Tandem glycosyl iodide glycosylation and regioselective enzymatic acylation affords 6-o-tetradecanoyl-α- d -cholesterylglycosides

Article abstract of DOI:10.1021/jo501371h

A generalized synthesis of α-d-cholesterylglycosides has been achieved using one-pot per-O-trimethylsilyl glycosyl iodide glycosidation. Both cholesteryl α-d-glucopyranoside (αCG) and cholesteryl α-d-galactopyranoside were prepared in high yield. These compounds were further esterified using regioselective enzymatic acylation with tetradecanoyl vinyl ester to afford 6-O-tetradecanoyl-α-d-cholesteryl glucopyranoside (αCAG) of Helicobacter pylori and the corresponding galactose analogue in 66-78% overall yields from free sugars. The tandem step-economy sequence provides novel analogues to facilitate glycolipidomic profiling.

Full text of DOI:10.1021/jo501371h

Note
pubs.acs.org/joc
Open Access on 08/05/2015
Tandem Glycosyl Iodide Glycosylation and Regioselective Enzymatic
Acylation Affords 6‑O‑Tetradecanoyl-α‑D‑cholesterylglycosides
Ryan A. Davis, James C. Fettinger, and Jacquelyn Gervay-Hague*
Department of Chemistry, University of California, Davis, One Shields Avenue, Davis, California 95616, United States
S
* Supporting Information
ABSTRACT: A generalized synthesis of α-D-cholesterylglycosides has been achieved using one-pot per-O-trimethylsilyl glycosyl
iodide glycosidation. Both cholesteryl α-D-glucopyranoside (αCG) and cholesteryl α-D-galactopyranoside were prepared in high
yield. These compounds were further esterified using regioselective enzymatic acylation with tetradecanoyl vinyl ester to afford 6-
O-tetradecanoyl-α-D-cholesteryl glucopyranoside (αCAG) of Helicobacter pylori and the corresponding galactose analogue in 66−
78% overall yields from free sugars. The tandem step-economy sequence provides novel analogues to facilitate glycolipidomic
profiling.
ecently, three cholesterylglucosides (Figure 1) have been
isolated from Helicobacter pylori, a bacteria that is believed
host, a symbiotic relationship between microbe and man has
been suggested.
R
To date, biological studies involving these cholesterylglyco-
sides have mostly relied upon natural sources, which furnish
mixtures of all three glycolipids, making it difficult to determine
the independent roles of each constituent.7,8 We have initiated
a synthetic campaign directed toward developing step-economy
syntheses of cholesterylglycosides. As part of that effort, we are
interested in making both glucosyl and galactosyl analogues of
αCAG available to the biological community for structure
activity relationship and glycolipidomic profiling studies. Access
to these standards may also facilitate the identification of
naturally occurring analogues that have yet to be discovered.
We recently reported a five-step synthesis of αCAG (2),10
which relied upon enzymatic acylation of free glucose to afford
6-O-tetradecanoyl glucose (Scheme 1). The remaining
hydroxyls were protected as trimethylsilyl ethers, and the
resulting compound was treated with trimethylsilyl iodide to
generate the α-iodide. The C-6 acyl functionality had a
remarkable effect on the reaction rate, requiring 14 h to
quantitatively produce the iodide as compared to the same
reaction employing per-O-silyl glucose, which took only 30
min.10 Moreover, subsequent glycosidation proceeded in only
45% yield, whereas glycosidations with per-O-silyl glycosyl
iodides are typically much higher. This synthetic protocol was
Figure 1. Structures of H. pylori cholesterylglucosides.
to infect 50% of the world population. While in many cases, H.
pylori infection goes undetected, chronic infection can lead to
gastric ulcers or even cancer.1−8 The core glucoside structure
includes glucose α-linked to cholesterol (αCG (1)). One
analogue is acylated at the sugar C6-hydroxyl with
tetradecanoic acid and is referred to as αCAG (2). A third
analogue contains a phosphatidyl group instead of the fatty acid
chain and is abbreviated αCPG (3). The lipid portion on the
phosphatidyl glycerol may vary in its composition of three
different lipids.9 Together, these glucosides make up approx-
imately 25% of the lipid content of H. pylori and are a
distinguishing feature of these bacteria.5,6 Most notably, these
compounds have been shown to stimulate the human immune
system and, since H. pylori must obtain cholesterol from the
Received: June 19, 2014
Published: August 5, 2014
© 2014 American Chemical Society
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Scheme 1. First-Generation Synthesis of αCAG
Figure 2. Retrosynthesis of a generalized strategy for making acylated α-cholesterylglycosides.
further limited by the fact that enzymatic acylation failed when
attempted with free galactose.
Scheme 2. Synthesis of α-Cholesteryl Galactose
To address these shortcomings, we have implemented an
alternative strategy that relies upon the one-pot glycosidation of
cholesterol to give the corresponding α-cholesterylglycoside,
followed by enzymatic acylation (Figure 2). The synthesis of
αCG (1) had been previously reported to require eight steps
from free D-glucose with the key step requiring a metallic Lewis
acid promoted glycosylation, which afforded a 1:1 α:β ratio of
CG in 75% yield.11 Recent developments in our lab led us to
believe that the yield and α-selectivity of the glycosylation
reaction could be improved using the per-O-silyl glycosyl iodide
methodology, which had proven successful in the synthesis of
α-linked glycolipids.12−14 We were hopeful that the lipase-
catalyzed regioselective acylation of the resulting cholester-
ylglycosides would uneventfully produce the acylated choles-
terylglycosides for both glucosyl and galactosyl substrates,
addressing the inability to esterify free α-D-galactose. This
approach could also circumvent the low yields we previously
encountered with chemical acylation of cholesterylglycosides.14
The new strategy began with glycosylation of the
monosaccahrides. Work previously performed in our laboratory
had already accomplished the glycosidation of cholesterol (6)
with per-O-TMS galactosyl iodide to afford the α-cholester-
ylgalactoside 5 in 85% yield (Scheme 2).14 This protocol relies
upon using excess per-O-TMS glycosyl iodide, which can be
made quantitatively from relatively inexpensive D-galactose.15
After glycosidation, the TMS-protecting groups can be
removed with ease using Dowex-50WX8-200 acidic resin in
methanol. To assess the stereochemical integrity of the
reaction, the crude reaction material is typically acetylated,
which allows facile separation of anomers. However, the β-
linked product was not observed when using the per-O-TMS
galactosyl iodide donor.
Following this same strategy, attention turned to the
synthesis of αCG (1). First, per-O-TMS glucoside was reacted
with iodotrimethylsilane (TMSI) to generate the glucosyl
iodide, which was directly cannulated into a solution of
cholesterol (6), TBAI, and DIPEA, and the mixture was stirred
for 2 days at ambient temperature. The solvent was then
removed, and TBAI precipitated upon adding a 1:1 mixture of
ethyl acetate and hexanes with cooling. The resulting solid
TBAI was removed by suction filtration, and the filtrate was
concentrated to give a white solid, which was treated with
methanol and Dowex-50WX8-200 acidic resin to remove the
silyl protecting groups. Acetylation of the crude products
afforded a mixture of CG anomers in a 39:1 α:β ratio. The
anomers were separated by HPLC chromatography, and each
anomer was then deactylated using sodium methoxide in a
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methanol:dichloromethane (10:1) solution to yield αCG (1) in
78% along with 2% of the β-anomer (Scheme 3).
With the cholesterylglycosides 1 and 5 in hand, the
regioselective enzymatic acylation of the C6-hydroxyl group
was attempted using the protocol we published in 2012.10 We
were delighted to find that the enzymatic acylation allowed
smooth transformation of αCG to αCAG in a quantitative
manner using 3 equiv of myristic acid vinyl ester (Scheme 4).
The esterification proceeded in both a tetrahydrofuran:pyridine
(4:1 THF:pyr.) solvent system10 and in acetone, which had
proven effective in the regioselective acylation of amygdalin by
Vemula et al.16 However, the galactosyl analogue (5) failed to
give any acylated product in a variety of solvents, including
acetone, THF:pyridine (4:1), dioxane:pyridine (4:1), dioxane:-
pyridine (1:1), dioxane:pyridine (3:7), THF:pyridine (1:1),
and THF:pyridine (3:7). Aqueous solvent was not investigated
because the cholesterylglycosides were not water-soluble and
the lipase enzyme required anhydrous conditions.17,18 We
initially thought that the enzyme was specific for glucose until
we learned of one example that reported C-6 acetylation of α-D-
methyl galactoside using 22 equiv of vinyl acetate in 74%
yield.19 Believing the reaction to be independent of solvent, we
again subjected α-cholesterylgalactoside to enzymatic acylation
using increasing amounts of myristic acid vinyl ester in the
THF:pyr (4:1) solvent system (Scheme 4). No reaction was
observed using 6 equiv of fatty acid vinyl ester, even after 4
days. However, we were encouraged to find that doubling the
amount of vinyl ester to 12 equiv yielded 51% of 4. The
reaction was further optimized using 18 equiv of fatty acid vinyl
ester to afford 4 in 77% isolated yield after 4 days. Although the
reaction was considerably slower than the glucose analogue,
and required a large excess of vinyl ester, the yield compares
favorably with previous chemical acylation processes14,20 and, as
far as we are aware, represents the first example of lipase
catalyzed regioselective fatty acid acylation of galactose.
Scheme 3. Glycosidation of Glucosyl Iodide with
Cholesterol
The glycosylation/deprotection protocol provided a step-
economy approach to α-linked cholesteryl monosaccharides,
and the acetylated analogues turned out to be crystalline. X-ray
crystallography revealed distinguishing structural features of the
two cholesterylglucoside anomers. The carbohydrate head-
group of αCG orients perpendicular to the cholesterol
backbone, whereas, in the β-anomer, the pyranose is parallel
and the carbohydrate group is in an extended oritentation
(Figure 3). Understanding the biological relevance of different
presentation geometries will require further investigation.
A step-economy synthetic protocol involving the glycosida-
tion of cholesterol prior to enzymatic regioselective acylation
has allowed expansion of the acylated α-cholesterylglycoside
inventory to include galactose analogues. The glycosidation of
per-O-silylated glucose provides better α-selectivity (39:1) than
past syntheses (8:1 α-selectivity)10,11 and higher glycosylation
yields due to the armed nature of per-O-silyl donors. Moreover,
unlike free galactose, which failed to undergo regioselective
esterification, α-cholesteryl galactose was a substrate for lipase,
giving a 77% yield of the C-6 ester. The lipase catalyzed process
was highly regioselective and gave increased yields compared to
previous chemical esterification (43% yield) methods.14 The
new methodolgy has allowed for the preparation of αCG (1)
and αCAG (2) in 78% overall yields from D-glucose (compared
Figure 3. X-ray crystal structures of per-O-acetylated αCG (left) and
per-O-acetylated βCG (right) displaying different carbohydrate
orientations.
Scheme 4. Enzymatic Regioselective Acylation Using Novozym 435
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Note
to 45% previously)10 as well as the synthesis of 6-O-
tetradecanoyl-α-D-cholesterylgalactoside in an overall yield of
66% from D-galactose. Little is known about the biological roles
of cholesterylglycosides in general. While the galactose
analogues reported herein have not yet been isolated from
natural sources, αCG and αCAG are known to inhibit T-cell
activation and thus aid in immune evasion of H. pylori.7
5H), 1.56−1.62 (m, 3H), 1.79−1.90 (m, 3H), 1.96−2.06 (m, 2H),
2.00 (s, 3H), 2.01 (s, 3H), 2.04 (s, 3H), 2.07 (s, 3H), 2.16−2.20 (m,
1H), 2.25 (ddd, J = 2.4, 4.8, 13.3 Hz, 1H), 3.48 (dp, J = 0.6, 4.8, 9.6
Hz, 1H), 3.67 (ddd, J = 2.4, 4.8, 9.6 Hz, H5, 1H), 4.11 (dd, J = 2.4,
12.6 Hz, H6, 1H), 4.25 (dd, J = 4.8, 12.6 Hz, H6′, 1H), 4.59 (d, J = 7.8
Hz, H1, 1H), 4.95 (dd, J = 8.0, 9.6 Hz, H2, 1H), 5.07 (app. t, J = 9.6
Hz, H4, 1H), 5.19 (app. t, J = 9.6, Hz, H3, 1H), 5.35−5.36 (m, 1H).
13C NMR (200 MHz, CDCl3): δ 170.8, 170.5, 169.5, 169.4, 140.5,
122.3, 99.8 (C1), 80.2, 73.0 (C3), 71.8 (C5), 71.6 (C2), 68.7 (C4),
62.2 (C6), 56.9, 56.3, 50.3, 42.5, 39.9, 39.7, 39.1, 37.3, 36.9, 36.3, 35.9,
32.1, 32.0, 29.6, 28.4, 28.2, 24.4, 24.0, 23.0, 22.7, 21.2, 20.9, 20.87,
20.8, 20.76, 19.5, 18.9, 12.0. HRMS (ESI-Ion Trap) m/z: [M + NH4]+
calcd for C41H68 NO10 734.4843; found 734.4843.
EXPERIMENTAL SECTION
■
Cholesteryl-α-D-glucopyranose (1). In a flame-dried argon-
purged round-bottom flask containing 4 Å molecular sieves (300 mg)
were added TBAI (140 mg, 0.38 mmol, 4.5 equiv), cholesterol (32 mg,
0.08 mmol, 1.0 equiv), and anhydrous dichloromethane (2.5 mL).
Alternatively, the crude mixture could be purified by flash column
chromatography using a 9:1 dichloromethane:methanol solvent system
to afford compound (1) (35 mg, 0.06 mmol, 78% yield).
Hunig’s base (90 μL, 0.51 mmol, 6.0 equiv) was then added to the
̈
solution, and the reaction was stirred for 30 min. In a separate flame-
dried argon-purged round-bottom flask was placed per-O-trimethylsi-
lylated glucose (140 mg, 0.25 mmol),15 which was azeotroped with
anhydrous benzene (2 × 3 mL), and then anhydrous chloroform was
added and removed under reduced pressure (2 × 3 mL). The per-O-
TMS sugar15 was placed under vacuum for at least 2 h prior to use, but
typically was dried on vacuum overnight. Once dried according to the
above protocol, the reaction was diluted with anhydrous dichloro-
methane (2.5 mL), and TMSI (40 μL, 0.28 mmol, 3.3 equiv) was
added and allowed to react for 10 min at rt. The in situ generated
glucosyl iodide was then transferred via cannula into the acceptor flask
and allowed to stir for 2 days at rt. The solvent was then filtered to
remove the molecular sieves and concentrated under reduced pressure.
A 1:1 ratio of ethyl acetate and hexanes (20 mL) was then added to
the round-bottom flask, and the flask was cooled in a dry ice/acetone
bath, causing the excess TBAI to precipitate. The solid was filtered,
and the solution was again concentrated to give an oil. Methanol (5
mL) and Dowex 50WX8-200 acidic resin (300 mg) were then added,
and the reaction was stirred for 2 h. Initially, the crude reaction
mixture was acetylated to separate the anomers and to obtain an
accurate α:β ratio.
Cholesteryl-2,3,4,6-tetra-O-acetyl-α-D-glucopyranose. The
acetylated mixture was purified by HPLC using a silica microsorb
normal phase 250 × 10 mm column and a gradient mobile phase
starting with 20% ethyl acetate in hexanes with gradual polarity
increasing to 40% ethyl acetate in hexanes over a 23 min time period
and a flow rate of 4 mL per min. The α-anomer had an HPLC
retention time of 14.4 min and was obtained as a white powder. The
purified compound was then recrystallized from diethyl ether and
methanol to afford glasslike, rod-shaped crystals. mp = 196.4−196.8
°C. Rf = 0.72 (hexanes:acetone 6:4). [α]2D0 +70.2 (c 0.95, CH2Cl2). 1H
NMR (800 MHZ, CDCl3): δ 0.67 (s, 3H), 0.85 (d, J = 6.6 Hz, 3H),
0.86 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 1.02 (s, 3H), 1.03−
1.17 (m, 9H), 1.23−1.29 (m, 2H), 1.32−1.39 (m, 2H), 1.41−1.58 (m,
9H), 1.75−1.85 (m, 2H), 1.95−2.00 (m, 2H), 2.01 (s, 3H), 2.03 (s,
3H), 2.05 (s, 3H), 2.08 (s, 3H), 2.30 (ddd, J = 2.2, 4.9, 13.2 Hz, 1H),
3.36−2.38 (m, 1H), 3.40−3.44 (m, 1H), 4.11 (dd, J = 2.2, 12.2 Hz,
H6, 1H), 4.14 (ddd, J = 2.2, 4.8 10.2 Hz, H5, 1H), 4.23 (dd, J = 4.8,
12.2 Hz, H6′, 1H), 4.80 (dd, J = 3.8, 10.2 Hz, H2, 1H), 5.03 (app. t, J
= 10.2 Hz, H4, 1H), 5.22 (d, J = 3.8 Hz, H1, 1H), 5.33−5.34 (m, 1H),
5.48 (app. t, J = 10.0 Hz, H3, 1H). 13C NMR (200 MHz, CDCl3): δ
170.9, 170.4, 170.3, 169.9, 140.4, 122.3, 94.3 (C1), 78.9, 71.2 (C2),
70.3 (C3), 68.8 (C4), 67.3 (C5), 62.2 (C6), 56.8, 56.2, 50.2, 42.4,
40.1, 39.8, 39.6, 37.1, 36.8, 36.3, 35.9, 32.1, 32.0, 28.4, 28.2, 28.0, 24.4,
24.0, 23.0, 22.7, 21.2, 20.94, 20.91, 20.9, 20.8, 19.5, 18.8, 12.0. HRMS
(ESI-Ion Trap) m/z: [M + Na]+ calcd for C41H64O10Na 739.4397;
found 739.4404.
[α]2D3 +30.2 (c 0.54, CHCl3:MeOH). Rf = 0.26 (dichloromethane:-
methanol 9:1). mp = 220.1−221.8 °C. 1H NMR NOESY presaturation
1D to suppress water (4.23 ppm) and the spectrum was locked to
CD3OD (800 MHZ, CDCl3: CD3OD (3:1)): δ 0.49 (s, 3H), 0.67 (d, J
= 6.6 Hz, 3H), 0.68 (d, J = 6.6 Hz, 3H), 0.74 (d, J = 6.6 Hz, 3H),
0.78−0.81 (m, 1H), 0.82 (s, 3H), 0.83−0.99 (m, 8H), 1.05−1.13 (m,
1H), 1.14−1.18 (m, 3H), 1.24−1.39 (m, 8H), 1.64−1.66 (m, 1H),
1.67−1.69 (m, 1H), 1.72−1.74 (m, 1H), 1.76−1.79 (m, 1H), 1.82−
1.83 (m, 1H), 2.14−2.17 (m, 2H), 3.22 (dd, J = 3.8, 10.2 Hz, H2, 1H),
3.23 (app. t, J = 9.6 Hz, H4, 1H), 3.28−3.32 (m, 1H), 3.47 (app. t, J =
9.6 Hz, H3, 1H), 3.46−3.49 (m, H5, 1H), 3.58−3.60 (m, H6, H6′,
2H), 4.79 (d, J = 3.8 Hz, H1, 1H), 5.15−5.20 (m, 1H). 13C NMR (200
MHz, CDCl3: CD3OD (3:1)): δ 140.4, 121.8, 96.7 (C1), 77.5, 73.8
(C3), 71.8 (C2), 71.5 (C5), 70.0 (C4), 61.3 (C6), 56.6, 55.9, 49.9,
42.1, 39.8, 39.6, 39.3, 36.8, 36.5, 36.0, 35.6, 31.7, 31.6, 28.0, 27.8, 27.4,
24.0, 23.6, 22.5, 22.2, 20.8, 19.0, 18.4, 11.6. HRMS (MALDI-TOF) m/
z: [M + Na]+ calcd for C33H56O6Na 571.3969; found 571.3937.
Cholesteryl-6-O-tetradecanoyl-α-D-glucopyranoside (2).
Compound 1 (20.0 mg, 0.036 mmol) was placed into an oven-dried
screw cap vial. Then, anhydrous acetone (0.9 mL) and commercial
lipase Novozym 435 (36 mg on solid support) were added to the vial,
followed by myristic vinyl ester (6.0 equiv, 60 μL). The vial was then
placed on a thermoplate shaker at 40 °C for 18 h. Upon completion of
the reaction, the solution was decanted into a round-bottom flask and
the enzyme was rinsed twice with methanol, followed by chloroform.
The organic solvents were combined and concentrated to afford a
white solid. This solid was purified by flash chromatography using
100% hexanes, and the characterization data matched that of what was
previously reported.14
Cholesteryl-6-O-tetradecanoyl-α-D-galactopyranoside (4).
Compound 5 (10.0 mg, 0.018 mmol) was placed into an oven-dried
screw cap vial. Then, anhydrous pyridine (60 μL), followed by THF
(240 μL), was added to give a M = 0.1 concentration of 13. Then,
commercial lipase Novozym 435 (10 mg on solid support) was added
to the vial, followed by myristic vinyl ester (18.0 equiv, 95 μL, 0.32
mmol). The vial was then placed on a thermoplate shaker at 40 °C for
4 days, after which the solution was diluted with chloroform and
decanted into a round-bottom flask. The enzyme was rinsed twice with
methanol, followed by chloroform. The organic solvents were
combined and concentrated to afford a white solid, which was purified
by flash chromatography on a 3.6 cm diameter glass column that was
25.5 cm long plugged with cotton and filled with 150 mL of silica. A
gradient solvent system of 100% hexanes (100 mL) with increasing
polarity to 1:1 hexanes:ethyl acetate (100 mL), followed by 100% ethyl
acetate (100 mL) to remove the excess fatty acid, which does not stain
on the TLC plate, was used. After using 100 mL of each solvent ratio, a
9:1 ethyl acetate:methanol solvent system was used to elute compound
4 (10 mg, 0.014 mmol) in 77% yield. Rf = 0.73 (ethyl acetate:methanol
Cholesteryl-2,3,4,6-tetra-O-acetyl-β-D-glucopyranose. The β-
anomer had an HPLC retention time of 16.8 min and was obtained as
a white powder and was then recrystallized from diethyl ether and
methanol to afford glasslike, rod-shaped crystals. mp = 149.7−152.3
9:1). [α]2D4 +41.1 (c 0.48, CH2Cl2). H NMR (800 MHZ, CDCl3): δ
1
0.67 (s, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.87 (d, J = 6.6 Hz, 3H), 0.89 (t,
J = 7.2 Hz, 3H), 0.92 (d, J = 6.6 Hz, 3H), 0.96−0.99 (m, 1H), 1.00 (s,
3H), 1.02−1.17 (m, 7H), 1.25−1.27 (m, 22H), 1.27−1.36 (m, 2H),
1.42−1.62 (m, 13H), 1.81−1.89 (m, 4H), 1.95−2.02 (m, 2H), 2.31−
2.39 (m, 4H), 3.48−3.50 (m, 1H), 3.74−3.76 (m, H2, H3, 2H), 3.95−
°C. Rf = 0.70 (hexanes:acetone 6:4). [α]2D0 −10.0 (c 1.0, CH2Cl2). H
1
NMR (800 MHZ, CDCl3): δ 0.66 (s, 3H), 0.85 (d, J = 6.6 Hz, 3H),
0.86 (d, J = 6.0 Hz, 3H), 0.91 (d, J = 6.0 Hz, 3H), 0.98 (s, 3H), 1.00−
1.17 (m, 9H), 1.22−1.28 (m, 2H), 1.32−1.37 (m, 2H), 1.41−1.54 (m,
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3.96 (m, H4, 1H), 4.07 (app. t, J = 4.8 Hz, H5, 1H), 4.21 (dd, J = 8.0,
12.0 Hz, H6, 1H), 4.41 (dd, J = 4.8, 12.0 Hz, H6′, 1H), 5.06 (d, J = 4.0
Hz, H1, 1H), 5.35−5.36 (m, 1 H). 13C NMR (200 MHz, CDCl3): δ
174.1, 140.4, 122.4, 97.3 (C1), 78.6, 71.3 (C3), 69.6 (C2), 69.0 (C4),
68.3 (C5), 63.5(C6), 56.8, 56.3, 50.2, 42.4, 40.2, 39.8, 39.6, 37.1, 36.8,
36.3, 35.9, 34.4, 32.1, 32.0, 31.9, 29.9, 29.89, 29.85, 29.84, 29.8, 29.53,
29.5, 29.4, 28.4, 28.2, 28.1, 25.1, 24.4, 24.0, 23.0, 22.9, 22.7, 21.6, 19.5,
18.8, 14.3, 12.0. HRMS (ESI-Ion Trap) m/z: [M + Na]+ calcd for
C47H82O7Na 781.5958; found 781.6014.
Cholesteryl-α-D-galactopyranose (5). In a flame-dried argon-
purged round-bottom flask containing 4 Å molecular sieves (300 mg)
were added TBAI (140 mg, 0.38 mmol, 1.5 equiv), cholesterol (32 mg,
0.083 mmol, 0.3 equiv), and anhydrous dichloromethane (2.5 mL).
Hz, 3H), 0.76−0.79 (m, 1H), 0.85 (s, 3H), 0.87−1.03 (m, 8H), 1.08−
1.13 (m, 5H), 1.16−1.23 (m, 3H), 1.27−1.44 (m, 8H), 1.66−1.68 (m,
1H), 1.69−1.72 (m, 1H), 1.72−1.73 (m, 1H), 1.76−1.79 (m, 1H),
1.80−1.87 (m, 1H), 2.19−2.20 (m, 2H), 3.31−3.35 (m, 1H), 3.57−
3.70 (m, H3, H6, H6′, H2, 4H), 3.75 (app. t, J = 6.0 Hz, H5, 1H),
3.83−3.84 (m, H4, 1H), 4.87 (d, J = 3.5 Hz, H1, 1H), 5.18−5.19 (m,
1H). 13C NMR (200 MHz, CDCl3: CD3OD (3:1)): δ 140.4, 121.9,
97.1 (C1), 77.6, 70.3 (C3), 69.9 (C5), 69.7 (C4), 68.9 (C2), 61.6
(C6), 56.6, 56.0, 50.0, 42.2, 39.9, 39.6, 39.4, 36.9, 36.5, 36.0, 35.7, 31.7,
31.6, 28.1, 27.9, 27.6, 24.1, 23.6, 22.6, 22.3, 20.9, 19.1, 18.5, 11.6.
HRMS (MALDI-TOF) m/z: [M + Na]+ calcd for C33H56O6Na
571.3969; found 571.3968.
Hunig’s base (DIPEA) (90 μL, 0.51 mmol, 2.0 equiv) was then added
̈
ASSOCIATED CONTENT
■
to this solution, which was stirred for 30 min. In a separate flame-dried
argon-purged round-bottom flask was placed per-O-trimethylsilylated
galactose made from the synthetic protocol of Bhat and Gervay-
Hague15 (140 mg, 0.25 mmol) and azeotroped with anhydrous
chloroform (2 × 3 mL). After azeotroping, the sugar was placed under
vacuum for at least 2 h prior to use. The resulting residue was then
diluted with anhydrous dichloromethane (2.5 mL), and TMSI (40 μL,
0.28 mmol, 1.1 equiv) was added and allowed to react for 10 min. The
in situ generated galactosyl iodide was then transferred via cannula
into the acceptor flask and allowed to stir for 2 days at rt. The solvent
was then filtered to remove the molecular sieves, and the solvent was
removed under reduced pressure. A 1:1 ratio of ethyl acetate and
hexanes (20 mL) was then added to the round-bottom flask, and the
flask was cooled using dry ice. The cooling helped solidify the TBAI,
which was then filtered off, and the solution was again concentrated.
Methanol (5 mL) and Dowex 50WX8-200 acidic resin (300 mg) were
then added, and the mixture was stirred for 2 h. The solution was then
filtered to remove the acidic resin and concentrated under reduced
pressure. The crude reaction mixture was then acetylated to identify
the α:β-ratio of the glycosylation. However, no β-anomer was
observed or isolated.
S
* Supporting Information
NMR, MS, and X-ray diffraction data. This material is available
free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMATION
■
Corresponding Author
*E-mail: jgervayhague@ucdavis.edu (J.G.-H.).
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This work was supported by NIH R01GM090262, NSF CHE-
0196482, NSF CRIF Program (CHE-9808183), and NSF
OSTI 97-24412. Grants for 600 and 800 MHz instruments
include NSF DBIO 722538 and NIH PR1973. J.G.-H. gratefully
acknowledges support from the National Science Foundation
for independent research and development during her tenure as
Divison Director of Chemistry.
Cholesteryl-2,3,4,6-tetra-O-acetyl-α-D-galactopyranose. The
α-anomer of the cholesteryl galactoside was further purified by HPLC
using a silica microsorb normal phase 250 × 10 mm column and a
gradient mobile phase starting with 20% ethyl acetate in hexanes with
gradual polarity increasing to 50% ethyl acetate in hexanes over a 20
min time period and a flow rate of 4 mL per min. It had an HPLC
retention time of 9.6 min and was obtained as a white foam. The white
foam was also recrystallized from diethyl ether and methanol. Rf = 0.72
(hexanes:acetone 6:4). [α]2D0 +8.6 (c 0.40, CH2Cl2). mp = 153.6−
REFERENCES
■
(1) Algood, H. M. S.; Cover, T. L. Clin. Microbiol. Rev. 2006, 19, 597.
(2) Lee, S. J.; Lee, B. I.; Suh, S. W. Proteins 2011, 79, 2321.
(3) Peek, R. M.; Blaser, M. J. Nat. Rev. Cancer 2002, 2, 28.
(4) Kobayashi, M.; Lee, H.; Nakayama, J.; Fukuda, M. Curr. Drug
Metab. 2009, 10, 29.
(5) Hirai, Y.; Haque, M.; Yoshida, T.; Yokota, K.; Yasuda, T.; Oguma,
K. J. Bacteriol. 1995, 177, 5327.
(6) Haque, M.; Hirai, Y.; Yokota, K.; Mori, N.; Jahan, I.; Ito, H.;
Hotta, H.; Yano, I.; Kanemasa, Y.; Oguma, K. J. Bacteriol. 1996, 178,
2065.
(7) Wunder, C.; Churin, Y.; Winau, F.; Warnecke, D.; Vieth, M.;
Lindner, B.; Zahringer, U.; Mollenkopf, H.-J.; Heinz, E.; Meyer, T. F.
Nat. Med. 2006, 12, 1030.
(8) Beigier-Bompadre, M.; Moos, V.; Belogolova, E.; Allers, K.;
Schneider, T.; Churin, Y.; Ignatius, R.; Meyer, T. F.; Aebischer, T. J.
Infect. Dis. 2011, 204, 1339.
(9) Lebrun, A.-H.; Wunder, C.; Hildebrand, J.; Churin, Y.; Zahringer,
U.; Lindner, B.; Meyer, T. F.; Heinz, E.; Warnecke, D. J. Biol. Chem.
2006, 281, 27765.
1
155.9 °C. H NMR (800 MHZ, CDCl3): δ 0.68 (s, 3H), 0.85 (d, J =
6.6 Hz, 3H), 0.86 (d, J = 6.6 Hz, 3H), 0.91 (d, J = 6.6 Hz, 3H), 0.96−
1.01 (m, 1H), 1.02 (s, 3H), 1.03−1.14 (m, 8H), 1.23−1.28 (m, 2H),
1.31−1.38 (m, 2H), 1.44−1.52 (m, 7H), 1.53−1.56 (m, 2H), 1.74−
1.77 (m, 1H), 1.81−1.85 (m, 2H), 1.96−2.01 (m, 1H), 1.99 (s, 3H),
2.04 (s, 3H), 2.07 (s, 3H), 2.13 (s, 3H), 2.30 (ddd, J = 2.2, 4.9, 13.2
Hz, 1H), 3.34−2.38 (m, 1H), 3.40−3.44 (m, 1H), 4.07 (dd, J = 7.2,
11.2 Hz, H6, 1H), 4.11 (dd, J = 5.6, 11.2 Hz, H6′, 1H), 4.34 (app t, J =
7.2 Hz, H5, 1H), 5.06 (dd, J = 4.0, 11.2 Hz, H2, 1H), 5.25 (d, J = 4.0
Hz, H1, 1H), 5.35 (dd, J = 3.2, 11.2 Hz, H3, 1H), 5.35−5.36 (m, 1H),
5.45 (dd, J = 1.6, 3.2 Hz, H4, 1H). 13C NMR (200 MHz, CDCl3): δ
170.6, 170.5, 170.4, 170.2, 140.5, 122.3, 95.0 (C1), 78.9, 68.5 (C2),
68.4 (C4), 67.8 (C3), 66.4 (C5), 62.1 (C6), 56.8, 56.3, 50.3, 42.5,
40.1, 39.9, 39.7, 37.1, 36.8, 36.3, 35.9, 32.1, 32.0, 29.8, 28.4, 28.2, 24.4,
24.0, 23.0, 22.7, 21.2, 21.0, 20.9, 20.8, 20.8, 19.5, 18.9, 12.0. HRMS
(ESI-Ion Trap) m/z: [M + Na]+ calcd for C41H64O10Na 739.4397;
found 739.4404.
̈
(10) Davis, R. A.; Lin, C.-H.; Gervay-Hague, J. Chem. Commun. 2012,
48, 9083.
(11) Lee, H.; Wang, P.; Hoshino, H.; Ito, Y.; Kobayashi, M.;
Nakayama, J.; Seeberger, P. H.; Fukuda, M. Glycobiology 2008, 18, 549.
(12) Du, W.; Kulkarni, S. S.; Gervay-Hague, J. Chem. Commun. 2007,
2336.
(13) Schombs, M.; Park, F. E.; Du, W.; Kulkarni, S. S.; Gervay-
Hague, J. J. Org. Chem. 2010, 75, 4891.
(14) Kulkarni, S. S.; Gervay-Hague, J. Org. Lett. 2008, 10, 4739.
(15) Bhat, A. S.; Gervay-Hague, J. Org. Lett. 2001, 3, 2081.
(16) Vemula, P. K.; Li, J.; John, G. J. Am. Chem. Soc. 2006, 128, 8932.
(17) Waldmann, H.; Sebastian, D. Chem. Rev. 1994, 94, 911.
Alternatively, the crude solid after removing the trimethylsilyl
protecting groups could be purified with a 9:1 dichloromethane:me-
thanol solvent system, and the white powder was collected to afford
1
the target compound (5) (39 mg, 0.071 mmol, 85%). The H NMR
utilized NOESY presaturation 1D to suppress water (4.23 ppm) and
the spectrum was locked to CD3OD. Rf = 0.25 (dichloromethane:-
methanol 9:1). [α]2D5 +55.6 (c 0.52, CHCl3:MeOH). mp = 223.6−
1
224.3 °C. H NMR (800 MHZ, CDCl3: CD3OD (3:1)): δ 0.53 (s,
3H), 0.70 (d, J = 6.6 Hz, 3H), 0.71 (d, J = 6.6 Hz, 3H), 0.77 (d, J = 6.6
8451
dx.doi.org/10.1021/jo501371h | J. Org. Chem. 2014, 79, 8447−8452

The Journal of Organic Chemistry
Note
(18) Gayot, S.; Santarelli, X.; Coulon, D. J. Biotechnology 2003, 101,
29.
(19) Danieli, B.; Luisetti, M.; Sampognaro, G.; Carrea, G.; Riva, S. J.
Mol. Catal. B: Enzym. 1997, 3, 193.
(20) Twibanire, J. K.; Omran, R. P.; Grindley, T. B. Org. Lett. 2012,
14, 3909.
8452
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