Rearrangement reactions in the fluorination of D-glucopyranoside at?the C-4 position by DAST

Article abstract of DOI:10.1016/j.tet.2016.06.075

Attempts to synthesize 4-deoxy-4-fluoro-KRN-7000 (2), a potential immune adjuvant, by fluorination of 4-hydroxy of α-D-glucopyranoside using DAST, were not successful. Instead, the unusual 5-deoxy-5-fluoro β-L-altrofurnoside and the corresponding 4-deoxy-4-fluoro α-D-glucopyranoside with retained configuration were obtained. In the presence of polar solvents, the reaction afforded the epoxide product, suggesting the bicyclic oxiranium ion intermediate to be involved in this reaction. Compound 2 was eventually achieved by treating 4-methanesufonated glucopyranoside with fluoride ion and found to be a weak agonist for CD1d and NKT cell activation.

Full text of DOI:10.1016/j.tet.2016.06.075

Tetrahedron 72 (2016) 5571e5577
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Rearrangement reactions in the fluorination of -glucopyranoside
D
at the C-4 position by DAST
*
Tzung-Sheng Lin, Wei-Tse Tsai, Pi-Hui Liang
School of Pharmacy, College of Medicine, National Taiwan University, No. 17, Xu-Zhou Road, Taipei 100, Taiwan
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 20 May 2016
Received in revised form 27 June 2016
Accepted 29 June 2016
Available online 30 June 2016
Attempts to synthesize 4-deoxy-4-fluoro-KRN-7000 (2), a potential immune adjuvant, by fluorination of
4-hydroxy of
a
-
D
-glucopyranoside using DAST, were not successful. Instead, the unusual 5-deoxy-5-
-glucopyranoside with retained
fluoro -altrofurnoside and the corresponding 4-deoxy-4-fluoro a-D
b
-
L
configuration were obtained. In the presence of polar solvents, the reaction afforded the epoxide product,
suggesting the bicyclic oxiranium ion intermediate to be involved in this reaction. Compound 2 was
eventually achieved by treating 4-methanesufonated glucopyranoside with fluoride ion and found to be
a weak agonist for CD1d and NKT cell activation.
Keywords:
DAST
Fluorinated carbohydrate
Ring constrain
Glycolipid
Ó 2016 Elsevier Ltd. All rights reserved.
1. Introduction
the most selective Th1-biased compound.7 Thus, it is believed that
the stability of glycosidic bond of the glycolipid strong correlates
Fluorine is the most electronegative of the elements, and its
powerful electron-withdrawing properties can profoundly affect
the reactivity of the molecules that contain it. For example, sub-
stitution of hydrogen or hydroxyl groups for fluorine can change
the hydrophobicity of molecules.1 Some fluorine-containing com-
pounds in pharmaceuticals are used for disease treatment, such as
fluorouracil and capecitabine.2 The presence of a fluorine atom in
carbohydrate often increases the chemical and metabolic stability
of the glycosidic bond, which in turn can enhance the therapeutic
value of sugar- or nucleoside-based pharmaceuticals.3
with the cytokine secretion profile.
As part of our ongoing interest in the synthesis of cytokine bi-
ased
a
-GalCer-related derivatives,8 the synthesis of 4-deoxy-4-
fluoro-KRN-7000 (2, Scheme 1) was pursued. The presence of
fluorine at the 4-position of compound 2 was expected to both
enhance the metabolic stability of the glycosidic bond and allow
the formation of H-bonds with TCR
a-chain and main chain of Phe
29a
in CD1d protein pocket.9 During the course of this work, the 4-
deoxy-4-fluoro-KRN-7000 related sphinganine analog (3) was re-
ported to have similar potency as KRN-7000 in the TH2 cytokine
secretion.10 This study revealed that compound 3 cannot activate
a
-GalCer (alpha-galactosyl ceramide, also known as KRN7000, 1,
Scheme 1), a glycolipid composed of -linked galactose and
ceramide, is the most potent ligand for invariant natural killer T
cells (iNKT cells).4 Interaction of
-GalCer and with iNKT cells re-
sults in the secretion of T helper 1 (TH1, e.g., IFN- ) and T helper 2
a
TH1 cytokines. Compound 3 did not have 4-a-hydroxy group at
ceramide portion which was also important for cytokine stimula-
tion,9 thus promoted us to investigate the activity of compound 2.
The nucleophilic fluorinating agent-DAST (diethylaminosulfur
trifluoride) is widely used in the preparation of deoxyfluoro
sugars.11 Fluorination with DAST usually occurs with inversion of
configuration (SN2 mechanism), and can be used to convert the 4-
hydroxy group of glucoside to 4-deoxy-4-fluoro-galactoside with
moderate yield.12 Introduction of a fluorine atom to 2 was in-
vestigated by treating intermediate 4 with DAST; however, the
desired product 5 was not obtained (Scheme 1). Careful examina-
tion of the reaction mixture revealed furanoside 6 to be the major
by-product in 34% yield. The unexpected nature of this discovery
prompted us to investigate its formation. An alternative route to 2
was also developed and is reported herein.
a
g
(TH2, e.g., IL-4, IL-10) cytokines.5 Both TH1 and TH2 cytokines have
important biological functions. TH1 cytokines are associated with
anti-infection and antitumor activity and TH2 cytokines are corre-
lated with immunosuppression and immunomodulation.5 Due to
the reciprocal effects of TH1 and TH2 cytokines,6 compounds with
biased cytokine profiles are highly desirable.
a-C-GalCer, the C-
glycosidic bond of which is resistance to hydrolysis, was found to be
* Corresponding author. Tel.: þ886 2 33668694; fax: þ886 2 23919098; e-mail
address: phliang@ntu.edu.tw (P.-H. Liang).
http://dx.doi.org/10.1016/j.tet.2016.06.075
0040-4020/Ó 2016 Elsevier Ltd. All rights reserved.

5572
T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
OH
F
OH
OH
OH
HO
F
O
O
O
O
O
O
C25H51
OH
C25H51
C25H51
HO
HO
HN
HN
OH
HN
OH
OH
OH
O
O
O
C14H29
C14H29
C14
H
29
OH
OH
OH
α-GalCer (1)
2
3
OBn
F
OBn
O
O
DAST, CH2Cl2.
DAST, CH2Cl2.
HO
BnO
N3 OBn
C14H29
BnO
N3 OBn
C14H29
BnO
O
BnO
O
OBn
4
5
OBn
O
F
N3 OBn
O
C14H29
BnO
BnO
OBn
OBn
6
Scheme 1. Structures of a-GalCer derivatives and our attempt to synthesis of compound 2 by using DAST as a fluorinating agent.
2. Results and discussion
starting material-7). The SN2 type reaction to compound 7 was de-
duced to be of negligible significance because no configurationally
inverted fluoride was detected.
2.1. Fluorination at the C-4 position of
using DAST
D-glucopyranoside
Raising the reaction temperature was found to shorten the re-
action (entry 1 vs 2; and 10 vs 11, Table 1). Increasing or reducing
the number of equivalents of DAST resulted in lower yields (entry
3e5), therefore, three-equivalents of DAST was conclude to be the
optimized amount. Addition of either dimethylaminopyridine
(DMAP), diisopropyl ethylamine (DIPEA) or tetrabutyl ammonia
fluorine (TBAF) disabled the reaction (entry 6e8). Changing the
solvent to diethyl ether or acetonitrile and incorporating methanol
into the work-up was found to increase the ratio of furanose 9. To
our surprise, another epoxide derivative 10 (15% in diethyl ether,
entry 9; 42% in acetonitrile, entry 10 and 11) was formed. Use of the
To investigate the formation of compound 6 from compound 4
(Scheme 1), allyl 2,3,6 -tri-O-benzyl-
-glucopyranoside (7)13 was
a
used as a model compound. To understand the scope of this reaction,
it was repeated under a variety of reaction conditions (Table 1). First,
compound 7 was subjected to the standard procedure, which was
three-equivalents of DAST in refluxing dichloromethane (DCM) for
3 h (entry 1, Table 1). Once all the starting material had been con-
sumed (by TLC), careful separation of products was accomplished
using high performance liquid chromatography (HPLC) to give both
the configuration-retained 8 and the ring-contracted furanose 9 in
a ratio of 5:6 and an isolated yield of 59% (88% yield after recovery of
anomeric
b
form of 7 (allyl 2,3,6-tri-O-benzyl
b
-glucopyranoside)
resulted in similar outcome (entry 12).
Table 1
Results on attempted fluorination at 4-position of glucopyranose 7 in different conditions
OBn
OBn
O
F
DSAT
O
HO
BnO
O
O
F
+
BnO
BnO
BnO
BnO
OBn
O
BnO
O
7
8
9
Entry
Reagents (equiv.)
Solvent
Temp (time)
Product ratioa
Yieldb
1
2
3
4
5
6
7
8
DAST (3)
DAST (3)
DAST (1)
DAST (5)
DAST (10)
DAST (3), DMAP (1)
DAST (3), DIPEA (1)
DAST (3), TBAF (1)
DAST (3)
DCM
DCM
DCM
DCM
DCM
DCM
DCM
DCM
Reflux (3 h)
RT (16 h)
RT (16 h)
RT (16 h)
RT (16 h)
RT (16 h)
RT (16 h)
RT (16 h)
RT (16 h)
40 ꢀC (3 h)
RT (16 h)
Reflux (3 h)
5:6
5:6
d
59
63
NRc
55
7:9
1:1
d
33
NR
NR
NR
45d
46d
48d
60
d
d
9
Diethyl ether
ACN
ACN
1:5
2:7
2:7
5:6
10
11
12e
DAST (3)
DAST (3)
DAST (3)
DCM
a
Ratio of 8: 9 which was determined by HPLC of reaction mixture.
Isolated yields of all products being purified by HPLC.
NR (no reaction, almost starting material was recovered).
Additional epoxide 10 was obtained in a yield of 15e42%.
b
c
d
e
Reaction of
b form of 7 (allyl 2,3,6-tri-O-benzyl b-glucopyranoside).

T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
5573
OBn OMe
intermediate that accounts for the formation of these three
products is compound 12 (Scheme 2). Upon formation of 11, the
1,3-trans relationship between the ring oxygen and sulfonyloxyl
group caused the lone pair from ring oxygen to attack the C-4
position (Scheme 2). Departure of the equatorial disposed O-
SF2NEt2 took place with concomitant formation of the bicyclic
oxiranium ion intermediate 12 which was stabilized in presence of
acetonitrile (or diethyl ether). Nucleophilic attack of 12 by fluoride
ion results in compound 8 (route a; attack at C-4) and 9 (route b;
attack at C-5). The reaction ratio of 8 and 9 is 1:5 and 2:7 in ether
and acetonitrile, respectively, indicating that the formation of 9 is
a stereoelectronic effect. A lone pair of electrons located in an n-
molecular orbital of nitrogen atom from acetonitrile overlaps with
O
BnO
OAllyl
OBn
10
The structure of 9 was determined by NOEs experiments; the
protons interactions of 9 are shown in Fig. 1 (the NOESY spectrum
itself is presented in the Supplemental data). The stereochemis-
try at C-4 of 9 is R form, determined by a positive NOE interaction
between H-1 and H-2; H-1 and H-4; H-2 and H-4; H-3 and H-5;
and H-4 and H-5 (Fig. 1). Morishima N. et al. reported that
treatment of methyl 2,3-di-O-benzyl-6-deoxy-
side with DAST resulted in the formation of methyl 2,3-di-O-
benzyl-5,6-di-deoxy-5-fluoro- -altrofurnoside, in which the
a-D-glucopyrano-
the antibonding
localization of electrons favored the electron of fluorine attack at
side of 12 (route b, a kinetic route). Thus, ‘route a’ could be
s* orbital of the OeC bond of oxirane. The de-
b-L
stereo-formation of the 5-fluoro group was verified by synthesis
of its enantiomer.14 Comparing our NMR spectrums with re-
ported values found they were comparable, suggesting the C-5 to
have the S configuration (Fig. 1).
a
considered a themodynamic route. The reaction was monitored by
TLC and quenched only once all the starting material 7 had been
consumed. However, after work-up, compound 7 was again
detected by TLC. For the purpose of capturing the reaction in-
termediate 12, addition of a few drops of methanol in the work-up
step resulted in isolation of 10, obtained by nucleophilic attack by
methanol on C-1 (route c, Scheme 2). This result is further evi-
dence for the formation of intermediate 12 during this reaction. It
is noteworthy that an analog of compound 10 has been reported as
H4
H1
O
H5
H
OBn
2
F
OAll
an important intermediate in the synthesis of 5-thio-L-altrose,
being obtained from
D-xylose in 12 steps and overall yield of
BnO
w6%.16 In contrast, our process delivered compound 11 from
glucose in only five steps and w20% yield.
D-
H3
OBn
Fig. 1. Observed NOE relationships of compound 9.
When the reaction is performed using DCM; ‘route b’ is pre-
sumed to dominate. There are two reasons for this assumption.
First, the ratio of 5-fluoro-furanose 9 was higher than 4-fluoro-
pyranose 8. Second, there was no reaction in the presence of base
(either DMAP or DIPEA), therefor SNi pathway is less plausible.14
2.2. Plausible mechanism for the formation of side products
It was reported that when saccharides bearing an unprotected,
somewhat hindered hydroxyl group with an electron-rich group at
the vicinal position, are treated with DAST, a product with retained
configuration and side-products showing migration of the neigh-
boring group can be obtained.15 In our case, the anomeric config-
uration of a saccharide 7 seems to be uninvolved in the formation of
products (entry 1 vs 12, Table 1) and the 4-hydroxy groups in
glucopyranoside 4 and 7 are not hindered. Accordingly, it is sug-
gested that the electron-rich groups at the C-3 and C-6 positions of
compounds 4 and 7 may be the contributory factors. The O-SF2NEt2
leaving group in compound 11 must adopt a quasi-axial orientation
in a half-chair conformation of the pyranose ring (Fig. 2), resulting
a disfavorable 1,3-diaxial interaction between the C-3 and C-6
positions.
2.3. Synthesis of compound 2
Since DAST cannot afford the configurationally inverted fluoride
from 7, installation of the axial fluoride was accomplished by a two-
step process (Table 2). Treating 4-OH of glucoside with triflic an-
hydride (Tf2O) and then tri(dimethylamino) sulfonium difluoro-
trimethylsilicate (TASF)17 was reported to able to afford the
configurationally inverted fluoride; however, in our hands, a com-
plex mixture was obtained (entry 2, Table 2, two steps¼25% yield).
Use of TBAF in ACN slightly increased the reaction yield (33%, entry
4). The low yield was hypothesized to be due to the low stability of
the triflic group of intermediate 14, so the use of methanesulfonyl
(Ms) group was pursued instead. Treatment of 7 with MsCl afforded
4-methanesulfonate intermediate 15, which was attacked by a nu-
cleophilic addition of fluoride ion from TBAF (entry 8) in ACN, gave
a complete conversion of inverted fluoride in a satisfactory yield
(75% for two steps). Use of DAST (entry 5) or TASF (entry 6) in place
of TBAF did not result in any reaction.
BnO
F
OBn
O
With an optimized method for the fluorination of 7 in hand, the
desired compound 2 was pursued (Scheme 3). 4-tert-butyldime-
O
F
thylsilyl-2,3,6-tri-O-benzyl 1-thiol-
-glucopyranoside 188 was ac-
D
BnO
S
OAllyl
tivated with Me2S2/Tf2O at ꢁ10 ꢀC and then reacted with the
F
Et2N
ceramide acceptor 198 to afford 20 in 54% yield. Deprotection of 20
11
with TBAF gave 2,3,6-tri-O-benzyl-D-a-glucosyl ceramide 21 in 82%
yield. Treatment of 21 with methanesulfonic chloride (MsCl)
afforded 4-methanesulfonate glucosyl ceramide 22, which was
attacked by TBAF to give configuration-inversed 23 in 83% yield.
Debenzylation of 23 in the presence of 10% Pd(OH)2 under H2 at RT
gave 4-deoxy-4-fluoro-KRN-7000 (2).
Fig. 2. Disfavored transition state for the reaction of
configuration.
7 to give fluorine inverted
Reaction of 7 with DAST in polar solvents gave stereospecific
configuration-retained 4-fluorine glucopyranose 8, 5-fluorine
altrofurnose 9, and 4,5 epoxide open-chain 10. One plausible
In order to ascertain the ability of compound 2 to activate NKT
cells, a mouse B lymphoma cell line (A20-CD1d) was selected as

5574
T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
OBn
O
F
δ-
N
BnO
BnO
a
BnO
OAllyl
OBn
O
BnO
F
b
8
F
O
DAST
Et2N
HO
BnO
BnO
S
O
O
O
F
OAllyl
F
BnO
OBn
BnO
F
a
OAllyl
OAllyl
OBn
b
BnO
OAllyl
7
12
BnO
BnO
11
9
c
δ-
N
OBn
O
BnO
BnO
OBn OMe
O
c
HOMe
O
BnO
BnO
OAllyl
BnO
13
BnO
OAllyl
OBn
OAllyl
10
12
Scheme 2. Plausible mechanism for the formation of side reactions in entries 10e11 of Table 1.
Table 2
Attempts to effect the fluorination at the 4-position of glucopyranose 7 via an SN2 mechanism
OBn
O
TfO
BnO
Fluorination
Protocol A
OBn
Tf2O, pyridine
BnO
O
F
OBn
O
O
14
HO
BnO
BnO
BnO
OBn
O
O
BnO
O
16
MsO
BnO
7
MsCl, pridine
BnO
Protocol B
O
15
Entry
Protocol
Reagent (equiv.)
Solvent
Temp (time)
Yielda
1
2
3
4
5
6
7
8
A
A
A
A
B
B
B
B
DAST (3)
TASF (3)
TBAF (3)
TBAF (3)
DAST (3)
TASF (3)
TBAF (3)
TBAF (3)
DCM
DCM
DCM
ACN
DCM
DCM
DCM
ACN
Reflux (16 h)
Reflux (16 h)
Reflux (4 h)
Reflux (4 h)
Reflux (24 h)
Reflux (24 h)
Reflux (24 h)
Reflux (24 h)
NRb
25
23
33
NRb
NRb
NRb
75b
a
Yield of two steps.
NR (no reaction, almost starting material was recovered).
b
O
HN
C25H51
OBn
O
O
OBn
O
OBn
OBn
O
HO
HN
C25H51
OBn
O
HN
C25H51
OBn
C14H29
TBDMSO
BnO
HO
BnO
b
19
TBDMSO
BnO
OBn
BnO
BnO
O
O
STol
BnO
18
C14H29
C14H29
a
OBn
OBn
20
21
O
O
OBn
OH
O
F
OBn
O
F
MsO
BnO
HN
C25H51
OBn
HN
C25H51
OH
O
O
e
c
d
HN
C25H51
OBn
HO
BnO
BnO
HO
O
O
BnO
O
C14H29
C14H29
C14H29
OBn
OH
OBn
22
23
2
Scheme 3. Alternative route to give 4-deoxy-4fluoro-KRN-7000 (2). a) Me2S2, Tf2O, DCM, 54%; b) TBAF, THF, 50 ꢀC, 82%; c) MsCl, pyridine, 96%; d) TBAF, ACN, reflux, 83%; e) H2,
Pd(OH)2/C, rt, 64%.

T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
5575
a source of antigen-presenting cells for loading with the glycolipid.
34%) as hyaline oils: [
a
]
25þ12 (c 0.0017, CHCl3); IR (neat) nmax 2923,
D
After co-incubation with V
a
14-TCR-expressing mNK1.2 cells as ef-
2846,1460,1104 cmꢁ1; 1H NMR (CDCl3, 600 MHz)
d
7.39e7.29 (25H,
fector cells, IL-2 secretion was measured by ELISA.8 Compound 2
induced much less IL-2 cytokine secretions than KRN-7000 (1),
which was not found to have a significant effect (data not shown).
m, Ph), 4.97 (1H, t, J¼3.6 Hz, H-1), 4.67 (1H, dm, J¼48.6 Hz, H-5),
4.74e4.51 (10H, m, CH2Ph), 4.40 (1H, t, J¼6.0 Hz, H-3), 4.14 (1H, m,
H-4), 4.12 (1H, dd, J¼6.0, 3.6 Hz, H-2), 4.05 (1H, dd, J¼10.8, 2.4 Hz,
H2-7), 3.84 (1H, m, H-8), 3.67 (2H, m, H2-6), 3.66 (1H, m, H-10), 3.65
(1H, m, H-9), 3.59 (1H, dd, J¼10.2, 7.8 Hz, H2-7), 1.59e1.28 (26H, m,
eCH2CH2e), 0.92 (3H, t, J¼6.6 Hz, -CH3); 13C NMR (CDCl3, 150 MHz)
Since a
-C-GalCer was reported inactive in vitro and active in vivo,7b
whether compound 2 is able to activate iNKT cells in vivo is cur-
rently being investigated, and the results will be reported in a due
course.
d
138.3, 138.0, 137.9, 137.9, 137.7 (C-Ph), 128.4e127.6 (CH-Ph), 101.5
(C-1), 92.5 (d, J¼175.5 Hz, C-5), 84.1 (C-2), 82.8 (C-3), 79.6 (C-10),
79.0 (C-9), 78.8 (d, J¼28.5 Hz, C-4), 73.5, 73.5, 72.4, 72.4, 72.1
(CH2Ph), 69.0 (d, J¼19.5 Hz, C-6), 68.3 (C-7), 62.4 (C-8), 31.9e22.7
(eCH2CH2-), 14.1 (eCH3); HR-ESIMS m/z 980.5667 (MþNa)þ (calcd
for C59H76FN3Na O7, 980.5565).
3. Conclusion
Treatment of 4-hydroxy group of
gave 5-deoxy-5-fluoro - altrofurnoside 9 as a major product, and
the corresponding 4-deoxy-4-fluoro -glucopyranoside 8 with
a-D-glucoside 7 with DAST
b-L
a-D
retained configuration as the minor product. When the reaction
was undertaken in the presence of polar solvents, the epoxide side
product 10 was isolated, suggesting the bicyclic oxiranium ion in-
termediate 12 to be involved in this reaction. A novel method to
synthesize compounds 9 and 10 from the relatively simple starting
4.4. Fluorination of compound 7
material-
D
-glucose in a few steps and with acceptable yields was
4.4.1. General procedure. DAST (47.7 mL, 0.39 mmol) was added to
developed. However, synthesis of 4-deoxy-4-fluoro-KRN-7000 (2)
was achieved by treating 4-methanesufonated glucopyranoside
with SN2 attack of fluoride from TBAF. Though compound 2 was
found to a weak agonist for CD1d and NKT cell activation, it might
be of use in the immune-compromise therapy.
a solution of compound 713 (63.6 mg, 0.13 mmol) in either dry DCM
(8 mL), dry ACN, dry diethyl ether under N2 (Entry 1e11 of Table 1).
The reaction was heated at 40 ꢀC for 3 h or at RT for 16 h, with
stirring. The mixture was diluted with a few drops of methanol and
then immediately poured onto iced saturated aqueous sodium
hydrogen carbonate. The aqueous layer was extracted with DCM
and the combined organic layers were washed with brine, dried
(MgSO4) and concentrated. The ratio for the products was moni-
tored by the reaction residue by HPLC (C18 column (4.6
4. Experimental
4.1. General methods
mmꢂ250 mm, 5 m; Thermo), MeOH: H2O¼80: 20, flow¼1 mL/
m
All reagents and solvents were reagent grades and were used
without further purification unless otherwise stated. The progress
of the reaction was monitored by analytical TLC on 0.25 mm E.
Merck silica gel 60 F254 using p-anisaldehyde, ninhydrine, and ce-
rium molybdate as visualizing agents. Bruker-AV-400 (400 MHz)
and Bruker-AV-600 (600 MHz) with TCI cryo probe and a BACS 60
min, UV 214 nm). For further purification, the reaction mixture was
evaporated under vacuum and purified by column chromatography
on silica gel (Hexane: EtOAc, 9: 1) to afford the residue. The residue
was purified by HPLC (syncronis C18 column (4.6 mmꢂ250 mm,
5
mm), MeOH: H2O¼80: 20, flow¼4.5 mL/min to get products 8
(retention time: 36.8), 9 (retention time 43.9 min), 10 (retention
sample changer. Chemical shift (d) are given in parts per million
time: 28 min).
relative to 1H: 7.24 ppm, 13C: 77.0 ppm for CDCl3 and 1H: 8.74 ppm,
13C: 150.35 ppm for pyridine-D5. The splitting patterns are re-
ported as s (singlet), bs (broad singlet), d (doublet), bd (broad
doublet), t (triplet), q (quartet), dd (double doublet), m (multiplet).
Coupling constant (J) was given in Hz. Peak assignments were
confirmed using 2DNMR (COSY, HSQC) techniques. Exact mass
measurements were performed on VG platform electrospray ESI/
MS or BioTOF II (Taiwan). Melting point was measured by a melting
point apparatus (MP-1D), infrared spectroscopy was measure by
FT-IR spectrometer Nicolet iS5 and optical rotation was measured
by a polarimeter (Jasco DIP-370).
4.4.2. Allyl 4-deoxy-4-fluoro-2,3,6-tri-O-benzyl 1-a-D-glucopyrano-
side (9). Clear oils; [
a
]
30þ36 (c 0.04, CHCl3); IR (neat) nmax 3023,
D
1559, 1198, 1164 cmꢁ1; 1H-NMR (CDCl3, 400 MHz)
d
7.39e7.23 (15H,
m, Ph), 5.93 (1H, m), 5.31 (1H, dd, J¼17.2, 1.6 Hz), 5.21 (1H, dd,
J¼10.4, 1.6 Hz),4.84 (2H, s), 4.79 (1H, d, J¼2.4 Hz),4.77 (1H, d,
J¼6.4 Hz), 4.63 (2H, d, J¼12 Hz), 4.57 (2H, d, J¼9.6 Hz), 4.52 (1H,
ddd, J¼36.8, 9.6, 8.4 Hz), 4.17 (1H, ddd, J¼14.4, 5.2, 1.6 Hz),
4.07e3.98 (2H, m), 3.89 (1H, m), 3.67 (1H, dd, J¼7.2, 2.4 Hz), 3.51
(1H, dd, J¼9.6, 3.6 Hz); 13C NMR (CDCl3, 100 MHz)
d 138.5, 138.0,
137.8, 133.5(CH-8), 128.4e127.5, 118.3(CH2-9), 95.7 (C-1), 89.7 (d,
J¼182 Hz, C-4), 79.8 (d, J¼17 Hz, C-3), 78.5, 75.2, 73.5, 73,4, 68.53 (d,
J¼18 Hz, C-5), 68.5, 68.0; HR-ESIMS m/z 493.2395 (MþH)þ (calcd
for C30H34FO5:493.2390).
4.2. Biological assay
Both a-GalCer and compound 2 were dissolved in DMSO (100%)
at concentrations of 1 mM. Before use, the solutions were diluted
with phosphate-buffered saline (PBS, pH 7.4) to obtain final con-
centrations. Cytokine release from the activation of A20-CD1d
system was conducted using the procedure reported previously.8
4.4.3. Allyl 5-deoxy-5-fluoro-2,3,6-tri-O-benzyl b-L-altrofurnoside
(10). Hyaline oils; [
a]
25þ20 (c 0.001, CHCl3); IR (neat) nmax 2924,
D
2873, 1105, 1027, 697 cmꢁ1; 1H NMR (CDCl3, 600 MHz)
d 7.33e7.25
(15H, m, Ph), 5.84 (1H, m, H-8), 5.25 (1H, dd, J¼17.2, 1.2 Hz, H2-9),
5.17 (1H, dd, J¼10.8, 1.2 Hz, H2-9), 4.87 (1H, dd, J¼4.2, 3.6 Hz, H-1),
4.66 (1H, ddt, J¼36.8, 6.0, 2.0, H-5), 4.63e4.54 (6H, m, CH2Ph), 4.33
(1H, dd, J¼6.4, 5.6 Hz, H-3), 4.10 (1H, q, J¼2.4 Hz, H2-7), 4.07 (1H, d,
J¼3.2 Hz, H-4), 4.05 (1H, dd, J¼6.4, 4.2 Hz, H-2), 3.88 (1H, dd,
J¼12.8, 6.4 Hz, H2-7), 3.78 (1H, ddd, J¼28.4, 11.6, 2.0 Hz, H2-6), 3.67
(1H, ddd, J¼26.4, 11.6, 5.6 Hz, H2-6); 13C NMR (CDCl3, 150 MHz)
4.3. 2-Azido-3,4-di-O-benzyl-1-O-(50-fluoro-20,30,60-tri-O-ben-
zyl-b-L-altrofurnosyl) D-ribo- 1,3,4-octadecan-triol (6)
To a solution of compound 4 (100 mg, 0.104 mmol, see
Supplemental data for its preparation) in DCM (10 mL) was added
DAST (38.4
mL, 0.31 mmol) and stirred at reflux for 3 h to afford the
d 138.0, 137.9, 137.6 (C-Ph), 133.6 (CH-8), 128.4e127.7 (CH-Ph), 117.9
mixture residue. The residue was separated by column chroma-
tography (Hexane: EtOAc, 8: 1 to 7: 1) again to get pure 6 (34 mg,
(CH2-9), 99.7 (CH-1), 92.8 (d, J¼175.5 Hz, CH-5), 84.2 (CH-2), 83.4
(CH-3), 78.9 (d, J¼28.5 Hz, CH-4), 73.5, 72.6, 72.4 (CH2Ph), 69.2 (t,

5576
T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
J¼19.5 Hz, CH2-6), 68.5 (CH2-7); HR-ESIMS m/z 515.22104 [MþNa]þ
0.056 mmol) in THF (1 mL) and warmed to 50 ꢀC. After the mix-
ture was stirred for 3 h at 50 ꢀC, it was diluted with DCM and
extracted three times with brine and water. The organic layers
were separated, dried over MgSO4, filtered, and concentrated. The
residue was purified by column chromatography (Hexane: EtOAc,
(calcd for C30H33FO5 Na, 515.2210).
4.4.4. 1-Allyl-1-methyl-2,3,6-tri-O-benzyl-4,5-epoxy-a-D-glucoside
(11). Hyaline oils; [
a
]
D
25þ30 (c 0.001, CHCl3); IR (neat) nmax 2918,
2850, 1724, 1272, 1097 cmꢁ1; 1H NMR (CDCl3, 400 MHz)
d
7.35e7.23
5: 1 to 4: 1) to afford 21 (60 mg, 82%) as a syrup; [
a]
25ꢁ15 (c 0.002,
D
(15H, m, Ph), 5.93 (1H, m), 5.29 (1H, dd, J¼17.2, 1.6 Hz), 5.16 (1H, dd,
J¼10.4, 1.6 Hz), 4.84 (1H, d, J¼11.2 Hz), 4.67e4.49 (6H, m), 4.18 (1H,
dt, J¼5.6, 1.6 Hz), 3.59 (1H, dd, J¼6.8, 2.4 Hz), 3.52 (1H, dd, J¼11.6,
2.8 Hz), 3.45 (1H, m), 3.29 (3H, s, -OCH3), 3.24 (1H, dd, J¼11.6,
6.4 Hz), 3.02 (1H, m), 3.98 (1H, dd, J¼6.4, 2.4 Hz); 13C NMR (CDCl3,
CHCl3); IR (neat) nmax 2923, 2853, 1455, 1057 cmꢁ1
;
1H NMR
(CDCl3, 400 MHz)
d
7.32e7.23 (25H, m, Ph), 6.00 (1H, d, J¼8.0 Hz,
NH), 4.97 (1H, d, J¼12.0 Hz), 4.81e4.45 (10H, m), 4.21 (1H, br s),
3.94 (1H, dd, J¼12.0, 8.0 Hz), 3.87 (1H, dd, J¼8.0, 4.0 Hz),
3.80e3.72 (3H, m), 3.62e3.50 (5H, m), 2.41 (1H, s, OH), 1.98e1.23
(74H, m, eCH2CH2e), 0.87 (6H, t, J¼6.0 Hz, eCH3); 13C NMR
100 MHz) d 138.4, 138.3, 138.0, 134.5, 128.4e127.6, 116.9, 103.7 (CH-
1), 80.1, 77.6, 77.3, 75.0, 73.3, 73.2, 70.2, 70.0, 57.0, 54.6, 54.3; HR-
(CDCl3, 100 MHz) d 172.8, 138.6, 138.5, 138.0, 137.9, 137.7,
ESIMS m/z 505.2564 [MþH]þ (calcd for C31H37O6, 505.2585).
128.5e127.6, 98.4, 81.3, 79.9, 79.6, 78.9, 73.6, 73.5, 73.0, 71.8, 71.0,
70.5, 69.7, 68.8, 50.3, 31.9e29.3, 14.1; HRMS (ESI) m/z 1308.9710
[MþH]þ (calcd for C85H130NO9, 1308.9746).
4.4.5. Allyl 4-deoxy-4-fluoro-2,3,6-tri-O-benzyl 1-
anoside (16). The solution of 7 (49 mg, 0.1 mmol) in pyridine (3 mL)
was cooled to 0 ꢀC MsCl (10
L) was added to the solution and
a-D-galactopyr-
m
4.5.3. 1-O-(40-fluoro-20,30,60-tri-O-benzyl-
hexacosanoylamino- -ribo-1,3,4-octadecantriol (23). The solution
of 21 (60 mg, 0.046 mmol) in pyridine (2 mL) was cooled to 0 ꢀC.
Methanesulfonyl chloride (50 L) was added to the solution and
D-galactopyranosyl)-2-
stirred for 8 h. The mixture was quenched with water, and
extracted with DCM for three times. The organic layers were sep-
arated and evaporated under vacuum to obtain compound 15 which
D
m
was added ACN (5 mL) and TBAF/THF(1.0 M, 306
m
L). The mixture
stirred for 4 h. The mixture was quenched with water, and
extracted with DCM for three times. The organic layers were
separated and evaporated under vacuum to obtain 4-O-meth-
was heated at reflux and stirred for 24 h. The mixture was
quenched with NaHCO3 (aq), and extracted with DCM for three
times. Organic phases were separated and evaporated. The residue
was purified by flash column chromatography (hexane: EtOAc, 9: 1)
to get compound 16 (37 mg, 75%) as a syrup: 1H NMR (CDCl3,
anesulfonyl-2,3,6-tri-O-benzyl-
96%) as hyaline oils. Compound 22 (32 mg, 0.022 mmol) in dry
ACN (5 mL) under N2 was subjected to TBAF/THF (1.0 M, 66 L,
a-GalCer (19, 64 mg, 0.044 mmol,
m
400 MHz)
d
7.40e7.26 (15H, m, Ph), 5.96e5.86 (1H, m, H-8), 5.32
0.108 mmol) and then heated to 80e90 ꢀC (reflux) and stirred for
1 d. The reaction mixture was diluted with EtOAc and extracted
with water. The organic phases were separated and evaporated.
The residue was purified by flash column chromatography (hex-
ane: EtOAc, 7: 1e4: 1) to afford compound 23 (24 mg, 83%) as
(1H, dd, J¼17.2, 1.4 Hz, H2-9), 5.17 (1H, dd, J¼10.3, 1.2 Hz, H2-9), 4.89
(1H, dd, J¼28.3, 2.2 Hz, H-4), 4.85 (1H, d, J¼3.6 Hz, H-1), 4.82e4.54
(6H, m, CH2Ph), 4.15 (1H, dd, J¼13.0, 5.1 Hz), 4.04e3.88(4H, m), 3.66
(1H, dd, J¼10.8, 7.2 Hz), 3.58 (1H, dd, J¼7.44, 2.4 Hz); 13C NMR
(CDCl3, 100 MHz)
d
138.3, 138.2, 137.8, (C-Ph), 133.6 (CH-8),
a syrup; [
a
;
]
25ꢁ10 (c 0.001, CHCl3); IR (neat) nmax 2924, 2853, 1455,
D
128.4e127.6 (CH-Ph), 118.1 (CH2-9), 96.26 (CH-1), 87.5 (d, J¼182 Hz,
CH-4), 76.0 (d, J¼18 Hz), 75.7 (CH-2), 73.6, 73.5, 72.7 (CH2Ph), 68.5
(CH2-7), 68.1 (d, J¼18 Hz), 67.9 (d, J¼6 Hz, CH-6); HR-ESIMS m/z
515.2222 [MþNa]þ (calcd for C30H33FO5 Na, 515.2210).
1112 cmꢁ1
1H NMR (CDCl3, 600 MHz)
d
7.37e7.23 (25H, m, Ph),
5.94 (1H, d, J¼7.2 Hz, NH), 4.82 (1H, d, J¼49.8 Hz, H-4), 4.81 (1H, s,
H-1), 4.81e4.72 (4H, m, CH2Ph), 4.63e4.43 (6H, CH2Ph), 4.21 (1H,
br s, H-8), 3.93e3.80 (6H, m, H-2, H-3, H-5, H2-7, H-9), 3.57 (2H,
ddd, J¼34.8, 7.2, 6.6 Hz, H2-6), 3.49 (1H, m, H-10), 1.92e1.22 (74H,
m, -CH2CH2-), 0.87 (6H, t, J¼6.6 Hz, -CH3); 13C NMR (CDCl3,
4.5. Synthesis of 4-deoxy-4-fluoro-KRN-7000 (2)
150 MHz)
d 172.8 (NHCO), 138.6, 138.5, 138.2, 138.0, 137.6 (C-Ph),
4.5.1. 1-O-(4-tert-butyldimethylsilyl-20,30,60-tri-O-benzyl-
D-glucopyr-
128.4e127.6 (CH-Ph), 99.0 (C-1), 87.2 (d, J¼183.0 Hz, C-4), 79.9 (C-
10), 78.9 (C-9), 75.9 (d, J¼18.0 Hz, C-3), 75.9 (C-2), 73.8, 73.6, 73.4,
72.3, 71.8 (CH2Ph), 68.9 (CH2-7), 68.5 (d, J¼18.0 Hz, CH-5), 68.1 (d,
J¼6.0 Hz, CH2-6), 50.2 (CH-8), 30.0e29.4 (eCH2CH2-), 14.1 (eCH3);
HR-ESIMS m/z 1310.9716 [MþH]þ (calcd. for C85H129FNO8,
1310.9702).
anosyl)-2-hexacosanoylamino-D-ribo-1,3,4-octadecantriol
(20). Compound 188 (100 mg, 0.15 mmol), ceramide 198 (109 mg,
ꢀ
0.125 mmol), and 4 A molecular sieve (100 mg) were dried under
vacuo for 2 h, and then DCM/THF (2/1, 6 mL) was added to the
mixture. The solution was cooled to ꢁ10 ꢀC, and then Me2S2-Tf2O
(1.0 M solution in DCM, 225 mL, 0.225 mmol) was added. After stirring
for 30 min, the mixture was quenched with triethylamine (1 mL),
diluted with DCM, filtered through Celite, and washed with DCM. The
solution was extracted with water and the organic layer was sepa-
rated and concentrated to get
by column chromatography (hexane: EtOAc, 8: 1 to 6: 1) to get pure
4. 5. 4. 1-O-(40-40-deoxy-fluoro-
hexacosanoylamino- -ribo-1,3,4-octadecantriol (2). 23 (24 mg,
D -galactopyranosyl)-2-
D
0.018 mmol) in a mixed solvent MeOH/DCM (4/1, 1.5 mL) was
hydrogenated in the presence of 10% Pd(OH)2 (2.5 mg) under H2 at
RT. After stirring overnight, the mixture was filtered through
Celite, and the filtrate was concentrated in vacuo. The residue was
purified by flash column chromatography (MeOH: DCM, 10: 1 to 9:
1) to get 2 (10 mg, 0.012 mmol, 64%) as white powders: mp
a/b mixture form which was purified
a
from 120 (96.5 mg, 54%) and its b form (48 mg, 27%) as clear oils.
20: [
a]
D
25þ27 (c 0.0086, CHCl3); IR (neat) nmax 2923, 2853, 1028,
1072 cmꢁ1; 1H NMR (CDCl3, 400 MHz)
d 7.29e7.18 (25H, m, Ph), 6.12
(1H, d, J¼8.0 Hz, NH), 5.03 (1H, d, J¼8.0 Hz), 4.81e4.43 (10 H, m), 4.18
(1 H, br s), 3.99 (1H, dd, J¼12.0, 4.0 Hz), 3.92 (1H, dd, J¼8.0, 4.0 Hz),
3.77e3.49 (8H, m), 1.99e1.24 (74H, m, eCH2CH2e), 0.87 (6H, t,
229e232 ꢀC; [
a
]
25ꢁ20 (c 0.001, MeOH); IR (neat) nmax 3458, 1728,
1070, 1025 cmꢁ1D
; d 8.50 (1H, d,
1H NMR (d5-pyridine, 600 MHz)
J¼8.9 Hz, NH), 5.59 (1H, d, J¼3.6 Hz, H-1), 5.41 (1H, dd, J¼51.0,
3.6 Hz, H-4), 5.27 (1H, m, H-8), 4.68 (1H, dd, J¼10.8, 5.4 Hz, H2-7),
4.56 (1H, dd, J¼3.6, 2.4 Hz, H-2), 4.54 (1H, m, H-5), 4.48 (1H, ddd,
J¼30.6, 10.2, 2.4 Hz, H-3), 4.39 (1H, dd, J¼10.8, 4.8 Hz, H2-7),
4.35e4.27 (4H, m, H-9, H-10, H2-6), 2.48e1.25 (74H, m,
eCH2CH2e), 0.89 (6H, t, J¼6.6 Hz, -CH3); 13C NMR (d5-pyridine,
J¼8.0 Hz, eCH3); 13C NMR (CDCl3, 100 MHz)
d 172.8, 139.0, 138.7,
138.7, 137.9, 137.7, 128.4e127.1, 98.2, 81.5, 80.7, 80.1, 78.6, 74.8, 73.6,
73.4, 72.9, 72.1, 71.8, 70.8, 69.3, 69.1, 31.9e29.3, 14.1; HR-ESIMS m/z
1423.0619 [MþH]þ (calcd for C91H144NO9Si, 1423.0605).
4. 5. 2. 1-O-(20, 30, 60-tri-O-benzyl-
hexacosanoylamino- -ribo-1,3,4-octadecantriol (21). TBAF (40.5
0.14 mmol) was subjected to the solution of 20 (80 mg,
D
-glucopyranosyl)-2-
L,
150 MHz)
d
173.6 (NHCO), 101.6 (C-1), 91.8 (d, J¼178.5 Hz, C-4),
D
m
76.9 (C-9), 72.8 (C-10), 72.0 (d, J¼18.0 Hz, C-5), 70.5 (C-2), 70.3 (d,
J¼18.0 Hz, C-3), 69.1 (C-7), 61.1 (CH2-6), 51.6 (C-8), 37.1e23.2

T.-S. Lin et al. / Tetrahedron 72 (2016) 5571e5577
5577
(eCH2CH2e), 14.6 (eCH3); HR-ESIMS m/z 860.7368 [MþH]þ (calcd
ꢁ
ꢁ
~
2. (a) Wang, J.; Sanchez-Rosello, M.; Acena, J. L.; del Pozo, C.; Sorochinsky, A. E.;
Fustero, S.; Soloshonok, V. A.; Liu, H. Chem. Rev. 2014, 114, 2432; (b) Hagmann,
W. K. J. Med. Chem. 2008, 51, 4359.
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Acknowledgements
This work was financially supported by the Ministry of Science
and Technology (MOST 104-2320-B-002 -008 -MY3), Taiwan. We
thank Dr. Jung-Tung Hung and Prof. Alice L. Yu in Institute of Stem
Cell and Translational Cancer Research, Chang Gung Memorial
Hospital at Linkou, Taiwan for the biological evaluations.
8. Hsieh, M. H.; Hung, J. T.; Liw, Y. W.; Lu, Y. J.; Wong, C. H.; Yu, A. L.; Liang, P. H.
Chembiochem. 2012, 13, 1689.
Supplementary data
9. Borg, N. A.; Wun, K. S.; Kjer-Nielsen, L.; Wilce, M. C.; Pellicci, D. G.; Koh, R.
Nature 2007, 448, 44.
Supplementary data (Synthesis of compound 4 and NMR spectra
for new compounds are provided.) associated with this article can
be found in the online version, at http://dx.doi.org/10.1016/
j.tet.2016.06.075.
10. Raju, R.; Castillo, B. F.; Richardson, S. K.; Thakur, M.; Severins, R.; Kronenberg,
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References and notes
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