic layer was dried over Na2SO4, concentrated. The residue was purified by silica gel
column chromatography with EtOAc and toluene (1:16) as the eluent to give 22 (66 mg, 67% for
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two steps) as syrup. 1H NMR (600 MHz, CDCl3) δ 7.40ꢀ6.98 (m, 112H), 6.58 (d, J = 8.4 Hz, 2H),
5.39 (s, 1H, ManA Hꢀ1), 5.29 (s, 1H, ManF Hꢀ1), 5.09 (s, 1H, ManE Hꢀ1), 5.02 (d, J = 11.4 Hz,
1H), 5.00 (s, 1H, ManC Hꢀ1), 4.94 (s, 1H, ManB Hꢀ1), 4.93ꢀ4.27 (m, 46H, ManD Hꢀ1, 45 ×
BnꢀCH2ꢀ), 4.05 (t, J = 9.6 Hz, 1H), 4.03ꢀ3.78 (m, 21H), 3.73 (s, 1H), 3.68 (d, J = 9.6 Hz, 2H),
3.62ꢀ3.54 (m, 6H), 3.53ꢀ3.48 (m, 3H), 3.46ꢀ3.43 (m, 2H), 3.38 (d, J = 9.0 Hz, 1H), 3.35ꢀ3.25 (m,
6H), 3.17 (d, J = 11.4 Hz, 1H), 3.10 (d, J = 10.8 Hz, 1H), 0.85 (s, 9H), ꢀ0.002 (s, 3H), ꢀ0.01 (s,
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3H). C NMR (150 MHz, CDCl3) δ 159.4, 139.1, 138.9, 138.8, 138.7, 138.6, 138.5, 138.3,
138.2, 138.1, 137.9, 129.5, 129.0, 128.5, 128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6,
127.5, 127.4, 127.3, 127.2, 127.1, 126.9, 126.8, 113.9, 99.1 (ManA Cꢀ1), 98.6 (ManB Cꢀ1), 98.4
(ManC Cꢀ1), 98.2 (ManD Cꢀ1), 98.15 (ManE Cꢀ1), 98.10 (ManF Cꢀ1), 82.0, 81.4, 80.6, 79.6, 79.4,
79.3, 79.2, 79.1, 78.9, 76.0, 75.9, 75.7, 75.6, 75.0, 74.92, 74.90, 74.8, 74.7, 74.67, 74.62, 74.52,
74.50, 74.4, 74.2, 73.9, 73.8, 73.6, 73.2, 72.8, 72.63, 72.61, 72.5, 72.4, 72.3, 72.2, 72.1, 71.9,
71.8, 71.5, 71.4, 71.26, 71.22, 71.20, 71.1, 71.0, 69.0, 65.8, 65.7, 65.6, 62.2, 55.0, 53.4, 25.9,
18.3, ꢀ5.1, ꢀ5.4.HR MS (ESIꢀTOF) m/z: calcd for C210H226O37SiNa2 [M + 2Na]2+ 1706.7684;
found, 1706.7743. MS (MALDIꢀTOF) m/z: calcd for C210H226O37SiNa [M + Na]+ 3390.5; found,
3291.2.
6ꢀOꢀ[(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannop
yranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)]ꢀ3,4,5ꢀtriꢀOꢀbenz
ylꢀ2ꢀOꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(paraꢀmethoxybenzyl)ꢀDꢀmyoꢀinositol
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(23): A solution of 22 (60.0 mg, 0.018 mmol) in THF and CH3CN (2:13, mL) and triethylamine
trihydrofluoride (1.0 mL) was stirred at room temperature overnight under Ar. The solution was
quenched with dropwise addition of saturated aq. NaHCO3 solution. The aq. phase was extracted
with CH2Cl2 (3×40 mL), and the organic layer was dried over Na2SO4, concentrated. The residue
was purified by silica gel column chromatography with EtOAc and toluene (1:10) as the eluent
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to give 23 (50.4 mg, 87%) as syrup. H NMR (600 MHz, CDCl3) δ 7.40ꢀ7.04 (m, 112H), 6.65
(dd, J = 7.8 Hz, 2H), 5.44 (d, J = 3.6 Hz, 1H, ManA Hꢀ1), 5.21 (d, J = 4.2 Hz, 1H, ManB Hꢀ1),
5.12 (d, J = 4.2 Hz, 1H, ManE Hꢀ1), 5.07ꢀ5.04 (d, J = 10.8 Hz, 1H), 5.02 (d, J = 3.6 Hz, 1H,
ManD Hꢀ1), 4.96ꢀ4.85 (m, 8H, ManC Hꢀ1, ManF Hꢀ1, 6 × BnꢀCH2ꢀ), 4.77 (d, J = 10.8 Hz, 1H),
4.72ꢀ4.29 (m, 38H), 4.07ꢀ3.42 (m, 36H), 3.40 (d, J = 8.4 Hz, 1H), 3.36ꢀ3.25 (m, 6H), 3.18 (d, J =
10.8 Hz, 1H), 3.12 (d, J = 10.8 Hz, 1H), 2.11 (s, 1H). 13C NMR (150 MHz,CDCl3) δ 159.4, 138.9,
138.8, 138.7, 138.6, 138.5, 138.4, 138.3, 138.2, 138.1, 138.0, 137.7, 129.2, 128.9, 128.5, 128.4,
128.3, 128.2, 128.1, 128.0, 127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 127.1, 127.0,
126.8, 113.9, 99.1 (ManA Cꢀ1), 99.0 (ManB Cꢀ1), 98.7 (ManC Cꢀ1), 98.4 (ManD Cꢀ1), 98.3 (ManE
Cꢀ1), 98.2 (ManF Cꢀ1), 81.8, 81.3, 80.7, 79.6, 79.4, 79.3, 79.2, 79.0, 78.7, 75.9, 75.8, 75.7, 75.1,
74.9, 74.8, 74.7, 74.6, 74.5, 74.2, 74.0, 73.9, 73.7, 73.2, 72.8, 72.6, 72.5, 72.4, 72.26, 72.21, 72.1,
72.0, 71.8, 71.6, 71.5, 71.3, 71.2, 71.1, 70.9, 69.1, 65.8, 65.7, 65.6, 62.1, 55.1. HR MS (ESIꢀTOF)
m/z: calcd for C204H212O37Na2 [M + 2Na]2+ 1649.7252; found, 1649.7317. MS (MALDIꢀTOF)
m/z: calcd for C204H212O37Na [M + Na]+ 3276.4; found, 3277.1.
6ꢀOꢀ[(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannop
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yranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)]ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀOꢀ
(2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀstearoylꢀαꢀDꢀmannopyranosyl)ꢀDꢀmyoꢀinositol (25): To a solution of
23 (49.0 mg, 0.015 mmol) in anhydrous CH2Cl2 (2 mL) was added stearic acid (21.3 mg, 0.075
mmol), DCC (15.5 mg, 0.075 mmol), and DMAP (9.2 mg, 0.075 mmol) at room temperature.
After having been stirred overnight, the mixture was filtered off through a Celite pad, and the
filtrate was concentrated to get a residue that was purified by silica gel column chromatography
with toluene and EtOAc (15:1) as the eluent to give 24 as a white solid. To a solution of 24 in
CH2Cl2 (1.5 mL) was added 20% TFA/CH2Cl2, giving a final concentration of about 10% of TFA.
The mixture was stirred for 3 h, at which time TLC indicated the completion of the reaction. The
solution was coꢀevaporated with toluene 3 times to remove TFA completely. Purification of the
residue by silica gel column chromatography with EtOAc and toluene (1:12) as the eluent gave
25 (35.3 mg, 69% from 23) as a white solid. 1H NMR (600 MHz, CDCl3) δ 7.31ꢀ7.01 (m, 110H),
5.36 (s, 1H, ManA Hꢀ1), 5.09 (s, 1H, ManB Hꢀ1), 5.04 (s, 1H, ManF Hꢀ1), 4.96 (s, 2H, ManC Hꢀ1,
ManD Hꢀ1), 4.85ꢀ4.73 (m, 8H, ManE Hꢀ1, 7 × BnꢀCH2ꢀ), 4.65 (d, J = 10.8 Hz, 1H), 4.60ꢀ4.27 (m,
36H), 4.16 (s, 1H), 4.08 (d, J = 9.6 Hz, 1H), 4.05 (dd, J = 12.0, 3.6 Hz, 1H), 3.96ꢀ3.64 (m, 24H),
3.60 (d, J = 12.0, 2.4 Hz, 1H), 3.58ꢀ3.53 (m, 2H), 3.50ꢀ3.35 (m, 9H), 3.30ꢀ3.27 (m, 1H), 3.22 (dd,
J = 9.6, 1.8 Hz, 1H), 3.12 (t, J = 9.6 Hz, 1H), 2.10 (t, J = 7.8 Hz, 2H), 1.48ꢀ1.41 (m, 2H),
1.24ꢀ1.12 (m, 28H), 0.80 (t, J = 7.2 Hz, 3H). 13C NMR (150 MHz, CDCl3) δ 173.6, 138.8, 138.7,
138.6, 138.5, 138.4, 138.3, 138.2, 138.1, 138.0, 137.9, 137.6, 128.5, 128.4, 128.3, 128.2, 128.1,
127.9, 127.8, 127.7, 127.6, 127.5, 127.4, 127.3, 127.2, 98.6 (ManA Cꢀ1), 98.5 (ManB Cꢀ1, ManC
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Cꢀ1, ManD Cꢀ1), 98.4 (ManE Cꢀ1), 98.2 (ManF Cꢀ1), 81.2, 80.4, 80.3, 79.4, 79.3, 79.2, 78.8, 78.3,
75.5, 75.3, 75.2, 75.1, 75.0, 74.9, 74.87, 74.81, 74.7, 74.6, 74.3, 74.1, 74.0, 73.9, 73.2, 73.0,
72.67, 72.61, 72.5, 72.2, 72.0, 71.9, 71.8, 71.7, 71.67, 71,61, 71.3, 71.28, 71.26, 71.20, 70.1, 69.1,
66.5, 65.8, 65.7, 65.6, 63.1, 34.1, 31.9, 29.7, 29.65, 29.60, 29.5, 29.3, 29.25, 29.20, , 24.8, 22.7,
14.1. HR MS (ESIꢀTOF) m/z: calcd for C214H238O37Na2 [M + 2Na]2+ 1722.8269; found,
1722.8318. MS (MALDIꢀTOF) m/z: calcd for C214H238O37Na [M + Na]+ 3422.6; found, 3423.0
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6ꢀOꢀ[(2,3,4,6ꢀtetraꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannop
yranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀ
mannopyranosyl)ꢀ(1→6)ꢀ(2,3,4ꢀtriꢀOꢀbenzylꢀαꢀDꢀmannopyranosyl)]ꢀ3,4,5ꢀtriꢀOꢀbenzylꢀ2ꢀOꢀ
(2,3,4ꢀtriꢀOꢀbenzylꢀ6ꢀOꢀstearoylꢀαꢀDꢀmannopyranosyl)ꢀ1ꢀOꢀ(1,2ꢀdiꢀOꢀstearoylꢀsnꢀglyceroꢀ3ꢀ
benzylphosphoryl)ꢀDꢀmyoꢀinositol (26): To a mixture of 25 (16.0 mg, 4.7 ꢂmol), freshly
prepared glycerylphosphoramidite 4 (20.2 mg, 0.024 mmol), and MS 4 Å in CH2Cl2 and CH3CN
(2:1, 3 mL) was added 1Hꢀtetrazole (0.45 M in CH3CN, 105 ꢂL, 0.047 mmol). After stirring at
room temperature under Ar for 40 min, the reaction mixture was cooled to ꢀ20 ˚C, and mꢀCPBA
(4.1 mg, 0.024 mmol) was added. The reaction mixture was slowly warmed to room temperature
in 1 h, and then quenched with saturated aq. NaS2O3 solution. The aq. layer was extracted with
CH2Cl2 (3×30 mL), and the organic phase, after being dried over Na2SO4, was concentrated, with
the residue purified by silica gel column chromatography with EtOAc and toluene (1:13) as the
eluent to give 26 (14.1 mg, 72%, mixture of two isomers in about 1:3 ratio) as a white solid. 1H
and 13C NMR spectroscopic data for the major stereoisomer: 1H NMR (600 MHz, CDCl3) δ 5.33
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(s, 1H, ManA Hꢀ1), 5.19 (s, 1H, ManB Hꢀ1), 5.03 (s, 1H, ManC Hꢀ1), 4.93 (s, 1H, ManD Hꢀ1),
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31P NMR (162 MHz, CDCl3) δ ꢀ0.42 (minor isomer), ꢀ0.19 (major isomer). HR MS (ESIꢀTOF)
m/z: calcd for C260H319O44PNa2 [M + 2Na]2+ 2111.1129; found, 2111.1270.
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6ꢀOꢀ[(αꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(αꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(αꢀDꢀmannopyranosyl)ꢀ(
1→6)ꢀ(αꢀDꢀmannopyranosyl)ꢀ(1→6)ꢀ(αꢀDꢀmannopyranosyl)]ꢀ2ꢀOꢀ[(6ꢀOꢀstearoylꢀαꢀDꢀmann
opyranosyl)]ꢀ1ꢀOꢀ(1,2ꢀdiꢀOꢀstearoylꢀsnꢀglyceroꢀ3ꢀphosphoryl)ꢀDꢀmyoꢀinositol (1): The mixture
of 26 (10 mg, 2.4 ꢂmol) and 10% Pd/C (15 mg) in CHCl3, MeOH and H2O (3:3:1, 3 mL) was
stirred under a 50 psi H2 atmosphere for 3 days. The reaction solution was filtered off through a
pad of Celite, and the filtrate was concentrated in vacuum to give the target compound 1 (3.5 mg,
69%) as a pale yellow solid. 1H NMR (600 MHz, CD3OD, CDCl3 and D2O 3:3:1) δ 5.33 (s, 1H,
ManA Hꢀ1), 5.19 (s, 1H, ManB Hꢀ1), 5.12 (s, 1H, ManC Hꢀ1), 4.90 (s, 1H, ManD Hꢀ1), 4.84 (s, 1H,
ManE Hꢀ1), 4.83 (s, 1H, ManF Hꢀ1), 4.60ꢀ2.98 (m, 47H), 2.38ꢀ2.16 (m, 6H), 1.65ꢀ1.55 (m, 6H),
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1.40ꢀ1.15 (m, 84H), 0.90ꢀ0.80 (m, 9H). P NMR (162 MHz, CD3OD/CDCl3/D2O 3:3:1) δ 0.54.
MS (MALDIꢀTOF) m/z: calcd for C99H180O44PK3 [M + 3K ꢀ H]2+ 1110.5; found, 1109.2.
Acknowledgments This work was supported in part by National High Technology Research and
Development (863) Program of China (No. 2012AA021504) and National Major Scientific and
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Technological Special Project for New Drugs Development (2012ZX09502001) of China and the
National Institutes of Health (R01 GM090270) of the United States.
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Supporting Information H, C, P, Hꢀ1H gCOSY, and Hꢀ13C gHMQC NMR spectra of all
new compounds. This material is available free of charge via the Internet at http://pubs.acs.org.
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