Facile synthesis toward the construction of an activity probe library for glycosidases

Article abstract of DOI:10.1016/j.carres.2005.12.005

Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-related research. We have previously demonstrated the design and synthesis of an activity probe for β-glucosidase adopting a cassette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the structure of a key intermediate. The recognition head of this library covers a series of six sugars, including α- and β-D-Glc, α- and β-D-Gal, α-D-Man, and α-L-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the first time to target α-D-glucosidase, α-D-galactosidase, and α-L-fucosidase, respectively.

Full text of DOI:10.1016/j.carres.2005.12.005

Carbohydrate
RESEARCH
Carbohydrate Research 341 (2006) 443–456
Facile synthesis toward the construction of an activity probe
library for glycosidases
Tong-Hong Shie,a Ying-Ling Chiang,a Jing-Jer Lin,b,* Yaw-Kuen Lic
and Lee-Chiang Loa,*
aDepartment of Chemistry, National Taiwan University, Taipei 106, Taiwan
bInstitute of Biopharmaceutical Sciences, National Yang-Ming University, Taipei 112, Taiwan
cDepartment of Applied Chemistry, The National Chiao Tung University, Hsinchu 300, Taiwan
Received 4 October 2005; received in revised form 29 November 2005; accepted 14 December 2005
Available online 18 January 2006
Abstract—Chemical probes that selectively label the glycoside hydrolase (GH) subfamilies have proven to be a powerful tool in GH-
related research. We have previously demonstrated the design and synthesis of an activity probe for b-glucosidase adopting a cas-
sette-like design in a model study. Herein we report an improved synthetic route using (4-hydroxyphenyl)acetic acid 2-cyanoethyl
ester as the precursor for the latent trapping device. Parallel syntheses were performed for the preparation of a library based on the
structure of a key intermediate. The recognition head of this library covers a series of six sugars, including a- and b-D-Glc, a- and b-
D-Gal, a-D-Man, and a-L-Fuc. Each member in this versatile intermediate library could serve as the building block in constructing
an activity probe for GHs. As demonstrated in this study, three probes that have the 1,2-cis configuration were thus prepared for the
first time to target a-D-glucosidase, a-D-galactosidase, and a-L-fucosidase, respectively.
ꢀ 2006 Elsevier Ltd. All rights reserved.
Keywords: Activity probe; Glycosidase; Proteomics; Hydrolase; Carbohydrate; Mechanism-based labeling; Biotin
1. Introduction
proven to be a powerful tool in modern chemical pro-
teomics.10–13 This approach is especially useful for the
hydrolase families as the labeling process matches well
with the catalytic mechanism. For example, activity
probes for GHs have been successfully applied in a wide
variety of research, such as the screening and selection
of catalytic antibodies, profiling of GH activity in
proteomics studies, and investigation of active-site
structures.14–19 In view of the great potential of activity
probes for GH families, we envision that a system-
atic preparation of these probes would open new oppor-
tunities and accelerate the progress in GH-related
research.
Recently, deoxyfluoro sugar derivatives bearing suit-
able reporter groups, which were able to form stable
glycosyl–enzyme intermediates, have been incorporated
into probe design to investigate glycosidases.18,19 How-
ever, this strategy is only applicable to the retaining glyco-
sidases. As an alternative, we have previously adopted
Glycoconjugates on the cell surface play important roles
in many biological events, including viral infection, cell–
cell recognition, and protein trafficking.1–4 The carbo-
hydrate signal molecules of these glycoconjugates are
produced by sequential actions of designated glycosyl-
transferases and glycosidases.5,6 Dysfunction of these
key enzymes often leads to detrimental situations. Due
to their biological significance, glycoside hydrolases
(GHs, glycosidases; EC 3.2.1) have become targets of
interest in numerous interdisciplinary research fields.7–9
Currently, researchers are interested not only in the rela-
tive amount of the GHs, but also in the level of their
activities. Chemical probes that selectively react with
protein subfamilies through covalent interactions have
*
Corresponding authors. Tel.: +886 2 33661669; fax: +886 2 23636359
(L.-C.L.); e-mail: lclo@ntu.edu.tw
0008-6215/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2005.12.005

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T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
Figure 1. Structures of probe 1 and the key intermediate 2.
a suicide substrate-based approach and developed
activity probes for hydrolases such as tyrosine phospha-
tases and GHs.16,20,21 Compound 1 was shown to be an
activity probe for b-glucosidase in a model study, and
compound 2, which already carries a recognition head
linked to the latent trapping device, was a key synthetic
intermediate (Fig. 1).16 In this report, we develop an im-
proved synthetic route to construct a versatile library
based on the structure of intermediate 2. The recogni-
tion head of the library covers commonly six sugars
and anomeric configurations, including both a and b
anomers of D-Glc and D-Gal as well as a-D-Man and
a-L-Fuc. It has to be noted that, due to synthetic conve-
nience, activity probes for GHs reported to date are lim-
ited to those that have the 1,2-trans configuration at the
anomeric carbon. Herein we demonstrate the usefulness
of such a library by preparing three activity probes for
GHs with the 1,2-cis configuration.
other glycosyl units. In the context, we used a letter fol-
lowing the compound number to specify both sugar
identity and anomeric configuration of the recognition
head for the series of compounds in the library synthe-
sis; a for a-D-Glc, b for a-D-Gal, c for a-L-Fuc, d for
b-D-Glc, e for b-D-Gal, and f for a-D-Man.
The design principle for an activity probe is similar to
that for a suicide substrate of enzymes, except that a
reporter group is attached to the activity probe. What
is unique in this approach is that the probes themselves
are also the substrates of the target hydrolases, resulting
in high specificity toward the targets. The activation and
alkylation process is depicted in Scheme 1. Generally, an
activity probe 3 is stable under incubation conditions in
the absence of the corresponding GH. However, when
the glycosidic bond between the sugar unit and the
latent trapping device is cleaved by the target GH, it
releases intermediate 4, which quickly undergoes 1,6-
elimination to generate quinone methide 5. The highly
reactive quinone methide 5 then reacts with suitable
nucleophiles on nearby GH molecules to produce
labeled adduct 6. As of this date, only a limited number
of chemical structures are eligible for the latent trapping
device of the activity probes.14–16,20,21 The quinone
methide 5 utilized in this study was previously shown
to preferably react with cysteine residues on target
proteins.22 However, no comparative study on the
performance of these latent trapping devices has ever
been reported.
2. Results and discussion
2.1. Library design and labeling mechanism
A typical activity probe carries two key structural
elements in its design, namely, a reactive unit and a
reporter group. Various reporter groups have been
incorporated in the probe design in order to achieve dif-
ferent experimental goals. For example, fluorescent dyes
and biotin are commonly used to monitor and help
characterize the labeled proteins. Probe 1 consists of
four structural units in its design, including a b-D-Glc
recognition head, a latent trapping device, and a biotin
reporter group connected through a linker bridge. The
synthesis of probe 1 was accomplished in a cassette-like
manner. Intermediate 2 is not only the core of the probe,
but is also a versatile building block for future applica-
tions, depending on the property of the chosen reporter
group. Therefore, the basic structure of intermediate 2 is
well suited for library construction. In this study, we
carried out parallel syntheses to prepare a building block
library by simply replacing the b-D-Glc moiety with
2.2. Retrosynthetic analysis
The synthesis of the building block library 7 was divided
into two stages. The retrosynthetic analysis is shown
in Scheme 2. The first stage involves the preparation of
glycosidation products 8. In recent years, progress in
glycosidation reactions with a high degree of control of
the anomeric configuration has been made by manipul-
ating the properties of the participants in the glycosida-
tion step, including the glycosyl donor, protective group,
solvent and promoter.23–27 However, in order to achieve
this high selectivity, special reagents needed for the reac-

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
445
Scheme 1. General design of an activity probe 3 for GHs and the mechanism for the selective labeling event.
bromide peracetate in the presence of AgOTf.16 How-
ever, the benzyl ester had to be removed and converted
to the b-cyanoethyl ester before the introduction of the
benzylic functionality. In this study, we have improved
on the procedure by carrying out the glycosidation
directly with b-cyanoethyl ester 11, which was obtained
in 65% yield by treating acid 10 with b-cyanoethyl alco-
hol in the presence of concentrated H2SO4 catalyst
(Scheme 3). Ester 11 was then subjected to two different
glycosidation conditions; BF3-mediated glycosidation
and Koenigs–Knorr method. In the former series, ester
11 reacted with peracetylated sugar donors 9 in the pres-
ence of BF3 to give compounds 8a–c in 37%, 31%, and
40% yields, respectively. Coupling constants of 3.6 Hz
between H-1 and H-2 of the sugar moiety in this series
of products supported the 1,2-cis configuration. For
the 1,2-trans products, the corresponding peracetates 9
were first converted to peracetylated glycosyl bromides
12 by treatment with HBr–HOAc,31–33 followed by cou-
pling with b-cyanoethyl ester 11 under Koenigs–Knorr
conditions with the assistance of AgOTf to give com-
pounds 8d–f in 40%, 32%, and 63% yields, respectively.
Although the yields for both glycosidation reactions
were only moderate, the results are reasonably accep-
table due to the simplicity in the operations. The 1,2-
trans stereochemistry for 8d and 8e was established by
the coupling constants of 7.4–7.9 Hz between H-1 and
H-2. The anomeric configuration of compound 8f was
established by the chemical shift of the anomeric proton
(5.40 ppm) and the anomeric carbon (95.4 ppm), both in
support of the a-D-configuration.
Scheme 2. Retrosynthetic pathway for the construction of building
block library 7.
tions had to be prepared prior to the synthesis. Since the
recognition head carries only a monosaccharide, and
both anomeric products are desired in this study, we
chose the shortest possible synthetic route, beginning
with the easily accessible peracetylated sugars 9. Two
different glycosidation conditions, each in favor of one
type of anomer, were employed. BF3-mediated condi-
tions gave mainly the 1,2-cis products, whereas the clas-
sical Koenigs–Knorr method led to the 1,2-trans
products.23,28 The desired glycosidation products could
be obtained after chromatographic purifications. The
peracetylated glycosyl bromides needed for the latter
method could be derived from the peracetylated sugars
9. The benzylic functionalization was achieved at the
second stage in three steps: bromination, hydrolysis,
and fluorination, to give compounds 7a–f as the building
block library.
2.3. Synthesis of glycosidation products 8
The library synthesis begins with p-hydroxyphenylacetic
acid 10, from which the latent trapping device was
derived. The design of this latent trapping device has
been utilized in many biochemical applications that
required covalent labeling of target proteins.16,21,22,29,30
In the previous synthesis of probe 1, the critical glyco-
sidation step was achieved by Koenigs–Knorr method
using the benzyl ester of compound 10 and the glucosyl
2.4. Synthesis of building block library 7a–f
Glycosidation products 8a–f were then subjected to ben-
zylic functionalization (Scheme 4). Benzylic bromination
was achieved by treatment with NBS and AIBN under
irradiation to give benzylic bromides 13a–f in high
yields (73–95%). These benzylic bromides underwent

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T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
Scheme 3. The structure of compounds 8a–f and two glycosidation conditions for their formation.
Scheme 4. Benzylic functionalization of compounds 8a–f for the syntheses of building blocks 7a–f.
hydrolysis with AgNO3 and Ag2CO3 in acetone–H2O to
afford benzylic alcohols 14a–f. Although the reactions
were sluggish, moderate yields were obtained (30–
56%). The hydroxyl group of 14a–f was smoothly con-
verted to fluoride by reaction with DAST to give the fi-
nal products 7a–f (56–94%). It must be noted that all the
compounds prepared after benzylic functionalization
are present as diastereomeric pairs. However, the newly
generated stereocenter has a negligible effect on the
NMR spectra, giving rise to mainly only one set of sig-
nals for each diastereomeric pair.
tion. Here we utilize three members of the above library
7 that possess 1,2-cis configuration to prepare the GH
probes 18a–c to target a-D-glucosidase, a-D-galactosi-
dase, and a-L-fucosidase, respectively (Scheme 5). Com-
pounds 7a–c were treated with DBU in CH2Cl2 to give
a-fluorocarboxylates 15a–c in excellent yields (94–
100%). Due to the instability of the free acids, they were
isolated and stored as their triethylammonium salts. a-
Fluorocarboxylates 15a–c were coupled with a biotin
derivative 1634 carrying a disulfide bridge by EDCI–
HOBt method to give fully protected probes 17a–c in
65%, 65%, and 67% yields, respectively. Compounds
17a–c were eventually subjected to deacetylation by
treatment with Na2CO3–MeOH to give probes 18a–c.
A cleavable disulfide linker is incorporated in the probes
18a–c. This cleavable linker would enable us to recover
the labeled targets by treatment of a reducing agent such
as DTT.
2.5. Synthesis of probes 18a–c
The sugar moiety and the carboxylate of building blocks
7a–f are orthogonally protected; the former is peracetyl-
ated and the latter is protected as its b-cyanoethyl ester.
For future applications, the b-cyanoethyl group of com-
pounds 7 could be selectively removed under alkaline
conditions to release carboxylic acid to which a suitable
reporter group is attached through amide bond forma-
In summary, we have adopted a cassette-like design
for the systematic preparation of a versatile building
block library targeting GH families. An improved syn-

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
447
Scheme 5. The synthesis of probes 18a–c.
thetic route beginning with (4-hydroxyphenyl)-acetic
acid 2-cyano-ethyl ester was developed. It could react
with the designated glycosyl donors under two different
coupling conditions to preferentially afford the a and b
anomers, respectively. Parallel syntheses were performed,
and a series of sugars including a- and b-D-Glc, a- and
b-D-Gal, a-D-Man, and a-L-Fuc were attached as the
recognition head. The synthetic route presented here
offers an efficient means to rapidly assemble the building
block library in a straightforward manner. These versa-
tile intermediates are the key building blocks in con-
structing activity probes for GH families and have
numerous potential applications. As an example, in this
study we have further utilized the building block library
and demonstrated the preparation of three activity
probes that have the 1,2-cis configuration, which have
never been reported before. These three probes will be
used for the screening of a-D-glucosidase, a-D-galactosi-
dase, and a-L-fucosidase from various biological sources.
or phosphomolybdic acid–EtOH. Column chromato-
graphy was performed with Kiesegel 60 (70–230 mesh)
silica gel (E. Merck). Melting points are reported with-
out correction.
3.2. Preparation of b-cyanoethyl ester 11
To a solution of 4-hydroxyphenylacetic acid (10.0 g,
65.8 mmol), b-cyanoethanol (4.71 g, 65.8 mmol), and
10.0 g of anhyd Na2SO4 in 50 mL of anhyd MeCN
was added 1.75 mL of concentrated H2SO4 (32.9 mmol)
as a catalyst. The reaction mixture was heated under
reflux for 16 h. The solvent was removed under reduced
pressure, and the residual oil was dissolved in 100 mL
of EtOAc. The EtOAc solution was washed consecu-
tively with satd NaHCO3 (·3), H2O and brine, followed
by drying over anhyd Na2SO4. It was filtered and con-
centrated to give b-cyanoethyl ester 11 (8.91 g) in 66%
1
yield. Rf 0.50 (4:1 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.10 (d, J 6.5 Hz, 2H, aromatic), 6.74
(d, J 6.5 Hz, 2H, aromatic), 5.94 (br s, 1H), 4.25 (t,
J 6.2 Hz, 2H), 3.57 (s, 2H, benzylic), 2.66 (t, J 6.2 Hz,
2H); 13C NMR (CDCl3, 100 MHz): d 171.7 (C), 155.1
(C), 130.5 (CH), 125.0 (C), 116.7 (C), 115.6 (CH), 59.0
(CH2), 40.0 (CH2), 17.9 (CH2). FABMS: m/z (%) 205
(M+, 8), 136 (100); HRMS: calcd for C11H11NO3
(M+), 205.0739; found, 205.0741.
3. Experimental
3.1. General methods
All reagents and starting materials were obtained from
commercial suppliers (Acros, Aldrich and E. Merck)
and were used without further purification. 1H and
13C NMR were recorded using a Brucker AC-300 or
Bruker Avance 400 spectrometer. The proton and car-
bon chemical shifts are given in ppm using CDCl3 (dH
7.24 and 77.0) as internal standard. High-resolution
mass spectra were recorded with a JEOL-102A mass
spectrometer. Analytical TLC (silica gel, 60F-54, E.
Merck) and spots were visualized under UV light and/
3.3. General procedure for the preparation of
glycosidation products 8a–c
To a solution of b-cyanoethyl ester 11 (1.40 mmol) and
the corresponding sugar peracetate 9 (1.02 mmol) in
anhyd MeCN was slowly added BF3Æetherate
(2.25 mmol). The reaction mixture was kept at room

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T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
temperature for 24 h and quenched by adding
2.25 mmol of Et3N. The solvent was removed under
reduced pressure, and the residual oil was subjected to
silica gel column chromatography. The desired glycosi-
dation products 8a–c were eluted with 1:1 hexane–
EtOAc and were obtained in 31–40% yields.
5.24 (dd, J 10.9, 3.6 Hz, 1H, H-2), 4.27 (t, J 6.3 Hz,
2H), 4.24 (q, J 6.5 Hz, 1H, H-5), 3.60 (s, 2H, benzylic),
2.68 (t, J 6.3 Hz, 2H), 2.16 (s, 3H, OAc), 2.03 (s, 3H,
OAc), 1.99 (s, 3H, OAc), 1.09 (d, J 6.5 Hz, 3H, CH3);
13C NMR (CDCl3, 100 MHz): d 171.1 (C), 170.5 (C),
170.4 (C), 170.0 (C), 155.8 (C), 130.5 (CH), 127.3 (C),
116.6 (CH), 116.6 (C), 94.8 (CH), 70.9 (CH), 67.9
(CH), 67.7 (CH), 65.4 (CH), 59.0 (CH2), 40.0 (CH2),
20.7 (CH3), 20.7 (CH3), 20.6 (CH3), 17.9 (CH2), 15.8
(CH3). IR (neat): 1746, 1520, 1381, 1222, 1156,
1076 cmꢀ1. FABMS: m/z (%) 478 (M+1+, 4), 273
(100); HRMS: calcd for C23H28NO10 (M+1)+,
478.1714; found, 478.1716.
3.3.1. [(4-Tetra-O-acetyl-a-D-glucopyranosyloxy)phenyl]-
acetic acid 2-cyanoethyl ester (8a). Yield 37% (oil), Rf
0.20 (1:1 n-hexane–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.16 (d, J 8.6 Hz, 2H, aromatic), 7.00 (d,
J 8.6 Hz, 2H, aromatic), 5.65 (d, J 3.6 Hz, 1H, H-1),
5.62 (dd, J 10.2, 9.6 Hz, 1H, H-3), 5.09 (dd, J 10.0,
9.6 Hz, 1H, H-4), 4.97 (dd, J 10.2, 3.6 Hz, 1H, H-2),
4.23 (t, J 6.2 Hz, 2H), 4.19 (dd, J 12.3, 4.4 Hz, 1H, H-
6), 4.03 (m, 1H, H-5), 3.98 (dd, J 12.3, 1.9 Hz, 1H, H-
60), 3.57 (s, 2H), 2.64 (t, J 6.2 Hz, 2H), 2.01 (s, 3H,
OAc), 1.99 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.97 (s,
3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.9 (C),
170.3 (C), 170.0 (C), 170.0 (C), 169.4 (C), 155.2 (C),
130.3 (CH), 127.7 (C), 116.6 (CH), 115.3 (C), 94.1
(CH), 70.2 (CH), 69.8 (CH), 68.1 (CH), 67.8 (CH),
61.4 (CH2), 58.9 (CH2), 39.8 (CH2), 20.6 (CH3), 20.5
(CH3), 20.5 (CH3), 20.4 (CH3), 17.8 (CH2). IR (neat):
1759, 1514, 1375, 1222, 1162, 1043 cmꢀ1. FABMS: m/z
(%) 558 (M+Na+, 4), 536 (M+1+, 12), 331 (100);
HRMS: calcd for C25H30NO12 (M+1)+, 536.1768;
found, 536.1762.
3.4. General procedure for the preparation of
glycosidation products 8d–f
To an ice-cooled solution of the corresponding sugar
peracetates 9 (10.25 mmol) in 5 mL of anhyd CH2Cl2
was slowly added 10 mL of HBr–HOAc. The reaction
mixture was stirred for 15 min, and then it was poured
into 100 mL of ice water. A white cloudy precipitate
was observed. It was extracted with 100 mL of CHCl3.
The organic layer was collected and washed with ice
water (100 mL · 3), satd NaHCO3 (50 mL · 2), and
brine. The organic layer was separated and dried over
anhyd Na2SO4. It was filtered and concentrated to give
the corresponding glycosyl bromides 12, which were
used without further purification. To an ice-cooled solu-
tion of b-cyanoethyl ester 11 (7.0 mmol) and AgOTf
(6.9 mmol) in 20 mL of anhyd CH2Cl2 was slowly added
a solution of glcosyl bromide 12 (5.4 mmol) in 10 mL of
CH2Cl2. The reaction mixture was kept in the dark and
stirred for 2.5 h. It was quenched by adding 3 mL of
DIEA, followed by filtration. The filtrate was then con-
centrated under reduced pressure, and the residual oil
was subjected to silica gel column chromatography.
The desired glycosidation products 8d–f were obtained
in 32–63% by using the same conditions described above
for the products 8a–c.
3.3.2. [(4-Tetra-O-acetyl-a-D-galactopyranosyloxy)phenyl]-
acetic acid 2-cyanoethyl ester (8b). Yield 31% (oil), Rf
0.40 (4:6 n-hexane–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.18 (d, J 8.5 Hz, 2H, aromatic), 6.99 (d,
J 8.5 Hz, 2H, aromatic), 5.71 (d, J 3.6 Hz, 1H, H-1),
5.52 (dd, J 10.7, 3.4 Hz, 1H, H-3), 5.48 (m, 1H, H-4),
5.23 (dd, J 10.7, 3.6 Hz, 1H, H-2), 4.30 (dd, J 6.5,
6.7 Hz, 1H, H-5), 4.25 (t, J 6.3 Hz, 2H), 4.10–3.99 (m,
2H, H-6+H-60), 3.59 (s, 2H, benzylic), 2.66 (t, J
6.3 Hz, 2H), 2.12 (s, 3H, OAc), 2.03 (s, 3H, OAc), 1.98
(s, 3H, OAc), 1.91 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 170.7 (C), 170.0 (C), 169.9 (C), 169.8 (C),
169.6 (C), 155.3 (C), 130.2 (CH), 127.6 (C), 116.7
(CH), 116.5 (C), 94.7 (CH), 67.5 (CH), 67.4 (CH), 67.2
(CH), 66.8 (CH), 61.1 (CH2), 58.8 (CH2), 39.6 (CH2),
20.6 (CH3), 20.3 (CH3), 20.2 (CH3), 20.2 (CH3), 17.5
(CH2). IR (neat): 2939, 2369, 1753, 1514, 1428, 1375,
1222, 1156, 1063 cmꢀ1. FABMS: m/z (%) 536 (M+1+,
7), 331 (100); HRMS: calcd for C25H30NO12 (M+1)+,
536.1768; found, 536.1766.
3.4.1. [(4-Tetra-O-acetyl-b-D-glucopyranosyloxy)phenyl]-
acetic acid 2-cyanoethyl ester (8d). Yield 40% (oil), Rf
0.25 (8:2 CHCl3–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.15 (d, J 8.6 Hz, 2H, aromatic), 6.89 (d,
J 8.6 Hz, 2H, aromatic), 5.23 (dd, J 9.8, 9.4 Hz, 1H),
5.18 (dd, J 9.4, 7.4 Hz, 1H), 5.09 (dd, J 9.8, 9.2 Hz,
1H), 5.02 (d, J 7.4 Hz, 1H, H-1), 4.24–4.00 (m, 3H),
4.09 (dd, J 12.3, 2.2 Hz, 1H, H-6), 3.81 (m, 1H, H-5),
3.56 (s, 2H, benzylic), 2.63 (t, J 6.2 Hz, 2H), 2.00 (s,
3H, OAc), 1.98 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.97
(s, 3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.9
(C), 170.3 (C), 170.0 (C), 169.2 (C), 169.1 (C), 155.9
(C), 130.3 (CH), 128.0 (C), 116.9 (CH), 116.7 (C), 98.8
(CH), 72.4 (CH), 71.7 (CH), 70.8 (CH), 68.0 (CH),
61.7 (CH2), 58.9 (CH2), 39.8 (CH2), 20.5 (CH3), 20.4
3.3.3. [(4-Tri-O-acetyl-a-L-fucopyranosyloxy)phenyl]acet-
ic acid 2-cyanoethyl ester (8c). Yield 40% (oil), Rf 0.37
(8:2 CHCl3–EtOAc). 1H NMR (CDCl3, 400 MHz): d
7.19 (d, J 8.6 Hz, 2H, aromatic), 7.00 (d, J 8.6 Hz, 2H,
aromatic), 5.69 (d, J 3.6 Hz, 1H, H-1), 5.54 (dd, J
10.9, 3.3 Hz, 1H, H-3), 5.33 (d, J 3.3 Hz, 1H, H-4),

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
449
(CH3), 20.4 (CH3), 20.4 (CH3), 17.7 (CH2). IR (neat):
1752, 1514, 1381, 1229, 1149, 1043 cmꢀ1. FABMS: m/z
(%) 558 (M+Na+, 1), 536 (M+1+, 2), 331 (100); HRMS:
calcd for C25H30NO12 (M+1)+, 536.1768; found,
536.1772.
centrated. It was subjected to silica gel column chroma-
tography, eluting with 15:85 EtOAc–CHCl3 to give the
bromides 13a–f (73–95%) as white foams.
3.5.1. [(4-Tetra-O-acetyl-a-D-glucopyranosyloxy)phenyl]-
bromoacetic acid 2-cyanoethyl ester (13a). Yield 85%
1
3.4.2. [(4-Tetra-O-acetyl-b-D-galactopyranosyloxy)phenyl]-
acetic acid 2-cyanoethyl ester (8e). Yield 32% (oil), Rf
0.25 (8:2 CHCl3–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.15 (d, J 8.5 Hz, 2H, aromatic), 6.91 (d,
J 8.5 Hz, 2H, aromatic), 5.42–5.38 (m, 2H), 5.05 (dd, J
7.0, 3.4 Hz, 1H, H-3), 4.98 (d, J 7.9 Hz, 1H, H-1), 4.21
(t, J 6.2 Hz, 2H), 4.16 (dd, J 11.2, 7.1 Hz, 1H, H-6),
4.09 (dd, J 11.2, 6.0 Hz, 1H, H-60), 4.02 (dd, J 7.1,
6.0 Hz, 1H, H-5), 3.57 (s, 2H), 2.63 (t, J 6.2 Hz, 2H),
2.10 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H,
OAc), 1.94 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 170.9 (C), 170.2 (C), 170.1 (C), 169.9 (C),
169.2 (C), 156.0 (C), 130.3 (CH), 127.9 (C), 116.8
(CH), 116.6 (C), 99.3 (CH), 70.8 (CH), 70.6 (CH), 68.4
(CH), 66.7 (CH), 61.2 (CH2), 58.9 (CH2), 39.8 (CH2),
20.5 (CH3), 20.5 (CH3), 20.4 (CH3), 20.4 (CH3), 17.7
(CH2). IR (neat): 1752, 1235, 1076 cmꢀ1. FABMS: m/z
(%) 536 (M+1+, 3), 331 (100); HRMS: calcd for
C25H30NO12 (M+1)+, 536.1768; found, 536.1773.
(oil), Rf 0.52 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.46 (d, J 8.6 Hz, 2H, aromatic), 7.04 (d,
J 8.6 Hz, 2H, aromatic), 5.69 (d, J 3.6 Hz, 1H, H-1),
5.61 (dd, J 10.3, 9.8 Hz, 1H, H-3), 5.32 (s, 1H), 5.09
(dd, J 10.7, 9.8 Hz, 1H, H-4), 4.98 (dd, J 10.3, 3.6 Hz,
1H, H-2), 4.32 (t, J 6.2 Hz, 2H), 4.17 (dd, J 12.6,
4.2 Hz, 1H, H-6), 4.03–3.96 (m, 2H, H-5+H-60), 2.69
(t, J 6.2 Hz, 2H), 1.99 (s, 3H, OAc), 1.99 (s, 3H, OAc),
1.97 (s, 3H, OAc), 1.97 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 170.3 (C), 169.9 (C), 169.9 (C), 169.4 (C),
167.6 (C), 156.6 (C), 130.1 (CH), 129.3 (C), 116.7
(CH), 116.2 (C), 93.8 (CH), 70.1 (CH), 69.7 (CH), 68.0
(CH), 68.0 (CH), 61.3 (CH2), 60.2 (CH2), 44.9 (CH),
20.5 (CH3), 20.5 (CH3), 20.4 (CH3), 20.4 (CH3), 17.6
(CH2). IR (neat): 1759, 1514, 1375, 1235, 1042 cmꢀ1
.
FABMS: m/z (%) 616 (M+1+, 81Br, 60), 614 (M+1+,
79Br, 69), 391 (100); HRMS: calcd for C25H2979BrNO12
(M+1)+ 614.0873, found, 614.0872.
3.5.2. [(4-Tetra-O-acetyl-a-D-galactopyranosyloxy)phen-
yl]bromoacetic acid 2-cyanoethyl ester (13b). Yield 87%
(oil), Rf 0.45 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
3.4.3. [(4-Tetra-O-acetyl-a-D-mannopyranosyloxy)phenyl]-
acetic acid 2-cyanoethyl ester (8f). Yield 63% (oil), Rf
0.20 (1:1 n-hexane–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.11 (d, J 8.5 Hz, 2H, aromatic), 6.94 (d,
J 8.5 Hz, 2H, aromatic), 5.43–5.40 (m, 2H, H-1+H-3),
5.27 (br, 1H, H-2), 5.24 (dd, J 10.0, 9.9 Hz, 1H, H-4),
4.18–4.14 (m, 3H), 3.99–3.93 (m, 2H, H-5+H-6), 3.51
(s, 2H, benzylic), 2.59 (t, J 6.3 Hz, 2H), 2.59 (s, 3H,
OAc), 2.08 (s, 3H, OAc), 1.94 (s, 3H, OAc), 1.91 (s,
3H, OAc), 1.91 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 170.6 (C), 170.1 (C), 169.5 (C), 169.5 (C),
169.3 (C), 154.5 (C), 130.2 (CH), 127.6 (C), 116.6 (C),
116.3 (CH), 95.4 (CH), 68.9 (CH), 68.7 (CH), 68.5
(CH), 65.4 (CH), 61.7 (CH2), 58.8 (CH2), 39.6 (CH2),
20.5 (CH3), 20.3 (CH3), 20.3 (CH3), 20.3 (CH3), 17.6
1
400 MHz): d 7.48 (d, J 8.5 Hz, 2H, aromatic), 7.04 (d,
J 8.5 Hz, 2H, aromatic), 5.76 (d, J 3.6 Hz, 1H, H-1),
5.52 (dd, J 10.7, 3.3 Hz, 1H, H-3), 5.48 (m, 1H, H-4),
5.33 (s, 1H), 5.24 (dd, J 10.7, 3.6 Hz, 1H, H-2), 4.30
(t, J 6.3 Hz, 2H), 4.26 (dd, J 7.1, 5.8 Hz, 1H, H-5),
4.08 (dd, J 11.2, 5.8 Hz, 1H, H-6), 4.02 (dd, J 11.2,
7.1 Hz, 1H, H-60), 2.70 (t, J 6.3 Hz, 2H), 2.13 (s, 3H,
OAc), 2.03 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.90 (s,
3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.3 (C),
170.2 (C), 170.1 (C), 169.9 (C), 167.7 (C), 156.9 (C),
130.2 (CH), 129.3 (C), 116.9 (CH), 116.2 (C), 94.6
(CH), 67.6 (CH), 67.5 (CH), 67.3 (CH), 67.2 (CH),
61.2 (CH2), 60.3 (CH2), 45.0 (CH), 20.6 (CH3), 20.5
(CH3), 20.5 (CH3), 20.5 (CH3), 17.7 (CH2). IR (neat):
1745, 1520, 1374, 1228, 1175, 1129, 1063 cmꢀ1. FABMS:
m/z (%) 616 (M+1+, 81Br, 11), 614 (M+1+, 79Br, 10),
391 (100); HRMS: calcd for C25H2979BrNO12 (M+1)+,
614.0873; found, 614.0878.
(CH2). IR (neat): 1745, 1374, 1222, 1129, 1043 cmꢀ1
.
FABMS: m/z (%) 536 (M+1+, 8), 331 (100); HRMS:
calcd for C25H30NO12 (M+1)+, 536.1768; found,
536.1770.
3.5. General procedure for the preparation of benzylic
bromides 13a–f
3.5.3. [(4-Tri-O-acetyl-a-L-fucopyranosyloxy)phenyl]bromo-
acetic acid 2-cyanoethyl ester (13c). Yield 83% (oil),
Rf 0.45 (85:15 CHCl3–EtOAc). 1H NMR (CDCl3,
400 MHz): d 7.48 (d, J 8.8 Hz, 2H, aromatic), 7.04 (d,
J 8.8 Hz, 2H, aromatic), 5.72 (d, J 3.6 Hz, 1H, H-1),
5.53 (dd, J 10.9, 3.3 Hz, 1H, H-3), 5.33–5.32 (m, 2H),
5.24 (dd, J 10.9, 3.6 Hz, 1H, H-2), 4.38–4.33 (m, 2H),
4.20 (q, J 6.7 Hz, 1H, H-5), 2.72 (t, J 6.3 Hz, 2H), 2.16
(s, 3H, OAc), 2.03 (s, 3H, OAc), 2.00 (s, 3H, OAc),
To a solution of compound 8 (1.70 mmol) in 20 mL of
CCl4 were added NBS (1.90 mmol) and a catalytic
amount of AIBN. The reaction flask was equipped with
a condenser, and the apparatus was purged and filled
with N2 three times. It was irradiated with a 100-W
tungsten lamp for 1 h until a white precipitate formed.
The precipitate was filtered off and the filtrate was con-

450
T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
1.09 (d, J 6.7 Hz, 3H, CH3); 13C NMR (CDCl3,
100 MHz): d 170.5 (C), 170.4 (C), 170.1 (C), 167.8 (C),
157.4 (C), 130.3 (CH), 128.9 (C), 116.7 (CH), 116.2
(C), 94.6 (CH), 70.8 (CH), 67.8 (CH), 67.6 (CH), 65.6
(CH), 60.3 (CH2), 45.1 (CH), 20.7 (CH3), 20.7 (CH3),
20.6 (CH3), 17.7 (CH2), 15.8 (CH3). IR (neat): 1746,
1520, 1375, 1228, 1175, 1129, 1070 cmꢀ1. FABMS: m/z
(%) 558 (M+1+, 81Br, 1), 556 (M+1+, 79Br, 0.9), 273
(100).
400 MHz): d 7.45 (d, J 8.7 Hz, 2H, aromatic), 7.03 (d,
J 8.7 Hz, 2H, aromatic), 5.48 (d, J 1.6 Hz, 1H, H-1),
5.46 (dd, J 10.1, 3.5 Hz, 1H, H-3), 5.36 (dd, J 3.5,
1.6 Hz, 1H, H-2), 5.31 (br, 1H), 5.25 (dd, J 10.1,
10.0 Hz, 1H, H-4), 4.33–4.28 (m, 2H), 4.20 (dd, J
12.1, 6.2 Hz, 1H, H-6), 4.01–3.98 (m, 2H, H-5+H-60),
2.68 (t, J 6.3 Hz, 2H), 2.13 (s, 3H, OAc), 1.99 (s, 3H,
OAc), 1.97 (s, 3H, OAc), 1.96 (s, 3H, OAc); 13C NMR
(CDCl3, 100 MHz): d 170.5 (C), 169.9 (C), 169.7 (C),
167.8 (C), 167.8 (C), 156.2 (C), 130.2 (CH), 129.5 (C),
116.8 (CH), 116.3 (C), 95.6 (CH), 69.2 (CH), 69.1
(CH), 68.7 (CH), 65.7 (CH), 62.0 (CH2), 60.4 (CH2),
45.1 (CH), 20.8 (CH3), 20.8 (CH3), 20.7 (CH3), 20.7
(CH3), 17.7 (CH2). IR (neat): 1759, 1507, 1374,
1228, 1135, 1043 cmꢀ1. FABMS: m/z (%) 616 (M+1+,
81Br, 5), 614 (M+1+, 79Br, 5), 331 (100); HRMS: calcd
for C25H2979BrNO12 (M+1)+, 614.0873; found,
614.0876.
3.5.4. [(4-Tetra-O-acetyl-b-D-glucopyranosyloxy)phenyl]-
bromoacetic acid 2-cyanoethyl ester (13d). Yield 88%
1
(oil), Rf 0.38 (8:2 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.48 (d, J 8.6 Hz, 2H, aromatic), 6.96 (d,
J 8.6 Hz, 2H, aromatic), 5.33 (s, 1H), 5.30–5.21 (m,
2H), 5.15 (m, 1H), 5.09 (d, J 7.2 Hz, 1H, H-1), 4.40–
4.31 (m, 2H), 4.25 (dd, J 12.3, 5.2 Hz, 1H, H-6), 4.14
(dd, J 12.3, 2.3 Hz, 1H, H-60), 3.86 (m, 1H, H-5), 2.71
(t, J 6.3 Hz, 2H), 2.05 (s, 3H, OAc), 2.02 (s, 3H, OAc),
2.01 (s, 3H, OAc), 1.99 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 170.5 (C), 170.2 (C), 169.3 (C), 169.2 (C),
167.8 (C), 157.4 (C), 130.2 (CH), 129.7 (C), 117.1
(CH), 116.2 (C), 98.5 (CH), 72.5 (CH), 72.1 (CH), 71.0
(CH), 68.1 (CH), 61.8 (CH2), 60.3 (CH2), 45.0 (CH),
20.7 (CH3), 20.7 (CH3), 20.6 (CH3), 20.6 (CH3), 17.7
(CH2). IR (neat): 1759, 1388, 1229, 1043 cmꢀ1. FABMS:
m/z (%) 638 (M+Na+, 81Br, 79), 636 (M+Na+, 79Br,
83), 616 (M+1+, 81Br, 79), 614 (M+1+, 79Br, 100), 391
(96); HRMS: calcd for C25H2979BrNO12 (M+1)+,
614.0873; found, 614.0872.
3.6. General procedure for the preparation of benzylic
alcohols 14a–f
Benzylic bromide 13 (1.50 mmol) was dissolved in 5 mL
of acetone. A solution of AgNO3 (3.00 mmol) and
Ag2CO3 (0.75 mmol) in 5 mL of H2O was added. A dark
greenish precipitate formed immediately. The reaction
mixture was stirred in the dark at rt for 30 min. It was
filtered and the filtrate was concentrated to dryness.
The residual oil was dissolved in EtOAc and washed
with 5% citric acid (·3), satd NaHCO3 (·3) and brine.
The organic phase was dried over anhyd Na2SO4, fil-
tered, and concentrated. The resulting oil was chromato-
graphed on silica gel (4:6 EtOAc–CHCl3) to give
benzylic alcohols 14a–f in 30–56% yields.
3.5.5. [(4-Tetra-O-acetyl-b-D-galactopyranosyloxy)phenyl]-
bromoacetic acid 2-cyanoethyl ester (13e). Yield 73%
1
(oil), Rf 0.32 (8:2 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.48 (d, J 8.7 Hz, 2H, aromatic), 6.97 (d,
J 8.7 Hz, 2H, aromatic), 5.47–5.42 (m, 2H), 5.33 (s,
1H), 5.08 (dd, J 7.0, 3.4 Hz, 1H), 5.04 (d, J 7.9 Hz,
1H, H-1), 4.40–4.24 (m, 2H), 4.18 (dd, J 11.3, 7.1 Hz,
1H, H-6), 4.12 (dd, J 12.3, 6.0 Hz, 1H, H-60), 4.05 (dd,
J 7.1, 6.0 Hz, 1H, H-5), 2.72–2.65 (m, 2H), 2.14 (s,
3H, OAc), 2.02 (s, 3H, OAc), 2.00 (s, 3H, OAc) 1.98
(s, 3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.3
(C), 170.1 (C), 170.0 (C), 169.3 (C), 167.8 (C), 157.5
(C), 130.2 (CH), 129.6 (C), 117.1 (CH), 116.1 (C), 99.1
(CH), 71.1 (CH), 70.7 (CH), 68.4 (CH), 66.8 (CH),
61.3 (CH2), 60.3 (CH2), 45.0 (CH), 20.7 (CH3), 20.6
(CH3), 20.6 (CH3), 20.5 (CH3), 17.7 (CH2). IR (neat):
3.6.1. [(4-Tetra-O-acetyl-a-D-glucopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14a). Yield 38%
(oil), Rf 0.25 (1:1 CHCl3–EtOAc). H NMR (CDCl3,
1
400 MHz): d 7.32 (d, J 8.6 Hz, 2H, aromatic), 7.03 (d,
J 8.6 Hz, 2H, aromatic), 5.67 (d, J 3.6 Hz, 1H, H-1),
5.62 (dd, J 10.2, 9.6 Hz, 1H, H-3), 5.15 (d, J 5.6 Hz,
1H), 5.10 (dd, J 10.0, 9.6 Hz, 1H, H-4), 4.97 (dd, J
10.2, 3.6 Hz, 1H, H-2), 4.32 (m, 1H), 4.21 (m, 1H),
4.17 (dd, J 12.9, 4.2 Hz, 1H, H-6), 4.05–3.96 (m, 2H,
H-5+H-6), 3.58 (d, J 5.6 Hz, 1H, OH), 2.63 (m, 2H),
1.99 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.97 (s, 3H,
OAc), 1.97 (s, 3H, OAc); 13C NMR (CDCl3,
100 MHz): d 172.8 (C), 170.4 (C), 170.1 (C), 170.0 (C),
169.5 (C), 156.1 (C), 132.2 (C), 127.9 (CH), 116.7
(CH), 116.2 (C), 94.0 (CH), 72.1 (CH), 70.2 (CH), 69.8
(CH), 68.0 (CH), 67.8 (CH), 61.4 (CH2), 59.8 (CH2),
20.5 (CH3), 20.5 (CH3), 20.5 (CH3), 20.4 (CH3), 17.7
(CH2). IR (neat): 3489, 1752, 1507, 1374, 1228,
1043 cmꢀ1. FABMS: m/z (%) 552 (M+1+, 5), 331
(100); HRMS: calcd for C25H30NO13 (M+1)+,
552.1714; found, 552.1721.
1746, 1235, 1076 cmꢀ1
.
FABMS: m/z (%) 638
(M+Na+, 81Br, 79), 636 (M+Na+, 79Br, 83), 616
(M+1+, 81Br, 1), 614 (M+1+, 79Br, 1), 331 (100);
HRMS: calcd for C25H2979BrNO12 (M+1)+, 614.0873;
found, 614.0878.
3.5.6. [(4-Tetra-O-acetyl-a-D-mannopyranosyloxy)phenyl]-
bromoacetic acid 2-cyanoethyl ester (13f). Yield 95%
1
(oil), Rf 0.55 (1:1 CHCl3–EtOAc). H NMR (CDCl3,

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
451
3.6.2. [(4-Tetra-O-acetyl-a-D-galactopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14b). Yield 45%
(oil), Rf 0.30 (1:1 CHCl3–EtOAc). H NMR (CDCl3,
(CH), 71.1 (CH), 68.2 (CH), 61.9 (CH2), 60.0 (CH2),
20.7 (CH3), 20.6 (CH3), 20.6 (CH3), 20.6 (CH3), 17.9
(CH2). IR (neat): 3481, 2969, 2259, 1746, 1517, 1372,
1227, 1182, 1044 cmꢀ1. FABMS: m/z (%) 552 (M+1+,
5), 331 (100); HRMS: calcd for C25H30NO13 (M+1)+,
552.1714; found, 552.1721.
1
400 MHz): d 7.31 (d, J 8.7 Hz, 2H, aromatic), 6.99 (d,
J 8.7 Hz, 2H, aromatic), 5.69 (d, J 3.2 Hz, 1H, H-1),
5.49–5.45 (m, 2H, H-3+H-4), 5.19 (dd, J 10.6, 3.2 Hz,
1H, H-2), 5.14 (d, J 5.5 Hz, 1H), 4.31–4.16 (m, 3H),
4.083–3.97 (m, 2H, H-6+H-60), 3.33 (s, 1H, OH),
2.63–2.58 (m, 2H), 2.09 (s, 3H, OAC), 1.99 (s, 3H,
OAC), 1.94 (s, 3H, OAC), 1.86 (s, 3H, OAC); 13C
NMR (CDCl3, 100 MHz): d 172.6 (C), 170.2 (C),
170.2 (C), 170.0 (C), 169.9 (C), 156.2 (C), 132.2 (C),
127.8 (CH), 116.8 (CH), 116.3 (C), 94.7 (CH), 72.1
(CH), 67.6 (CH), 67.5 (CH), 67.2 (CH), 66.9 (CH),
61.2 (CH2), 59.6 (CH2), 20.5 (CH3), 20.4 (CH3), 20.4
(CH3), 20.3 (CH3), 17.6 (CH2). IR (neat): 3489, 2985,
1752, 1613, 1513, 1434, 1374, 1228, 1175, 1129,
1076 cmꢀ1. FABMS: m/z (%) 552 (M+1+, 10), 331
(100); HRMS: calcd for C25H30NO13 (M+1)+,
552.1714; found, 552.1722.
3.6.5. [(4-Tetra-O-acetyl-b-D-galactopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14e). Yield 40%
1
(oil), Rf 0.25 (1:1 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.33 (d, J 7.5 Hz, 2H, aromatic), 6.96 (d,
J 8.6 Hz, 2H, aromatic), 5.45–5.40 (m, 2H), 5.17 (d, J
5.2 Hz, 1H), 5.07 (dd, J 7.0, 3.4 Hz, 1H), 5.01 (d, J
7.9 Hz, 1H, H-1), 4.38–4.21 (m, 2H), 4.17 (dd, J 11.3,
7.1 Hz, 1H, H-6), 4.11 (dd, J 11.3, 6.1 Hz, 1H, H-60),
4.03 (dd, J 7.1, 6.1 Hz, 1H, H-5), 3.47 (d, J 4.8 Hz,
1H), 2.68–2.60 (m, 2H), 2.13 (s, 3H, OAc), 2.01 (s, 3H,
OAc), 2.01 (s, 3H, OAc), 1.80 (s, 3H, OAc); 13C NMR
(CDCl3, 100 MHz): d 172.9 (C), 170.4 (C), 170.3 (C),
170.1 (C), 169.4 (C), 157.1 (C), 132.6 (C), 127.9 (CH),
117.1 (CH), 116.3 (C), 99.4 (CH), 72.3 (CH), 71.1
(CH), 70.8 (CH), 68.6 (CH), 66.9 (CH), 61.4 (CH2),
59.9 (CH2), 20.7 (CH3), 20.7 (CH3), 20.6 (CH3), 20.6
(CH3), 17.9 (CH2). IR (neat): 3443, 1752, 1235,
1063 cmꢀ1. FABMS: m/z (%) 552 (M+1+, 6), 331
(100); HRMS: calcd for C25H30NO13 (M+1)+,
552.1714; found, 552.1721.
3.6.3. [(4-Tri-O-acetyl-a-L-fucopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14c). Yield
30%, (oil); Rf 0.17 (7:3 CHCl3–EtOAc). 1H NMR
(CDCl3, 400 MHz): d 7.33 (d, J 8.7 Hz, 2H, aromatic),
7.03 (d, J 8.7 Hz, 2H, aromatic), 5.70 (d, J 3.5 Hz, 1H,
H-1), 5.53 (dd, J 10.9, 3.4 Hz, 1H, H-3), 5.32 (d, J
3.4 Hz, 1H, H-4), 5.23 (dd, J 10.9, 3.5 Hz, 1H, H-2),
5.18 (d, J 4.2 Hz, 1H), 4.37–4.21 (m, 2H), 4.30 (q, J
6.7 Hz, 1H, H-5), 3.42 (br s, 1H, OH), 2.67–2.63 (m,
2H), 2.16 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.99 (s, 3H,
OAc), 1.08 (d, J 6.7 Hz, 3H, CH3); 13C NMR (CDCl3,
100 MHz): d 173.0 (C), 170.6 (C), 170.5 (C), 170.2 (C),
156.9 (C), 131.9 (C), 128.0 (CH), 116.8 (CH), 116.2
(C), 94.8 (CH), 72.4 (CH), 70.9 (CH), 67.9 (CH),
67.8 (CH), 65.5 (CH), 59.9 (CH2), 20.8 (CH3), 20.7
(CH3), 20.7 (CH3), 17.9 (CH2), 15.9 (CH3). IR (neat):
3.6.6. [(4-Tetra-O-acetyl-a-D-mannopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14f). Yield 39%
1
(oil), Rf 0.25 (1:1 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.33 (d, J 8.7 Hz, 2H, aromatic), 7.05 (d,
J 8.7 Hz, 2H, aromatic), 5.51–5.48 (m, 2H, H-1+H-3),
5.38 (dd, J 3.4, 1.7 Hz, 1H, H-2), 5.32 (dd, J 10.1,
10.0 Hz, 1H, H-4), 5.16 (d, J 5.3 Hz, 1H), 4.37–4.31
(m, 1H), 4.27–4.20 (m, 2H), 4.04–4.00 (m, 2H, H-
5+H-60), 3.46 (br, 1H, OH), 2.64–2.60 (m, 2H), 2.15
(s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H, OAc),
1.87 (s, 3H, OAc); 13C NMR (CDCl3, 100 MHz): d
172.8 (C), 170.5 (C), 169.7 (C), 169.6 (C), 169.6 (C),
155.8 (C), 132.2 (C), 127.9 (CH), 116.7 (CH), 116.1
(C), 95.7 (CH), 72.2 (CH), 69.2 (CH), 69.1 (CH), 68.7
(CH), 65.8 (CH), 62.0 (CH2), 59.8 (CH2), 20.7 (CH3),
20.7 (CH3), 20.5 (CH3), 20.5 (CH3), 17.7(CH2). IR
3483, 1759, 1514, 1381, 1229, 1189, 1076 cmꢀ1
.
FABMS: m/z (%) 494 (M+1+, 6), 273 (100); HRMS:
calcd for C23H28NO11 (M+1)+, 494.1662; found,
494.1670.
3.6.4. [(4-Tetra-O-acetyl-b-D-glucopyranosyloxy)phenyl]-
hydroxyacetic acid 2-cyanoethyl ester (14d). Yield 56%
(neat): 3489, 1752, 1513, 1374, 1228, 1182, 1043 cmꢀ1
.
1
(oil), Rf 0.20 (1:1 CHCl3–EtOAc). H NMR (CDCl3,
FABMS: m/z (%) 552 (M+1+, 7), 331 (100); HRMS:
calcd for C25H30NO13 (M+1)+, 552.1714; found,
552.1716.
400 MHz): d 7.35 (d, J 8.6 Hz, 2H), 6.99 (d, J 8.6 Hz,
2H), 5.30–5.21 (m, 2H), 5.19 (d, J 5.6 Hz, 1H), 5.14
(dd, J 9.8, 9.5 Hz, 1H), 5.07 (d, J 7.6 Hz, 1H, H-1),
4.38 (m, 1H), 4.30–4.22 (m, 2H), 4.14 (dd, J 12.4,
1.9 Hz, 1H, H-6), 3.84 (m, 1H, H-5), 3.30 (d, J 5.6 Hz,
1H, OH), 2.67–2.62 (m, 2H), 2.05 (s, 3H), 2.03 (s, 3H),
2.02 (s, 3H), 2.01 (s, 3H); 13C NMR (CDCl3,
100 MHz): d 172.9 (C), 170.6 (C), 170.2 (C), 169.4 (C),
169.3 (C), 157.1 (C), 132.5 (C), 127.9 (CH), 117.2
(CH), 117.1 (C), 98.9 (CH), 72.6 (CH), 72.2 (CH), 72.0
3.7. General procedure for the preparation of fluorinated
products 7a–f
To an ice-cooled solution of compound 14 (0.67 mmol)
in 10 mL of anhyd CH2Cl2 was added 213 lL
(1.40 mmol) of DAST. The reaction mixture was
allowed to warm to rt, and it was stirred for 15 h. It

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T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
was then quenched by adding small amount of silica gel
and 1.5 mL of MeOH. It was concentrated and the fluo-
rinated products 7a–f were obtained as white foam after
silica gel column chromatography, eluting with 2:8
EtOAc–CHCl3.
3.6 Hz, 1H, H-1), 5.49 (dd, J 10.9, 3.3 Hz, 1H, H-3),
5.30 (d, J 3.3 Hz, 1H, H-4), 5.21 (dd, J 10.9, 3.6 Hz,
1H, H-2), 4.38–4.26 (m, 2H), 4.18 (q, J 6.5 Hz, 1H, H-
5), 2.73–2.64 (m, 2H), 2.13 (s, 3H, OAc), 2.00 (s, 3H,
OAc), 1.97 (s, 3H, OAc), 1.06 (d, J 6.5 Hz, 3H, CH3);
13C NMR (CDCl3, 100 MHz): d 170.4 (C), 170.3 (C),
170.0 (C), 167.9 (d, J 29 Hz, C), 157.6 (C), 128.4
(CH), 127.5 (d, J 20 Hz, C), 116.7 (CH), 116.2 (C),
94.6 (CH), 88.4 (d, J 185 Hz, CHF), 70.6 (CH), 67.6
(CH), 67.5 (CH), 65.4 (CH), 59.6 (CH2), 20.6 (CH3),
20.5 (CH3), 20.4 (CH3), 17.7 (CH2), 15.7 (CH3). IR
3.7.1. [(4-Tetra-O-acetyl-a-D-glucopyranosyloxy)phenyl]-
fluoroacetic acid 2-cyanoethyl ester (7a). Yield 89%
1
(oil), Rf 0.37 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.34 (d, J 8.4 Hz, 2H, aromatic), 7.06 (d,
J 8.4 Hz, 2H, aromatic), 5.72 (d, J 47.1 Hz, 1H, CHF),
5.67 (d, J 3.6 Hz, 1H, H-1), 5.59 (dd, J 10.3, 9.7 Hz,
1H, H-3), 5.07 (dd, J 11.1, 9.7 Hz, 1H, H-4), 4.95 (dd,
J 10.3, 3.6 Hz, 1H, H-2), 4.33–4.24 (m, 2H), 4.15 (dd,
J 12.3, 4.4 Hz, 1H, H-6), 4.01–3.93 (m, 2H, H-5+H-6),
2.56–2.61 (m, 2H), 1.96 (s, 3H, OAc), 1.96 (s, 3H,
OAc), 1.94 (s, 3H, OAc), 1.93 (s, 3H, OAc); 13C NMR
(CDCl3, 100 MHz): d 170.2 (C), 169.9 (C), 169.9 (C),
169.3 (C), 167.7 (d, J 29 Hz, C), 156.9 (C), 128.3
(CH), 127.9 (d, J 21 Hz, C), 116.7 (CH), 116.1 (C),
93.8 (CH), 88.3 (d, J 185 Hz, CHF), 70.0 (CH), 69.6
(CH), 67.9 (CH), 67.9 (CH), 61.2 (CH2), 59.6 (CH2),
20.4 (CH3), 20.3 (CH3), 20.3 (CH3), 20.3 (CH3), 17.6
(CH2). IR (neat): 2972, 1745, 1619, 1520, 1374, 1228,
1043 cmꢀ1. FABMS: m/z (%) 576 (M+Na+, 4), 554
(M+1+, 14), 331 (100); HRMS: calcd for C25H29FNO12
(M+1)+, 554.1674; found, 554.1671.
(neat): 1759, 1613, 1520, 1381, 1229, 1076, 983 cmꢀ1
.
FABMS: m/z (%) 496 (M+1+, 3), 273 (100); HRMS:
calcd for C23H27FNO10 (M+1)+, 496.1619; found,
496.1617.
3.7.4. [(4-Tetra-O-acetyl-b-D-glucopyranosyloxy)phenyl]-
fluoroacetic acid 2-cyanoethyl ester (7d). Yield 86%
1
(oil), Rf 0.38 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.39 (d, J 8.4 Hz, 2H), 7.00 (d, J 8.4 Hz,
2H), 5.76 (d, J 47.1 Hz, 1H, CHF), 5.30–5.22 (m, 2H),
5.14 (dd, J 9.2, 8.6 Hz, 1H), 5.09 (d, J 7.6 Hz, 1H, H-
1), 4.38 (m, 1H), 4.31 (m, 1H), 4.25 (dd, J 12.3,
5.2 Hz, 1H, H-6), 4.14 (dd, J 12.3, 2.0 Hz, 1H, H-60),
3.85 (m, 1H, H-5), 2.70–2.66 (m, 2H), 2.04 (s, 3H,
OAc), 2.02 (s, 3H, OAc), 2.02 (s, 3H, OAc), 2.00 (s,
3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.5 (C),
170.1 (C), 169.3 (C), 169.2 (C), 167.9 (C), 157.8 (C),
128.4 (CH), 128.3 (C), 117.2 (CH), 116.1 (C), 98.6
(CH), 88.5 (J 185 Hz, CHF), 72.5 (CH), 72.1 (CH),
71.0 (CH), 68.1 (CH), 61.8 (CH2), 59.7 (CH2), 20.6
(CH3), 20.6 (CH3), 20.5 (CH3), 20.5 (CH3), 17.8
(CH2). IR (neat): 1759, 1235, 1043 cmꢀ1. FABMS: m/z
(%) 576 (M+Na+, 25), 554 (M+1+, 100); HRMS: calcd
for C25H29FNO12 (M+1)+, 554.1674; found, 554.1678.
3.7.2. [(4-Tetra-O-acetyl-a-D-galactopyranosyloxy)phenyl]-
fluoroacetic acid 2-cyanoethyl ester (7b). Yield 94%
1
(oil), Rf 0.40 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.37 (d, J 8.7 Hz, 2H, aromatic), 7.07 (d,
J 8.7 Hz, 2H, aromatic), 5.75 (d, J 47.2 Hz, 1H, CHF),
5.75 (d, J 3.6 Hz, 1H, H-1), 5.48 (dd, J 10.7, 3.4 Hz,
1H, H-3), 5.46 (m, 1H, H-4), 5.23 (dd, J 10.7, 3.6 Hz,
1H, H-2), 4.36 (m, 1H), 4.32–4.26 (m, 2H), 4.10–4.00
(m, 2H, H-6+H-60), 2.69–2.65 (m, 2H), 2.11 (s, 3H,
OAc), 2.01 (s, 3H, OAc), 1.97 (s, 3H, OAc), 1.88 (s,
3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.2 (C),
170.1 (C), 170.0 (C), 169.9 (C), 167.7 (d, J 28 Hz, C),
157.3 (C), 128.4 (CH), 127.9 (d, J 21 Hz, C), 117.0
(CH), 116.1 (C), 94.7 (CH), 88.3 (d, J 190 Hz, CHF),
67.6 (CH), 67.5 (CH), 67.3 (CH), 67.2 (CH), 61.2
(CH2), 59.6 (CH2), 20.6 (CH3), 20.5 (CH3), 20.5
(CH3), 20.4 (CH3), 17.7 (CH2). IR (neat): 1752, 1606,
1513, 1374, 1235, 1189, 1129, 1069 cmꢀ1. FABMS: m/z
(%) 576 (M+Na+, 3), 554 (M+1+, 7), 331 (100); HRMS:
calcd for C25H29FNO12 (M+1)+, 554.1674; found,
554.1678.
3.7.5. [(4-Tetra-O-acetyl-b-D-galactopyranosyloxy)phenyl]-
fluoroacetic acid 2-cyanoethyl ester (7e). Yield 52%
1
(oil), Rf 0.32 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
400 MHz): d 7.38 (d, J 8.4 Hz, 2H, aromatic), 7.01 (d,
J 8.4 Hz, 2H, aromatic), 5.76 (d, J 47.2 Hz, 1H, CHF),
5.47–5.42 (m, 2H), 5.08 (dd, J 7.0, 3.4 Hz, 1H), 5.04
(d, J 7.9 Hz, 1H, H-1), 4.41–4.27 (m, 2H), 4.24 (dd, J
12.1, 5.2 Hz, 1H, H-6), 4.13 (dd, J 12.1, 2.1 Hz, 1H,
H-60), 4.05 (m, 1H, H-5), 2.69–2.65 (m, 2H), 2.14 (s,
3H, OAc), 2.02 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.97
(s, 3H, OAc); 13C NMR (CDCl3, 100 MHz): d 170.3
(C), 170.1 (C), 170.0 (C), 169.3 (C), 167.8 (C), 157.9
(C), 128.4 (CH), 128.2 (C), 117.1 (CH2), 116.1 (C),
99.1 (CH), 88.5 (d, J 185 Hz, CHF), 71.1 (CH), 70.6
(CH), 68.4 (CH), 66.8 (CH), 61.3 (CH2), 59.7 (CH2),
20.6 (CH3), 20.6 (CH3), 20.6 (CH3), 20.5 (CH3), 17.8
(CH2). IR (neat): 1752, 1514, 1235, 1083 cmꢀ1. FABMS:
m/z (%) 554 (M+1+, 8), 331 (100); HRMS: calcd for
C25H29FNO12 (M+1)+, 554.1674; found, 554.1685.
3.7.3. [(4-Tri-O-acetyl-a-L-fucopyranosyloxy)phenyl]fluo-
roacetic acid 2-cyanoethyl ester (7c). Yield 85% (oil), Rf
0.50 (7:3 CHCl3–EtOAc). 1H NMR (CDCl3, 400 MHz):
d 7.35 (d, J 8.5 Hz, 2H, aromatic), 7.05 (d, J 8.5 Hz, 2H,
aromatic), 5.74 (d, J 47.2 Hz, 1H, CHF), 5.70 (d, J

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
453
3.7.6. [(4-Tetra-O-acetyl-a-D-mannopyranosyloxy)phenyl]-
fluoroacetic acid 2-cyanoethyl ester (7f). Yield 85%
(oil), Rf 0.37 (7:3 CHCl3–EtOAc). H NMR (CDCl3,
3.8.2. Triethylammonium [(4-tetra-O-acetyl-a-D-galacto-
pyranosyloxy)phenyl]fluoroacetate (15b). Yield 94%
(oil), Rf 0.37 (8:2 CHCl3–MeOH). H NMR (CDCl3,
1
1
400 MHz): d 7.39 (d, J 8.3 Hz, 2H, aromatic), 7.11 (d,
J 8.3 Hz, 2H, aromatic), 5.76 (d, J 47.2 Hz, 1H, CHF),
5.52–5.49 (m, 2H, H-1+H-3), 5.40 (dd, J 3.4, 1.8, 1H,
H-2), 5.34 (dd, J 10.0, 9.9 Hz, 1H, H-4), 4.41–4.28 (m,
2H), 4.23 (dd, J 12.9, 5.9 Hz, 1H, H-6), 4.04–4.01 (m,
2H, H-5+H-60), 2.70–2.66 (m, 2H), 2.16 (s, 3H, OAc),
2.01 (s, 3H, OAc), 2.00 (s, 3H, OAc), 1.99 (s, 3H,
OAc); 13C NMR (CDCl3, 100 MHz): d 170.4 (C),
169.9 (C), 169.9 (C), 169.6 (C), 167.9 (C), 156.7 (C),
128.4 (CH), 128.0 (d, J 21 Hz, C), 116.8 (CH), 116.1
(C), 95.6 (CH), 88.5 (d, J 185 Hz, CHF), 69.2 (CH),
69.2 (CH), 68.7 (CH), 65.7 (CH), 61.9 (CH2), 59.7
(CH2), 20.8 (CH3), 20.6 (CH3), 20.6 (CH3), 20.6
(CH3), 17.8 (CH2). IR (neat): 1759, 1368, 1228, 1043
cmꢀ1. FABMS: m/z (%) 554 (M+1+, 5), 331 (100);
HRMS: calcd for C25H29FNO12 (M+1)+, 554.1674;
found, 554.1667.
400 MHz): d 7.42 (d, J 8.4 Hz, 2H, aromatic), 7.00 (d,
J 8.4 Hz, 2H, aromatic), 5.71 (d, J 3.5 Hz, 1H, H-1),
5.60 (d, 1H, J 50.8 Hz, CHF), 5.53–5.48 (m, 2H, H-
3+H-4), 5.23 (dd J 10.7, 3.5 Hz, 1H, H-2), 4.29 (dd, J
6.7, 6.3 Hz, 1H, H-5), 4.09–3.98 (m, 2H, H-6+H-60),
3.04 (q, J 7.3 Hz, 6H, TEA), 2.12 (s, 3H, OAc), 2.02
(s, 3H, OAc), 1.98 (s, 3H, OAc), 1.89 (s, 3H, OAc),
1.19 (t, J 7.3 Hz, 9H, TEA); 13C NMR (CDCl3,
100 MHz): d 173.8 (d, J 25 Hz, C), 170.3 (C), 170.1
(C), 170.1 (C), 170.0 (C), 156.4 (C), 131.9 (d, J 21 Hz,
C), 128.3 (CH), 116.6 (CH), 94.8 (CH), 90.5 (d, J
182 Hz, CHF), 67.8 (CH), 67.7 (CH), 67.4 (CH), 67.0
(CH), 61.4 (CH2), 45.0 (CH2), 20.6 (CH3), 20.6 (CH3),
20.6 (CH3), 20.5 (CH3), 8.3 (CH3). IR (neat): 1759,
1222, 1076 cmꢀ1. FABMS: m/z (%) 602 (M+TEA+1+,
89), 523 (M+Na+, 100), 501 (M+1+, 90); HRMS:
calcd for C28H41FNO12 (M+1)+, 602.2613; found,
602.2620.
3.8. General procedure for the deprotection of b-cyano-
ethyl esters
3.8.3. Triethylammonium [(4-tri-O-acetyl-a-L-fucopyran-
osyloxy)phenyl]fluoroacetate (15c). Yield 100% (oil),
Rf 0.37 (8:2 CHCl3–MeOH). 1H NMR (CDCl3,
400 MHz): d 7.35 (d, J 8.4 Hz, 2H, aromatic), 6.92 (d,
J 8.4 Hz, 2H, aromatic), 5.60 (d, J 3.6 Hz, 1H, H-1),
5.52 (d, J 49.6 Hz, 1H, CHF), 5.44 (dd, J 10.9, 3.3 Hz,
1H, H-3), 5.23 (d, J 3.3 Hz, 1H, H-4), 5.14 (dd, J 10.9,
3.6 Hz, 1H, H-2), 4.14 (q, J 6.4 Hz, 1H, H-5), 2.98 (q,
J 7.3 Hz, 6H, TEA), 2.07 (s, 3H, OAc), 1.96 (s, 3H,
OAc), 1.90 (s, 3H, OAc), 1.15 (t, J 7.3 Hz, 9H, TEA),
1.00 (d, J 6.4 Hz, 3H, CH3); 13C NMR (CDCl3,
100 MHz): d 174.3 (C), 170.3 (C), 170.2 (C), 169.9 (C),
156.3 (C), 131.5 (C), 128.2 (CH), 116.1 (CH), 94.5
(CH), 90.8 (d, J 180 Hz, CHF), 70.6 (CH), 67.7 (CH),
67.5 (CH), 65.1 (CH), 45.0 (CH2), 20.5 (CH3), 20.5
(CH3), 20.4 (CH3), 15.6 (CH3), 8.1 (CH3). IR (neat):
3417, 2999, 1753, 1619, 1381, 1229 cmꢀ1. FABMS: m/z
(%) 544 (M + TEA + 1+, 8), 273 (100); HRMS:
calcd for C26H39FNO10 (M+1)+, 544.2558; found,
544.2561.
To a solution of fluoride 7 (0.26 mmol) in 5 mL of
CH2Cl2 was added 57 lL (0.52 mmol) of DBU. The
reaction mixture was stirred at rt for 30 min. EtOAc
(20 mL) was then added, and the organic extract was
washed with 5% citric acid (·3), H2O (·2), and brine
(·1). The organic phase was collected, dried over anhyd
Na2SO4, and filtered. Since the a-fluoroacids were not
stable, they were stored as the triethylammonium salts.
This was achieved by adding 1 mL of TEA to the fil-
trate. The triethylammonium salts 15a–c were obtained
in high yields (94–100%) after concentration.
3.8.1. Triethylammonium [(4-tetra-O-acetyl-a-D-gluco-
pyranosyloxy)phenyl]fluoroacetate (15a). Yield 94%
1
(oil), Rf 0.33 (8:2 CHCl3–MeOH). H NMR (CDCl3,
400 MHz): d 7.40 (d, J 8.4 Hz, 2H, aromatic), 7.03 (d,
J 8.4 Hz, 2H, aromatic), 5.69–5.65 (m, 2H, H-1+H-3),
5.58 (d, J 37.4 Hz, 1H, CHF), 5.11 (dd, J 10.1, 9.5 Hz,
1H, H-4), 5.00 (dd, J 10.1, 3.5 Hz, 1H, H-2), 4.22 (dd,
J 12.3, 4.2 Hz, 1H, H-6), 4.09–3.98 (m, 2H, H-5+H-
60), 3.04 (q, J 7.3 Hz, 6H, TEA), 2.02 (s, 3H, OAc),
2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.01 (s, 3H,
OAc), 1.21 (t, J 7.3 Hz, 9H, TEA); 13C NMR (CDCl3,
100 MHz): d 173.9 (C), 170.6 (C), 170.1 (C), 170.1 (C),
169.6 (C), 156.1 (C), 132.0 (C), 128.4 (CH), 116.4
(CH), 94.1 (CH), 90.7 (CH, J 183 Hz, CHF), 70.4
(CH), 70.0 (CH), 68.2 (CH), 67.9 (CH), 61.5 (CH2),
44.9 (CH2), 20.7 (CH3), 20.6 (CH3), 20.6 (CH3), 20.6
3.9. General procedure for the preparation of fully
protected probes 17a–c
To a solution of the triethylammonium salt of a-fluoro-
acid 15 (0.16 mmol) and compound 16 (0.20 mmol) in
3 mL of DMF were added HOBt (27.5 mg, 0.2 mmol),
TEA (0.25 mmol), and EDCI (38.9 mg, 0.20 mmol).
The reaction mixture was stirred at rt for 24 h. DMF
was then removed under high vacuum. The residual oil
was dissolved in 20 mL of EtOAc and washed with 5%
citric acid (·3), satd NaHCO3 (·3), and brine. The
organic phase was dried over anhyd Na2SO4, filtered,
and concentrated. The biotinylated products 17 were
(CH3), 8.4 (CH3). IR (neat): 1759, 1235, 1043 cmꢀ1
.
FABMS: m/z (%) 602 (M+TEA+1+, 80), 523
(M+Na+, 90), 501 (M+1+, 100); HRMS: calcd for
C28H41FNO12 (M+1)+, 602.2613; found, 602.2623.

454
T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
obtained after silica gel column chromatography, elut-
ing with 5:95 MeOH–CHCl3.
7.32 (br, 1H, NH), 7.05 (d, J 8.5 Hz, 2H, aromatic),
6.86 (br, 1H, NH), 6.52 (d, J 7.6 Hz, 1H, NH), 5.72
(d, J 3.3 Hz, 1H, H-1), 5.72 (d, J 48.1 Hz, 1H, CHF),
5.63 (br, 1H, NH), 5.53 (dd, J 10.9, 3.3 Hz, 1H, H-2),
5.33 (d, J 3.5 Hz, 1H, H-4), 5.23 (dd, J 10.9, 3.5 Hz,
1H, H-3), 4.45 (dd, J 7.0, 5.0 Hz, 1H), 4.27 (m, 1H),
4.20 (q, J 6.5 Hz, 1H), 3.68–3.63 (m, 2H), 3.52–3.47
(m, 2H), 3.09 (m, 1H), 2.88–2.79 (m, 5H), 2.67 (d, J
12.8 Hz, 1H), 2.20–2.18 (m, 2H), 2.16 (s, 3H, OAc),
2.02 (s, 3H, OAc), 1.99 (s, 3H, OAc), 1.66–1.62 (m,
4H), 1.41–1.37 (m, 2H), 1.08 (d, J 6.5 Hz, 3H, CH3);
13C NMR (CDCl3, 100 MHz): d 173.7 (C), 170.5 (C),
170.4 (C), 170.1 (C), 169.1 (d, J 23 Hz, C), 164.0 (C),
157.4 (C), 128.9 (C), 128.6 (CH), 116.5 (CH), 94.6
(CH), 91.3 (d, J 192 Hz, CHF), 70.8 (CH), 67.8 (CH),
67.7 (CH), 65.5 (CH), 61.7 (CH), 60.1 (CH), 55.6
(CH), 40.5 (CH2), 38.2 (CH2), 38.0 (CH2), 37.2 (CH2),
35.7 (CH2), 29.6 (CH2), 28.0 (CH2), 25.5 (CH2), 20.7
(CH3), 20.7 (CH3), 20.6 (CH3), 15.8 (CH3). HRMS:
calcd for C34H48FN4O11S3 (M+1)+803.2466, found,
803.2455.
3.9.1. N-[4-(Tetra-O-acetyl-a-D-glucopyranosyloxy)phenyl]-
fluoroacetyl-N0-biotinylcystamine (17a). Yield 65%, Rf
0.30 (9:1 CHCl3–MeOH), mp 105–110 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.37 (d, J 8.3 Hz, 2H, aromatic),
7.34 (br, 1H, NH), 7.08 (d, J 8.3 Hz, 2H, aromatic),
6.84 (br, 1H, NH), 6.50 (br, 1H, NH), 5.77–5.62 (m,
4H), 5.12 (dd, J 9.8, 9.4 Hz, 1H, H-4), 5.00 (dd, J
10.3, 3.6 Hz, 1H, H-2), 4.45 (m, 1H), 4.27 (m, 1H),
4.20 (dd, J 12.0, 3.9 Hz, 1H, H-6), 4.06–3.99 (m, 2H),
3.64–3.61 (m, 2H), 3.50–3.48 (m, 2H), 3.09 (m, 1H),
2.86–2.78 (m, 5H), 2.67 (d, J 12.7 Hz, 1H), 2.17–2.16
(m, 2H), 2.02 (s, 3H, OAc), 2.01 (s, 3H, OAc), 2.00 (s,
3H, OAc), 2.00 (s, 3H, OAc), 1.65–1.62 (m, 4H), 1.23–
1.20 (m, 2H); 13C NMR (CDCl3, 100 MHz): d 173.6
(C), 170.5 (C), 170.1 (C), 170.1 (C), 169.5 (C), 164.0
(C), 156.8 (C), 129.6 (C), 128.6 (CH), 116.6 (CH), 94.0
(CH), 91.3 (d, J 185 Hz, CHF), 70.3 (CH), 69.9 (CH),
68.1 (CH), 68.1 (CH), 61.7 (CH), 61.4 (CH2), 60.1
(CH), 55.6 (CH), 40.5 (CH2), 38.2 (CH2), 37.9 (CH2),
37.2 (CH2), 35.7 (CH2), 29.6 (CH2), 28.1 (CH2), 27.9
(CH2), 25.5 (CH2), 20.6 (CH3), 20.6 (CH3), 20.6
(CH3), 20.5 (CH3). HRMS: calcd for C36H50FN4O13S3
(M+1)+, 861.2520; found, 861.2517.
3.10. General procedure for the preparation of probes
18a–c
To a solution of biotinylated compound 17 (0.10 mmol)
in 3 mL of MeOH was added 50 mg of Na2CO3. The
mixture was stirred at rt for 1 h and filtered through Cel-
ite 535, and the filtrate was concentrated. The target
probes 18a–c were obtained after silica gel column chro-
matography eluting with CHCl3–MeOH (7:3).
3.9.2. N-[4-(Tetra-O-acetyl-a-D-galactopyranosyloxy)-
phenyl]fluoroacetyl-N0-biotinylcystamine (17b). Yield
65%, Rf 0.17 (9:1 CHCl3–MeOH), mp 88–93 ꢁC. 1H
NMR (CDCl3, 400 MHz): d 7.36 (d, J 8.3 Hz, 2H, aro-
matic), 7.29 (br, 1H, NH), 7.05 (d, J 8.3 Hz, 2H, aro-
matic), 6.93 (br, 1H, NH), 6.61 (br, 1H, NH), 5.75–
5.53 (m, 3H), 5.51 (dd, J 10.4, 2.7 Hz, 1H, H-3), 5.48
(m, 1H, H-4), 5.23 (dd, J 10.4, 3.2 Hz, 1H, H-2), 4.44
(br, 1H), 4.28–4.25 (m, 2H), 4.09–4.00 (m, 2H), 3.69–
3.55 (m, 2H), 3.54–3.39 (m, 2H), 3.08 (m, 1H), 2.89–
2.72 (m, 5H), 2.66 (d, J 11.9 Hz, 1H), 2.22–2.15 (m,
2H), 2.12 (s, 3H, OAc), 2.02 (s, 3H, OAc), 1.98 (s, 3H,
OAc), 1.89 (s, 3H, OAc), 1.74–1.51 (m, 4H), 1.48–1.31
(m, 2H); 13C NMR (CDCl3, 100 MHz): d 173.7 (C),
170.3 (C), 170.3 (C), 170.1 (C), 170.0 (C), 169.0 (d, J
34 Hz, C), 164.1 (C), 157.0 (C), 129.4 (d, J 20 Hz, C),
128.6 (CH), 116.8 (CH), 94.6 (CH), 91.2 (d, J 186 Hz,
CHF), 67.6 (CH), 67.6 (CH), 67.3 (CH), 67.1 (CH),
61.6 (CH), 61.3 (CH2), 60.1 (CH), 55.6 (CH), 40.5
(CH2), 38.2 (CH2), 37.9 (CH2), 37.2 (CH2), 35.7 (CH2),
29.6 (CH2), 28.0 (CH2), 27.9 (CH2), 25.5 (CH2), 20.6
(CH3), 20.6 (CH3), 20.5 (CH3), 20.5 (CH3). HRMS:
calcd for C36H50FN4O13S3 (M+1)+, 861.2520; found,
861.2517.
3.10.1. N-Biotinyl-N0-[4-(a-D-glucopyranosyloxy)phenyl]-
fluoroacetylcystamine (18a). Yield 45%, Rf 0.30 (7:3
1
CHCl3–MeOH), mp 144–147 ꢁC. H NMR (DMSO-d6,
400 MHz): d 8.62 (br, 1H, exchangeable H), 8.00 (br,
1H, exchangeable H), 7.36 (d, J 8.1 Hz, 2H, aromatic),
7.11 (d, J 8.1 Hz, 2H, aromatic), 6.43 (br, 1H, exchange-
able H), 6.37 (br, 1H, exchangeable H), 5.80 (d, J
47.8 Hz, 1H, CHF), 5.40 (br, 1H, H-1), 5.09 (br, 1H,
exchangeable H), 5.05–4.91 (br, 2H, exchangeable H),
4.48 (br, 1H, exchangeable H), 4.29 (m, 1H), 4.11 (m,
1H), 3.64–3.22 (m, 9H), 3.17 (m, 1H), 3.08 (m, 1H),
2.85–2.73 (m, 5H), 2.56 (d, J 12.4 Hz, 1H), 2.05 (t, J
6.8 Hz, 2H), 1.65–1.39 (m, 4H), 1.38–1.21 (m, 2H); 13C
NMR (DMSO-d6, 100 MHz): d 172.5 (C), 168.4 (d, J
24 Hz, C), 162.9 (C), 158.1 (C), 129.3 (C), 129.1 (CH),
117.0 (CH), 98.0 (CH), 90.9 (d, J 187 Hz, CHF), 74.0
(CH), 73.2 (CH), 71.7 (CH), 70.1 (CH), 61.3 (CH),
60.9 (CH2), 59.4 (CH), 55.7 (CH), 40.6 (CH2), 38.8
(CH2), 38.5 (CH2), 38.1 (CH2), 37.5 (CH2), 35.4
(CH2), 28.4 (CH2), 28.3 (CH2), 25.5 (CH2); 19F
3.9.3. N-[4-(Tri-O-acetyl-a-L-fucopyranosyloxy)phenyl]-
fluoroacetyl-N0-biotinylcystamine (17c). Yield 67%, Rf
0.25 (9:1 CHCl3–MeOH), mp 96–99 ꢁC. 1H NMR
(CDCl3, 400 MHz): d 7.34 (d, J 8.5 Hz, 2H, aromatic),
NMR (DMSO-d6, 400 MHz):
d
ꢀ165.2 (d,
J
53.6 Hz), ꢀ165.5 (d, J 50.4 Hz). FABMS: m/z (%) 715
(M+Na+, 37), 693 (M+1+, 26), 605 (100); HRMS:

T.-H. Shie et al. / Carbohydrate Research 341 (2006) 443–456
455
calcd for C28H42FN4O9S3 (M+1)+, 693.2098; found,
Acknowledgements
693.2098.
This work was supported by the National Science
Council (NSC 93-3112-B-002-001 to L.-C.L.). We thank
Professor Yulin Lam for reading the manuscript.
3.10.2.
N-Biotinyl-N0-[4-(a-D-galactopyranosyloxy)-
phenyl]fluoroacetylcystamine (18b). Yield 50%, Rf 0.30
(7:3 CHCl3–MeOH), mp 124–127 ꢁC. 1H NMR
(DMSO-d6, 400 MHz): d 8.62 (br, 1H, exchangeable
H), 7.99 (br, 1H, exchangeable H), 7.35 (d, J 8.4 Hz,
2H, aromatic), 7.09 (d, J 8.4 Hz, 2H, aromatic), 6.43
(br, 1H, exchangeable H), 6.36 (br, 1H, exchangeable
H), 5.80 (d, J 47.9 Hz, 1H, CHF), 5.74 (br, 1H), 4.91
(d, J 5.5 Hz, 1H, exchangeable H), 4.75 (br, 1H,
exchangeable H), 4.56 (br, 1H, exchangeable H), 4.53
(br, 1H, exchangeable H), 4.29 (m, 1H), 4.11 (m, 1H),
3.76–3.73 (m, 2H), 3.65–3.28 (m, 8H), 3.09 (m, 1H),
2.85–2.73 (m, 5H), 2.56 (d, J 8.1 Hz, 1H), 2.06 (t, J
7.3 Hz, 2H), 1.49 (m, 1H), 1.45–1.40 (m, 3H), 1.37–
1.22 (m, 2H); 13C NMR (DMSO-d6, 100 MHz): d
172.7 (C), 168.7 (d, J 24 Hz, C), 163.2 (C), 158.5 (C),
129.3 (C), 129.3 (CH), 117.2 (CH), 98.5 (CH), 91.2 (d,
J 185 Hz, CHF), 72.8 (CH), 69.8 (CH), 68.9 (CH),
68.3 (CH), 61.5 (CH), 60.6 (CH2), 59.6 (CH), 55.9
(CH), 40.3 (CH2), 38.3 (CH2), 38.2 (CH2), 37.7 (CH2),
37.2 (CH2), 35.6 (CH2), 28.6 (CH2), 28.5 (CH2), 25.7
(CH2); 19F NMR (DMSO-d6, 400 MHz): d ꢀ165.0 (d,
J 50.4 Hz), ꢀ165.3 (d, J 50.4 Hz). FABMS: m/z (%)
693 (M+1+, 57), 605 (100); HRMS: calcd for
C28H42FN4O9S3 (M+1)+, 693.2098; found, 693.2098.
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.carres.
2005.12.005.
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