Synthesis of 1,5-Anhydro-D-fructose derivatives and evaluation of their inflammasome inhibitors

Article abstract of DOI:10.1016/j.bmc.2017.11.041

Synthesis of several 1,5-Anhydro-D-fructose (1,5-AF) derivatives to evaluate inhibitory activities of the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome, although with only low activity. We focused on the hydration of

Full text of DOI:10.1016/j.bmc.2017.11.041

Accepted Manuscript
Synthesis of 1,5-Anhydro-D-fructose derivatives and evaluation of their in-
flammasome inhibitors
Kohtaro Goto, Hiroko Ideo, Akiko Tsuchida, Yuriko Hirose, Ikuro Maruyama,
Satoshi Noma, Takashi Shirai, Junko Amano, Mamoru Mizuno, Akio Matsuda
PII:
S0968-0896(17)32119-3
https://doi.org/10.1016/j.bmc.2017.11.041
BMC 14093
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry
Received Date:
Revised Date:
Accepted Date:
31 October 2017
27 November 2017
28 November 2017
Please cite this article as: Goto, K., Ideo, H., Tsuchida, A., Hirose, Y., Maruyama, I., Noma, S., Shirai, T., Amano,
J., Mizuno, M., Matsuda, A., Synthesis of 1,5-Anhydro-D-fructose derivatives and evaluation of their inflammasome
inhibitors, Bioorganic & Medicinal Chemistry (2017), doi: https://doi.org/10.1016/j.bmc.2017.11.041
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Synthesis of 1,5-Anhydro-D-fructose derivatives and evaluation of their
inflammasome inhibitors
a
a
a
a
b
b
Kohtaro Goto , Hiroko Ideo , Akiko Tsuchida , Yuriko Hirose , Ikuro Maruyama , Satoshi Noma ,
a
a
a
a
Takashi Shirai , Junko Amano , Mamoru Mizuno , Akio Matsuda*
a
The Noguchi Institute. 1-9-7, Kaga, Itabashi-Ku, Tokyo 173-0003 JAPAN
b
Department of Systems Biology in Thromboregulation, Kagoshima University, Graduate School of
Medical and Dental Sciences. 8-35-1, Sakuragaoka, Kagoshima-City, Kagoshima 890-8520 JAPAN
*Corresponding author. Tel: +81 3 3961 3255
E-mail address: matsuda.ab@noguchi.or.jp (A. Matsuda)
Abstract
Synthesis of several 1,5-Anhydro-D-fructose (1,5-AF) derivatives to evaluate inhibitory activities of
the inflammasome was carried out. Recently, 1,5-AF reported to suppress the inflammasome,
although with only low activity. We focused on the hydration of 2-keto form of 1,5-AF and
speculated that this hydration was the cause of low activity. Therefore, we synthesized some 1,5-AF
derivatives that would not be able to form the dimer conformation and can be expected to have high
activity against inflammasome, and then evaluated their inhibitory activities with respect to the
NLRP3 inflammasome by using mouse bone marrow–derived macrophages and human THP-1 cells.
As a result, some synthesized 2-keto form compounds had much higher inhibitory activities with
respect to the NLRP3 inflammasome than did 1,5-AF.
1
. Introduction
Natural immunity is a biological defense mechanism present in lower organisms through to higher
organisms, and central to the innate immune response are many pattern recognition receptors (PRRs).

S2
Inflammasomes are protein complexes consisting of particular PRRs, ASC, and caspase-1 which are
formed intracellularly in response to various stimulating factors such as pathogenic components,
1
, 2
endogenous factors, and exogenous factors. The mechanism of inflammasome formation can be
explained as follows. First, a PPR such as NLRP3, AIM2, or NLRC4 recognizes a particular
stimulating factor (agonist). This recognition triggers structural changes in the PRR and encourages
the assembly of complexes with proteins such as ASC and caspase-1. Finally, the PRRs form large
multiprotein oligomers called inflammasomes. In inflammasomes, caspase-1 (which is a kind of
protease) is activated, and the active caspase-1 then induces inflammation by promoting the
3-6
maturation and secretion of inflammatory cytokines such as IL-1β and IL-18. Inflammatory
responses based on the formation of inflammasomes are often effective for defense against
infections. However, negative aspects of this inflammasome response are also known. Excessive
inflammasome formation induced by certain kinds of stimulator can be inappropriately raised,
resulting in persistent inflammation. This mechanism is implicated in the development of various
inflammatory responses such as seen in infection, diabetes mellitus, arteriosclerosis, and
autoimmune diseases. Therefore, elucidating the mechanisms underlying the control of
inflammasome formation or activity will lead to a deeper understanding of the mechanisms of
pathogenesis, and is expected to contribute to diagnosis and treatment.
As the mechanisms of inflammasome formation have been gradually revealed, many studies
into inflammasome inhibitors have been carried out with the aim of developing diagnostic and
7
therapeutic medicines.
1,5-Anhydro-D-fructose (1,5-AF) is a functional monosaccharide that can be formed directly
8
from starch and glycogen in vivo by α-1,4-glucan lyase (Scheme 1). It occurs in edible morels, red
seaweeds, and certain mammalian tissues, and has been revealed to exhibit strong anti-oxidant
9
activity and antibacterial activity. Therefore, 1,5-AF is expected to find use in various fields such as
health foods and medicines.

S3
Scheme 1. Synthesis of 1,5-AF from starch.
10
Recently, 1,5-AF was reported to suppress the inflammasome, although with only low activity.
Herein, we show the synthesis and the evaluation of inflammasome inhibitors of which 1,5- AF is a
lead compound.
2
. Results and Discussion
We focused first on the 2-keto form of 1,5-AF. 1,5-AF has never been observed in purely
monomeric form in H O. Owing to its presumably hygroscopic nature, it has only been isolated as
2
mixtures of monomeric and dimeric forms or as mixtures of the monomeric (2-keto) and the
hydrated forms (Scheme 2). As a result, the abundance ratio of the 2-keto form of 1,5-AF is only a
few percent.
Scheme 2. Conformations of 1,5-AF.

S4
We inferred that this was one of the reasons that 1,5-AF had low inhibitory activity with respect
to inflammasomes. We carried out the synthesis of several 1,5-AF derivatives that we expected
would not be able to form the dimer conformation, and we evaluated the inhibitory activities of those
derivatives with respect to inflammasomes.
2
.1. Synthesis and evaluation of 3-deoxy derivative 6 and enone 7
To suppress dimer formation and increase the abundance ratio of the keto form, we first synthesized
11
3
-deoxy-1,5-AF (6) (Scheme 3). Compound 1, which had a furanose conformation, was treated
with 0.1 M H SO , and subsequent acetylation of all hydroxyl groups afforded compound 2, which
2
4
had a pyranose conformation, in 90%. Bromination of the anomer position of compound 2 was
conducted with HBr-AcOH solution; this was followed by removal of the bromine under reductive
3 3
conditions by using (TMS) SiH and Et B to give compound 3 in 63%. All acetyl groups of
compound 3 were removed with NaOMe, and the subsequent formation of the benzylidene acetal
produced alcohol 4 in 79%. Oxidation of the hydroxyl group of 4 was performed using Dess–Martin
12
oxidation to afford compound 5 in 98%. Finally, the removal of the benzylidene acetal was
conducted with 70% aq. AcOH to give the 3-deoxy derivative 6 in 85%. In this step, enone
derivative 7 was obtained as a byproduct. As expected, the abundance ratio of the 2-keto form in
13
H
2
O of compound 6 was improved up to about 20% . We then evaluated the inhibitory activity of
compounds 6 and 7 with respect to the inflammasome.

S5
Scheme 3. Synthesis of 3-deoxy derivative 6 and enone 7. Reagents and conditions: a) 0.1 M H
2
SO
B, Tol-1,4-dioxane, 2
2
, CSA, MeCN, 2 steps 79%; g) Dess-Martin periodinane,
4
, 60
o
2 2 2 3 3
C; b) Ac O, Pyridine, 2 steps 90%; c) HBr-AcOH, CH Cl ; d) (TMS) SiH, Et
steps 63%; e) NaOMe, MeOH; f) PhCH(OMe)
o
CH
2
Cl
2
, 98%; h) 70% aq. AcOH 70 C, 85%(6), 10%(7).
2
.2. In vitro assay of compounds 6 and 7
We measured the amount of IL-1β produced by bone marrow–derived macrophages (BMDMs)
stimulated with lipopolysaccharide (LPS) and the NLRP3 activator Nigericin. The
NLRP3 inflammasome–dependent events follow a two-stage process. In the first priming stage,
IL-1β expression is induced through NF-κB-mediated signaling, and in the second stage, the NLRP3
inflammasome is assembled and activated by different stimuli. We found that both 1,5-AF and
compound 6 inhibited IL-1β secretion.
Compound 6 dose-dependently inhibited the amount of IL-1β in culture supernatants produced
by BMDMs (Fig. 1a). The half-maximal inhibitory concentration (IC50) of compound 6 in BMDMs
was approximately 2 mM, which was much smaller than that of 1,5-AF (Table 1). Activation of the
NLRP3 inflammasome causes pyroptosis (a caspase-1-dependent programmed cell death) in
BMDMs. The anti-pyroptotic effects of compound 6 were also determined by measuring the amount
of intracellular ATP (Fig. 1b). Without compound 6 pre-incubation, Nigericin treatment caused cell
death and resulted in a pronounced decrease in intracellular ATP. Pre-treatment with compound 6
dose-dependently restored the intracellular ATP level, preventing the NLRP3-dependent pyroptosis
of BMDMs. As was the case for compound 6, the enone derivative 7 also dose-dependently inhibited
IL-1β secretion and pyroptosis (data not shown). The half-maximal inhibitory concentration (IC50) of
compound 7 in BMDMs was approximately 15 µM, which was much smaller than that of
compound 6 (Table 1). Inhibition of IL-1β release was also confirmed by western blotting of IL-1β.
Although almost equal amounts of pro-IL-1β (37 kDa) were produced in all of the LPS-primed cells

S6
regardless of treatment, compounds 6 and 7 prevented their processing to the mature 17 kDa form
caused by Nigericin stimulation (Fig. 2a).
Figure 1. 3-Deoxy-1,5-AF (6) inhibits IL-1β secretion and pyroptosis in response
to Nigericin-induced NLRP3 inflammasome activation.
a) Concentration of IL-1β in supernatants from BMDMs stimulated with LPS and
Nigericin and treated with compound 6 (0.45–7.5 mM) measured by ELISA.
b) Amount of intracellular ATP in BMDMs stimulated with LPS and Nigericin and
treated with compound 6 (measured by CellTiter-Glo reagents). Intracellular ATP was
measured after aliquots of supernatants were removed from BMDM cultures. Data are
expressed as mean ± standard deviation (n = 3). This data is representative of at least
three independent experiments.

S7
Figure 2. Synthesized compounds inhibit Nigericin-induced NLRP3
inflammasome activation.
a) BMDMs primed with LPS (100 ng/mL) for 4 h were treated with compound 7
(
100 µM), compound 6 (6.8 mM), or 1,5-AF (60 mM) for 1 h prior to stimulation with
Nigericin (5 µM) for 1 h. The cells were lysed with lysis buffer and the collected
supernatants were concentrated with 10 K MWCO centrifugal filters (Amicon Ultra).
Immunoblots of pro-IL-1β and mature-IL-1β in culture supernatants (Sup.) and
immunoblots of pro-IL-1β, mature-IL-1β, and β-actin in cell lysates are shown.
b) THP-1 cells primed with LPS (100 ng/mL) for 3 h were treated with compound 7
(
150 µM) or compound 6 (3.4 mM) for 1 h prior to stimulation with Nigericin (2.5 µM)
for 1 h. The cell lysates and collected supernatants were prepared as for BMDMs above.
Immunoblots of pro-IL-1β and mature-IL-1β in culture supernatants and immunoblots
of pro-IL-1β, mature-IL-1β, and β-actin in cell lysates are shown. All data shown are
representative of three independent experiments.

S8
2
.3. Synthesis of enone derivatives
Based on this result, we designed and synthesized compound 8-10 and fluorinated enones 17 and
3, assuming that the structure of the enone is important. First, we attempted the synthesis enone
2
derivatives 8, 9, and 10 which can be easily converted from the compound 6. Each enone derivative
was obtained by acylation of compound 6, with good yield of the corresponding anhydride of
carboxylic acids (Scheme 4).
2
Scheme 4. Synthesis of acyl enones 8, 9 and 10. Reagents and conditions: a) (RC(O)) O, Pyridine.
14
Next, we synthesized the fluorinated enone derivative 17. Compound 11 was treated with N,
N-diethylaminosulfur trifluoride (DAST) to afford fluoride 12 in 90%. Compound 12 was reacted
2 4
with 0.1 M H SO , followed by acetylation to give compound 13 in quant. Compound 13 was
brominated by treating with HBr-AcOH solution; subsequent removal of bromine by the radical
reaction produced compound 14 in 90%. Removal of the acetyl groups of compound 14 was
conducted by using NaOMe. The formation of the benzylidene acetal produced compound 15 in
15, 16
88%. Compound 15 was oxidized with 1-Me-AZADO
2
in the presence of PhI(OAc) to give
compound 16 in 75%. The removal of the benzylidene group of compound 16 was conducted by
hydrogenation; this was followed by acetylation to give the desired compound 17 in 87% (Scheme
5).

S9
o
Scheme 5. Synthesis of 3-F derivative 17. Reagents and conditions: a) DAST, CH
2
Cl , 40 C, 90%; b) 0.1
2
o
M H
Tol-1,4-dioxane, 2 steps 90%; f) NaOMe, MeOH; g) PhCH(OMe) , CSA, MeCN, 2 steps 88%; h)
2
-Me-AZADO CH Cl , 75%; i) H gas, Pd/C, EtOH-EtOAc; j) Ac O, Pyridine, 2 steps 87%.
2
SO
4
, 60 C; c) Ac
2
O, Pyridine, 2 steps quant.; d) HBr-AcOH, CH
2
Cl
2
; e) (TMS) SiH, Et3B,
3
2
1
2
2
2
We also synthesized fluorinated levoglucosenone 23. Removal of the acetyl groups of
compound 13, followed by formation of the benzylidene acetal provided compound 18 in 56%.
Compound 18 was acetylated to prepare compound 19 in 96%. Regioselective ring opening of the
benzylidene acetal yielded compound 20 in 49%, which was then tosylated to give 21 in 91%.
Compound 21 was then treated with 3.03 M NaOH solution to prepare bicyclic compound 22 in 69%.
Compound 22 was reacted with 1-Me-AZADO to afford the corresponding ketone, followed by the
hydrogenationin in the presence of Pd/C was conducted to remove benzyl ether. Finally, acetylation
could afford the desired compound 23 in 39% with enol acetate 24 in 8% as a byproduct (Scheme
6).

S10
Scheme 6. Synthesis of bicyclo derivative 23. Reagents and conditions: a) NaOMe, MeOH; b)
PhCH(OMe) , CSA, MeCN, 2 steps 56%; c) Ac O, Pyridine, 96%; d) Et SIH, PhBCl , CH Cl
9%; e) TsCl, Pyridine, 91%; f) 3.03N aq. NaOMe, Pyridine, 69%; g) 1-Me-AZADO, PhI(OAc)
2%; h) H gas, Pd/C, EtOH-EtOAc, 76%; i) Ac O, Pyridine, 3 steps 39%.
o
2
2
3
2
2
2
, -78 C,
4
5
2
, CH
2 2
Cl ,
2
2
2
.4. In vitro assay of enone derivatives (compounds 8–10, 17, 23, and 24) and
levoglucosenone
We measured the inhibitory activity of newly synthesized compounds (8–10, 17, 23, and 24) and
17
levoglucosenone with respect to Nigericin in the same way as we did for compound 6. These
compounds dose-dependently inhibited IL-1β secretion and pyroptosis as was the case for
compound 6. The half-maximal inhibitory concentration (IC50) of 1,5-AF and the synthesized
compounds (8–10, 17, 23, and 24) and levoglucosenone with respect to NLRP3 stimuli are listed in
Table 1. It is clear that the IC50s of compound 7, levoglucosenone, and their derivatives are much
lower than those of 1,5-AF and compound 6.
Furthermore, fluorination and/or O-acylation of compound 7 increased the inhibitory activity by
several times that of 6. It is not known whether macrophages have a specific transport system for
1,5-AF and its derivatives. However, it is expected that fluorination and/or O-acylation would elicit
hydrophobicity in these compounds, and because it is well known that the membrane permeability of

S11
a compound increases with its hydrophobicity, this may be part of the reason that the inhibitory
activity is enhanced.
The effects of various compounds on IL-1β release were also evaluated in human macrophage
cells (THP-1 cells). Cells were pretreated with several inhibitors and then cultured with Nigericin;
consequent IL-1β production was then estimated by cell-based assay and western blotting. As was
shown in BMDMs, all of the tested compounds except for 1,5-AF significantly inhibited IL-1β
secretion in a dose-dependent manner (data not shown). These compounds also restored the ATP
levels in THP-1 cells in a dose-dependent manner (data not shown), revealing that the synthesized
compounds prevented NLRP3-dependent pyroptotic cell death triggered by Nigericin in THP-1 cells.
The IC50 values of the tested compounds with respect to Nigericin are summarized in Table 1.
Compound 6 exhibited an IC50 value of 2.3 ± 0.03 mM, and the modified enone compounds (8–10,
17, 23, 24, and levoglucosenone) exhibited slightly improved potency, with IC50 values in THP-1
cells that ranged from 3.7 ± 1.4 to 6.6 ± 2.8 µM.
We also confirmed the reduction of IL-1β in concentrated supernatants by immunoblots with
anti-IL-1β monoclonal antibody in THP-1 cells (Fig. 2b).

S12
Table 1. Inhibitory effect (IC50) of compounds on release of IL-1β from BMDMs and THP-1 cells
triggered by Nigericin stimulation.
3
. Conclusion
1,5-AF derivatives and related compounds were designed and synthesized, and their inhibitory
activities with respect to the NLRP3 inflammasome were evaluated in vitro by using mouse BMDMs
and human THP-1 cells. Synthesized compounds (6–10, 17, 23, 24) and levoglucosenone have much
higher inhibitory activities with respect to the NLRP3 inflammasome than does 1,5-AF. In vivo
evaluations of the effects of the newly synthesized compounds and investigation of target molecules
are ongoing.
From these results, it is suggested that the enone structure may be an important factor in their
inhibitory activities with respect to the NLRP3 inflammasome.

S13
4
4
. Experimental Section
.1. General methods for chemical synthesis
All reagents were purchased from commercial sources and were used without further purification
unless noted. All reactions were carried out under positive pressure of air at room temperature unless
specified and were monitored using TLC on silica gel 60-F254 (Merck KGaA, Darmstadt, Germany).
Organic solvents were evaporated under reduced pressure and the products were purified using
column chromatography on silica gel (silica-gel 60N, spherical, neutral, 40-50 m, KANTO
1
CHEMICAL CO., INC., Tokyo, Japan). The yields reported are after purification. H NMR spectra
were recorded at 600 MHz and Chemical shifts are reported in ppm and calibrated using
1
13
tetramethylsilane (0.00 ppm for H in CDCl
3
and CD
3
OD), HOD (4.76 ppm for 1H in D
2
O). C
NMR spectra were recorded at 150 MHz and chemical shifts were referenced to CDCl
3
(77.0 ppm)
1
9
or CD
PhCF
3
OD (49.0 ppm). F NMR were recorded at 600 MHz and chemical shifts were referenced to
3
(-63.2 ppm). Assignments of NMR spectra were based on two-dimensional experiments
1
1
(
H- H COSY, HMQC and HMBC). High resolution mass spectra were recorded on an
JMS-T100LP (JEOL Ltd., Tokyo, JAAPN), JMS-T100GCv (JEOL Ltd., Tokyo, JAAPN), Exactive
(
Thermo Scientific, MA, USA) and LTQ Orbitrap XL (Thermo Scientific, MA, USA).
1
8
1
,2,4,6-Tetra-O-acetyl-3-deoxy-D-glucopyranose (2) : 0.1 M sulfulic acid (500 mL, 0.05 mmol)
was added to 3-deoxy-1,2,5,6-di-O-isopropylidene--D-glucofuranose (1; 13.9 g, 56.9 mmol). After
o
stirring at 60 C for 20 h, the reaction was cooled to room temperature and neutralized with NaHCO
3
(
9.24 g, 0.11 mmol). The reaction solution was concentrated under reduced pressure. The resulting
residue was dried under high vacuum for 3 hours and used without any further purification.
Acetic anhydride (100 mL) and catalytic amount of DMAP were added to a solution of the oil from
o
above in pyridine (200 mL) at 0 C. After stirred at room temperature for 14 h, MeOH (150 mL) was
o
added to the reaction mixture at 0 C, followed by concentrated under reduced pressure. The residual
2 3
oil was diluted with AcOEt and washed with H O, 1 N HCl, saturated aq. NaHCO , and saturated aq.

S14
NaCl. The organic layer was dried over anhydrous MgSO
was purified by column chromatography on silica-gel (n-Hexane : AcOEt = 3 : 2) to give compound
(17.1 g, 90%, / = 33/67, inseparable) as a color less oil. R = 0.37 (n-Hexane : AcOEt = 1 : 1).
Spectrum data were consistent with those of known compound.
4
, filtered and concentrated. The residue
2
f
2
,4,6-Tri-O-acetyl-1,5-anhydro-3-deoxy-D-glucitol (3): HBr (33% in acetic acid, 40.8 mL) was
added to a mixture of 2 (16.8 g, 50.6 mmol) in dry CH Cl (70 mL) at room temperature. After
stirring at room temperature for 3 h, ice chips were added to the reaction mixture and very carefully
extracted with CH Cl . The organic layer was washed twice with H O, saturated aq. NaHCO , and
saturated aq. NaCl. The organic layer was dried over anhydrous MgSO , filtered and concentrated to
afford the anomeric bromide (16.4 g) which was used without any further purification.
2
2
2
2
2
3
4
To a mixture of the bromide from above and TTMSS (46.8 mL, 15.2 mmol) in toluene (160 mL) and
o
1
,4-dioxane (80 mL) was added 1.0 M triethylborane in THF (15.2 mL, 15.2 mmol) at 0 C. After
stirring for 16 h at room temperature, the reaction mixture was concentrated. The residue was
purified by column chromatography on silica-gel (n-Hexane : AcOEt = 2 : 1) to give compound 3
1
(
8.72 g, 2steps 63%) as a color less oil. Rf = 0.34 (n-Hexane : AcOEt = 2 : 1). H NMR (600 MHz,
CDCl
3
):  = 4.94-4.86 (m, 1H, H-2), 4.79 (dt, J = 9.6, 4.8 Hz, 1H, H-4), 4.20 (dd, J = 12.4, 2.8 Hz,
H, H-6a), 4.16 (dd, J = 12.4, 5.5 Hz, 1H, H-6b), 4.14-4.07 (m, 1H, H-1a), 3.49 (ddd, J = 9.6, 5.5,
.8 Hz, 1H, H-5), 3.22 (t, J = 10.3 Hz, 1H, H-1b), 2.62-2.55 (m, 1H, H-3a), 2.10 (s, 3H, Ac), 2.06 (s,
1
2
3
1
6
13
H, Ac), 2.04 (s, 3H, Ac), 1.56 (q, J = 11.0 Hz, 1H, H-3b) ; C NMR (150 MHz, CDCl ):  =
3
70.87 (C=O), 169.78 (C=O), 169.57 (C=O), 77.16 (C-5), 68.91 (C-1), 66.08 (C-4), 66.04 (C-2),
2.81 (C-2), 34.80 (C-3), 20.95 (Ac), 20.92 (Ac), 20.84 (Ac); HRMS (ESI-TOF MS.): Calcd for
+
12
C H
18
O
7
Na m/z [M+Na] : 297.0945, Found: 297.0939.
1,5-Anhydro-4,6-O-benzylidene-3-deoxy-D-glucitol (4): To a solution of the compound 3 (7.94 g,
28.9 mmol) in dry MeOH (160 mL) was added sodium methoxide (160 L, 28% in MeOH solution)
at room temperature. After stirring for 5 h at room temperature, the reaction mixture was treated with

S15
+
Amberlite IR-120 (H form). After filtration, the filtrate was concentrated to afford the
corresponding triol (4.19 g) which was used without any further purification.
Benzaldehyde dimethylacetal (12.8 mL, 86.8 mmol) and (±)-10-Camphorsulfonic acid (600 mg, 2.58
mmol) were added to a solution of the triol from above in MeCN (300 mL). After stirred at room
3
temperature for 20 h, the reaction mixture was quenched by Et N (3 mL), followed by concentrated
under reduced pressure. The residue was purified by column chromatography on silica-gel
(
(
n-Hexane : AcOEt = 1 : 1) to give compound 3 (5.39 g, 2steps 79%) as a white powder. Rf = 0.30
1
n-Hexane : AcOEt = 1 : 1). H NMR (600 MHz, CDCl
3
):  = 7.51-7.47 (m, 2H, Ph), 7.40-7.33 (m,
3H, Ph), 5.53 (s, 1H, PhCH-), 4.32 (dd, J = 11.0, 4.8 Hz, 1H, H-6a), 4.08-4.02 (m, 1H, H-2), 3.67 (t,
J = 10.3 Hz, 1H, H-6b), 3.58-3.51 (m, 1H, H-4), 3.28 (dt, J = 10.3, 4.8 Hz, 1H, H-5), 3.21 (t, J =
1
1
1
0.3 Hz, 1H, H-1b), 2.56-2.49 (m, 1H, H-3a), 1.65 (q, J = 11.7 Hz, 1H, H-3b), 1.56 (d, J = 6.9 Hz,
13
H, 2-OH); C NMR (150 MHz, CDCl ):  = 137.30 (Ph), 129.14 (Ph), 128.37 (Ph), 126.17 (Ph),
3
01.72 (PhCH-), 76.39 (C-4), 73.15 (C-5), 72.48 (C-1), 69.25 (C-6), 65.63 (C-2), 38.46 (C-3);
+
HRMS (APCI-TOF MS.): Calcd for C13
H
17
O
4
m/z [M+H] : 237.1121, Found: 237.1126.
1
,5-Anhydro-4,6-O-benzylidene-3-deoxy-D-fructose (5): Dess-Martin Periodinane (9.28 g, 21.9
mmol) was added to a solution of 4 (4.62 g, 19.6 mmol) in dry CH Cl (200 mL) under Ar
atmosphere. After stirring for 2 h at room temperature, saturated aq. NaHCO (100 mL) was added
to the reaction mixture and stirred for 0.5 h. The resulting mixture was diluted with CH Cl and
washed with saturated aq. NaHCO , and saturated aq. NaCl. The organic layer was dried over
anhydrous Na SO , filtered and concentrated. The residue was purified by column chromatography
on silica-gel (Toluene : AcOEt = 10 : 1) to give compound 5 (4.47 g, 98%) as a white powder. Rf =
2
2
3
2
2
3
2
4
1
1
0
7
.41 (Toluene : AcOEt = 10 : 1). H NMR (600 MHz, CDCl3):  = H NMR (600 MHz, CDCl
3
):  =
.53-7.46 (m, 2H, Ph), 7.42-7.34 (m, 3H, Ph), 5.58 (s, 1H, PhCH-), 4.45 (dd, J = 10.3, 4.8 Hz, 1H,
H-6a), 4.17 (dd, J = 16.5, 1.4 Hz, 1H, H-1a), 4.04 (d, J = 16.5 Hz, 1H, H-1b), 3.97-3.90 (m, 1H,
H-4), 3.79 (t, J = 10.3 Hz, 1H, H-6b), 3.63 (dt, J = 9.6, 4.8 Hz, 1H, H-5), 3.07 (dd, J = 16.5, 5.5 Hz,
13
1
3
H, H-3a), 2.66 (dd, J = 16.5, 11.7 Hz, 1H, H-3b); C NMR (150 MHz, CDCl ):  = 204.03 (C-2),

S16
136.89 (Ph), 129.31 (Ph), 128.42 (Ph), 126.14 (Ph), 101.41 (PhCH-), 75.82 (C-4), 74.78 (C-1), 71.58
+
(
C-5), 69.08 (C-6), 44.77 (C-3) ; HRMS (APCI-TOF MS.): Calcd for C13
H
15
O
4
m/z [M+H] :
235.0965, Found: 235.0971.
1,5-Anhydro-3-deoxy-D-fructose (6) and 1,5-Anhydro-3-deoxy-D-arabino-hex-3-en-2-ulose (7):
2 2
70% aq. acetic acid (50 mL) was added to a solution of 5 (475 mg, 2.03 mmol) in dry CH Cl (10
o
mL). After stirring for 1.5 h at 70 C, the reaction mixture was concentrated. The residue was
3 2
purified by column chromatography on silica-gel (CHCl : MeOH : H O = 85 : 15 : 1 and n-Hexane :
AcOEt = 1 : 2) to give the desired compound 6 (251 mg, 85%) and compound 7 (25.1 mg, 10%) as a
color less oil respectively.
1
Data for 1,5-Anhydro-3-deoxy-D-fructose (6): Rf = 0.41 (CHCl
NMR (600 MHz, CDCl
H, H-6a), 3.96 (d, J = 16.5 Hz, 1H, H-1b), 3.91-3.85 (m, 1H, H-6b), 3.59-3.54 (m, 1H, H-5), 2.98
dd, J = 16.5, 5.5 Hz, 1H, H-3a), 2.50 (dd, J = 16.5, 7.6 Hz, 1H, H-3b), 2.25 (brs, 1H, 4-OH), 1.96
3
: MeOH : H
2
O = 8 : 2 : 0.2). H
3
):  = 4.24-4.19 (m, 1H, H-4), 4.15 (d, J = 16.5 Hz, 1H, H-1a), 3.98-3.91 (m,
1
(
(
(
1
3
brs, 1H, 6-OH); C NMR (150 MHz, CDCl
3
):  = 206.28 (C-2), 80.40 (C-5), 73.40 (C-1), 67.12
+
C-4), 63.23 (C-6), 46.21 (C-3) ; HRMS (ESI-TOF MS.): Calcd for C
69.0471, Found: 169.0472.
6
H
10
O
4
Na m/z [M+Na] :
1
Data for 1,5-Anhydro-3-deoxy-D-arabino-hex-3-en-2-ulose (7): Rf = 0.30 (n-Hexane : AcOEt = 1 :
1
3
1
). H NMR (600 MHz, CDCl
3
):  = 7.01 (dd, J = 10.3, 2.1 Hz, 1H, H-3), 6.24 (dd, J = 10.3, 2.7 Hz,
H, H-4), 4.49-4.43 (m, 1H, H-5), 4.34 (d, J = 15.8 Hz, 1H, H-1a), 4.17 (dd, J = 15.8, 2.1 Hz, 1H,
13
H-1b), 3.90-3.83 (m, 1H, H-6a), 3.82-3.76 (m, 1H, H-6b); C NMR (150 MHz, CDCl
3
):  = 194.43
(
C-2), 148.40 (C-3), 128.35 (C-4), 74.47 (C-5), 71.28 (C-1), 64.09 (C-6) ; HRMS (APCI-TOF MS.):
+
6 9 3
Calcd for C H O m/z [M+H] : 129.0546, Found: 129.0550.
6-O-Acetyl-1,5-anhydro-3-deoxy-D-arabino-hex-3-en-2-ulose (8): Acetic anhydride (1.0 mL) was
added to a solution of the compound 6 (47.5 mg, 312 mol) in pyridine (2.0 mL) at room
temperature. After stirred at room temperature for 20 h, the reaction mixture was concentrated. The

S17
residue was purified by column chromatography on silica-gel (n-Hexane : AcOEt = 2 : 1) to give
1
compound 9 (42.5 mg, 82%) as a white powder. R
MHz, CDCl
.60-4.56 (m, 1H, H-5), 4.36 (d, J = 15.8 Hz, 1H, H-1a), 4.35 (dd, J = 11.7, 6.2 Hz, 1H, H-6a), 4.27
f
= 0.23 (n-Hexane : AcOEt = 2 : 1); H NMR (600
3
):  = 6.97 (dd, J = 11.0, 2.1 Hz, 1H, H-3), 6.24 (dd, J = 11.0, 2.1 Hz, 1H, H-4),
4
1
3
(
(
(
dd, J = 11.7, 4.1 Hz, 1H, H-6b), 4.16 (dd, J = 16.5, 1.4 Hz, 1H, H-1b), 2.12 (s, 3H, Ac); C NMR
150 MHz, CDCl
3
):  = 193.91 (C-2), 170.66 (C=O), 146.88 (C-3), 128.47 (C-4), 71.92 (C-5), 71.13
+
C-1), 64.38 (C-6), 20.76 (Ac) ; HRMS (APCI-TOF MS.): Calcd for C
8
H
11
O
4
m/z [M+H] : 170.0652,
Found: 170.0658.
1
2
6
,5-Anhydro-6-O-butanoyl-3-deoxy-D-arabino-hex-3-en-2-ulose (9): Butylic anhydride (335 L,
.05 mmol) and catalytic amount of DMAP were added to a solution of the compound 6 (100 mg,
84 mol) in pyridine (1 mL) at room temperature. After stirred at room temperature for 18 h,
o
MeOH (5 mL) was added to the reaction mixture at 0 C, followed by diluted with AcOEt and
washed with H
2
O, 5% aq. citiric acid, saturated aq. NaHCO
3 ,
, and saturated aq. NaCl dried over
anhydrous MgSO
4
, filtered and concentrated. The residue was purified by column chromatography
f
on silica-gel (n-Hexane : AcOEt = 3 : 1) to give compound 9 (85.6 mg, 63%) as a yellow oil. R =
1
0
.36 (n-Hexane : AcOEt = 2 : 1); H NMR (600 MHz, CDCl
3
):  = 6.97 (dd, J = 10.3, 2.1 Hz, 1H,
H-3), 6.24 (dd, J = 10.3, 2.1 Hz, 1H, H-4), 4.59-4.55 (m, 1H, H-5), 4.37 (dd, J = 11.7, 6.2 Hz, 1H,
H-6a), 4.35 (d, J = 16.5 Hz, 1H, H-1a), 4.26 (dd, J = 11.7, 4.1 Hz, 1H, H-6b), 4.15 (dd, J = 16.5, 2.1
Hz, 1H, H-1b), 2.35 (t, J = 7.6 Hz, 2H, -CH
2
CH
); C NMR (150 MHz, CDCl
C=O), 147.07 (C-3), 128.43 (C-4), 72.01 (C-5), 71.16 (C-1), 64.16 (C-6), 35.93 (-CH
2
CH
3
), 1.67 (sext, J = 7.6 Hz, 2H, -CH
):  = 194.00 (C-2), 173.35
CH CH
2 2 3 2 2 3 15 4
8.37 (-CH CH CH ), 13.63 (-CH CH CH ) ; HRMS (APCI-TOF MS.): Calcd for C10H O m/z
2 2 3
CH CH ),
13
0.96 (t, J =7.6 Hz, 3H, -CH
2
CH
2
CH
3
3
(
2
2
3
),
1
+
[
M+H] : 199.0965, Found: 199.0972.
1,5-Anhydro-3-deoxy-6-O-octanoyl-D-arabino-hex-3-en-2-ulose (10): n-Octanoic anhydride (628
L, 2.11 mmol) and catalytic amount of DMAP were added to a solution of the compound 6 (103

S18
mg, 705 mol) in pyridine (1 mL) at room temperature. After stirred at room temperature for 22 h,
o
MeOH (5 mL) was added to the reaction mixture at 0 C, followed by diluted with AcOEt and
washed with H O, 5% aq. citiric acid, saturated aq. NaHCO , and saturated aq. NaCl. The organic
layer was dried over anhydrous MgSO , filtered and concentrated. The residue was purified by
2
3
4
column chromatography on silica-gel (n-Hexane : AcOEt = 4 : 1) to give compound 10 (109 mg,
1
61%) as a color less oil. R
f
= 0.29 (n-Hexane : AcOEt = 3 : 1); H NMR (600 MHz, CDCl
3
):  = 6.97
(
dd, J = 10.3, 2.1 Hz, 1H, H-3), 6.24 (dd, J = 10.3, 2.1 Hz, 1H, H-4), 4.60-4.55 (m, 1H, H-5),
.39-4.32 (m, 2H, H-1a, H-6a), 4.26 (dd, J = 11.7, 4.1 Hz, 1H, H-6b), 4.16 (dd, J = 16.5, 1.4 Hz, 1H,
H-1b), 2.36 (t, J = 7.6 Hz, 2H, -C 15), 1.68-1.59 (m, 2H, -C 15), 1.38-1.20 (m, 8H, -C 15), 0.88
15); C NMR (150 MHz, CDCl ):  = 193.99 (C-2), 173.56 (C=O), 147.08
C-3), 128.43 (C-4), 72.01 (C-5), 71.17 (C-1), 64.16 (C-6), 34.07 (-C 15), 31.64 (-C 15), 29.03
-C 15), 28.89 (-C 15), 24.86 (-C 15), 22.56 (-C 15), 14.07 (-C 15); HRMS (ESI-TOF MS.):
Na m/z [M+Na] : 277.1410, Found: 277.1414.
4
7
H
7
H
7
H
13
(
(
(
t, J =7.6 Hz, 3H, -C
7
H
3
7
H
7
H
7
H
7
H
7
H
7
H
7
H
+
22 4
Calcd for C14H O
1
9
3
-Deoxy-3-fluoro-1,2-5,6-di-O-isopropylidene--D-glucofuranose (12) : (Diethylamino)sulfur
trifluoride (4.83 mL, 49.5 mmol) was added to a solution of
1,2-5,6-di-O-isopropylidene--D-allofuranose (11; 6.44 g, 24.7 mmol) in THF (60 mL). After
o
stirring at 40 C for 15 h, the reaction was cooled to room temperature and quenched by MeOH (10
mL) and Et
saturated aq. NaHCO
filtered and concentrated. The residue was purified by column chromatography on silica-gel
n-Hexane : AcOEt = 4 : 1) to give compound 12 (5.81 g, 90%) as yellow oil. Rf = 0.50 (n-Hexane :
AcOEt = 4 : 1). Spectrum data were consistent with those of known compound.
3
N (5 mL). The reaction mixture was diluted with ethyl acetate and washed with H
2
O,
3
, and saturated aq. NaCl. The organic layer was dried over anhydrous MgSO
4
,
(
1
9
1
,2,4,6-Tetra-O-acetyl-3-deoxy-3-fluoro-D-glucopyranose (13) : 0.1 M sulfulic acid (180 mL,
o
1
8.0 mmol) was added to 12 (5.81 g, 22.2 mmol). After stirring at 60 C for 17 h, the reaction was
3
cooled to room temperature and neutralized with NaHCO (3.33 g, 39.6 mmol). The reaction

S19
solution was concentrated under reduced pressure. The resulting residue was dried under high
vacuum for 3 hours and used without any further purification.
o
Acetic anhydride (30 mL) was added to a solution of the oil from above in pyridine (60 mL) at 0 C.
o
After stirred at room temperature for 14 h, MeOH (30 mL) was added to the reaction mixture at 0 C,
followed by concentrated under reduced pressure. The residual oil was diluted with AcOEt and
washed with H
dried over anhydrous MgSO
chromatography on silica-gel (n-Hexane : AcOEt = 3 : 2) to give compound 13 (7.89 g, quant.) as a
color less oil. = 0.35 (n-Hexane : AcOEt = 3 : 2)
Spectrum data were consistent with those of known compound.
2
O, 1 N HCl, saturated aq. NaHCO
3
and saturated aq. NaCl. The organic layer was
4
, filtered and concentrated. The residue was purified by column
R
f
1
9
2
,4,6-Tri-O-acetyl-1,5-Anhydro-3-deoxy-3-fluoro-D-glucitol (14) : HBr (33% in acetic acid, 22.0
mL, 65.9 mmol) was added to a mixture of 13 (7.69 g, 22.0 mmol) in dry CH Cl (35 mL) at room
temperature. After stirring at room temperature for 3 h, ice chips were added to the reaction mixture
and very carefully extracted with CH Cl . The organic layer was washed with twice H O, saturated
aq. NaHCO and saturated aq. NaCl. The organic layer was dried over anhydrous MgSO , filtered
2
2
2
2
2
3
4
and concentrated to afford the anomeric bromide (8.20 g) which was used without any further
purification.
To a mixture of the bromide from above and TTMSS (16.9 mL, 54.9 mmol) in toluene (80 mL) and
1,4-dioxane (40 mL) was added 1.0 M triethylborane in THF (6.59 mL, 6.59 mmol) at room
temperature. After stirring for 16 h at room temperature, the reaction mixture was concentrated. The
residue was purified by column chromatography on silica-gel (n-Hexane : AcOEt = 2 : 1) to give
compound 14 (5.78 g, 2steps 90%) as a color less oil. Rf = 0.22 (n-Hexane : AcOEt = 2 : 1).
Spectrum data were consistent with those of known compound.
1
9
1
,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-D-glucitol (15) : To a solution of the
compound 14 (4.24 g, 14.5 mmol) in dry MeOH (80 mL) was added sodium methoxide (240 L,

S20
28% in MeOH solution) at room temperature. After stirring for 22 h at room temperature, the
+
reaction mixture was treated with Amberlite IR-120 (H form). After filtration, the filtrate was
concentrated to afford the corresponding triol (2.49 g) which was used without any further
purification.
Benzaldehyde dimethylacetal (6.43 mL, 43.5 mmol) and (±)-10-Camphorsulfonic acid (300 mg,
.29 mmol) were added to a solution of the triol from above in MeCN (150 mL). After stirred at
1
room temperature for 19 h, the reaction mixture was quenched by triethylamine (2 mL), followed by
concentrated under reduced pressure. The residue was purified by column chromatography on
silica-gel (n-Hexane : AcOEt = 3 : 2) to give compound 15 (3.26 g, 2steps 88%) as a white powder.
Rf = 0.35 (n-Hexane : AcOEt = 3 : 2). Spectrum data were consistent with those of known
compound.
1
,5-Anhydro-4,6-O-benzylidene-3-deoxy-3-fluoro-D-fructose (16): PhI(OAc)
mmol) was added to a mixture of the compound 15 (405 mg, 1.59 mmol) and 1-Me-AZADO (2.65
mg, 15.9 mol) in CH Cl (16 mL) at room temperature. After stirred for 22 h at room temperature,
the reaction was quenched by saturated aq. Na (4 mL) and saturated aq. NaHCO (4 mL). The
mixture was stirred for 0.5 h at room temperature, followed by diluted with AcOEt and washed with
O, saturated aq. NaHCO and saturated aq. NaCl. The organic layer was dried over anhydrous
MgSO , filtered and concentrated. The residue was purified by column chromatography on silica-gel
2
(770 mg, 2.40
2
2
2 2
S O
3
3
H
2
3
4
(
(
f
n-Hexane : AcOEt = 1 : 1) to give compound 16 (299 mg, 75%) as a white powder. R = 0.28
1
n-Hexane : AcOEt = 1 : 1). H NMR (600 MHz, CDCl3):  = 7.56-7.49 (m, 2H, Ph), 7.42-7.36 (m,
3
1
H, Ph), 5.61 (s, 1H, PhCH-), 5.11 (dd, J = 49.5, 10.3 Hz, 1H, H-3), 4.88 (ddd, J = 10.3, 4.8, 2.1 Hz,
H, H-6a), 4.31 (dd, J = 15.1, 4.1 Hz, 1H, H-1a), 4.12 (d, J = 15.1 Hz, 1H, H-1b), 4.12-4.03 (m, 1H,
13
H-4), 3.84 (t, J = 10.3 Hz, 1H, H-6b), 3.79 (dt, J = 8.9, 4.8 Hz, 1H, H-5); C NMR (150 MHz,
CDCl ):  = 197.48 (d, J = 13.0 Hz, C-2), 136.33 (Ph), 129.43 (Ph), 128.40 (Ph), 126.17 (Ph),
01.32 (PhCH-), 91.84 (d, J = 202.3 Hz, C-3), 80.65 (d, J = 17.3 Hz, C-4), 74.24 (C-1), 70.10 (d, J =
3
1
7
19
.2 Hz, C-5), 68.47 (C-6); F NMR (600 MHz, CDCl ):  = -202.08 (d, J = 43.6 Hz); HRMS
3

S21
+
(
14 4
APCI-TOF MS.): Calcd for C13H O F m/z [M+H] : 253.0871, Found: 253.0878.
1,5-Anhydro-3-deoxy-3-fluoro-D-arabino-hex-3-en-2-ulose (17): A solution of the compound 16
(
289 mg, 1.15 mmol) in EtOH (1.5 mL)–AcOEt (1.5 mL) was added to a suspension 10% Pd/C (243
mg) in EtOH (8.5 mL)–AcOEt (8.5 mL). After bubbling with hydrogen for 1 h at room temperature,
the reaction mixture was filtered on Celite and concentrated in vacuo, and used without any further
purification.
Acetic anhydride (2.0 mL) was added to a solution of the oil from above in pyridine (3.0 mL). After
o
stirred at room temperature for 18 h, MeOH (4 mL) was added to the reaction mixture at 0 C,
followed by diluted with AcOEt and washed with H
saturated aq. NaCl. The organic layer was dried over anhydrous MgSO
The residue was purified by column chromatography on silica-gel (n-Hexane : AcOEt = 2 : 1) to
2
O, twice 1 N HCl, saturated aq. NaHCO
3
and
4
, filtered and concentrated.
f
give compound 17 (164 mg, 2steps 76%) as a color less oil. R = 0.28 (n-Hexane : AcOEt = 2 : 1).
1
H NMR (600 MHz, CDCl
3
):  = 6.48 (dd, J = 12.4, 2.1 Hz, 1H, H-4), 4.81-4.76 (m, 1H, H-5), 4.46
(
dd, J = 16.5, 3.4 Hz, 1H, H-1a), 4.36 (dd, J = 11.7, 6.7 Hz, 1H, H-6a), 4.24 (dd, J = 12.4, 4.1 Hz,
1
3
1
1
H, H-6b), 4.23 (dd, J = 16.5, 1.4 Hz, 1H, H-1b), 2.13 (s, 3H, Ac); C NMR (150 MHz, CDCl
3
):  =
87.20 (d, J = 17.3 Hz, C-2), 170.55 (C=O), 152.15 (d, J = 274.5 Hz, C-3), 122.54 (d, J = 8.7 Hz,
C-4), 72.36 (d, J = 4.3 Hz, C-5), 71.80 (d, J = 4.3 Hz, C-1), 64.37 (d, J = 2.9 Hz, C-6), 20.73 (Ac) ;
19
F NMR (600 MHz, CDCl
3 8 9 4
):  = -129.13; HRMS (ESI-TOF MS.): Calcd for C H O FNa m/z
+
[
M+Na] : 211.0377, Found: 211.0378.
4,6-O-Benzylidene-3-deoxy-3-fluoro-D-glucopyranose (18): To a solution of the compound 13
(
8.92 g, 25.5 mmol) in dry MeOH (120 mL) was added sodium methoxide (480 L, 28% in MeOH
solution) at room temperature. After stirring for 24 h at room temperature, the reaction mixture was
+
treated with Amberlite IR-120 (H form). After filtration, the filtrate was concentrated to afford the
corresponding triol (4.89 g) which was used without any further purification.
Benzaldehyde dimethylacetal (11.3 mL, 76.4 mmol) and (±)-10-Camphorsulfonic acid (500 mg, 2.15

S22
mmol) were added to a solution of the tetraol from above in MeCN (250 mL). After stirred at room
temperature for 2 hours, the reaction mixture was quenched by Et N (2 mL), followed by
concentrated under reduced pressure. The residual oil was diluted with AcOEt and washed with H
saturated aq. NaHCO and saturated aq. NaCl. The organic layer was dried over anhydrous MgSO
filtered and concentrated. The residue was purified by column chromatography on silica-gel
3
2
O,
,
3
4
(
n-Hexane : AcOEt = 1 : 3) to give compound 18 (3.89 g, 2steps 56%, / = 60/40, inseparable) as a
1
white powder. Rf = 0.41 (n-Hexane : AcOEt = 1 : 3). H NMR (600 MHz, CDCl
3
): Data for 
isomer;  = 7.49-7.43 (m, 2H, Ph), 7.38-7.31 (m, 3H, Ph), 5.59 (s, 1H, PhCH-), 5.17 (t, J = 3.4 Hz,
1
1
7
1
H, H-1), 4.67 (dt, J = 55.7, 8.9 Hz, 1H, H-3), 4.21-4.18 (m, 1H, H-6a), 3.99 (dt, J = 10.3, 4.8 Hz,
H, H-5), 3.81-3.69 (m, 3H, H-2, H-4, H-6b); Data of  isomer;  = 7.49-7.43 (m, 2H, Ph),
.38-7.31 (m, 3H, Ph), 5.58 (s, 1H, PhCH-), 4.63 (d, J = 7.6 Hz, 1H, H-1), 4.49 (dt, J = 53.6, 8.9 Hz,
13
H, H-3), 4.30-4.26 (m, 1H, H-6a), 3.81-3.69 (m, 2H, H-4, H-6b), 3.52-3.42 (m, 2H, H-2, H-5); C
):  = 138.93 (Ph), 138.85 (Ph), 130.01 (Ph), 129.10 (Ph), 127.42 (Ph),
27.40 (Ph), 102.73 (-PhCH-), 102.60 (-PhCH-), 98.46 (d, J = 11.6 Hz, -C-1), 94.97 (d, J = 10.1
Hz, -C-1), 94.32 (d, J = 185.0 Hz, -C-3), 92.68 (d, J = 183.5 Hz, -C-3), 81.08 (d, J = 15.9 Hz,
-C-4), 80.39 (d, J = 17.3 Hz, -C-4), 75.60 (d, J = 17.3 Hz, -C-2), 72.76 (d, J = 17.3 Hz, -C-2),
NMR (150 MHz, CDCl
3
1

19
7
0.06 (-C-6), 69.61 (-C-6), 66.29 (d, J = 8.7 Hz, -C-5), 62.84 (d, J = 7.2 Hz, -C-5); F NMR
(
600 MHz, CDCl
3
):  = -196.25 (d, J = 43.6 Hz), -201.70 (d, J = 43.6 Hz) ; HRMS (ESI-TOF MS.):
+
Calcd for C13
H
15
O
5
FNa m/z [M+Na] : 293.0796, Found: 293.0803.
1,2-Di-O-acetyl-4,6-O-benzylidene-3-deoxy-3-fluoro-D-glucopyranose (19): Acetic anhydride (6.0
o
mL) was added to a solution of the compound 18 (3.83 g, 14.2 mmol) in pyridine (12 mL) at 0 C.
o
After stirred at room temperature for 16 h, MeOH (10 mL) was added to the reaction mixture at 0 C,
followed by concentrated under reduced pressure. The residual oil was diluted with AcOEt and
washed with H
was dried over anhydrous MgSO
chromatography on silica-gel (Toluene : AcOEt = 15 : 1) to give compound 19 (4.84 g, 96%, / =
2
O, twice 1 N HCl, saturated aq. NaHCO
3
and saturated aq. NaCl. The organic layer
4
, filtered and concentrated. The residue was purified by column

S23
1
6
0/40, inseparable) as a white powder. R
f
= 0.43 (Toluene : AcOEt = 10 : 1); H NMR (600 MHz,
CDCl ): Data for  isomer;  = 7.54-7.47 (m, 2H, Ph), 7.41-7.33 (m. 3H, Ph), 6.33 (t, J = 3.4 Hz,
3
1
4
1
7
1
3
H, H-1), 5.59 (s, 1H, PhCH-), 5.23-5.15 (m, 1H, H-2), 4.92 (dt, J = 54.3, 8.9 Hz, 1H, H-3),
.36-4.31 (m, 1H, H-6a), 3.97 (dt, J = 10.3, 4.8 Hz, 1H, H-5), 3.92-3.83 (m, 1H, H-4), 3.83-3.75 (m,
H, H-6b), 2.12 (s, 3H, Ac), 2.10 (s, 3H, Ac); Data of  isomer;  = 7.54-7.47 (m, 2H, Ph),
.41-7.33 (m, 3H, Ph), 5.73 (d, J = 8.3 Hz, 1H, H-1), 5.57 (s, 1H, PhCH-), 5.28 (dt, J = 13.7, 8.9 Hz,
H, H-2), 4.72 (dt, J = 52.9, 8.9 Hz, 1H, H-3), 4.44-4.37 (m, 1H, H-6a), 3.92-3.83 (m, 1H, H-4),
13
.83-3.75 (m, 1H, H-6b), 3.59 (dt, J = 9.6, 4.8 Hz, 1H, H-5), 2.17 (s, 3H, Ac), 2.12 (s, 3H, Ac); C
):  = 169.75 (C=O), 169.16 (C=O), 168.97 (C=O), 168.86 (C=O), 136.46
Ph), 136.40 (Ph), 129.37 (Ph), 128.37 (Ph), 126.19 (Ph), 101.77 (-PhCH-), 101.68 (-PhCH-),
NMR (150 MHz, CDCl
3
(
91.84 (d, J = 10.1 Hz, -C-1), 90.40 (d, J = 192.2 Hz, -C-3), 89.85 (d, J = 10.1 Hz, -C-1), 87.89
(
d, J = 190.7 Hz, -C-3), 78.96 (d, J = 17.3 Hz, -C-4), 78.44 (d, J = 17.3 Hz, -C-4), 71.02 (d, J =
18.83 Hz, -C-2), 70.13 (d, J = 17.3 Hz, -C-2), 68.43 (-C-6), 68.18 (-C-6), 65.81 (d, J = 8.7 Hz,
19

-C-5), 64.27 (d, J = 7.2 Hz, -C-5), 20.83 (Ac), 20.76 (Ac), 20.67 (Ac), 20.55 (Ac); F NMR (600
3
MHz, CDCl ):  = -196.41 (d, J = 43.6 Hz), -200.77 (d, J = 43.6 Hz); HRMS (ESI-TOF MS.): Calcd
+
for C17H
19
O
7
FNa m/z [M+Na] : 377.1007, Found: 377.1012.
1
1
,2-Di-O-acetyl-4-O-benzyl-3-deoxy-3-fluoro-6-D-glucopyranose (20): To a mixture of compound
3 2 2
9 (4.81 g, 13.6 mmol) and Et SiH (6.50 mL, 40.7 mmol) in dry CH Cl (100 mL) was added
o
o
PhBCl
2
(5.89 mL, 44.8 mmol) at -78 C under Ar atmosphere. After stirring for 30 min at -78 C, the
reaction was quenched by a cocktail of Et
stirring for 10 min at room temperature, the reaction mixture was diluted with CHCl
with saturated aq. NaHCO and saturated aq. NaCl. The organic layer was dried over anhydrous
MgSO , filtered and concentrated. The residue was purified by column chromatography on silica-gel
n-Hexane : EtOH = 10 : 1) to give compound 20 (2.35 g, 49%, / = 45/55, inseparable) as a color
3
N (25 mL), MeOH (50 mL) and CHCl
3
(50 mL). After
3
and washed
3
4
(
1
less oil. R
f 3
= 0.19 (n-Hexane : EtOH = 10 : 1); H NMR (600 MHz, CDCl ): Data for  isomer;  =
7.40-7.29 (m, 5H, Ph), 6.28 (t, J = 3.4 Hz, 1H, H-1), 5.13-5.07 (m, 1H, H-2), 4.94 (dt, J = 53.6, 8.9

S24
Hz, 1H, H-3), 4.89 (d, J = 11.7 Hz, 1H, PhCH
H, H-4, H-5, H-6a, H-6b), 2.12 (s, 3H, Ac), 2.10 (s, 3H, Ac), 1.87 (dd, J = 7.6, 5.5 Hz, 1H, -OH);
Data of  isomer;  = 7.40-7.29 (m, 5H, Ph), 5.63 (d, J = 8.2 Hz, 1H, H-1), 5.18 (dt, J = 13.6, 8.9
Hz, 1H, H-2), 4.86 (d, J = 11.0 Hz, 1H, PhCH -),4.70 (dt, J = 50.9, 8.2 Hz, 1H, H-3), 4.65 (d, J =
1.0 Hz, 1H, PhCH -), 3.91-3.70 (m, 3H, H-4, H-6a, H-6b), 3.52-3.47 (m, 1H, H-5), 2.12 (s, 3H, Ac),
.10 (s, 3H, Ac), 1.82 (dd, J = 7.6, 5.5 Hz, 1H, -OH); C NMR (150 MHz, CDCl
2 2
-), 4.67 (d, J = 13.1 Hz, 1H, PhCH -), 3.91-3.70 (m,
4
2
1
2
13
2
3
):  = 169.93
(
(
C=O), 169.36 (C=O), 169.16 (C=O), 168.99 (C=O), 137.37 (Ph), 137.31 (Ph), 128.55 (Ph), 128.24
Ph), 128.19 (Ph), 95.43 (d, J = 186.4 Hz, -C-3), 92.83 (d, J = 186.5 Hz, -C-3), 91.27 (d, J = 11.6
Hz, -C-1), 89.51 (d, J = 10.2 Hz, -C-1), 74.93 (d, J = 8.7 Hz, -C-5),74.84 (PhCH
15.9 Hz, -C-4), 74.67 (d, J = 17.3 Hz, -C-4), 74.56 (PhCH -), 72.84 (d, J = 7.2 Hz, -C-5),
0.52 (d, J = 18.8 Hz, -C-2), 69.79 (d, J = 18.8 Hz, -C-2), 61.03 (-C-6), 60.99 (-C-6), 20.77
2
-), 74.82 (d, J
=
2
7
19
(
Ac), 20.68 (Ac), 20.58 (Ac); F NMR (600 MHz, CDCl
3
):  = -192.28 (d, J = 43.6 Hz), -196.29 (d,
+
J = 43.6 Hz); HRMS (ESI-TOF MS.): Calcd for C17
H
21
O
7
FNa m/z [M+Na] : 379.1164, Found:
379.1167.
4-O-Benzyl-3-deoxy-3-fluoro-1,2-di-O-acetyl-6-O-p-toluenesulfonyl-D-glucopyranose (21):
p-toluenesulfonyl chloride (1.84 g, 9.64 mmol) was added to a solution of the compound 20 (2.29 g,
6.63 mmol) in pyridine (25 mL) at room temperature. After stirred for 19 hours, MeOH (10 mL) was
o
added to the reaction mixture at 0 C, followed by concentrated under reduced pressure. The residual
oil was diluted with AcOEt and washed with H
NaCl. The organic layer was dried over anhydrous MgSO
was purified by column chromatography on silica-gel (Toluene : AcOEt = 10 : 1) to give compound
2
O, 1 N HCl, saturated aq. NaHCO
3
and saturated aq.
4
, filtered and concentrated. The residue
2
1 (2.97 g, 91%, / = 47/53, inseparable) as a white powder. R
f
= 0.33 (Toluene : AcOEt = 10 : 1);
1
H NMR (600 MHz, CDCl
3
): Data for  isomer;  = 7.78 (d, J = 3.4 Hz, 1H, Ph), 7.39-7.23 (m, 7H,
Ph), 6.15 (t, J = 3.4 Hz, 1H, H-1), 5.03-4.95 (m, 1H, H-2), 4.88 (dt, J = 52.9, 8.9 Hz, 1H, H-3), 4.85
d, J = 11.0 Hz, 1H, PhCH -), 4.59 (d, J = 10.3 Hz, 1H, PhCH -), 4.32 (dd, J = 11.0 3.4 Hz, 1H,
H-6a), 4.20 (dt, J = 11.0 Hz, 1H, H-6b), 3.89 (brd, J = 10.3 Hz, 1H, H-5), 3.81-3.72 (m, 1H, H-4),
(
2
2

S25
2
1
4
.44 (s, 3H, PhCH
H, Ph), 7.39-7.23 (m, 7H, Ph), 5.54 (d, J = 8.3 Hz, 1H, H-1), 5.12 (dt, J = 13.7, 8.2 Hz, 1H, H-2),
.81 (d, J = 11.0 Hz, 1H, PhCH -), 4.63 (dt, J = 52.2, 8.9 Hz, 1H, H-3), 4.55 (d, J = 11.0 Hz, 1H,
-), 4.29 (dd, J = 11.0, 3.4 Hz, 1H, H-6a), 4.24 (d, J = 11.0 Hz, 1H, H-6b), 3.83-3.72 (m, 1H,
3
), 2.08 (s, 3H, Ac), 2.07 (s, 3H, Ac); Data of  isomer;  = 7.77 (d, J = 3.4 Hz,
2
PhCH
H-4), 3.58 (brd, J = 9.6 Hz, 1H, H-5), 2.43 (s, 3H, PhCH
NMR (150 MHz, CDCl ):  = 169.70 (C=O), 169.16 (C=O), 168.90 (C=O), 168.56 (C=O), 145.20
Ph), 145.08 (Ph), 136.96 (Ph), 136.94 (Ph), 132.58 (Ph), 129.89 (Ph), 129.86 (Ph), 128.57 (Ph),
2
13
3
), 2.09 (s, 3H, Ac), 2.09 (s, 3H, Ac); C
3
(
1
29.52(Ph), 128.34 (Ph), 128.25 (Ph), 128.23 (Ph), 128.05 (Ph). 128.02 (Ph), 95.26 (d, J = 189.3 Hz,
-C-3), 92.63 (d, J = 187.8 Hz, -C-3), 90.94 (d, J = 11.6 Hz, -C-1), 89.16 (d, J = 8.7 Hz, -C-1),
4.90 (d, J = 2.9 Hz, -PhCH -), 74.61 (d, J = 2.9 Hz, -PhCH -), 74.51 (d, J = 17.3 Hz, -C-4),
4.36 (d, J = 17.3 Hz, -C-4), 72.34 (d, J = 10.1 Hz, -C-5), 70.43 (d, J = 10.1 Hz, -C-5), 70.16 (d,
),
3
0.75 (Ac), 20.72 (Ac), 20.63 (Ac), 20.51 (Ac); F NMR (600 MHz, CDCl ):  = -191.93 (d, J =

7
7
2
2
J = 18.8 Hz, -C-2), 69.41 (d, J = 17.3 Hz, -C-2), 67.44 (-C-6), 67.16 (-C-6), 21.66 (Ph-CH
3
19
2
4
5
+
27 9
5.6 Hz), -196.21 (d, J = 45.6 Hz); HRMS (ESI-TOF MS.): Calcd for C24H O FNaS m/z [M+Na] :
33.1252, Found: 533.1259.
1,6-Anhydro-4-O-benzyl-3-deoxy-3-fluoro--D-glucopyranoside (22): 3.03 M aq. NaOH (6.27
mL, 19.0 mmol) was added to a solution of the compound 21 (2.94 g, 5.76 mmol) in pyridine (30
mL) at room temperature. After stirred for 17 h, the reaction mixture was diluted with AcOEt and
washed with four times 5% aq. citric acid, saturated aq. NaHCO
3
and saturated aq. NaCl. The
organic layer was dried over anhydrous MgSO , filtered and concentrated. The residue was purified
4
by column chromatography on silica-gel (Toluene : AcOEt = 5 : 1) to give compound 22 (1.01 g,
1
6
7
1
9%) as a white powder. R
f
= 0.33 (Toluene : AcOEt = 5 : 1); H NMR (600 MHz, CDCl
3
):  =
-), 4.66 (d, J = 12.3 Hz,
-), 4.64 (d, J = 44.7 Hz, 1H, H-3), 4.61 (d, J = 6.2 Hz, 1H, H-5), 3.98 (d, J = 7.6 Hz, 1H,
.40-7.30 (m, 5H, Ph), 5.45 (s, 1H, H-1), 4.70 (d, J = 12.3 Hz, 1H, PhCH
H, PhCH
2
2
H-6a), 3.78 (dt, J = 6.2, 2.1 Hz, 1H, H-6b), 3.64 (brt, J = 10.3 Hz, 1H, H-2), 3.51 (d, J = 13.1 Hz, 1H,
13
H-4), 2.65 (d, J = 10.3 Hz, 1H, -OH); C NMR (150 MHz, CDCl ):  = 136.82 (Ph), 128.68 (Ph),
3

S26
1
7
28.28 (Ph), 127.89 (Ph), 101.26 (C-1), 88.22 (d, J = 183.5 Hz, C-3), 74.17 (d, J = 27.5 Hz, C-4),
19
3.58 (C-5), 71.61 (PhCH
2
-), 67.37 (d, J = 23.1 Hz, C-2), 64.97 (d, J = 4.3 Hz, C-6); F NMR (600
MHz, CDCl
3
):  = -184.94 (d, J = 43.6 Hz); HRMS (ESI-TOF MS.): Calcd for C13 FNa m/z
15 4
H O
+
[
M+Na] : 277.0847, Found: 277.0850.
1
2
,6-anhydro-3,4-bisdeoxy-3-fluoro--D-glycero-hex-3-enopyranos-2-ulose (23) and
,4-di-O-acetyl-1,6-anhydro-3-deoxy-3-fluoro--D-glycero-hex-2-enopyranose (24): PhI(OAc)2
(
1.21 g, 3.74 mmol) was added to a mixture of the compound 22 (476 mg, 1.87 mmol) and
-Me-AZADO (93.4 mg, 562 mol) in CH Cl (20 mL) at room temperature. After stirred for 20 h at
1
2
2
room temperature, the reaction was quenched by saturated aq. Na S O (5 mL) and saturated aq.
2
2
3
NaHCO (5 mL). The mixture was stirred for 0.5 h at room temperature, followed by diluted with
3
AcOEt and washed with H O, saturated aq. NaHCO and saturated aq. NaCl. The organic layer was
2
3
dried over anhydrous MgSO , filtered and concentrated. The residue was purified by column
4
chromatography on silica-gel (Toluene : AcOEt = 3 : 2) to give the crude compound 23 (220 mg)
which was used without any further purification.
A solution of the oil from above (220) in AcOEt (3 mL) was added to a suspension 10% Pd/C (158
mg) in AcOEt (13 mL). After bubbling with hydrogen for 1.5 h at room temperature, the reaction
mixture was filtered on Celite and concentrated in vacuo, and used without any further purification.
Acetic anhydride (1.0 mL) was added to a solution of the oil from above in pyridine (1.5 mL). After
o
stirred at room temperature for 2 h, MeOH (2 mL) was added to the reaction mixture at 0 C,
followed by diluted with AcOEt and washed with three times of 5% aq. citiric acid, saturated aq.
NaHCO
3
and saturated aq. NaCl. The organic layer was dried over anhydrous MgSO
4
, filtered and
concentrated. The residue was purified by column chromatography on silica-gel (CHCl
3
) to give
compound 23 (106 mg, 3 steps 39%) and compound 24 (35.9 mg, 3 steps 8%) as a color less oil
respectively.
Data for 1,6-anhydro-3,4-bisdeoxy-3-fluoro--D-glycero-hex-3-enopyranos-2-ulose (23): Rf = 0.42
1
(
CHCl
3
). H NMR (600 MHz, CDCl
3
):  = 6.72(dd, J = 10.3, 5.5 Hz, 1H, H-4), 5.48 (d, J = 6.9 Hz,

S27
1H, H-1), 5.15 (q, J = 4.8 Hz, 1H, H-5), 3.95 (t, J = 5.5 Hz, 1H, H-6a), 3.85 (d, J = 6.9 Hz, 1H,
13
H-6b); C NMR (150 MHz, CDCl
3
):  = 182.69 (d, J = 18.8 Hz, C-2), 152.85 (d, J = 280.33 Hz,
C-3), 123.06 (d, J = 7.2 Hz, C-4), 101.22 (d, J = 7.2 Hz, C-1), 71.81 (d, J = 7.2 Hz, C-5), 67.16 (d, J
19
=
2.9 Hz, C-6); F NMR (600 MHz, CDCl ):  = -131.98; HRMS (APCI-TOF MS.): Calcd for
3
+
6
C H
6
O
3
F m/z [M+H] : 145.0296, Found: 145.0300.
Data for 2,4-di-O-acetyl-1,6-anhydro-3-deoxy-3-fluoro--D-glycero-hex-2-enopyranose (24): Rf =
1
0
.15 (CHCl
3
). H NMR (600 MHz, CDCl
3
):  = 5.68 (d, J = 7.6 Hz, 1H, H-1), 5.23 (dd, J = 6.9, 1.4
Hz, 1H, H-4), 4.77 (t, J = 6.9 Hz, 1H, H-5), 4.01 (t, J = 6.9 Hz, 1H, H-6a), 3.66 (dd, J = 8.2, 2.1 Hz,
1
3
1
1
7
H, H-6b), 2.25 (s, 3H, Ac), 2.19 (s, 3H, Ac); C NMR (150 MHz, CDCl
3
):  = 170.46 (C=O),
66.43 (C=O), 142.94 (d, J = 274.6 Hz, C-3), 131.30 (d, J = 4.3 Hz, C-2), 97.41 (d, J = 4.3 Hz, C-1),
19
5.31 (d, J = 7.2 Hz, C-5), 68.99 (d, J = 20.3 Hz, C-4), 63.24 (C-6), 20.85 (Ac), 20.28 (Ac); F
3
11 6
NMR (600 MHz, CDCl ):  = -137.93; HRMS (ESI-TOF MS.): Calcd for C10H O FNa m/z
+
[
M+Na] : 269.0432, Found: 269.0435.
4
.2. Biological studies
Cell culture and reagents
To generate bone marrow–derived macrophages (BMDMs), bone marrow cells were collected from
C57BL/6 mouse femurs and cultured for 6–8 days on petri dishes in DMEM/F12 supplemented with
10% FBS (Thermo Fisher Scientific, Waltham, MA USA), 100 U/mL penicillin, 100 µg/mL
streptomycin, and 30% L929 cell-conditioned medium.
The human monocyte cell line THP-1 was cultured in RPMI-1640 medium with 10% FBS,
10 mM glutamine, 100 U/mL penicillin, 100 µg/mL streptomycin, and 50 µM 2-ME (complete
RPMI medium) (Wako Pure Chemical Industries, Osaka, Japan).
Anti-murine IL-1β antibody (AF-401-NA) was purchased from R&D Systems (Minneapolis,
MN, USA), and anti-human IL-1β monoclonal antibody was purchased from Santa Cruz

S28
Biotechnology, Dallas, TX, USA). Ultrapure LPS from Escherichia coli 0111:B4 and Nigericin were
purchased from Invivogen (San Diego, CA, USA). 1,5-AF, 3-deoxy-1,5-AF, and enone compounds
were dissolved in sterile, endotoxin-free water, and other synthetic compounds were dissolved in
sterile, endotoxin-free DMSO; these were stored as stock solutions at −20°C and were diluted with
medium just before each experiment. The final DMSO concentration did not exceed 0.1%
throughout the study.
Cell stimulation and measurement of IL-1β
4
BMDMs were seeded onto 96-well plates at 5 × 10 cells per well and were primed with 100 ng/mL
LPS in DMEM/F12 supplemented with 10% FBS for 4 h. Cells were washed once with PBS and
were incubated with each compound for 1 h prior to stimulation with Nigericin (5 µM, 1 h) in
serum-free DMEM/F12 medium. The release of IL-1β in culture supernatants was determined by
anti-mouse IL-1β-specific ELISA (eBioscience, San Diego, CA, USA).
5
THP-1 cells were seeded at 1 × 10 cells per well in a 96-well plate. They were treated with
100 nM PMA (phorbol 12-myristate 13-acetate) for 48 h. LPS (100 ng/mL) was then added to
activate the PMA-differentiated THP-1 cells. THP-1 cells primed with LPS for 3 h were washed
once with PBS and were incubated with each compound for 1 h prior to stimulation with Nigericin
(
5 µM, 1 h) in serum-free RPMI medium. At the end of stimulation, supernatants were collected.
The levels of IL-1β in supernatants were determined by cell-based assay using HEK-blue-IL-1β cells
Invivogen) following the manufacturer’s instructions. In brief, HEK-blue-IL-1β cells were
(
incubated with collected supernatants and assayed for alkaline phosphatase activities by adding
QUANTI-Blue substrate (Invivogen) and reading the absorbance at 650 nm.
Intracellular ATP assay
To determine the number of viable cells in culture, we quantified the amount of ATP present, which
indicates the presence of metabolically active cells. Equal volumes of CellTiter-Glo reagents
(
Promega Corporation, Madison, MI, USA) were added directly to the wells. Plates were incubated
at room temperature for 10 min on a shaker and luminescence was measured by a luminometer

S29
2
(
TriStar LB 942; Berthold Technology, Bad Wildbad, Germany) according to the manufacturer’s
instructions.
Western blotting
6
BMDMs or THP-1 cells (1.5 × 10 cells per 3.5 ⌀ dish) primed with LPS were stimulated with
Nigericin (5 µM) in serum-free RPMI or DMEM/F12 medium for 1 h. At the end of stimulation,
supernatants were collected and concentrated with 10 K MWCO centrifugal filters (Amicon Ultra,
Merck Millipore, Bilerica, MA, USA). The cells were lysed with lysis buffer (20 mM HEPES–KOH,
pH 7.5, 150 mM KCl, 1% Nonidet P-40, 0.5% deoxycholate, 0.1 mM PMSF, ×100 protease inhibitor
mixture (CalbioChem, San Diego, CA, USA). Cell lysates and concentrated supernatants were
mixed and boiled with SDS sample buffer for 5 min at 99°C, and were resolved by 12.5% gel
SDS-PAGE and were transferred onto PVDF membranes. Generation of IL-1β was determined by
immunoblotting with anti-mouse or anti-human IL-1β antibodies followed with
peroxidase-conjugated secondary antibodies. Signal detection was achieved using SuperSignal West
Dura Substrate (Thermo Fisher Scientific) and blots were imaged using the ChemiDoc XRS Plus
system (Bio-Rad, Hercules, CA, USA).
Acknowledgements
We thank Miho Yamada, Nozomi Ogasawara, Tomohiro Hirose (Instrumental Analysis Division,
Equipment Management Center, Creative Research Institution, Hokkaido University), and Yoshiki
Oda (Technology Joint Management Office, Tokai University) for high resolution mass spectra
analysis.
Keywords
Carbohydrates, 1,5-Anhydro-D-fructose derivatives, enones, inflammasome, NLRP3