6-Azido-6-deoxy-l-idose as a Hetero-Bifunctional Spacer for the Synthesis of Azido-Containing Chemical Probes

Article abstract of DOI:10.1002/chem.201602044

The design of 6-azido-6-deoxy-l-idose for use as a hetero-bifunctional spacer is reported. The hemiacetal at one terminus is an equivalent of an aldehyde and can react with nucleophiles, such as amino groups and electron-rich aromatics. The azido group at the other terminus bio-orthogonally undergoes a Hüisgen [3+2] cycloaddition with an acetylene. The idose derivative exhibited a higher level of reactivity towards oxime formation than a corresponding glucose derivative. The¹³C NMR spectrum of the uniformly¹³C-labeled 6-azido-idose indicated that the acyclic forms of the sugar totaled 0.3 % of all the isomers, whereas those of glucose totaled 0.01 %. The larger population of the acyclic forms of the idose derivative would result in higher reactivity towards electrophilic addition in comparison with glucose derivatives. Finally, we prepared a C-idosyl epigallocatechin gallate (EGCG) that bears an azido group through C-glycosylation of EGCG with 6-azido-idose. This glycosyl form of the C-idosyl EGCG exhibited a cytotoxicity against U266 cells that was comparable to that of EGCG. These results suggested that the EGCG derivative could be used as an effective chemical probe for the elucidation of EGCG biological functions.

Full text of DOI:10.1002/chem.201602044

DOI: 10.1002/chem.201602044
Full Paper
&
Chemical Probes
6-Azido-6-deoxy-l-idose as a Hetero-Bifunctional Spacer for the
Synthesis of Azido-Containing Chemical Probes
Hiroki Hamagami,[a] Motofumi Kumazoe,[c] Yoshiki Yamaguchi,[b] Shinichiro Fuse,[a, d]
Hirofumi Tachibana,[c] and Hiroshi Tanaka*[a]
Abstract: The design of 6-azido-6-deoxy-l-idose for use as
a hetero-bifunctional spacer is reported. The hemiacetal at
one terminus is an equivalent of an aldehyde and can react
with nucleophiles, such as amino groups and electron-rich
aromatics. The azido group at the other terminus bio-or-
thogonally undergoes a Hüisgen [3+2] cycloaddition with
an acetylene. The idose derivative exhibited a higher level of
reactivity towards oxime formation than a corresponding
glucose derivative. The 13C NMR spectrum of the uniformly
13C-labeled 6-azido-idose indicated that the acyclic forms of
the sugar totaled 0.3% of all the isomers, whereas those of
glucose totaled 0.01%. The larger population of the acyclic
forms of the idose derivative would result in higher reactivity
towards electrophilic addition in comparison with glucose
derivatives. Finally, we prepared a C-idosyl epigallocatechin
gallate (EGCG) that bears an azido group through C-glycosy-
lation of EGCG with 6-azido-idose. This glycosyl form of the
C-idosyl EGCG exhibited a cytotoxicity against U266 cells
that was comparable to that of EGCG. These results suggest-
ed that the EGCG derivative could be used as an effective
chemical probe for the elucidation of EGCG biological func-
tions.
chemical probes from simple building blocks.[2] This approach
affords a high level of flexibility in the design of the chemical
probes. However, the de novo synthesis of chemical probes
frequently requires laborious efforts by well-trained synthetic
chemists. A second version is referred to as semisynthesis, and
it involves modifying natural products.[3] This approach omits
the synthesis of the structure of natural products and minimiz-
es the synthetic efforts required to synthesize the chemical
probes. However, incorporating spacers that possess bio-or-
thogonal functional groups in natural products that have mul-
tiple reactive functional groups is difficult to accomplish with-
out loss of their biological activities.
Introduction
Identifying the receptors of biologically active natural products
that exhibit unique biological activities is an effective strategy
for elucidating the roles of these biomolecules. Natural prod-
uct derivatives possessing functional devices, such as a bio-or-
thogonal functional group, a fluorescent dye, a photoaffinity
labeling unit, and affinity beads, are effective chemical probes
for the elucidation of the mechanism of the biological action.[1]
The synthesis of chemical probes that are based on natural
products can be categorized into two approaches. The first is
a de novo synthesis approach based on the preparation of
Hydrophilic hetero-bifunctional spacers that possess both
a chemoselectively reactable functional group and a bio-or-
thogonal functional group are effective tools when preparing
hybrid molecules by connecting two functional molecules.[4]
The hydrophilicity of the spacers is important to minimize non-
specific interactions with biomolecules. Reducing sugars that
possess a bio-orthogonal functional group, such as an azido
group or a terminal acetylene group, have served as useful
hetero-bifunctional spacers in chemical glycobiology.[5] These
sugars undergo enzymatic glycosidation in vivo to introduce
a bio-orthogonal functional group into the targeted biomole-
cules. The azido group can be stained with a fluorescent dye
possessing an alkyne moiety through a Hüisgen [3+2] cycload-
dition.[6] On the other hand, reducing sugars chemically react
with nucleophiles, such as amino groups in aqueous media.[7]
Imine formation is often applied to the synthesis of sugar-con-
taining materials and biochemical probes.[8] In addition, reduc-
ing sugars undergo electrophilic substitution of electrically rich
aromatics in the presence of a Lewis acid.[9] Rauter and co-
[a] H. Hamagami, Dr. S. Fuse, Dr. H. Tanaka
Department of Chemical Science and Engineering
School of Material and Chemical Technology
Tokyo Institute of Technology, 2-12-1-H101 Ookayama, Meguro
Tokyo, 152-8552 (Japan)
E-mail: thiroshi@apc.titech.ac.jp
[b] Dr. Y. Yamaguchi
RIKEN-Max-Planck Joint Research Center, for Systems Chemical Biology
RIKEN Global Research Cluster, 2-1 Hirosawa
Wako, Saitama, 351-0198 (Japan)
[c] Dr. M. Kumazoe, Prof. Dr. H. Tachibana
Department of Bioscience and Biotechnology
Faculty of Agriculture, Kyushu University
6-10-1 Hakozaki, Fukuoka, 812-8581 (Japan)
[d] Dr. S. Fuse
Present address:
Laboratory for Chemistry and Life Science
Tokyo Institute of Technology, 4259 Nagatsuta-cho, Midori-ku
Yokohama, 226-8503 (Japan)
Supporting information for this article can be found under
http://dx.doi.org/10.1002/chem.201602044.
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workers reported the synthesis of C-glycosyl flavanones
through C-glycosylation of flavanones with various sugars cata-
lyzed either by Sc(OTf)3 or Pr(OTf)3 in aqueous media.[10] One of
the important factors determining the reactivity of reducing
sugars towards electrophilic reactions is the population of the
acyclic forms involving acetals and aldehydes.[7,11] Serianni and
co-workers quantified the population of the acyclic forms of
glucose by using 13C NMR analysis and determined it to be
0.01%.[12] On the other hand, idose, a C5 epimer of glucose, in-
volved the acyclic forms in 0.8%, which was the maximum
among aldohexoses. These results suggested to us that idose
would exhibit a higher reactivity towards electrophilic reac-
tions than other aldohexoses. Herein, we report the use of 6-
azido-6-deoxy-l-idose[13] (1) as a hetero-bifunctional spacer and
its application to the synthesis of (À)-8-(6-azido-6-deoxy-b-l-
idopyranosyl)-3-(3,4,5-trihydroxybenzoyl)epigallocatechin (22)
as a chemical probe.
2-(bromomethyl)naphthalene under basic conditions to pro-
vide the ether 9, was followed by hydrolysis of the acetal to
provide the 1,2-diol 10 in 99% yield from compound 8. Selec-
tive acylation of the primary alcohol function of compound 10,
followed by mesylation of the remaining secondary alcohol of
compound 11 provided the mesylate 12. Exposure of com-
pound 12 under basic conditions resulted in the hydrolysis of
the benzoate, followed by intramolecular etherification to pro-
vide the epoxide 13 in 61% total yield from compound 10. We
next examined the incorporation of an azido group through
epoxide-opening of compound 13. Treatment of the epoxide
13 with TMSN3 (TMS=trimethylsilyl) in the presence of
Al(OiPr)3 at room temperature,[15] followed by hydrolysis of the
resultant TMS ether 14 with 1m HCl provided the azide 15
quantitatively. Oxidative acetalization of compound 15 with
2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ) under anhy-
drous conditions stereoselectively provided the acetal 16 in
82% yield. NOE experiments with compound 16 showed that
the irradiation of the proton Ha enhanced the signals of both
protons Hb (7.4%) and Hc (8.3%). These results revealed that
the stereochemistry of the generated chiral center in acetal 16
is S. Removal of all the protecting groups of compound 16
under acidic conditions provided the 6-azido-idose 1 in 86%
yield.
Results and Discussion
We designed the 6-azido-idose 1 as a hetero-bifunctional
spacer (Scheme 1). The spacer 1 was chemically coupled not
only with the electron-rich aromatic compounds 5 to provide
the C-glycosyl-type compounds 6, but also with the amines 3
to provide the imines 4. An enriched population of the acyclic
forms 2 of compound 1 resulted in a high reactivity of com-
pound 1 towards electrophilic addition. The remaining azido
group could be bio-orthogonally reacted with a terminal acety-
lene group through a Hüisgen [3+2] cycloaddition for the fur-
ther conjugation of a functional device. The 6-azido-idose
1 could be prepared from d-glucose (7) through epimerization
at the 5-position and incorporation of the azido group at the
6-position.
We first examined the oxime formation of the sugars 1, 7,
and 18 with hydroxylamine (Scheme 3). O-Benzylhydoxylamine
hydrochloride was treated with an equivalent of the sugars 1,
7, and 18 in a phosphate buffer (pH 7.0) at 08C for 22 h. The
Scheme 2 shows the synthesis of the 6-azido-idose 1 from
diacetone-d-glucose (8) by using the epoxide 13 as an inter-
mediate. The preparation of compound 13 was achieved
based on a reported method[14] for the synthesis of a related
compound possessing a benzyl ether group instead of a (2-
naphthyl)methyl ether. Etherification of the diacetonide 8 with
Scheme 1. Design of 6-azido-6-deoxy-l-idose (1) as a hetero-bifunctional
spacer.
Scheme 2. Synthesis of the 6-azido-idose 1. NAP = 2-naphthylmethyl, Ac =
acetyl, Bz = benzoyl, Py = pyridine, Ms = mesyl.
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reaction mixtures were analyzed by using HPLC based on UV
absorption at l=254 nm. The transformation rates of hydrox-
ylamine to the oximes 20 and 21 from d-glucose (7) and the
6-azido-glucose 18 were 5 and 15%, respectively. On the other
hand, the transformation rate to the oxime 19 from the 6-
azido-idose 1 was 91%. The yield of the isolated compound 19
was 85%. These results clearly indicated that the 6-azido-idose
1 exhibited a higher reactivity than the glucose derivatives 7
and 18.
Scheme 4. The six isomers of compound 1 involving the furanoses F-a and
F-b, the pyranoses P-a and P-b, and the acyclic acetal and aldehyde Acetal
and Alde.
cose.[12] The larger population of the acyclic forms may explain
the higher level of reactivity of the 6-azido-idose 1.
Scheme 3. Oxime formation. Bn=benzyl.
To demonstrate the utility of the 6-azido-idose 1, we plan-
ned the synthesis of (À)-8-(6-azido-6-deoxy-b-l-idopyranosyl)-
3-(3,4,5-trihydroxybenzoyl)epigallocatechin (22) through the
direct C-glycosylation of epigallocatechin gallate (EGCG, 23)
with the 6-azido-idose 1. The EGCG 23 is a phytochemical
found in tea leaves that exhibits various biological activities
(Scheme 5).[17] The gallate acts as a pharmacophore to bind
a 67 kDa laminin receptor (67 LR), which mediates anti-cancer
and anti-allergic actions.[18] In addition, the B-ring of the EGCG
was easily oxidized under physiological conditions to an ortho-
quinone moiety and was reacted with an amino group and
a thiol group on proteins.[19] The group of Nakayama reported
that the adduct of EGCG and proteins was stained with a func-
tionalized boronic acid through esterification of the catechol
moiety on the D-ring.[20] We expected the 6-azido-idose 1 to
undergo C-glycosylation of the EGCG 23 at the C8- or C6-posi-
tion under mildly acidic conditions. The resultant C-glycosyl
EGCG 22 possessing an azido group was an effective chemical
probe for exploring the mode of the biological action of the
EGCG.
Next, we estimated the population of the acyclic forms of
the 6-azido-idose 1 by using 13C NMR measurement of the uni-
formly 13C-labeled 6-azido-idose U-13C-1. The U-13C-6-azido-
idose U-13C-1 was prepared from uniformly 13C-labeled d-glu-
cose through our established method. Figure 1 shows the
13C NMR spectrum of the U-13C-6-azido-idose U-13C-1. The as-
signment of each signal was achieved based on a previous
report[16] by comparing the chemical shifts and the scalar cou-
pling constants. The populations of the six isomers of com-
pound 1 involving the furanoses F-a and F-b, the pyranoses P-
a and P-b, and the acyclic acetal and aldehyde Acetal and Alde
were 12.7, 16.4, 36.1, 34.4, 0.2, and 0.1%, respectively, based
on each 13C peak intensity (Scheme 4). The acyclic forms to-
taled 0.3% of all the isomers. The population of the acyclic
forms of compound 1 was more than ten times that of glu-
We first compared the reactivity of the 6-azido-idose 1 and
the 6-azido-glucose 17 (Scheme 6). The EGCG 23 was exposed
to either an equivalent of the 6-azido-idose 1 or the 6-azido-
glucose 18 in the presence of Sc(OTf)3 at 708C for 3 h.[21] The
1
Figure 1. 150 MHz H-coupled 13C NMR spectrum of the U-13C-6-azido-idose
U-13C-1 (anomeric carbon region) dissolved in D2O. The inset shows the ver-
tical scale expansion of the aldehyde signal. Each C1 signal appears in a quar-
1
1
tet, due to J(C1,C2) (ꢀ40 Hz) and J(C1,H1) (160–180 Hz) scalar couplings.
Scheme 5. Synthesis of the C-idosyl EGCG 22 from the EGCG 23 and the 6-
azido-idose 1.
The 1J(C1,H1) coupling constants are indicated for each peak.
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conversion of the reaction using compounds 1 and 18 was es-
timated to be 60 and 12%, respectively, through HPLC analysis
of the remaining EGCG 1 based on UV absorption at l=
254 nm. The EGCG was stable under the acidic conditions with-
out the sugars 1 and 18. These results clearly indicated that
the 6-azido-idose 1 would be more reactive towards electro-
philic substitution of the aromatic rings than the 6-azido-glu-
cose 18. To identify the major product of the reaction, we
treated the EGCG 23 with an equivalent of the 6-azido-idose
1 in the presence of Sc(OTf)3 at 708C for 9 h. HPLC-MS analysis
of the crude mixture indicated that the reaction mixture con-
tained several mono-glycosyl products (molecular weight
(MW)=645), and the compound (MW=1104). These results
suggested that the reaction mixture from the 6-azido-idose
1 might contain the a- and b-anomeric stereoisomers and the
pyran and furan derivatives as well as the regioisomers at C6-
and C8-positions. In addition, in the reaction a further electro-
philic addition of the benzylic cation 25 with the EGCG 23
might have occurred to provide the 1:2 complex 24 of the
sugar and the EGCG. A major product was purified by prepara-
tive HPLC based on a MS-triggered detection to provide the C-
glycosyl EGCG 22 in 20% yield as a single isomer. Although
the yield was not high, the single-step transformation of the
natural EGCG to the azido-containing EGCG 22 would be
useful for the synthesis of biochemical probes for the elucida-
tion of the mechanism of the function of the EGCG. It should
be noted that when the isolated C-glycosyl EGCG was exposed
to the reaction conditions, it provided several isomers possess-
ing the same molecular weight (i.e., MW=645). These results
indicated that the idose unit would be easily isomerized under
acidic conditions not only to an a-isomer but also to furan de-
rivatives.
Structure determination of the C-glycosyl product was ach-
1
ieved by H and 13C NMR spectroscopy as well as 2D NMR anal-
yses (i.e., COSY, HMQC, and HMBC experiments) (Figure 2). In
1
the H NMR spectra of compound 22 the signal for protons on
the A ring disappeared. A broad singlet at d=5.65 ppm as-
signed to H-1’’’, showed HMQC correlations with the 13C signal
at d=76.2 ppm and HMBC correlations with the 13C signals at
d=157.8, 153.0, and 103.2 ppm. These results indicated that C-
glycosylation of the EGCG 23 with the idose 1 occurred at the
C6- or C8-position. The stereochemistry of the glycosidic link-
age was determined by NOE observation between H-1’’’ and
H-5’’’ (6.4%) to be b. According to the report on the synthesis
of C-glycosyl flavonoids,[10,22] HMBC correlation between H-2
and aromatic carbon atoms enabled assignment of a 13C signal
at the 8a-position, which was critical information to determine
the position of the C-glycosylation. However, the requisite
HMBC correlation was not observed. We finally determined the
position of the C-glycosylation of the EGCG to be C8 based on
NOE observation between H-1’’’ and H-2’ (2.0%).
Figure 2. Key HMBC correlations and NOE observations for the structural as-
signment of compound 22.
Next, we investigated the anti-cancer effect of the C-idosyl
EGCG 22 in U266 cells expressing 67LR (a mouse melanoma
cell line) (Figure 3). The anti-cancer effects against U266 cells
served as an index for the affinity of EGCG-related compounds
to 67LR. EGCG was used as a positive control. The U266 cells
were incubated with compound 22 (0, 5.0, 10, or 25 mm) for
96 h at 378C. The relative number of viable cells was estimated
by using an ATPlite assay. Our data suggested that the C-idosyl
EGCG 22 exhibited a comparable cytotoxicity against U266
cells to that of EGCG. These results suggested that the azido-
bearing EGCG derivative could be used not only as a chemical
probe for the analysis of covalent adducts from EGCG, but also
as a precursor for the synthesis of chemical probes analyzing
EGCG-associated proteins involving 67LR.
Conclusion
In conclusion, 6-azido-6-deoxy-l-idose (1) was an effective
hetero-bifunctional spacer for the synthesis of azido-containing
chemical probes. The azido group can bio-orthogonally react
with terminal acetylene through a Hüisgen [3+2] cycloaddi-
tion. The masked aldehyde 1 was more reactive towards imina-
tion than either d-glucose (7) or the 6-azido-glucose 18 due to
its enhanced population of the acyclic forms involving the
acetal and the aldehyde. The population of the acylic forms of
Scheme 6. Direct C-glycosylation of the EGCG 23 with the hetero-bifunction-
al spacer 1.
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250 mm2, 5 mm, Inertsil ODS-3 column (GL Sciences Inc.). THF and
CH2Cl2 were dried by using a glass contour. tert-Butanol was dis-
tilled from CaH2. For 0.1m BnONH2 solution with pH 7.0 phosphate
buffer, BnONH2·HCl (80 mg, 0.50 mmol) was dissolved in phosphate
buffer solution (5.0 mL), which was prepared by dissolving
Na2HPO4 (200 mg) in water. d-Glucose-13C6 (99 atom% 13C) was pur-
chased from SI Science Co., Ltd.
1,2-O-Isopropylidene-3-O-(2-naphthyl)methyl-a-d-glucofuranose
(10): NaH (60% in mineral oil, 5.30 g) in a round bottom flask was
washed with hexane to remove the mineral oil. After decanting
the hexane, THF (134 mL) was added to the reaction vessel.
Then, 1,2:5,6-di-O-isopropylidene-a-d-glucofuranose (8) (17.4 g,
66.8 mmol), 2-(bromomethyl)naphthalene (15.5 g, 70.1 mmol), and
tetrabutylammonium iodide (1.20 g, 3.25 mmol) were sequentially
added to the reaction mixture at 08C. After being stirred at room
temperature for 3 h, the reaction mixture was poured into water at
08C and extracted with two portions of ethyl acetate. The com-
bined organic layers were washed with brine, dried over MgSO4, fil-
tered, and concentrated in vacuo. The residue was used in the
next reaction without further purification. The residue was dis-
solved in 70% aqueous acetic acid (200 mL, 2450 mmol). After
being stirred at 608C for 2 h, the reaction mixture was poured into
an aqueous solution (200 mL) of NaOH (88.0 g, 2200 mmol) and
NaHCO3 (21.0 g, 250 mmol) at 08C and extracted with two portions
of ethyl acetate. The combined organic layers were washed with
brine, dried over MgSO4, filtered, and concentrated in vacuo. The
residue was purified by column chromatography on silica gel
(ethyl acetate/hexane=2:1) to yield compound 10 (23.8 g,
66.0 mmol, 99% in two steps). [a]2D9 =À33.8 (c=0.755, CHCl3);
1H NMR (400 MHz, CDCl3): d=7.84–7.78 (m, 4H; aromatic), 7.49–
7.43 (m, 3H; aromatic), 5.94 (d, J=3.4 Hz 1H; H-1), 4.85 (d, J=
12.2 Hz, 1H; benzyl), 4.71 (d, J=12.2 Hz, 1H; benzyl), 4.65 (d, J=
3.9 Hz, 1H; H-2), 4.16–4.12 (m, 2H; H-3, H-4), 4.07–4.04 (m, 1H; H-
5), 3.81 (dd, J=11.7, 3.4 Hz, 1H; H-6a), 3.70 (dd, J=11.7, 5.8 Hz,
1H; H-6b), 1.47 (s, 3H; acetal), 1.30 ppm (s, 3H; acetal); 13C NMR
(100 MHz, CDCl3): d=134.7 (aromatic), 133.4 (aromatic), 133.3 (aro-
matic), 128.8 (aromatic), 128.1 (aromatic), 127.9 (aromatic), 127.0
(aromatic), 126.5 (aromatic), 126.4 (aromatic), 125.6 (aromatic),
112.0 (acetal), 105.3 (C-1), 82.3 (C-2), 82.1 (C-3), 80.2 (C-4), 72.4
(benzyl), 69.4 (C-5), 64.5 (C-6), 26.9 (CH3), 26.4 ppm (CH3); FTIR
(neat): n˜ =436, 2937, 1376, 1215, 1165, 1079, 1020, 752 cmÀ1; HRMS
(ESI-TOF): m/z calcd for C20H24O6: 383.1471 [M+Na]+; found:
383.1465.
Figure 3. Anti-cancer effect of the C-idosyl EGCG 22 in human multiple mye-
loma cells. U266 cells were treated with either EGCG or the C-idosyl EGCG
for 96 h and the relatively viable cell number was assessed through an AT-
Plite assay (n=3) repeated three independent times. Error bars represent
the standard deviation. (*, P<0.05 vs. control; corresponding to 0 mm). Stat-
istical analysis was performed with ANOVA followed by a Dunnett’s test (vs.
control; corresponding to 0 mm)).
compound 1 was estimated to be 0.3% by using the 13C NMR
spectrum of the U-13C-6-azido-idose U-13C-1, which was more
than ten times the reported data for glucose. We next applied
the 6-azido-idose 1 to the synthesis of a C-glycosyl EGCG as
a chemical probe. Treatment of EGCG with the 6-azido-idose
1 in the presence of Sc(OTf)3 provided the C8-idosyl EGCG 22
in 20% yield. The azido-containing EGCG 22 exhibited cytotox-
icity against U266 cells that was comparable to that of EGCG.
These results suggested that the azido-containing EGCG 22
could be used for biochemical probes to elucidate the mode
of the biological action. Biological research using compound
22 is now in progress.
Experimental Section
General information: NMR spectra were recorded on a JEOL
Model ECP-400 spectrometer (400 MHz for 1H, 100 MHz for 13C)
and a Bruker instrument in the indicated solvent. Chemical shifts
were reported in parts per million [ppm] relative to the signal (d=
0.00 ppm) for internal tetramethylsilane solutions in CDCl3. The
1H NMR spectral data are reported as follows: CDCl3 (d=7.26 ppm),
CD3OD (d=3.31 ppm). The 13C NMR spectral data are reported as
follows: CDCl3 (d=77.16 ppm), CD3OD (d=49.0 ppm). Multiplicities
are reported by the following abbreviation: s=singlet, d=doublet,
t=triplet, m=multiplet, br=broad, J=coupling constants in
Hertz. IR spectra were recorded on a Perkin–Elmer Spectrum One
FTIR spectrometer. Only the strongest and/or structurally important
peaks are reported as IR data given in [cmÀ1]. ESI-TOF mass spectra
were measured with a Waters LCT PremierTM XE. HRMS (ESI-TOF)
were calibrated by using Leu-enkephalin. Optical rotations were
measured on a JASCO model P-1020 polarimeter. All reactions
were monitored by thin-layer chromatography carried out on
0.2 mm E. Merck silica gel plate (60F-254) with UV light (l=
254 nm), visualized by using a p-anisaldehyde solution. Column
chromatography separations were performed by using silica gel
(Merck, silica gel). Analytical HPLC was carried out on a SSC-3461
pump with a SSC-5410 UV detector by using a 4.6”250 mm2,
3 mm, Inertsil ODS-3 column (GL Sciences Inc.). Preparative HPLC
5,6-O-Anhydro-1,2-O-isopropylidene-3-O-(2-naphthyl)methyl-b-l-
idofuranose (13): Pyridine (57.0 mL, 708 mmol) and benzoyl chlo-
ride (8.25 mL, 71.1 mmol) were sequentially added to a solution of
compound 10 (23.8 g, 66.0 mmol) in CH2Cl2 (200 mL) at 08C. After
being stirred for 2 h at room temperature, methanesulfonyl chlo-
ride (8.25 mL, 107 mmol) and triethylamine (30 mL, 216 mmol)
were sequentially added to the solution at 08C. After being stirred
at room temperature for 3 h, the reaction mixture was poured into
3m aqueous HCl solution, and the mixture was extracted with two
portions of ethyl acetate. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was used in the next reaction without further
purification. Potassium tert-butoxide (15.0 g, 134 mmol) was added
to a solution of the obtained residue in tert-butanol (200 mL) and
CH2Cl2 (200 mL) at 08C. After being stirred at room temperature for
5 h, the reaction mixture was poured into a saturated aqueous
NH4Cl solution and the obtained mixture was extracted with two
portions of ethyl acetate. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on
was carried out on
a
Waters AutoPurificationTM System (a
Waters 600 controller pump and a Waters 2767 sample manager)
with a Waters 2767 Dual l absorbance detector by using a 20”
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silica gel (ethyl acetate/hexane=1/3) and recrystallized to give
compound 13 (13.8 g, 40.3 mmol, 61% in two steps). [a]2D6 =À64.9
133.7 (aromatic), 132.9 (aromatic), 128.4 (aromatic), 128.1 (aromat-
ic), 127.7 (aromatic), 126.5 (aromatic), 126.2 (aromatic), 125.6 (aro-
matic), 123.5 (aromatic), 112.2 (acetal), 105.8 (C-1), 99.3 (benzyl),
83.2 (C-2), 79.6 (C-3), 75.7 (C-5), 72.3 (C-4), 51.7 (C-6), 26.7 (CH3),
26.2 ppm (CH3); FTIR (neat): n˜ =2102, 1165, 1141, 1096, 1073, 1015,
859, 823 cmÀ1; HRMS (ESI-TOF): m/z calcd for C20H22N3O5: 384.1559
[M+H]+; found: 384.1538.
1
(c=0.385, CHCl3); H NMR (400 MHz, CDCl3): d=7.84–7.76 (m, 4H;
aromatic), 7.49–7.42 (m, 3H; aromatic), 6.03 (d, J=3.4 Hz, 1H; H-1),
4.89 (d, J=12.7 Hz, 1H; benzyl), 4.68–4.64 (m, 2H; H-2, benzyl),
4.00 (d, J=3.9 Hz, 1H; H-3), 3.81 (dd, J=5.9, 3.4 Hz, 1H; H-4), 3.33–
3.31 (m, 1H; H-5), 2.76 (t, J=5.0 Hz, 1H; H-6a), 2.51 (dd, J=4.9,
2.9 Hz, 1H; H-6b), 1.44 (s, 3H; acetal), 1.32 ppm (s, 3H; acetal);
13C NMR (100 MHz, CDCl3): d=134.7 (aromatic), 133.2 (aromatic),
133.2 (aromatic), 128.5 (aromatic), 127.9 (aromatic), 127.8 (aromat-
ic), 126.7 (aromatic), 126.4 (aromatic), 126.3 (aromatic), 125.5 (aro-
matic), 112.0 (acetal), 105.6 (C-1), 82.6 (C-3), 82.5 (C-4), 82.2 (C-2),
72.0 (benzyl), 50.3 (C-5), 43.3 (C-6), 26.9 (CH3), 26.4 ppm (CH3); FTIR
(neat): n˜ =2989, 1164, 1076, 1028, 856 cmÀ1; HRMS (ESI-TOF): m/z
calcd for C20H22O5: 365.1365 [M+Na]+; found: 365.1379.
6-Azido-6-deoxy-l-idose (1):
A 1m aqueous H2SO4 solution
(2.80 mL) was added to a solution of compound 16 (0.353 g,
0.921 mmol) in 1,4-dioxane (2.80 mL) at room temperature. After
being stirred for 11 h at 708C, Amberliteꢁ IRA-67 free base was
added to the solution at 08C. The mixture was filtered through
a pad of Celite and concentrated in vacuo. The residue was puri-
fied by reverse-phase column chromatography on Bondesil-C18
(water) to yield compound 1 (0.163 g, 0.795 mmol, 86%). [a]3D0 = +
1
12.7 (c=0.565, CH3OH); H NMR (400 MHz, CD3OD): d=5.36 (d, J=
6-Azido-6-deoxy-1,2-O-isopropylidene-3-O-(2-naphthyl)methyl-b-
l-idofuranose (15): A mixture of Al(OiPr)3 (0.358 g, 1.75 mmol) and
Me3SiN3 (1.65 mL, 12.6 mmol) in CH2Cl2 (15.0 mL) was stirred at
room temperature for 2 h. A solution of compound 13 (0.955 g,
2.79 mmol) in CH2Cl2 (4.2 mL) was added to the mixture at room
temperature. After being stirred at room temperature for 29 h, the
reaction mixture was poured into a biphasic solution composed of
1m aqueous HCl solution and ethyl acetate. After being stirred at
room temperature for 12 h, the mixture was extracted with two
portions of ethyl acetate. The combined organic layers were
washed with brine, dried over MgSO4, filtered, and concentrated in
vacuo. The residue was purified by column chromatography on
silica gel (ethyl acetate/toluene=1/4) to yield compound 15
3.9 Hz), 5.09 (s), 4.99 (d, J=2.9 Hz), 4.98 (s), 4.32–4.28 (m), 4.16 (t,
J=4.9 Hz), 4.10–3.97 (m), 3.79 (t, J=4.9 Hz), 3.61–3.52 (m), 3.46–
3.45 (m), 3.41–3.35 ppm (m); 13C NMR (100 MHz, CD3OD): d=104.1,
97.5, 96.3, 94.0, 83.3, 82.9, 79.8, 78.4, 77.5, 77.3, 74.5, 72.1, 71.5,
71.5, 71.2, 71.1, 71.0, 70.0, 68.9, 54.9, 54.6, 52.6, 52.0 ppm; FTIR
(neat): n˜ =3436, 2535, 2117, 1634 cmÀ1; HRMS (ESI-TOF): m/z calcd
for C6H11N3O5: 206.0777 [M+H]+; found: 206.0815.
(À)-8-(6-Azido-6-deoxy-b-l-idopyranosyl)-3-(3,4,5-trihydroxyben-
zoyl)-epigallocatechin (22): Scandium trifluoromethanesulfonate
(12.0 mg, 0.0244 mmol) was added to a mixture of (À)-epigalloca-
techin-3-O-gallate (23) (55.9 mg, 0.122 mmol) and compound
1 (25.0 mg, 0.122 mmol) in acetonitrile/water (2:1; 0.364 mL) at
room temperature. After being stirred at 708C for 9 h, the reaction
mixture was diluted with water and purified by reverse-phase
column chromatography (VARIAN Bond ELUTꢁ C18) to remove un-
reacted monosaccharide and scandium trifluoromethaesulfonate.
The organic layer was concentrated in vacuo. The residue was sub-
jected to chromatography by using a preparative 20”250 mm2,
5 mm, Inertsil ODS-3 column (GL Sciences Inc.). Chromatography
was performed by using 20% acetonitrile/water containing 0.1%
formic acid as the eluent. After the solvent was removed in vacuo,
the residue was suspended in water, and freeze-dried to afford
compound 22 (15.7 mg, 20%). [a]2D9 =À46.9 (c=0.55, CH3OH);
1H NMR (400 MHz, CD3OD): d=6.94 (s, 2H; H-2’’), 6.51 (s, 2H; H-2’),
5.96 (s, 1H; H-6), 5.65 (s, 1H; H-1’’’), 5.55 (brs, 1H; H-3), 5.02 (s, 1H;
H-2), 4.12 (t, J=5.8 Hz, 1H; H-5’’’), 3.99 (t, J=2.9 Hz, 1H; H-3’’’),
3.72–3.66 (m, 2H; H-2’’’, H-6’’’a), 3.60 (s, 1H; H-4’’’), 3.52 (dd, J=
13.2, 5.4 Hz, 1H; H-6’’’b), 3.00 (dd, J=17.6, 4.9 Hz, 1H; H-4a),
2.86 ppm (dd, J=17.6, 2.4 Hz, 1H; H-4b); 13C NMR (100 MHz,
CD3OD): d=167.7 (carbonyl), 157.8 (C-7), 157.2 (C-5), 153.0 (C-8a),
146.8 (C-3’), 146.3 (C-3’’), 139.8 (C-4’’), 133.8 (C-4’), 130.6 (C-1’),
121.5 (C-1’’), 110.3 (C-2’’), 106.6 (C-2’), 103.2 (C-8), 99.3 (C-5a), 97.3
(C-6), 78.5 (C-2), 77.0 (C-5’’’), 76.2 (C-1’’’), 73.1 (C-2’’’), 70.1 (C-4’’’),
69.7 (C-3’’’), 69.5 (C-3), 49.6 (C-6’’’), 26.7 ppm (C-4); FTIR (neat): n˜ =
3271, 2114, 1679, 1451, 1338, 1239, 1146, 1041 cmÀ1; HRMS (ESI-
TOF): m/z calcd for C28H27N3O15: 668.1340 [M+Na]+; found:
668.1346.
1
(1.09 g, 2.83 mmol, 100%). [a]2D8 =À60.3 (c=0.115, CHCl3); H NMR
(400 MHz, CDCl3): d=7.87–7.82 (m, 3H; aromatic), 7.75 (s, 1H; aro-
matic), 7.52–7.48 (m, 2H; aromatic), 7.42 (dd, J=8.3, 1.4 Hz, 1H; ar-
omatic), 6.02 (d, J=3.9 Hz, 1H; H-1), 4.89 (d, J=12.2 Hz, 1H;
benzyl), 4.71 (d, J=3.9 Hz, 1H; H-2), 4.62 (d, J=12.2 Hz, 1H;
benzyl), 4.16–4.12 (m, 2H; H-4, H-5), 4.05 (d, J=3.4 Hz, 1H; H-3),
3.36 (dd, J=12.7, 6.3 Hz, 1H; H-6a), 3.28 (dd, J=13.2, 4.4 Hz, 1H;
H-6b), 1.49 (s, 3H; acetal), 1.34 ppm (s, 3H; acetal); 13C NMR
(100 MHz, CDCl3): d=133.9 (aromatic), 133.3 (aromatic), 133.2 (aro-
matic), 128.9 (aromatic), 128.0 (aromatic), 127.9 (aromatic), 127.3
(aromatic), 126.6 (aromatic), 126.5 (aromatic), 125.6 (aromatic),
112.2 (acetal), 105.1 (C-1), 82.8 (C-3), 82.4 (C-2), 79.8 (C-4), 72.1
(benzyl), 69.8 (C-5), 53.2 (C-6), 26.9 (CH3), 26.4 ppm (CH3); FTIR
(neat): n˜ =2929, 2103, 1164, 1075, 1028 cmÀ1; HRMS (ESI-TOF): m/z
calcd for C20H23N3O5: 386.1716 [M+H]+; found: 386.1715.
(S)-6-Azido-6-deoxy-1,2-O-isopropylidene-3,5-O-(2-naphthyl)me-
thylidene-b-l-idofuranose (16): Activated molecular sieve (4 Š)
(0.540 g) and DDQ (0.670 g, 2.95 mmol) were sequentially added to
a solution of compound 15 (1.03 g, 2.67 mmol) in CH2Cl2 (16.0 mL)
at room temperature. After being stirred at room temperature for
12 h, the reaction mixture was poured into a saturated aqueous so-
lution of NaHCO3. After being stirred at room temperature for 11 h,
the reaction mixture was extracted with two portions of dichloro-
methane. The combined organic layers were washed with brine,
dried over MgSO4, filtered, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (ethyl ace-
tate/hexane=1/3) to yield compound 16 (0.840 g, 2.20 mmol,
NMR experiments: Quantitative 13C NMR spectra were obtained by
using an AVANCE 600 spectrometer equipped with a TXI probe
(BrukerBioSpin). The probe temperature was set at 258C and the
13C NMR spectra were obtained with 16000 scans at a recycle time
of 11 s. To avoid 13C–1H heteronuclear NOEs, 1H-decoupling was
not applied throughout the measurement. 13C chemical shifts (in
[ppm]) were calibrated by using an exterior standard chemical shift
(4,4-dimethyl-4-silapentane-1-sulfonic acid, set to d=0 ppm). NMR
data processing and analysis were performed by using TOPSPIN
(version 2.1, BrukerBioSpin).
1
82%). [a]2D6 = +2.8 (c=0.310, CHCl3); H NMR (400 MHz, CDCl3): d=
7.99 (s, 1H; aromatic), 7.87–7.82 (m, 3H; aromatic), 7.58 (d, J=
8.3 Hz, 1H; aromatic), 7.51–7.47 (m, 2H; aromatic), 6.05 (d, J=
3.9 Hz, 1H; H-1), 5.71 (s, 1H; benzyl), 4.69 (d, J=3.9 Hz, 1H; H-2),
4.51 (d, J=2.0 Hz, 1H; H-3), 4.28 (ddd, J=8.8, 4.4, 2.0 Hz, 1H; H-5),
4.12 (t, J=2.4 Hz, 1H; H-4), 3.83 (dd, J=13.2, 8.3 Hz, 1H; H-6a),
3.46 (dd, J=13.2, 4.4 Hz, 1H; H-6b), 1.53 (s, 3H; acetal), 1.35 ppm
(s, 3H; acetal); 13C NMR (100 MHz, CDCl3): d=134.3 (aromatic),
Chem. Eur. J. 2016, 22, 12884 – 12890
www.chemeurj.org
12889
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim

Full Paper
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This work was supported by a Grant-in-Aid for Scientific Re-
search on Innovative Areas (Grant-in-aid No. 26102720).
Keywords: aldehydes · C-glycosylation · Lewis acids · oximes ·
phytochemistry
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Received: May 2, 2016
Published online on August 2, 2016
Chem. Eur. J. 2016, 22, 12884 – 12890
www.chemeurj.org
12890
ꢀ 2016 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim