Synthesis and biological activities of novel isoxazoline-linked pseudodisaccharide derivatives

Article abstract of DOI:10.1016/j.carres.2011.11.025

A series of novel isoxazoline linked pseudodisaccharide derivatives were regiospecifically synthesized by 1,3-dipolar cycloaddition of α-allyl-C-glycopyranosides and sugar-derived nitrile oxides with good yields. The structures of the compounds were chara

Full text of DOI:10.1016/j.carres.2011.11.025

Carbohydrate Research 351 (2012) 7–16
Contents lists available at SciVerse ScienceDirect
Carbohydrate Research
journal homepage: www.elsevier.com/locate/carres
Synthesis and biological activities of novel isoxazoline-linked
pseudodisaccharide derivatives
Pingzhu Zhang a, Chao Wei a, Enkai Wang a, Wei Wang a, Ming Liu a, Qingmei Yin a, Hua Chen a,
a
Kerang Wang a, Xiaoliu Li a,b, , Jinchao Zhang
⇑
a Key Laboratory of Chemical Biology of Hebei Province, School of Chemistry and Environmental Science, Hebei University, Baoding, Hebei 071002, China
b State Key Laboratory of Natural and Biomimetic Drugs, Peking University, Beijing 100191, China
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of novel isoxazoline linked pseudodisaccharide derivatives were regiospecifically synthesized by
Received 14 September 2011
Received in revised form 25 November 2011
Accepted 27 November 2011
Available online 4 December 2011
1,3-dipolar cycloaddition of a-allyl-C-glycopyranosides and sugar-derived nitrile oxides with good yields.
The structures of the compounds were characterized by NMR spectroscopy and MS spectrometry and
confirmed by the X-ray crystallographic analysis of compound ((5S)-3-(2,3-O-isopropylidene-5-deoxy-
D
-lyxofuranose-4-yl)isoxazoline-5-yl) methyl
glycosidases ( -amylase, -glucosidase, and b-glucosidase) and HIV-RT, and antitumor activity were pre-
liminarily evaluated. Some of them exhibited potent inhibitory activity to HIV-RT.
Ó 2011 Elsevier Ltd. All rights reserved.
a- C-D-galactopyranoside. Their biological activities against
a
a
Keywords:
Pseudodisaccharide
1,3-Dipolar cycloaddition
Nitrile oxide
Isoxazoline
HIV-RT inhibition
1. Introduction
2. Results and discussion
It is well known that carbohydrates play crucial roles in a wide
variety of biological systems,1,2 and considerable attention has
been paid to the design and synthesis of carbohydrate analogues
for developing therapeutic potentials in the treatment of the
diseases related to the carbohydrate metabolism, such as diabetes,
human immunodeficiency virus (HIV) infection, metastatic cancer,
and lysosomal storage diseases.3–6 For example, the non-oxygen
linked disaccharide analogues, in which the interglycosidic oxygen
in the naturally occurring O-disaccharide is replaced by
other atoms (e.g., carbon,7 nitrogen,8 and sulfur9,10), have been
extensively studied due to their promising bioactivities and very
good enzymatic stability. Furthermore, the heterocycle-linked
disaccharide analogues which constructed a novel nucleoside-like
pseudodisaccharide bearing two sugar moieties on the heterocycle
have recently been synthesized,11–17 and were found to be of good
antitumor and/or anti HIV-RT activities.15–17 As the further study
on the synthesis and biological activity of such new pseudodisac-
charide analogues, we would like to report herein a convenient
synthesis of novel isoxazoline linked pseudodisaccharide
analogues via 1,3-dipolar cycloaddition18 of sugar-derived nitrile
2.1. The synthesis of isoxazoline linked pseudodisaccharide
derivatives
The 3-(2,3,4,6-tetra-O-benzyl-
(1–3) were readily prepared from the corresponding methyl
-glycopyranosides following the reported procedures.19,20 The
a-D-glycopyranosyl)-1-propene
D
sugar-derived nitrile oxides (4a–c) were synthesized from the
corresponding sugar oxime according to the literatures15,21,22, and
were used in situ to the 1,3-dipolar cycloaddition with the
a-
allyl-C-glycoside (1–3) based on our previous study.15 Thus, taken
the reaction of 1 and 4a as the example (Scheme 1), the solution of
the sugar oxime (1.0 mmol) and N-chlorosuccinimide (NCS)
(1.0 mmol) in 1,2-dichloroethane (DCE) was refluxed for 20 min.
to form the intermediate of chlorated sugar oxime. Then cooled to
room temperature, to the solution the benzylated
a-allyl-C-
glucoside (1) (1.2 mmol) was added, and a solution of DMAP
(1.0 mmol) in DCE was added dropwise within 2 h under refluxing
to generate the sugar-derived nitrile oxide 4a, and then the reaction
mixture was refluxed for 1 h to complete the cycloaddition. In order
to minimize the formation of the N-oxide dimer of the nitrile oxide
under the basic condition15, the solution of DMAP was slowly added
dropwise to control the generation of the nitrile oxide 4a for keeping
the dipolarophile (1) excess. After common workup and silica gel
column chromatography, the mixture of two diastereoisomers of
the isoxazoline linked pseudodisaccharides (5a and 6a) was
oxides to a-allyl-C-glycosides and the evaluation of their inhibitory
activities against glycosidase and HIV-RT, and antitumor activity.
⇑
Corresponding author. Tel./fax: +86 312 5971116.
E-mail address: lixl@hbu.cn (X. Li).
0008-6215/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.carres.2011.11.025

8
P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
Cl
ref.15
i
Sugar
BnO
R
C
N
R
OH
R
C
H
N
OH
1
O
12
6
11
10
O
O
O
O
5
O
BnO
BnO
BnO
O
2
N
7
N
3
N
R
N
iii
4
O
N
C
+
+
BnO
8
OBn
BnO
OBn
OBn
9
R
R
R
OBn
5a-c
7a-c
9a-c
OBn
6a-c
8a-c
10a-c
OBn
4a-c
1: D-Glucoside
2: D-Mannoside
3: D-Galactoside
11a-c
iv
iv
O
O
O
OBn
O
1' OMe
1
O
12
6
5
O
O
O
11
5'
O
a
c
R =
b
BnO
2'
HO
HO
HO
(S)
(R)
2
N
7
BnO
OBn
N
4'
O
3'
O
O
O
HO10
4
OH
OBn
3
8
OH
9
R'
O
R'
OH
OH
13a-c
15a-c
17a-c
O
OMe
OH
OH
12a-c
14a-c
16a-c
O
c
a
b
R' =
HO
O
O
HO
OH
Scheme 1. Reagents and conditions: (i) DCE, NCS, reflux, 20 min; (ii) DMAP, DCE; (iii) DCE, DMAP, reflux, 3 h; (iv) MeOH, HCl, Pd(OH)2/C, H2, 2 h.
obtained in total yield of 90%, accompanied with the trace byproduct
of nitrile oxide dimer (11a) and the recovery of benzylated -allyl-
oxide cycloaddition with terminal alkenes and alkynes.25 The X-ray
crystallographic structure of compound 16a (Fig. 1)26 suggested the
configuration of C-5 to be of S form. Accordingly, its diastereoisomer
17a should possess the R form at C-5. Furthermore, as shown in
Table 2, each diastereoisomers of 5, 7, 9, 12, 14, 16 and 6, 8, 10,
13, 15, 17 exhibited similar NMR signals in H-5 and C-5 and optical
rotations, respectively. For instance, the signals of H-5 in the less
polar diastereoisomers 5, 7, 9 and 12, 14, 16 appeared in more
downfield than those in the more polar diastereoisomers 6, 8, 10
and 13, 15, 17. In contrast, the absorptions of C-5 in 5, 7, 9 and 12,
14, 16 were in more upfield than those in 6, 8, 10 and 13, 15, 17.
In addition, all of 5, 7, 9 and 12, 14, 16 had more positive optical
rotations than their diastereoisomers 6, 8, 10 and 13, 15, 17. Conse-
quently, according to the consistencies of the spectral data, the
polarities and the optical rotations of each diastereoisomers and
the crystallographical structure of 16a, the configurations of C-5
in 5, 7, 9 and 12, 14, 16, and their diastereoisomers 6, 8, 10 and
13, 15, 17 were tentatively assigned as S and R, respectively.
a
C-glucoside (1) (8%). The diastereoisomeric mixture was separated
by silica gel column chromatography again to afford the less polar
isomer 5a (49%) and the more polar isomer 6a (41%), respectively.
Similarly, the 1,3-dipolar cycloaddition of 1 to 4b–c, and 2–3 to
4a–c were carried out following the procedure (as shown in
Scheme 1) and afforded the corresponding cycloadducts as the less
polar products 5, 7, 9 and the more polar diastereoisomers 6, 8, 10
in good yields (listed in Table 1) and the nitrile oxide dimers
11a–c,15 respectively.
Subsequently, the cycloadducts 5–10 were debenzylated by cat-
alytic hydrogenation with Pd(OH)2/C in MeOH–HCl (cHCl = 0.3%)
solution at room temperature, and the corresponding isoxazoline
linked pseudodisaccharides 12–17 were produced in high yields
of 74–95%, respectively.
2.2. The structures of compounds 5–10 and 12–17
The structures of compounds 5–10 and 12–17 were character-
ized by the analyses of 1H NMR and 13C NMR spectra, 1H–13C corre-
lation, HRMS(ESI), optical rotations, and the X-ray crystallographic
structure of compound 16a. Theoretically, the 1,3-dipolar cycload-
dition of nitrile oxide to alkene should form four stereoisomers of
3,4-disubstituted (syn) and 3,5-disubstituted (anti) isoxazolines.
However, observed in their 1H NMR spectra (the table of data can
be found in supporting information), the typical 3,5-disubstituted
isoxazoline signals,23,24 such as the 3J values of 7–11 Hz for
5-H/4-Ha,b and the germinal coupling 1J of ca. 17–18 Hz for
4-Ha/4-Hb, were found, which indicated that the 1,3-dipolar cyclo-
addition underwent in anti-form and regiospecifically produced
the 3,5-disubstituted isoxazolines. This regioselectivity was in
accordance with the previously reported results based on the nitrile
2.3. Biological activities
The inhibitions against HIV-1 reverse transcriptase (HIV-RT) and
glycosidase, and the antitumor activity of some of compounds 12–
17 were preliminarily evaluated. The HIV-RT inhibitory activities
of compounds 14, 15, 16a and 17a were measured using colorimet-
ric reverse transcriptase assay by comparison with AZT.27 As shown
in Table 3, compounds 14b, 15b, 16a, and 17a exhibited very good
HIV-RT inhibitory activity with the IC50 values of 7.17, 9.61, 4.31,
and 4.64 lM, respectively, better than that of the positive control
AZT. The glycosidase inhibitory activities of 12a, 13a, 14a, 14b,
15a, 15b, 16c, and 17c were determined with hydrolytic reactions
of
a
a
-amylase,
a-glucosidase, and b-glucosidase using acarbose as
control, respectively. However, the compounds showed
a
Table 1
The yields of 1,3-dipolar cycloaddition reactions
a-Allyl-C-glycosides
1
1
1
2
2
2
3
3
3
Nitrile oxides
Adducts
4a
5a
6a
49
41
1.2:1
90
4b
5b
6b
46
31
1.5:1
77
4c
5c
6c
40
30
1.3:1
70
4a
7a
8a
49
29
1.7:1
78
4b
7b
8b
42
35
1.2:1
77
4c
7c
8c
46
38
1.2:1
84
4a
9a
10a
47
30
4b
9b
10b
57
19
4c
9c
10c
43
36
Yields (%)
Ratio of isomers
Total yield (%)
1.6:1
77
3.0:1
76
1.2:1
79

P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
9
Table 3
Anti-HIV-RT activity
Compounds IC50
(lM)
Compounds IC50 (lM)
(a colorimetric reverse
transcriptase assay)
(a colorimetric reverse
transcriptase assay)
14a
15a
14b
15b
AZT
>50
>50
7.17 1.82
9.61 2.54
21.41 1.93
14c
15c
16a
17a
>50
>50
4.31 1.64
4.64 0.63
glycosidase and HIV-RT and anti-tumor activity were measured on
a TU-1901 UV–vis spectrophotometer and a Bio-Rad Model 3550
microplate spectrophotometer, respectively. Thin-layer chroma-
tography (TLC) was performed on precoated plates (Qingdao
GF254) with detection by UV light or with phosphomolybdic acid
in EtOH–H2O followed by heating. Column chromatography was
performed using SiO2 (Qingdao 300–400 mesh).
Figure 1. The X-ray crystallographic structure of compound 16a.
selectively moderate inhibition to b-glucosidase, such as the inhibi-
tion of 14b, and 15b were 20% and 24% at the concentration of
2.7 lmol/mL, respectively. The cytotoxicities of the compounds
3.2. General procedure for the synthesis of compounds 5–10
12–17 against breast cancer cell line were examined by the modi-
fied Mosmann protocol,28 and only a few of them was found to be
of a slight cytotoxicity, such as 13c (13%), 15c (13%), and 16c
(27%) at the concentration of 10 mmol/L.
A solution of sugar aldehyde oxime (293 mg, 1.0 mmol) and N-
chlorosuccinimide (NCS) (134 mg, 1.0 mmol) in ClCH2CH2Cl
(10 mL) was refluxed for 20 min under N2 atmosphere, and cooled
In summary, we have regiospecifically synthesized a series of
novel isoxazoline linked pseudodisaccharide analogues by 1,3-
dipolar cycloaddition of a-allyl-C-glycosides and sugar-derived ni-
trile oxides, and followed by deprotection with Pd (OH)2/C catalytic
hydrogenation. The structures of the compounds were elucidated
by NMR and MS spectral analyses with the aid of X-ray crystallo-
graphical structure of compound 16a. Their biological activities
to room temperature. To the solution
a-allyl-C-glucoside 1
(678 mg, 1.2 mmol) was added and heated to reflux, then a solu-
tion of DMAP (122 mg, 1.0 mmol) in ClCH2CH2Cl (10 mL) was
added dropwise within 2 h, and the mixture was refluxed for an-
other 1 h. The solution was concentrated under reduced pressure,
the residue was submitted on a silica gel column chromatography
using petroleum ether/ethyl acetate (v/v = 6:1) as the eluent to ob-
against glycosidases (a-amylase, a-glucosidase, and b-glucosidase)
tain the recovered a-allyl-C-glucoside (1, 8%) and the correspond-
and HIV-RT, and antitumor were preliminarily evaluated, and some
of them exhibited potent inhibitory activity to HIV-RT. The further
study of the synthesis and biological activities of novel heterocycle
linked pseudodisaccharide analogues is underway.
ing adducts mixture of 5a and 6a (90%, based on 1) and the dimer
(11a) of the sugar nitrile oxide (4a). Then the mixture of 5a and 6a
was separated by silica gel column chromatography using cyclo-
hexane/ethyl acetate (v/v = 10:1) as the eluent to obtain the less
polar isomer 5a (49%, based on 1) and the more polar isomer 6a
(41%, based on 1). Following the above procedure, the 1,3-dipolar
3. Experimental
cycloadditions of the
a-allyl-C-glycosides (1–3) and the sugar ni-
trile oxides (4a–c) were carried out and afforded the corresponding
cycloadducts of the less polar products 5, 7, 9 and the more polar 6,
8, 10, and the dimers (11a–c) of the sugar nitrile oxides (4a–c).
3.1. General methods
Melting points were measured on an SGWÒX-4 micro melting
point apparatus and are uncorrected. Optical rotations were deter-
mined on an SGWÒ-1 automatic polarimeter. 1H NMR, 13C NMR
3.2.1. ((5S)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
D-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
spectra were measured on
a RT-NMR Bruker AVANCE 400
benzyl-a-C-D-glucopyranoside (5a)
(400 MHz) spectrometer using tetramethylsilane (Me4Si) as the
internal standard. High-resolution mass spectra (HRMS) were car-
ried out on a FTICR-MS (Ionspec 7.0 T) mass spectrometer in the
electrospray-ionization (ESI) mode. X-ray crystallographic mea-
surements were made on a Bruker SMART CCD diffractometer.
The optical densities for examining the inhibitory activities against
Colorless syrup; ½a D25
ꢀ
+82.1 (c 1.0, CHCl3); Rf = 0.34 (silica gel,
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.33 (s,
3H, CH3), 1.42 (s, 3H, CH3), 1.94 (m, 1H, 6-H), 2.08 (m, 1H, 6-H),
2.86 (dd, 1H, J = 17.3 Hz, J = 6.8 Hz, CH2, 4-H), 3.28 (dd, 1H,
J = 17.0 Hz, J = 10.1 Hz, CH2, 4-H), 3.59–3.76 (m, 6H, 8-H, 7-H, 10-
Table 2
The chemical shifts of H-5 and C-5 and the optical rotations of the adducts 5–10 and the deprotected products 12–17
Compounds
H-5 (ppm)
C-5 (ppm)
a 2D5
½ ꢀ
5a
6a
5b
6b
5c
6c
7a
8a
7b
8b
7c
8c
4.77
78.1
82.1
4.76
78.3
9.00
4.70
78.0
46.5
4.70
78.3
ꢁ21.1
4.62
78.0
113
4.59
78.1
16.5
4.84
76.3
47.6
4.83
76.6
ꢁ11.8
4.73
76.8
32.3
4.67
76.9
ꢁ30.5
4.63
76.6
88.3
4.58
76.7
ꢁ4.60
Compounds
H-5 (ppm)
C-5 (ppm)
a 2D5
½ ꢀ
9a
10a
9b
10b
9c
10c
12a
13a
12b
4.80
77.7
90.3
13b
12c
13c
4.79
77.5
68.7
4.78
78.4
6.80
4.75
76.8
59.7
4.69
76.9
ꢁ22.4
4.63
76.0
77.1
4.58
76.2
6.00
4.80
77.7
69.9
4.76
79.1
ꢁ41.3
4.75
79.1
ꢁ25.7
4.80
77.8
108
4.78
79.6
38.7
Compounds
H-5 (ppm)
C-5 (ppm)
a 2D5
½ ꢀ
14a
15a
14b
4.82
77.8
33.3
15b
14c
15c
16a
17a
16b
4.75
78.0
68.0
17b
16c
17c
4.76
76.7
80.0
4.74
78.6
ꢁ49.2
4.75
79.2
ꢁ37.7
4.79
78.5
84.4
4.78
79.0
18.6
4.75
78.0
66.9
4.74
79.8
ꢁ29.9
4.70
79.7
ꢁ36.3
4.82
78.2
108
4.79
79.2
47.0

10
P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
H, 9-H, 12-H, 12-H), 4.36 (m, 1H, 11-H), 4.48 (d, 1H, J = 3.9 Hz, 20-H),
4.38–4.69 (m, 7H, CH2Ph), 4.76–4.93 (m, 3H, CH2Ph), 4.77 (m, 1H, 5-
H), 4.78 (d, 1H, J = 3.7 Hz, 30-H), 4.89 (d, 1H, J = 5.4 Hz, 40-H), 5.14 (s,
1H, 10-H), 7.12–7.31 (m, 25H, Ar); 13C NMR (CDCl3): d 24.9 (CH3),
26.4 (CH3), 30.6 (6-C), 41.4 (4-C), 69.6 (12-C), 69.7 (CH2Ph), 71.6
(11-C), 72.3 (CH2Ph), 73.6 (CH2Ph), 74.1 (CH2Ph), 75.4 (CH2Ph),
75.8 (4-C), 76.3 (7-C), 78.2 (10-C), 78.5 (5-C), 79.9 (8-C), 82.1 (9-
C), 82.5 (30-C), 85.5 (20-C), 105.8 (10-C), 113.4 (C, 13-C), 128.1 (Ar),
128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.4 (Ar), 128.50 (Ar), 128.5
(Ar), 128.8 (Ar), 128.9 (Ar), 128.9 (Ar), 137.5 (Ar), 138.5 (Ar), 138.6
(Ar), 138.7 (Ar), 139.1 (Ar), 156.9 (C@N); HRMS(ESI): calcd for
3.81 (m, 3H, 9-H, 12-H, 12-H), 4.09 (m, 1H, 11-H), 4.13 (d, 1H,
J = 3.4 Hz, 30-H), 4.42–4.62 (m, 8H, CH2Ph), 4.70 (m, 1H, 5-H),
4.78 (d, 1H, J = 3.3 Hz, 20-H), 4.76–4.89 (m, 2H, CH2Ph), 5.10 (d,
1H, J = 3.3 Hz, 40-H), 6.01 (d, 1H, J = 3.6 Hz, 10-H), 7.04–7.37 (m,
25H, Ar); 13C NMR (CDCl3): d 26.7 (CH3), 27.3 (CH3), 31.4 (6-C),
41.5 (4-C), 69.3 (12-C), 72.1 (11-C), 72.2 (CH2Ph), 72.6 (CH2Ph),
73.5 (CH2Ph), 73.9 (CH2Ph), 75.3 (CH2Ph), 75.7 (7-C), 77.1 (40-C),
77.1 (10-C), 78.4 (5-C), 79.1 (20-C), 80.0 (30-C), 82.7 (8-C), 85.0 (9-
C), 105.6 (10-C), 112.6 (C, 13-C), 127.9 (Ar), 128.0 (Ar), 128.2 (Ar),
128.2 (Ar), 128.3 (Ar), 128.5 (Ar), 128.7 (Ar), 128.5 (Ar), 128.7
(Ar), 129.0 (Ar), 137.4 (Ar), 138.4 (Ar), 138. (Ar), 138.7 (Ar), 139.1
C
52H57NO10 ([M]+), 855.3982, found: 855.3978.
(Ar), 157.4 (C@N); HRMS(ESI): calcd for C
52H57NO10 ([M]+),
855.3982, found: 855.3986.
3.2.2. ((5R)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
D
-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
3.2.5. ((5S)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
a-D-
benzyl-a-C-D-glucopyranoside (6a)
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
White solid, mp: 122–123 °C; ½a D25
ꢀ
+9.00 (c 1.0, CHCl3); Rf = 0.30
benzyl-a-C-D-glucopyranoside (5c)
(silica gel, cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d
1.22 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.96 (m, 1H, 6-H), 2.26 (m, 1H,
6-H), 2.92 (dd, 1H, J = 17.0 Hz, J = 8.4 Hz, CH2, 4-H), 3.20 (dd, 1H,
J = 17.1 Hz, J = 10.3 Hz, CH2, 4-H), 3.65–3.71 (m, 6H, 8-H, 7-H, 10-
H, 9-H, 12-H, 12-H), 4.22 (m, 1H, 11-H), 4.43–4.49 (m, 3H, CH2Ph),
4.61–4.71 (m, 5H, CH2Ph), 4.63 (d, 1H, J = 3.5 Hz, 20-H), 4.75–4.82
(m, 2H, CH2Ph), 4.76 (m, 1H, 5-H), 4.79 (d, 1H, J = 3.1 Hz, 30-H),
4.90 (d, 1H, J = 3.2 Hz, 40-H), 5.16 (s, 1H, 10-H), 7.12–7.35 (m,
25H, Ar); 13C NMR (CDCl3): d 24.8 (CH3), 26.3 (CH3), 30.8 (6-C),
40.9 (4-C), 69.3 (12-C), 69.5 (11-C), 71.9 (CH2Ph), 72.1 (CH2Ph),
73.5 (CH2Ph), 73.9 (CH2Ph), 75.3 (CH2Ph), 75.8 (40-C), 76.3 (7-C),
78.4 (5-C), 79.2 (10-C), 80.06 (8-C), 82.1 (9-C), 82.6(30-C), 85.4
(20-C), 105.7 (10-C), 113.3 (C, 13-C), 128.0 (Ar), 128.1 (Ar), 128.2
(Ar), 128.3 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.7 (Ar),
128.8 (Ar), 128.9 (Ar), 137.4 (Ar), 138.4 (Ar), 138.5 (Ar), 138.6
(Ar), 139.1 (Ar), 156.7 (C@N); HRMS(ESI): calcd for C52H57NO10
([M]+), 855.3982, found: 855.3976.
White solid, mp: 118–119 °C; ½a D25
ꢀ
+113 (c 1.0, CHCl3); Rf = 0.45
cyclohexane/dichlormethane/diethyl ether v/
(silica
gel,
v = 3:8:0.5); 1H NMR (CDCl3): d 1.82 (m, 1H, 6-H), 2.02 (m, 1H, 6-
H), 2.51 (dd, 2H, J = 7.8 Hz, J = 2.1 Hz, CH2, 4-H), 3.35 (s, 3H,
OCH3), 3.46 (t, 1H, J = 9.6 Hz, CH), 3.51 (dd, 1H, J = 9.6 Hz,
J = 3.4 Hz, CH), 3.56 (d, 2H, J = 9.6 Hz, CH), 3.64 (m, 2H, CH2, 12-
H), 3.67 (m, 1H, CH, 9-H), 3.72 (m, 1H, CH, 20-H), 4.05 (t, 1H,
J = 9.2 Hz, 30-H), 4.33 (m, 1H, CH, 11-H), 4.48–4.66 (m, 6H, CH2Ph),
4.55 (d, 1H, J = 3.4 Hz, 50-H), 4.62 (m, 1H, 5-H), 4.63 (d, 1H,
J = 3.9 Hz, 10-H), 4.77 (m, 5H, CH2Ph), 4.84 (d, 1H, J = 10.8 Hz,
CH2Ph), 4.89 (d, 1H, J = 11.0 Hz, CH2Ph), 4.99 (d, 1H, J = 10.8 Hz,
CH2Ph), 7.12–7.35 (m, 35H, Ar); 13C NMR (CDCl3): d 31.0 (6-C),
39.4 (4-C), 56.0 (OCH3), 66.9 (CH2Ph), 69.7 (12-C), 71.3 (11-C),
72.2 (7-C), 73.3 (CH2Ph), 73.8 (CH2Ph), 74.0 (50-C), 74.8 (CH2Ph),
75.4 (CH2Ph), 75.7 (CH2Ph), 76.3 (CH2Ph), 78.0 (5-C), 78.4 (10-C),
78.6 (40-C), 79.8 (20-C), 80.2 (8-C), 82.3 (9-C), 82.3 (30-C), 98.6 (10-
C), 128.1 (Ar), 128.1 (Ar), 128.3 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5
(Ar), 128.5 (Ar), 128.8 (Ar), 128.9 (Ar), 128.9 (Ar), 138.4 (Ar),
138.4 (Ar), 138.6 (Ar), 138.6 (Ar), 139.1 (Ar), 156.9 (C@N); HRMS(E-
SI): calcd for C65H69NO11 ([M]+), 1039.4871, found: 1039.4879.
3.2.3. ((5S)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
xylofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
D-
benzyl-a-C-D-glucopyranoside (5b)
Colorless syrup; ½a D25
ꢀ
+46.5 (c 1.0, CHCl3); Rf = 0.32 (silica gel,
3.2.6. ((5R)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
a-D-
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.36 (s,
3H, CH3), 1.59 (s, 3H, CH3), 1.96 (m, 1H, 6-H), 2.13 (m, 1H, 6-H),
2.87 (dd, 1H, J = 17.2 Hz, J = 7.5 Hz, CH2, 4-H), 3.25 (dd, 1H,
J = 17.3 Hz, J = 10.2 Hz, CH2, 4-H), 3.67–3.88 (m, 6H, 8-H, 7-H, 10-
H, 9-H, 12-H, 12-H), 4.19 (d, 1H, J = 3.4 Hz, 20-H), 4.39 (d, 1H,
J = 3.6 Hz, 30-H), 4.47 (m, 1H, 11-H), 4.54–4.58 (m, 2H, CH2Ph),
4.61–4.70 (m, 6H, 5-H, CH2Ph), 4.78 (d, 1H, J = 3.3 Hz, 20-H),
4.85–4.99 (m, 2H, CH2Ph), 5.29 (d, 1H, J = 3.6 Hz, 40-H), 6.05 (d,
1H, J = 3.2 Hz, 10-H), 7.04–7.37 (m, 25H, Ar); 13C NMR (CDCl3): d
26.8 (CH3), 27.4 (CH3), 30.9 (6-C), 41.7 (4-C), 69.6 (12-C), 74.4
(11-C), 72.4 (CH2Ph), 72.8 (CH2Ph), 73.2 (CH2Ph), 74.0 (CH2Ph),
75.5 (CH2Ph), 75.7 (7-C), 76.8 (40-C), 77.7 (10-C), 78.0 (5-C), 80.0
(20-C), 82.6 (30-C), 82.8 (8-C), 85.2 (9-C), 105.7 (10-C), 112.6 (C,
13-C), 127.9 (Ar), 128.0 (Ar), 128.2 (Ar), 128.2 (Ar), 128.3 (Ar),
128.5 (Ar), 128.74] (Ar), 128.53] (Ar), 128.7 (Ar), 129.0 (Ar), 137.4
(Ar), 138.5 (Ar), 138.5 (Ar), 138.7 (Ar), 139.1 (Ar), 157.7 (C@N);
benzyl-a-C-D-glucopyranoside (6c)
Colorless syrup; ½a D25
ꢀ
+16.5 (c 1.0, CHCl3); Rf = 0.40 (silica gel,
cyclohexane/ dichlormethane/diethyl ether v/v = 3:8:0.5); 1H
NMR (CDCl3): d 1.90 (m, 1H, 6-H), 2.16 (m, 1H, 6-H), 2.65 (dd,
1H, J = 17.0 Hz, J = 9.0 Hz, CH2, 4-H), 3.01 (dd, 1H, J = 16.7 Hz,
J = 10.4 Hz, CH2, 4-H), 3.36 (s, 3H, OCH3), 3.50–3.68 (m, 8H, CH),
4.04 (m, 2H, CH, 30-H, 11-H), 4.48–4.66 (m, 6H, CH2Ph), 4.57 (d,
1H, J = 3.4 Hz, 50-H), 4.58 (d, 1H, J = 3.9 Hz, 10-H), 4.59 (m, 1H, 5-
H), 4.77 (m, 5H, CH2Ph), 4.82 (d, 1H, J = 10.8 Hz, CH2Ph), 4.87 (d,
1H, J = 11.0 Hz, CH2Ph), 4.96 (d, 1H, J = 10.8 Hz, CH2Ph), 7.12–7.45
(m, 35H, Ar); 13C NMR (CDCl3): d 30.3 (6-C), 39.2 (4-C), 56.0
(OCH3), 67.2 (CH2Ph), 69.6 (12-C), 71.8 (11-C), 72.2 (7-C), 73.7
(CH2Ph), 73.8 (CH2Ph), 73.9 (50-C), 75.1 (CH2Ph), 75.4 (CH2Ph),
75.8 (CH2Ph), 76.3 (CH2Ph), 78.1 (5-C), 79.3 (10-C), 79.8 (40-C),
80.2 (20-C), 80.2 (8-C), 82.2 (9-C), 82.7 (30-C), 98.7 (10-C), 128.1
(Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.4 (Ar), 128.9 (Ar),
129.0 (Ar), 129.3 (Ar), 129.3 (Ar), 129.3 (Ar), 138.6 (Ar), 138.6
(Ar), 138.6 (Ar), 139.1 (Ar), 139.2 (Ar), 157.0 (C@N); HRMS(ESI):
calcd for C65H69NO11 ([M]+), 1039.4871, found: 1039.4867.
HRMS(ESI): calcd for
855.3989.
C
52H57NO10 ([M]+), 855.3982, found:
3.2.4. ((5R)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
D-
xylofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
benzyl-
a
-C-
D
-glucopyranoside (6b)
3.2.7. ((5S)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
D-
Colorless syrup; ½a D25
ꢁ21.8 (c 1.0, CHCl3); Rf = 0.29 (silica gel,
ꢀ
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.33 (s,
3H, CH3), 1.50 (s, 3H, CH3), 1.87 (m, 1H, 6-H), 2.19 (m, 1H, 6-H),
2.77 (dd, 1H, J = 17.2 Hz, J = 8.9 Hz, CH2, 4-H), 3.16 (dd, 1H,
J = 17.3 Hz, J = 10.3 Hz, CH2, 4-H), 3.64 (m, 3H, 8-H, 7-H, 10-H),
benzyl-a-C-D-mannopyranoside (7a)
Colorless syrup; ½a D25
ꢀ
+47.7 (c 1.0, CHCl3); Rf = 0.38 (silica gel,
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.32 (s,
3H, CH3), 1.49 (s, 3H, CH3), 1.86 (m, 1H, 6-H), 2.00 (m, 1H, 6-H),

P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
11
2.92 (dd, 1H, J = 17.4 Hz, J = 7.1 Hz, CH2, 4-H), 3.55 (dd, 1H,
J = 17.4 Hz, J = 10.3 Hz, CH2, 4-H), 3.72–3.92 (m, 6H, 8-H, 7-H, 10-
H, 9-H, 12-H, 12-H), 4.32 (m, 1H, 11-H), 4.52–4.67 (m, 7H, CH2Ph),
4.66 (d, 1H, J = 3.6 Hz, 20-H), 4.70–4.80 (m, 3H, CH2Ph), 4.84 (m, 1H,
5-H), 4.85 (t, 1H, J = 3.8 Hz, 30-H), 4.95 (d, 1H, J = 3.6 Hz, 40-H), 5.22
(s, 1H, 10-H), 7.20–7.44 (m, 25H, Ar); 13C NMR (CDCl3): d 24.6 (CH3),
26.4 (CH3), 36.1 (6-C), 41.7 (4-C), 69.5 (12-C), 69.8 (CH2Ph), 70.6
(11-C), 72.0 (CH2Ph), 72.5 (CH2Ph), 73.9 (CH2Ph), 74.1 (CH2Ph),
74.4 (40-C), 75.4 (7-C), 76.3 (10-C), 76.7 (5-C), 77.2 (8-C), 78.6 (9-
C), 82.1 (30-C), 85.5 (20-C), 105.9 (10-C), 113.4 (C, 13-C), 127.9
(Ar), 128.1 (Ar), 128.1 (Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar),
128.5 (Ar), 128.8 (Ar), 128.8 (Ar), 128.9 (Ar), 137.5 (Ar), 138.6
(Ar), 138.7 (Ar), 138.7 (Ar), 138.9 (Ar), 157.0 (C@N); HRMS(ESI):
calcd for C52H57NNaO10 ([M+Na]+), 878.3880, found: 878.3877
3.72 (m, 3H, 7-H, 12-H, 10-H), 3.82 (m, 2H, 9-H, 12-H), 4.01 (m,
1H, 11-H), 4.12 (d, 1H, J = 3.5 Hz, 30-H), 4.46 (d, 1H, J = 2.1 Hz, 20-
H), 4.49–4.64 (m, 10H, CH2Ph), 4.67 (m, 1H, 5-H), 5.10 (d, 1H,
J = 3.4 Hz, 40-H), 5.98 (d, 1H, J = 3.6 Hz, 10-H), 7.19–7.29 (m, 25H,
Ar); 13C NMR (CDCl3): d 26.7 (CH3), 27.3 (CH3), 35.6 (6-C), 41.2
(4-C), 69.3 (12-C), 69.7 (11-C), 71.9 (CH2Ph), 72.7 (CH2Ph), 72.8
(CH2Ph), 73.7 (CH2Ph), 73.8 (CH2Ph), 74.2 (7-C), 75.2 (40-C), 76.5
(10-C), 76.7 (20 -C), 76.9 (5-C), 78.3 (30-C), 82.7 (8-C), 85.2 (9-C),
105.6 (10-C), 112.6 (C, 13-C), 127.9 (Ar), 128.0 (Ar), 128.1 (Ar),
128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.7 (Ar), 128.8 (Ar), 128.8
(Ar), 129.0 (Ar), 137.4 (Ar), 138.6 (Ar), 138.6 (Ar), 138.7 (Ar),
138.8 (Ar), 157.4 (C@N); HRMS(ESI): calcd for C52H57NO10 ([M]+),
855.3982, found: 855.3988.
3.2.11. ((5S)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
a-D-
3.2.8. ((5R)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
D-
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
benzyl-a-C-D-mannopyranoside (7c)
benzyl-
a
-C-
D
-mannopyranoside (8a)
White solid, mp: 69–70 °C; ½a D25
ꢀ
+88.4 (c 1.0, CHCl3); Rf = 0.35
Colorless syrup; ½a D25
ꢀ
ꢁ11.8 (c 1.0, CHCl3); Rf = 0.36 (silica gel,
(silica gel, cyclohexane/ dichlormethane/ diethyl ether v/
v = 3:4:0.5); 1H NMR (CDCl3): d 1.63 (m, 1H, 6-H), 1.93 (m, 1H, 6-
H), 2.53 (d, 2H, J = 3.1 Hz, CH2, 4-H), 3.37 (s, 3H, OCH3), 3.45 (t,
1H, J = 9.6 Hz, CH), 3.51 (dd, 1H, J = 9.6 Hz, J = 3.5 Hz, CH), 3.58
(m, 1H, 7-H), 3.71 (m, 3H, CH2, 12-H, 10-H), 3.77 (m, 2H, CH, 9-
H, 20-H), 4.03 (t, 1H, J = 9.3 Hz, 30-H), 4.16 (m, 1H, CH, 11-H),
4.46–4.66 (m, 10H, CH2Ph), 4.48 (d, 1H, J = 3.3 Hz, 50-H), 4.53 (d,
1H, J = 3.9 Hz, 10-H), 4.63 (m, 1H, 5-H), 4.77 (m, 2H, CH2Ph), 4.84
(d, 1H, J = 10.8 Hz, CH2Ph), 4.98 (d, 1H, J = 10.8 Hz, CH2Ph), 7.19–
7.36 (m, 35H, Ar); 13C NMR (CDCl3): d 36.4 (6-C), 39.6 (4-C), 55.9
(OCH3), 66.8 (CH2Ph), 69.5 (12-C), 70.0 (11-C), 71.9 (7-C), 72.5
(CH2Ph), 73.8 (CH2Ph), 73.8 (50-C), 74.4 (CH2Ph), 74.7 (CH2Ph),
75.3 (CH2Ph), 76.2 (CH2Ph), 76.6 (5-C), 76.6 (10-C), 78.4 (40-C),
78.5 (20-C), 78.5 (8-C), 80.1 (9-C), 82.2 (30-C), 98.6 (10-C), 127.9
(Ar), 128.0 (Ar), 128.2 (Ar), 128.3 (Ar), 128.3 (Ar), 128.4 (Ar),
128.4 (Ar), 128.4 (Ar), 128.7 (Ar), 128.9 (Ar), 138.3 (Ar), 138.5
(Ar), 138.7 (Ar), 138.8 (Ar), 139.0 (Ar), 156.8 (C@N); HRMS(ESI):
calcd for C65H69NO11 ([M]+), 1039.4871, found: 1039.4864.
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.28 (s,
3H, CH3), 1.46 (s, 3H, CH3), 1.90 (m, 1H, 6-H), 2.16 (m, 1H, 6-H),
3.01 (dd, 1H, J = 17.1 Hz, J = 7.9 Hz, CH2, 4-H), 3.21 (dd, 1H,
J = 17.0 Hz, J = 10.2 Hz, CH2, 4-H), 3.59–4.02 (m, 6H, 8-H, 7-H, 10-
H, 9-H, 12-H, 12-H), 4.16 (m, 1H, 11-H), 4.52–4.64 (m, 8H, CH2Ph),
4.67 (d, 1H, J = 3.6 Hz, 20-H), 4.73 (m, 2H, CH2Ph), 4.83 (m, 1H, 5-H),
4.84 (t, 1H, J = 3.6 Hz, 30-H), 4.98 (d, 1H, J = 3.6 Hz, 40-H), 5.22 (s, 1H,
10-H), 7.26–7.41 (m, 25H, Ar); 13C NMR (CDCl3): d 24.8 (CH3), 26.3
(CH3), 35.2 (6-C), 40.6 (4-C), 69.3 (12-C), 69.5 (CH2Ph), 72.0 (11-C),
72.8 (CH2Ph), 73.7 (CH2Ph), 74.3 (CH2Ph), 74.3 (CH2Ph), 75.2 (40-C),
76.3 (7-C), 76.6 (10-C), 76.6 (5-C), 76.7 (8-C), 78.5 (9-C), 82.3 (30-C),
85.4 (20-C), 105.8 (10-C), 113.3 (C, 13-C), 128.0 (Ar), 128.1 (Ar),
128.2 (Ar), 128.3 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.8
(Ar), 128.9 (Ar), 129.0 (Ar), 137.4 (Ar), 138.6 (Ar), 138.7 (Ar),
138.7 (Ar), 138.9 (Ar), 157.1 (C@N); HRMS(ESI): calcd for
C
52H57NNaO10 ([M+Na]+), 878.3880, found: 878.3884.
3.2.9. ((5S)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
D-
xylofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
3.2.12. ((5R)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
a-D-
benzyl-a-C-D-mannopyranoside (7b)
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
Colorless syrup; ½a D25
ꢀ
+32.4 (c 1.0, CHCl3); Rf = 0.40 (silica gel,
benzyl-a-C-D-mannopyranoside (8c)
cyclohexane/ dichlormethane/ diethyl ether v/v = 3:8:0.5); 1H
NMR (CDCl3): d 1.32 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.69 (m, 1H,
6-H), 1.91 (m, 1H, 6-H), 2.76 (dd, 1H, J = 17.6 Hz, J = 7.5 Hz, CH2,
4-H), 3.18 (dd, 1H, J = 17.5 Hz, J = 7.2 Hz, CH2, 4-H), 3.59 (m, 1H,
8-H), 3.74 (m, 3H, 7-H, 12-H, 10-H), 3.81 (m, 2H, 9-H, 12-H), 4.12
(d, 1H, J = 3.4 Hz, 30-H), 4.20 (m, 1H, 11-H), 4.49 (d, 1H, J = 3.6 Hz,
20-H), 4.46–4.66 (m, 10H, CH2Ph), 4.73 (m, 1H, 5-H), 5.12 (d, 1H,
J = 3.4 Hz, 40-H), 5.97 (d, 1H, J = 3.4 Hz, 10-H), 7.18–7.35 (m, 25H,
Ar); 13C NMR (CDCl3): d 26.7 (CH3), 27.3 (CH3), 36.4 (6-C), 44.9
(4-C), 69.6 (12-C), 70.1 (11-C), 72.0 (CH2Ph), 72.5 (CH2Ph), 72.9
(CH2Ph), 73.8 (CH2Ph), 73.9 (CH2Ph), 74.4 (7-C), 75.2 (40-C), 76.7
(10-C), 76.8 (20-C), 76.8 (5-C), 78.1 (30-C), 82.8 (8-C), 85.2 (9-C),
105.7 (10-C), 112.6 (C, 13-C), 127.9 (Ar), 128.1 (Ar), 128.3 (Ar),
128.1 (Ar), 128.2 (Ar), 128.3 (Ar), 128.4 (Ar), 128.5 (Ar), 128.7
(Ar), 129.0 (Ar), 137.4 (Ar), 138.6 (Ar), 138.6 (Ar), 138.7 (Ar),
138.9 (Ar), 157.7 (C@N); HRMS(ESI): calcd for C52H57NO10 ([M]+),
855.3982, found: 855.3985.
Colorless syrup; ½a D25
ꢁ4.60 (c 1.0, CHCl3); Rf = 0.42 (silica gel,
ꢀ
cyclohexane/dichlormethane/diethyl ether v/v = 3:4:0.5); 1H NMR
(CDCl3): d 1.63 (m, 2H, 6-H), 2.53 (dd, 1H, J = 19.6 Hz, J = 9.4 Hz,
CH2, 4-H), 2.96 (dd, 1H, J = 19.6 Hz, J = 10.4 Hz, CH2, 4-H), 3.37 (s,
3H, OCH3), 3.53 (m, 3H, CH, 10-H, 8-H, 7-H), 3.65 (m, 1H, 40-H),
3.72 (m, 2H, CH2, 12-H), 3.77 (t, 1H, J = 9.3 Hz, 9-H), 3.86 (m, 1H,
CH, 20-H), 3.98 (m, 1H, CH, 30-H), 4.22 (m, 1H, CH, 11-H), 4.45–
4.67 (m, 10H, CH2Ph), 4.48 (d, 1H, J = 3.3 Hz, 50-H), 4.56 (d, 1H,
J = 3.9 Hz, 10-H), 4.58 (m, 1H, 5-H), 4.75 (m, 2H, CH2Ph), 4.82 (d,
2H, J = 10.8 Hz, CH2Ph), 4.99 (d, 1H, J = 10.8 Hz, CH2Ph), 7.18–7.52
(m, 35H, Ar); 13C NMR (CDCl3): d 35.1 (6-C), 39.0 (4-C), 55.9
(OCH3), 67.1 (CH2Ph), 69.4 (12-C), 69.4 (11-C), 72.0 (7-C), 72.8
(CH2Ph), 73.6 (CH2Ph), 73.8 (50-C), 74.2 (CH2Ph), 74.8 (CH2Ph),
76.3 (CH2Ph), 76.5 (CH2Ph), 76.7 (5-C), 78.6 (10-C), 78.7 (40-C),
79.4 (20-C), 80.1 (8-C), 80.1 (9-C), 82.3 (30-C), 98.7 (10-C), 128.0
(Ar), 128.0 (Ar), 128.2 (Ar), 128.3 (Ar), 128.3 (Ar), 128.4 (Ar),
128.4 (Ar), 128.5 (Ar), 128.8 (Ar), 128.9 (Ar), 138.5 (Ar), 138.6
(Ar), 138.7 (Ar), 138.8 (Ar), 139.1 (Ar), 157.1 (C@N); HRMS(ESI):
calcd for C65H69NO11 ([M]+), 1039.4871, found: 1039.4868.
3.2.10. ((5R)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
D-
xylofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
benzyl-
a
-C-
D
-mannopyranoside (8b)
3.2.13. ((5S)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
D-
Colorless syrup; ½a D25
ꢁ30.5 (c 1.0, CHCl3); Rf = 0.50 (silica gel,
ꢀ
cyclohexane/dichlormethane/diethyl ether v/v = 3:8:0.5); 1H NMR
(CDCl3): d 1.31 (s, 3H, CH3), 1.49 (s, 3H, CH3), 1.63 (m, 1H, 6-H),
2.09 (m, 1H, 6-H), 2.78 (dd, 1H, J = 17.3 Hz, J = 8.7 Hz, CH2, 4-H),
3.12 (dd, 1H, J = 17.4 Hz, J = 10.4 Hz, CH2, 4-H), 3.55 (m, 1H, 8-H),
benzyl-a-C-D-galactopyranoside (9a)
Colorless syrup; ½a D25
ꢀ
+68.7 (c 1.0, CHCl3); Rf = 0.30 (silica gel,
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.26 (s,
3H, CH3), 1.42(s, 3H, CH3), 1.86 (m, 2H, 6-H), 2.82 (dd, 1H,

12
P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
J = 17.4 Hz, J = 7.3 Hz, CH2, 4-H), 3.28 (dd, 1H, J = 17.2 Hz,
J = 10.3 Hz, CH2, 4-H), 3.71 (m, 3H, CH, 8-H, 7-H, 10-H), 3.94 (m,
2H, 9-H, 12-H), 4.09 (m, 1H, 12-H), 4. 25 (m, 1H, 11-H), 4.45–
4.75 (m, 10H, CH2Ph), 4.67 (d, 1H, J = 3.6 Hz, 20-H), 4.79 (m, 1H,
5-H), 4.80 (t, 1H, J = 3.8 Hz, 40-H), 4.90 (d, 1H, J = 3.6 Hz, 30-H),
5.15 (s, 1H, 10-H), 7.19–7.31 (m, 25H, Ar); 13C NMR (CDCl3): d
24.9 (CH3), 26.4 (CH3), 34.4 (6-C), 41.7 (4-C), 67.8 (12-C), 68.3
(CH2Ph), 69.5 (11-C), 73.2 (CH2Ph), 73.5 (CH2Ph), 73.5 (CH2Ph),
73.7 (CH2Ph), 73.9 (40-C), 74.6 (7-C), 76.3 (10-C), 77.5 (5-C), 77.6
(8-C), 78.6 (9-C), 82.1 (30-C), 85.52 (20-C), 105.8 (10-C), 113.3 (C,
13-C), 128.0 (Ar), 128.0 (Ar), 128.2 (Ar), 128.2 (Ar), 128.3 (Ar),
128.4 (Ar), 128.5 (Ar), 128.8 (Ar), 128.9 (Ar), 128.9 (Ar), 137.5
(Ar), 138.6 (Ar), 138.9 (Ar), 138.9 (Ar), 138.9 (Ar), 157.1 (C@N);
2.74 (dd, 1H, J = 17.4 Hz, J = 9.1 Hz, CH2, 4-H), 3.06 (dd, 1H,
J = 17.3 Hz, J = 10.4 Hz, CH2, 4-H), 3.64 (dd, 1H, J = 10.1 Hz,
J = 5.5 Hz, 8-H), 3.71 (m, 2H, J = 10.8 Hz, J = 3.6 Hz, 10-H, 7-H),
3.79 (m, 1H, 12-H), 3.98 (m, 3H, 9-H, 12-H, 11-H), 4.10 (d, 1H,
J = 3.4 Hz, 30-H), 4.48 (d, 1H, J = 3.5 Hz, 20-H), 4.38–4.72 (m, 10H,
CH2Ph), 4.69 (m, 1H, 5-H), 5.09 (d, 1H, J = 3.3 Hz, 40-H), 5.98 (d,
1H, J = 3.6 Hz, 10-H), 7.18–7.32 (m, 25H, Ar); 13C NMR (CDCl3): d
26.7 (CH3), 27.3 (CH3), 33.1 (6-C), 41.2 (4-C), 67.8 (12-C), 68.9
(11-C), 72.7 (CH2Ph), 72.8 (CH2Ph), 73.3 (CH2Ph), 73.3 (CH2Ph),
73.7 (CH2Ph), 73.7 (7-C), 74.6 (40-C), 76.5 (10-C), 76.9 (5-C), 77.4
(20-C), 78.8 (30-C), 82.6 (8-C), 85.0 (9-C), 105.6 (10-C), 112.6 (C,
13-C), 127.3 (Ar), 128.0 (Ar), 128.1 (Ar), 128.2 (Ar), 128.3 (Ar),
128.3 (Ar), 128.5 (Ar), 128.7 (Ar), 128.8 (Ar), 129.0 (Ar), 137.4
(Ar), 138.5 (Ar), 138.7 (Ar), 138.9 (Ar), 138.9 (Ar), 157.4 (C@N);
HRMS(ESI): calcd for C52H57NNaO10 ([M+Na]+), 878.3880, found:
878.3876
HRMS(ESI): calcd for
856.4057
C
52H58NO10 ([M+H]+), 856.4061, found:
3.2.14. ((5R)-3-(Benzyl 2,3-O-isopropylidene-4-C-5-deoxy-
D-
lyxofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
3.2.17. ((5S)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
a-D-
benzyl-a-C-D-galactopyranoside (10a)
Colorless syrup; ½a D25
ꢀ
+6.80 (c 1.0, CHCl3); Rf = 0.29 (silica gel,
benzyl-a-C-D-galactopyranoside (9c)
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.21 (s,
3H, CH3), 1.35 (s, 3H, CH3), 1.80 (m, 1H, 6-H), 2.18 (m, 1H, 6-H),
2.89 (dd, 1H, J = 16.9 Hz, J = 8.1 Hz, CH2, 4-H), 3.10 (dd, 1H,
J = 16.9 Hz, J = 10.1 Hz, CH2, 4-H), 3.65 (m, 1H, 7-H), 3.73 (dd, 1H,
8-H), 3.81 (m, 2H, 10-H, 12-H), 4.01 (m, 2H, 9-H, 12-H), 4.12 (m,
1H, 11-H), 4.45–4.78 (m, 10H, CH2Ph), 4.62 (d, 1H, J = 3.1 Hz, 20-
H), 4.78 (m, 1H, 5-H), 4.79 (t, 1H, J = 3.8 Hz, 30-H), 4.90 (d, 1H,
J = 3.6 Hz, 40-H), 5.15 (s, 1H, 10-H), 7.21–7.34 (m, 25H, Ar); 13C
NMR (CDCl3): d 24.7 (CH3), 26.2 (CH3), 32.5 (6-C), 40.6 (4-C), 67.9
(12-C), 69.5 (CH2Ph), 72.1 (11-C), 72.3 (CH2Ph), 72.9 (CH2Ph),
73.4 (CH2Ph), 73.7 (CH2Ph), 74.7 (40-C), 76.2 (7-C), 76.7 (10-C),
78.4 (5-C), 78.9 (8-C), 79.6 (9-C), 82.2 (30-C), 85.4 (20-C), 105.7
(10-C), 113.2 (C, 13-C), 128.0 (Ar), 128.1 (Ar), 128.3 (Ar), 128.3
(Ar), 128.5 (Ar), 128.7 (Ar), 128.7 (Ar), 128.8 (Ar), 128.8 (Ar),
128.9 (Ar), 137.4 (Ar), 138.5 (Ar), 138.7 (Ar), 138.7 (Ar), 138.9
(Ar), 156.9 (C@N); HRMS(ESI): calcd for C52H58NO10 ([M+H]+),
856.4061, found: 856.4056.
Colorless syrup; ½a D25
ꢀ
+77.1 (c 1.0, CHCl3); Rf = 0.45 (silica gel,
cyclohexane/dichlormethane/diethyl ether v/v = 3:10:0.5); 1H
NMR (CDCl3): d 1.75 (m, 2H, 6-H), 2.53 (d, 2H, J = 8.8 Hz, CH2, 4-
H), 3.35 (s, 3H, OCH3), 3.46 (t, 1H, J = 9.6 Hz, CH), 3.50 (dd, 1H,
J = 9.6 Hz, J = 3.4 Hz, CH), 3.68 (m, 3H, CH, CH2, 40-H, 7-H, 12-H),
3.90 (m, 2H, CH, CH2, 12-H, 9-H), 4.04 (m, 2H, CH, 20-H, 11-H),
4.05 (t, 1H, J = 9.2 Hz, 30-H), 4.20 (m, 1H, CH, 11-H), 4.42–4.67
(m, 10H, CH2Ph), 4.50 (d, 1H, J = 3.5 Hz, 50-H), 4.55 (d, 1H,
J = 3.3 Hz, 10-H), 4.63 (m, 1H, 5-H), 4.79 (d, 1H, J = 11.1 Hz, CH2Ph),
4.84 (d, 1H, J = 12.1 Hz, CH2Ph), 4.86 (d, 1H, J = 11.0 Hz, CH2Ph),
4.98 (d, 1H, J = 10.8 Hz, CH2Ph), 7.20–7.39 (m, 35H, Ar); 13C NMR
(CDCl3): d 27.3 (6-C), 39.6 (4-C), 55.9 (OCH3), 66.9 (CH2Ph), 67.6
(12-C), 68.0 (11-C), 70.9 (7-C), 73.0 (CH2Ph), 73.4 (CH2Ph), 73.8
(50-C), 73.8 (CH2Ph), 73.8 (CH2Ph), 74.4 (CH2Ph), 74.7 (CH2Ph),
76.0 (5-C), 76.3 (10-C), 78.5 (40-C), 78.5 (20-C), 80.1 (8-C), 82.2 (9-
C), 82.2 (30-C), 98.6 (10-C), 127.9 (Ar), 128.1 (Ar), 128.2 (Ar), 128.4
(Ar), 128.4 (Ar), 128.5 (Ar), 128.5 (Ar), 128.7 (Ar), 128.9 (Ar),
129.1 (Ar), 138.2 (Ar), 138.4 (Ar), 138.8 (Ar), 138.8 (Ar), 139.0
3.2.15. ((5S)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
xylofuranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
D-
(Ar), 156.9 (C@N); HRMS(ESI): calcd for
C
65H69NO11 ([M]+),
1039.4871, found: 1039.4863.
benzyl-a-C-D-galactopyranoside (9b)
Colorless syrup; ½a D25
ꢀ
+59.8 (c 1.0, CHCl3); Rf = 0.28 (silica gel,
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.31 (s,
3H, CH3), 1.49 (s, 3H, CH3), 1.80 (m, 2H, 6-H), 2.77 (dd, 1H,
J = 17.6 Hz, J = 7.3 Hz, CH2, 4-H), 3.18 (dd, 1H, J = 17.5 Hz,
J = 10.4 Hz, CH2, 4-H), 3.65 (s, 1H, 8-H, 10-H), 3.70 (dd, 1H,
J = 10.8 Hz, J = 3.6 Hz, 12-H), 3.95 (m, 2H, 7-H, 9-H), 4.08 (m, 1H,
12-H), 4.10 (d, 1H, J = 3.4 Hz, 30-H), 4.23 (m, 1H, 11-H), 4.46 (d,
1H, J = 3.6 Hz, 20-H), 4.45–4.73 (m, 10H, CH2Ph), 4.75 (m, 1H, 5-
H), 5.11 (d, 1H, J = 3.3 Hz, 40-H), 5.97 (d, 1H, J = 3.4 Hz, 10-H),
7.19–7.30 (m, 25H, Ar); 13C NMR (CDCl3): d 26.8 (CH3), 27.3
(CH3), 34.7 (6-C), 41.9 (4-C), 67.7 (12-C), 67.9 (11-C), 72.8 (CH2Ph),
73.1 (CH2Ph), 73.1 (CH2Ph), 73.5 (CH2Ph), 73.5 (CH2Ph), 73.8 (7-C),
74.5 (40-C), 76.1 (10-C), 76.8 (5-C), 77.4 (20-C), 78.3 (30-C), 82.8 (8-
C), 85.1 (9-C), 105.7 (10-C), 112.6 (C, 13-C), 128.0 (Ar), 128.0 (Ar),
128.0 (Ar), 128.2 (Ar), 128.2 (Ar), 128.5 (Ar), 128.8 (Ar), 128.8
(Ar), 128.8 (Ar), 129.1 (Ar), 137.4 (Ar), 138.5 (Ar), 138.9 (Ar),
138.9 (Ar), 139.0 (Ar), 157.7 (C@N); HRMS(ESI): calcd for
3.2.18. ((5R)-3-(Methyl 2,3,4-tri-O-benzyl-5-C-6-deoxy-
glucopyranosidyl)isoxazoline-5-yl) methyl 2,3,4,6-tetra-O-
a-D-
benzyl-a-C-D-galactopyranoside (10c)
Colorless syrup; ½a D25
ꢀ
+6.00 (c 1.0, CHCl3); Rf = 0.50 (silica gel,
cyclohexane/ dichlormethane/ diethyl ether v/v = 3:10:0.5); 1H
NMR (CDCl3): d 1.68 (m, 1H, 6-H), 2.15 (m, 1H, 6-H), 2.43 (dd,
1H, J = 16.9 Hz, J = 9.5 Hz, CH2, 4-H), 2.92 (dd, 1H, J = 16.9 Hz,
J = 9.5 Hz, CH2, 4-H), 3.37 (s, 3H, OCH3), 3.49 (m, 2H, CH, 10-H, 8-
H), 3.60 (dd, 1H, J = 10.1 Hz, J = 4.7 Hz, CH, 40-H), 3.65 (dd, 1H,
J = 7.2 Hz, J = 2.2 Hz, CH, 7-H), 3.74 (m, 2H, CH2, 12-H), 3.97 (dd,
2H, J = 10.7 Hz, J = 4.1 Hz, CH, 20-H, 9-H), 4.02 (m, 2H, CH, 30-H,
11-H), 4.40–4.74 (m, 11H, CH2Ph), 4.48 (d, 1H, J = 3.5 Hz, 50-H),
4.58 (m, 1H, 5-H), 4.59 (d, 1H, J = 3.4 Hz, 10-H), 4.75 (d, 1H,
J = 11.1 Hz, CH2Ph), 4.82 (d, 1H, J = 10.8 Hz, CH2Ph), 4.97 (d, 1H,
J = 10.8 Hz, CH2Ph), 7.20–7.39 (m, 35H, Ar); 13C NMR (CDCl3): d
27.3 (6-C), 39.1 (4-C), 55.9 (OCH3), 67.0 (CH2Ph), 68.0 (12-C),
69.2 (11-C), 72.6 (7-C), 73.3 (CH2Ph), 73.6 (CH2Ph), 73.6 (50-C),
73.8 (CH2Ph), 73.8 (CH2Ph), 74.7 (CH2Ph), 74.9 (CH2Ph), 76.2 (5-
C), 76.7 (10-C), 79.2 (40-C), 79.4 (20-C), 80.0 (8-C), 82.1 (9-C), 82.1
(30-C), 98.6 (10-C), 127.9 (Ar), 128.0 (Ar), 128.1 (Ar), 128.2 (Ar),
128.3 (Ar), 128.3 (Ar), 128.5 (Ar), 128.7 (Ar), 128.8 (Ar), 128.9
(Ar), 138.4 (Ar), 138.5 (Ar), 138.6 (Ar), 138.9 (Ar), 139.1 (Ar),
156.9 (C@N); HRMS(ESI): calcd for C65H69NO11 ([M]+), 1039.4871,
found: 1039.4866.
C
52H57NNaO10 ([M+Na]+), 878.3880, found: 878.3875.
3.2.16. ((5R)-3-(1,2-O-Isopropylidene-3-benzyl-4-C-5-deoxy-
xylofuranosidyl)isoxazoline-5- yl) methyl 2,3,4,6-tetra-O-
D-
benzyl-a-C-D-galactopyranoside (10b)
Colorless syrup; ½a D25
ꢁ22.3 (c 1.0, CHCl3); Rf = 0.26 (silica gel,
ꢀ
cyclohexane/ethyl acetate v/v = 3:1); 1H NMR (CDCl3): d 1.32 (s,
3H, CH3), 1.49 (s, 3H, CH3), 1.71 (m, 1H, 6-H), 2.13 (m, 1H, 6-H),

P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
13
3.3. General procedure for the synthesis of compounds 12–17
(dd, 1H, J = 9.8 Hz, J = 1.9 Hz, 8-H), 4.10 (m, 1H, 11-H), 4.27 (d,
1H, J = 2.8 Hz, 30-H), 4.56 (d, 1H, J = 3.5 Hz, 20-H), 4.75 (m, 1H, 5-
H), 4.94 (d, 1H, J = 2.8 Hz, 40-H), 5.99 (d, 1H, J = 3.5 Hz, 10-H); 13C
NMR (CD3OD): d 25.4 (CH3), 26.1 (CH3), 30.1 (CH2, 6-C), 40.4
(CH2, 4-C), 62.0 (CH2, 12-C), 71.2 (CH, 8-C), 71.7 (CH, 9-C), 73.5
(CH, 7-C), 74.1 (CH, 10-C), 74.1 (CH, 40-C), 76.9 (CH, 30-C), 77.4
(CH, 11-C), 79.1 (CH, 5-C), 85.6 (CH, 20-C), 105.4 (CH, 10-C), 112.1
To a solution of the cycloadduct (5a) (171 mg, 0.2 mmol) in
MeOH (6 mL) was added a catalytic amount of Pd(OH)2/C. The mix-
ture was stirred under H2 atmosphere for 2 h. The catalyst was fil-
tered off, and the solvent was removed by evaporation. The residue
was applied on silica gel column chromatography with the eluent
of MeOH/ethyl acetate (v/v = 1:9) to afford the corresponding deb-
enzylated product 12a (64 mg, 79%). Under the same condition, the
debenzylation of the cycloadducts 5–10 was carried out to afford
the corresponding debenzylated products 12–17.
(C, 13-C), 158.0 (C@N); HRMS(ESI): calcd for
C17H28NO10
([M+H]+), 406.1713, found: 406.1716.
3.3.5. ((5S)-3-(Methyl-6-deoxy-a-D-glucopyranoside-5-
yl)isoxazoline-5-yl) methyl a-C-D-glucopyranoside (12c)
3.3.1. ((5S)-3-(2,3-O-Isopropylidene-5-deoxy-
D
-lyxofuranose-4-
White solid, mp: 103–105 °C; Yield: 80%; ½a D25
ꢀ
+108 (c 1.0, H2O);
yl)isoxazoline-5-yl) methyl
a
-C-
D
-glucopyranoside (12a)
1H NMR (D2O): d 1.95 (m, 2H, 6-H), 2.84 (dd, 1H, J = 17.2 Hz,
J = 7.1 Hz, CH2, 4-H), 3.26 (dd, 1H, CH2, J = 16.7 Hz, J = 7.3 Hz, 4-
H), 3.30 (s, 3H, OCH3), 3.43 (m, 2H, 10-H, 9-H), 3.52 (m, 2H, 7-H,
12-H), 3.57 (m, 2H, 11-H, 12-H), 3.67 (dd, 1H, J = 9.3 Hz,
J = 3.2 Hz, 20-H), 3.73 (m, 1H, 8-H), 3.76 (d, 1H, J = 1.9 Hz, 30-H),
4.00 (d, 1H, J = 10.5 Hz, 40-H), 4.32 (d, 1H, J = 10.5 Hz, 50-H), 4.73
(d, 1H, J = 3.5 Hz, 10-H), 4.78 (m, 1H, 5-H); 13C NMR (D2O): d 33.3
(CH2, 6-C), 39.2 (CH2, 4-C), 55.9 (OCH3), 61.7 (CH2, 12-C), 67.4
(CH, 8-C), 67.8 (CH, 40-C), 71.1 (CH, 20-C), 71.4 (CH, 50-C), 71.5 (9-
CH), 72.0 (CH, 7-C), 73.2 (CH, 30-C), 74.5 (CH, 10-C), 75.6 (CH, 11-
C), 77.8 (CH, 5-C), 100.3 (CH, 10-C), 159.6 (C@N); HRMS(ESI): calcd
for C16H27NNaO11 ([M+Na]+), 432.1482, found: 432.1479.
White solid, mp: 90–91 °C; Yield: 79%; ½a D25
ꢀ
+69.9 (c 1.0, H2O);
1H NMR (CD3OD): d 1.29 (s, 3H, CH3), 1.42 (s, 3H, CH3), 1.90 (m,
2H, 6-H), 2.87 (dd, 1H, J = 17.3 Hz, J = 6.2 Hz, CH2, 4-H), 3.30 (m,
3H, 4-H, 2CH), 3.45 (m, 2H, CH), 3.58 (m, 2H, CH), 3.79 (dd, 1H,
J = 11.7 Hz, J = 2.2 Hz, CH), 4.12 (m, 1H, CH), 4.60 (d, 1H,
J = 5.4 Hz, 20-H), 4.80 (m, 1H, 5-H), 4.94 (m, 2H, 30-H, 40-H), 5.44
(s, 1H, 10-H); 13C NMR (CD3OD): d 23.8 (CH3), 25.3 (CH3), 29.9
(CH2, 6-C), 41.0 (CH2, 4-C), 62.1 (CH2, 12-C), 71.2 (CH, 8-C), 71.5
(CH, 10-C), 73.1 (CH, 40-C), 73.8 (CH, 7-C), 74.1 (CH, 9-C), 76.1
(CH, 5-C), 77.7 (CH, 11-C), 81.8 (CH, 30-C), 86.1 (CH, 20-C), 101.3
(CH, 10-C), 113.1 (C, 13-C), 157.6 (C@N); HRMS(ESI): calcd for
C
17H28NO10 ([M+H]+), 406.1713, found: 406.1708.
3.3.6. ((5R)-3-(Methyl-6-deoxy-a-D-glucopyranoside-5-
3.3.2. ((5R)-3-(2,3-O-Isopropylidene-5-deoxy- -lyxofuranose-4-
D
yl)isoxazoline-5-yl) methyl a-C-D- glucopyranoside (13c)
yl)isoxazoline-5-yl) methyl
a
-C-
D
-glucopyranoside (13a)
White solid, mp: 140 °C (decomposed); Yield: 86%; ½a D25
ꢀ
+38.7 (c
White solid, mp: 84–86 °C; Yield: 80%; ½a D25
ꢀ
ꢁ41.4 (c 1.0, H2O);
1.0, H2O); 1H NMR (D2O): d 1.93 (m, 1H, 6-H), 2.16 (m, 1H, 6-H),
2.92 (dd, 1H, J = 17.2 Hz, J = 8.4 Hz, CH2, 4-H), 3.18 (dd, 1H, CH2,
J = 17.0 Hz, J = 10.3 Hz, 4-H), 3.34 (s, 3H, OCH3), 3.46–3.53 (m, 5H,
10-H, 8-H, 7-H, 40-H, 12-H), 3.63 (m, 3H, 12-H, 9-H, 20-H), 3.81
(d, 1H, J = 10.0 Hz, 30-H), 4.10 (d, 1H, J = 10.5 Hz, 11-H), 4.33 (d,
1H, J = 9.9 Hz, 50-H), 4.71 (d, 1H, J = 3.5 Hz, 10-H), 4.80 (m, 1H, 5-
H); 13C NMR (D2O): d 29.8 (CH2, 6-C), 37.9 (CH2, 4-C), 54.9
(OCH3), 61.9 (CH2, 12-C), 67.8 (CH, 8-C), 71.2 (CH, 40-C), 71.6 (CH,
20-C), 71.8 (CH, 50-C), 72.3 (9-CH), 73.5 (CH, 7-C), 73.6 (CH, 30-C),
74.0 (CH, 10-C), 74.2 (CH, 11-C), 79.6 (CH, 5-C), 100.6 (CH, 10-C),
1H NMR (CD3OD): d 1.32 (s, 3H, CH3), 1.51 (s, 3H, CH3), 1.88 (m, 1H,
6-H), 2.14 (m, 1H, 6-H), 2.95 (dd, 1H, J = 17.2 Hz, J = 7.9 Hz, CH2, 4-
H), 3.19 (dd, 1H, J = 17.1 Hz, J = 10.1 Hz, CH2, 4-H), 3.26 (m, 1H, CH),
3.52 (m, 2H, CH), 3.60 (m, 2H, CH), 3.79 (dd, 1H, J = 11.7 Hz,
J = 2.2 Hz, CH), 4.01 (m, 1H, CH), 4.59 (d, 1H, J = 6.4 Hz, 20-H),
4.76 (m, 1H, 5-H), 4.88 (t, 1H, J = 6.4 Hz, 30-H), 4.94 (d, 1H,
J = 3.7 Hz, 40-H), 5.31 (s, 1H, 10-H); 13C NMR (CD3OD): d 23.6
(CH3), 25.3 (CH3), 30.2 (CH2, 6-C), 40.4 (CH2, 4-C), 62.0 (CH2, 12-
C), 71.2 (CH, 8-C), 71.7 (CH, 10-C), 73.5 (CH, 40-C), 74.1 (CH, 7-C),
74.2 (CH, 9-C), 76.0 (CH, 5-C), 79.1 (CH, 11-C), 82.2 (CH, 30-C),
86.1 (CH, 20-C), 101.4 (CH, 10-C), 113.0 (C, 13-C), 157.7 (C@N);
158.1 (C@N); HRMS(ESI): calcd for
C
16H27NNaO11 ([M+Na]+),
432.1482, found: 432.1478.
HRMS(ESI): calcd for
406.1710.
C
17H28NO10 ([M+H]+), 406.1713, found:
3.3.7. ((5S)-3-(2,3-O-Isopropylidene-5-deoxy-D-lyxofuranose-4-
yl)isoxazoline-5-yl) methyl a-C-D-mannopyranoside (14a)
3.3.3. ((5S)-3-(1,2-O-Isopropylidene-5-deoxy-
D
-xylofuranose-4-
White solid, mp: 116–117 °C; Yield: 93%; ½a D25
ꢀ
+80.0 (c 1.0,
yl)isoxazoline-5-yl) methyl
a
-C-
D
-glucopyranoside (12b)
H2O); 1H NMR (CD3OD): d 1.25 (s, 3H, CH3), 1.34 (s, 3H, CH3),
1.70 (m, 1H, 6-H), 1.97 (m, 1H, 6-H), 2.78 (dd, 1H, J = 17.5 Hz,
J = 5.9 Hz, CH2, 4-H), 3.21 (dd, 1H, J = 17.5 Hz, J = 10.0 Hz, CH2, 4-
H), 3.45 (m, 1H, CH), 3.54 (t, 1H, J = 9.2 Hz, CH), 3.64 (m, 2H, CH),
3.74 (m, 2H, CH), 4.00 (d, 1H, J = 9.2 Hz, CH), 4.69 (d, 1H,
J = 5.4 Hz, 20-H), 4.76 (m, 1H, 5-H), 4.97 (d, 1H, J = 3.8 Hz, 40-H),
5.09 (t, 1H, J = 5.4 Hz, 30-H, 40-H), 5.38 (s, 1H, 10-H); 13C NMR
(CD3OD): d 24.1 (CH3), 25.3 (CH3), 33.0 (CH2, 6-C), 41.1 (CH2, 4-
C), 61.6 (CH2, 12-C), 67.7 (CH, 8-C), 71.1 (CH, 10-C), 72.0 (CH, 40-
C), 74.4 (CH, 7-C), 75.6 (CH, 9-C), 76.7 (CH, 5-C), 78.2 (CH, 11-C),
80.8 (CH, 30-C), 85.3 (CH, 20-C), 100.5 (CH, 10-C), 114.1 (C, 13-C),
158.5 (C@N); HRMS(ESI): calcd for C17H27NO10 ([M]+), 405.1635,
found: 405.1631.
White solid, mp: 78–80 °C; Yield: 80%; ½a D25
ꢀ
+90.3 (c 1.0, H2O);
1H NMR (CD3OD): d 1.33 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.98 (m,
2H, 6-H), 2.87 (dd, 1H, J = 17.5 Hz, J = 7.2 Hz, CH2, 4-H), 3.30 (m,
2H, CH, 4-H, 10-H), 3.46 (m, 2H, 9-H, 12-H), 3.63 (m, 2H, 7-H,
12-H), 3.81 (dd, 1H, J = 9.8 Hz, J = 1.9 Hz, 8-H), 4.10 (m, 1H, 11-
H), 4.27 (d, 1H, J = 2.8 Hz, 30-H), 4.56 (d, 1H, J = 3.5 Hz, 20-H), 4.80
(m, 1H, 5-H), 4.94 (d, 1H, J = 2.8 Hz, 40-H), 5.99 (d, 1H, J = 3.5 Hz,
10-H); 13C NMR (CD3OD): d 25.4 (CH3), 26.2 (CH3), 30.0 (CH2, 6-
C), 41.0 (CH2, 4-C), 62.1 (CH2, 12-C), 71.2 (CH, 8-C), 71.5 (CH, 9-
C), 73.1 (CH, 7-C), 73.9 (CH, 10-C), 74.0 (CH, 40-C), 76.8 (CH, 30-C),
77.3 (CH, 11-C), 77.7 (CH, 5-C), 85.6 (CH, 20-C), 105.4 (CH, 10-C),
112.1 (C, 13-C), 158.1 (C@N); HRMS(ESI): calcd for C17H28NO10
([M+H]+), 406.1713, found: 406.1709.
3.3.8. ((5R)-3-(2,3-O-Isopropylidene-5-deoxy-D-lyxofuranose-4-
3.3.4. ((5R)-3-(1,2-O-Isopropylidene-5-deoxy-
D
-xylofuranose-4-
yl)isoxazoline-5-yl) methyl a-C-D-mannopyranoside (15a)
yl)isoxazoline-5-yl) methyl
a
-C-
D
-glucopyranoside (13b)
White solid, mp: 72–74 °C; Yield: 76%; ½a D25
ꢁ49.2 (c 1.0, H2O);
ꢀ
White solid, mp: 67–69 °C; Yield: 90%; ½a D25
ꢀ
ꢁ25.7 (c 1.0, H2O);
1H NMR (D2O): d 1.29 (s, 3H, CH3), 1.43 (s, 3H, CH3), 1.73 (m, 1H, 6-
H), 2.18 (m, 1H, 6-H), 2.98 (dd, 1H, J = 17.3 Hz, J = 7.7 Hz, CH2, 4-H),
3.20 (dd, 1H, J = 17.2 Hz, J = 10.1 Hz, CH2, 4-H), 3.49 (m, 1H, CH),
3.62 (t, 1H, J = 8.2 Hz, CH), 3.68 (dd, 1H, J = 8.3 Hz, J = 2.9 Hz, CH),
1H NMR (CD3OD): d 1.33 (s, 3H, CH3), 1.48 (s, 3H, CH3), 1.98 (m, 2H,
6-H), 2.87 (dd, 1H, J = 17.5 Hz, J = 7.2 Hz, CH2, 4-H), 3.30 (m, 2H, CH,
4-H, 10-H), 3.46 (m, 2H, 9-H, 12-H), 3.63 (m, 2H, 7-H, 12-H), 3.81

14
P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
3.72 (t, 1H, J = 5.6 Hz, CH), 3.74 (s, 1H, CH), 3.79 (dd, 1H, J = 11.7 Hz,
J = 2.7 Hz, CH), 3.96 (m, 1H, CH), 4.59 (d, 1H, J = 5.7 Hz, 20-H), 4.74
(m, 1H, 5-H), 4.90 (t, 1H, J = 5.6 Hz, 30-H), 4.94 (d, 1H, J = 3.7 Hz, 40-
H), 5.32 (s, 1H, 10-H); 13C NMR (D2O): d 23.6 (CH3), 25.3 (CH3), 33.7
(CH2, 6-C), 40.3 (CH2, 4-C), 61.9 (CH2, 12-C), 68.4 (CH, 8-C), 71.6
(CH, 10-C), 71.7 (CH, 40-C), 74.4 (CH, 7-C), 75.5 (CH, 9-C), 75.9
(CH, 5-C), 78.6 (CH, 11-C), 82.2 (CH, 30-C), 86.1 (CH, 20-C), 101.4
(CH, 10-C), 113.0 (C, 13-C), 157.8 (C@N); HRMS(ESI): calcd for
11-H, 20-H), 3.81 (m, 2H, 8-H, 30-H), 4.09 (m, 1H, 40-H), 4.36 (d,
1H, J = 10.5 Hz, 50-H), 4.73 (d, 1H, J = 3.5 Hz, 10-H), 4.83 (m, 1H, 5-
H); 13C NMR (CD3OD): d 33.3 (CH2, 6-C), 37.8 (CH2, 4-C), 54.9
(OCH3), 61.5 (CH2, 12-C), 67.8 (CH, 8-C), 68.2 (CH, 40-C), 71.5 (CH,
20-C), 71.8 (CH, 50-C), 71.8 (9-CH), 72.2 (CH, 7-C), 73.6 (CH, 30-C),
74.7 (CH, 10-C), 75.3 (CH, 11-C), 79.0 (CH, 5-C), 100.6 (CH, 10-C),
158.1 (C@N); HRMS(ESI): calcd for
432.1482, found: 432.1485.
C
16H27NNaO11 ([M+Na]+),
C
17H27NO10 ([M]+), 405.1635, found: 405.1637.
3.3.13. ((5S)-3-(2,3-O-Isopropylidene-5-deoxy-
D
-lyxofuranose-
3.3.9. ((5S)-3-(1,2-O-Isopropylidene-5-deoxy-
D
-xylofuranose-4-
4-yl)isoxazoline-5-yl) methyl a-C-D-galactopyranoside (16a)
yl)isoxazoline-5-yl) methyl
a
-C-
D
-mannopyranoside (14b)
White solid, mp: 88–90 °C; Yield: 85%; ½a D25
ꢀ
+101 (c 1.0, H2O);
White solid, mp: 102–105 °C; Yield: 80%; ½a D25
ꢀ
+33.3 (c 1.0,
1H NMR (D2O): d 1.23 (s, 3H, CH3), 1.32 (s, 3H, CH3), 1.81 (m, 2H,
6-H), 2.76 (dd, 1H, J = 17.5 Hz, J = 5.8 Hz, CH2, 4-H), 3.22 (dd, 1H,
J = 17.4 Hz, J = 10.2 Hz, CH2, 4-H), 3. 62 (m, 4H, CH), 3.86 (m, 2H,
CH), 4.11 (m, 1H, CH), 4.67 (d, 1H, J = 4.5 Hz, 20-H), 4.75 (m, 1H,
5-H), 4.96 (d, 1H, J = 3.8 Hz, 40-H), 5.03 (t, 1H, J = 4.5 Hz, 30-H),
5.37 (s, 1H, 10-H); 13C NMR (D2O): d 24.1 (CH3), 25.2 (CH3), 29.2
(CH2, 6-C), 41.2 (CH2, 4-C), 61.5 (CH2, 12-C), 68.1 (CH, 8-C), 69.5
(CH, 10-C), 70.0 (CH, 40-C), 72.4 (CH, 7-C), 72.7 (CH, 9-C), 76.8
(CH, 11-C), 78.0 (CH, 5-C), 80.7 (CH, 30-C), 85.2 (CH, 20-C), 100.4
(CH, 10-C), 114.2 (C, 13-C), 158.5 (C@N); HRMS(ESI): calcd for
C17H27NO10 ([M]+), 405.1635, found: 405.1639.
H2O); 1H NMR (CD3OD): d 1.33 (s, 3H, CH3), 1.49 (s, 3H, CH3),
1.80 (m, 1H, 6-H), 2.00 (m, 1H, 6-H), 2.85 (dd, 1H, J = 17.5 Hz,
J = 6.9 Hz, CH2, 4-H), 3.32 (m, 1H, CH2, 4-H), 3.49 (m, 1H, 10-H),
3.65 (m, 2H, 9-H, 12-H), 3.78 (m, 3H, 7-H, 12-H), 4.10 (d, 1H,
J = 10.2 Hz, 11-H), 4.26 (d, 1H, J = 2.4 Hz, 30-H), 4.56 (d, 1H,
J = 3.3 Hz, 20-H), 4.82 (m, 1H, 5-H), 4.95 (d, 1H, J = 2.4 Hz, 40-H),
5.99 (d, 1H, J = 3.3 Hz, 10-H); 13C NMR (CD3OD): d 25.3 (CH3),
26.1 (CH3), 34.4 (CH2, 6-C), 41.0 (CH2, 4-C), 62.0 (CH2, 12-C), 68.4
(CH, 8-C), 71.6 (CH, 9-C), 72.0 (CH, 7-C), 74.5 (CH, 10-C), 75.4
(CH, 40-C), 76.8 (CH, 30-C), 77.3 (CH, 11-C), 77.8 (CH, 5-C), 85.6
(CH, 20-C), 105.4 (CH, 10-C), 112.1 (C, 13-C), 158.1 (C@N); HRMS(E-
SI): calcd for C17H27NO10 ([M]+), 405.1635, found: 405.1631.
3.3.14. ((5R)-3-(2,3-O-Isopropylidene-5-deoxy-D-lyxofuranose-
4-yl)isoxazoline-5-yl) methyl a-C-D-galactopyranoside (17a)
3.3.10. ((5R)-3-(1,2-O-Isopropylidene-5-deoxy-
D
-xylofuranose-
White solid, mp: 93–95 °C; Yield: 87%; ½a D25
ꢁ29.9 (c 1.0, H2O);
ꢀ
4-yl)isoxazoline-5-yl) methyl
a
-C-
D
-mannopyranoside (15b)
1H NMR (D2O): d 1.23 (s, 3H, CH3), 1.35 (s, 3H, CH3), 1.76 (m, 1H, 6-
H), 2.10 (m, 1H, 6-H), 2.85 (dd, 1H, J = 17.4 Hz, J = 7.9 Hz, CH2, 4-H),
3.17 (dd, 1H, J = 17.4 Hz, J = 10.1 Hz, CH2, 4-H), 3.59 (m, 2H, CH),
3.65 (dd, 1H, J = 9.8 Hz, J = 2.8 Hz, CH), 3.74 (t, 1H, J = 9.8 Hz, CH),
4.01 (m, 1H, CH), 4.67 (d, 1H, J = 4.2 Hz, 20-H), 4.75 (m, 1H, 5-H),
4.98 (d, 1H, J = 3.4 Hz, 40-H), 5.02 (t, 1H, J = 4.2 Hz, 30-H), 5.37 (s,
1H, 10-H); 13C NMR (D2O): d 24.0 (CH3), 25.2 (CH3), 29.3 (CH2, 6-
C), 40.7 (CH2, 4-C), 61.3 (CH2, 12-C), 68.3 (CH, 8-C), 69.3 (CH, 10-
C), 70.1 (CH, 40-C), 72.5 (CH, 7-C), 73.5 (CH, 9-C), 76.5 (CH, 11-C),
79.8 (CH, 5-C), 80.9 (CH, 30-C), 85.3 (CH, 20-C), 100.6 (CH, 10-C),
114.1 (C, 13-C), 158.3 (C@N); HRMS(ESI): calcd for C17H27NO10
([M]+), 405.1635, found: 405.1630.
White solid, mp: 72–74 °C; Yield: 74%; ½a D25
ꢁ37.7 (c 1.0, H2O);
ꢀ
1H NMR (D2O): d 1.28 (s, 3H, CH3), 1.44 (s, 3H, CH3), 1.73 (m, 1H, 6-
H), 2.16 (m, 1H, 6-H), 2.88 (dd, 1H, J = 17.5 Hz, J = 7.8 Hz, CH2, 4-H),
3.21 (dd, 1H, J = 17.5 Hz, J = 10.4 Hz, CH2, 4-H), 3.50 (m, 1H, 10-H),
3.57 (t, 1H, J = 9.3 Hz, 9-H), 3.64 (dd, 1H, J = 12.2 Hz, J = 5.6 Hz, 12-
H), 3.75 (m, 3H, 7-H, 12-H), 3.96 (d, 1H, J = 5.7 Hz, 11-H), 4.38 (S,
1H, 30-H), 4.68 (S, 1H, 20-H), 4.75(m, 1H, 5-H), 4.99 (S, 1H, 40-H),
6.03 (d, 1H, J = 3.0 Hz, 10-H); 13C NMR (D2O): d 25.5 (CH3), 26.1
(CH3), 32.7 (CH2, 6-C), 40.6 (CH2, 4-C), 61.6 (CH2, 12-C), 67.7 (CH,
8-C), 71.1 (CH, 9-C), 71.7 (CH, 7-C), 74.5 (CH, 10-C), 75.5 (CH, 40-
C), 75.9 (CH, 30-C), 77.8 (CH, 11-C), 79.2 (CH, 5-C), 84.8 (CH, 20-C),
105.0 (CH, 10-C), 113.4 (C, 13-C), 158.3 (C@N); HRMS(ESI): calcd
for C17H27NO10 ([M]+), 405.1635, found: 405.1632.
3.3.15. ((5S)-3-(1,2-O-Isopropylidene-5-deoxy-D-xylofuranose-
4-yl)isoxazoline-5-yl) methyl a- C-D-galactopyranoside (16b)
3.3.11. ((5S)-3-(Methyl-6-deoxy-
a
-
D
-glucopyranoside-5-
White solid, mp: 108–110 °C; Yield: 95%; ½a D25
ꢀ
+68.0 (c 1.0,
yl)isoxazoline-5-yl) methyl
a
-C-
D
- mannopyranoside (14c)
H2O); 1H NMR (D2O): d 1.25 (s, 3H, CH3), 1.40 (s, 3H, CH3), 1.83
(m, 2H, 6-H), 2.76 (dd, 1H, J = 17.5 Hz, J = 6.5 Hz, CH2, 4-H), 3.18
(dd, 1H, CH2, J = 17.6 Hz, J = 10.1 Hz, 4-H), 3.60 (m, 3H, 10-H, 9-H,
12-H), 3.66 (m, 1H, 7-H), 3.85 (m, 2H, 8-H, 12-H), 4.09 (m, 1H,
11-H), 4.35 (d, 1H, J = 2.0 Hz, 30-H), 4.65 (d, 1H, J = 3.3 Hz, 20-H),
4.74 (m, 1H, 5-H), 4.97 (s, 1H, 40-H), 5.99 (d, 1H, J = 3.2 Hz, 10-H);
13C NMR (D2O): d 25.5 (CH3), 26.0 (CH3), 29.3 (CH2, 6-C), 41.1
(CH2, 4-C), 61.5 (CH2, 12-C), 68.2 (CH, 8-C), 69.4 (CH, 9-C), 70.0
(CH, 7-C), 72.5 (CH, 10-C), 72.6 (CH, 40-C), 75.3 (CH, 30-C), 77.8
(CH, 11-C), 78.0 (CH, 5-C), 84.8 (CH, 20-C), 105.0 (CH, 10-C), 113.4
(C, 13-C), 158.5 (C@N); HRMS(ESI): calcd for C17H27NO10 ([M]+),
405.1635, found: 405.1641.
White solid, mp: 115–118 °C; Yield: 80%; ½a D25
ꢀ
+84.4 (c 1.0,
H2O); 1H NMR (CD3OD): d 1.72 (m, 1H, 6-H), 2.00 (m, 1H, 6-H),
2.79 (dd, 1H, J = 17.8 Hz, J = 6.8 Hz, CH2, 4-H), 3.19 (dd, 1H, CH2,
J = 17.6 Hz, J = 7.3 Hz, 4-H), 3.30 (s, 3H, OCH3), 3.43 (m, 2H, 10-H,
9-H), 3.52 (m, 2H, 7-H, 12-H), 3.57 (m, 2H, 11-H, 12-H), 3.67 (dd,
1H, J = 9.3 Hz, J = 3.2 Hz, 20-H), 3.73 (m, 1H, 8-H), 3.76 (d, 1H,
J = 1.9 Hz, 30-H), 4.00 (d, 1H, J = 10.5 Hz, 40-H), 4.32 (d, 1H,
J = 10.5 Hz, 50-H), 4.73 (d, 1H, J = 3.5 Hz, 10-H), 4.76 (m, 1H, 5-H);
13C NMR (CD3OD): d 33.3 (CH2, 6-C), 39.2 (CH2, 4-C), 55.9 (OCH3),
61.7 (CH2, 12-C), 67.4 (CH, 8-C), 67.8 (CH, 40-C), 71.1 (CH, 20-C),
71.4 (CH, 50-C), 71.5 (9-CH), 72.0 (CH, 7-C), 73.2 (CH, 30-C), 74.5
(CH, 10-C), 75.6 (CH, 11-C), 78.5 (CH, 5-C), 100.3 (CH, 10-C), 159.6
(C@N); HRMS(ESI): calcd for C16H27NNaO11 ([M+Na]+), 432.1482,
found: 432.1479.
3.3.16. ((5R)-3-(1,2-O-Isopropylidene-5-deoxy-D-xylofuranose-
4-yl)isoxazoline-5-yl) methyl a-C-D-galactopyranoside (17b)
White solid, mp: 95–97 °C; Yield: 94%; ½a D25
ꢁ36.3 (c 1.0, H2O);
ꢀ
3.3.12. ((5R)-3-(Methyl-6-deoxy-
a
-
D
-glucopyranoside-5-
1H NMR (D2O): d 1.24 (s, 3H, CH3), 1.39 (s, 3H, CH3), 1.74 (m, 1H, 6-
H), 2.08 (m, 1H, 6-H), 2.85 (dd, 1H, J = 17.5 Hz, J = 8.0 Hz, CH2, 4-H),
3.14 (dd, 1H, CH2, J = 17.6 Hz, J = 10.3 Hz, 4-H), 3.57 (m, 2H, 10-H,
9-H), 3.64 (dd, 1H, J = 9.2 Hz, J = 2.3 Hz, 12-H), 3.73 (m, 1H, 7-H),
3.85 (m, 2H, 8-H, 12-H), 4.03 (m, 1H, 11-H), 4.34 (d, 1H,
J = 1.7 Hz, 30-H), 4.64 (d, 1H, J = 3.3 Hz, 20-H), 4.76 (m, 1H, 5-H),
4.95 (s, 1H, 40-H), 5.98 (d, 1H, J = 3.2 Hz, 10-H); 13C NMR (D2O): d
yl)isoxazoline-5-yl) methyl a-C-D- mannopyranoside (15c)
White solid, mp: 137 °C (decomposed); Yield: 87%; ½a D25
ꢀ
+18.6 (c
1.0, H2O); 1H NMR (CD3OD): d 1.82 (m, 1H, 6-H), 2.24 (m, 1H, 6-H),
2.98 (dd, 1H, J = 17.2 Hz, J = 8.3 Hz, CH2, 4-H), 3.19 (dd, 1H, CH2,
J = 17.2 Hz, J = 6.6 Hz, 4-H), 3.43 (s, 3H, OCH3), (m, 2H, 10-H,
9-H), 3.52 (m, 2H, 7-H, 12-H), 3.56 (m, 1H, 12-H), 3.68 (m, 2H,

P. Zhang et al. / Carbohydrate Research 351 (2012) 7–16
15
25.5 (CH3), 26.0 (CH3), 29.2 (CH2, 6-C), 40.6 (CH2, 4-C), 61.3 (CH2,
12-C), 68.3 (CH, 8-C), 69.3 (CH, 9-C), 70.1 (CH, 7-C), 72.5 (CH, 10-
C), 73.5 (CH, 40-C), 75.4 (CH, 30-C), 77.8 (CH, 11-C), 79.7 (CH, 5-C),
84.8 (CH, 20-C), 105.0 (CH, 10-C), 113.4 (C, 13-C), 158.3 (C@N);
HRMS(ESI): calcd for
405.1641.
was added. After 10 min, 2 mL of 3,5-dinitrosalicylic acid was
added, and then the reaction mixture was heated in boiling water
for 5 min. The solution was finally diluted to 20 mL after cooling
down. Absorbance readings were taken on a TU-1901 UV–vis spec-
trophotometer at 540 nm using distilled deionized water as a blank
control.
C
17H27NO10 ([M]+), 405.1635, found:
The a-glucosidase assay was performed using PNPG (1 mg/mL)
3.3.17. ((5S)-3-(Methyl-6-deoxy-
a
-
D
-glucopyranoside-5-
as substrates in phosphate buffer, pH 6.8, at 37 °C. The enzyme
solution (0.1 mL, 10 mg of solid enzyme in 10 mL of pH 6.8 phos-
phate buffer), 0.1 mL of inhibitor (1 mg/mL), 1.9 mL of buffer, and
0.05 mL glutathione (reduced, 1 mg/mL) were incubated for
10 min, and then 0.15 mL of substrate was added. The reaction
was quenched with 10 mL of sodium carbonate (0.1 mol/L) after
10 min, and the solution was finally diluted to 20 mL after cooling
down. Absorbance readings were taken on a TU-1901 UV–vis spec-
trophotometer at 400 nm using distilled deionized water as a blank
control.
yl)isoxazoline-5-yl) methyl a-C-D-galactopyranoside (16c)
White solid, mp: 102–103 °C; Yield: 90%; ½a D25
ꢀ
+108 (c 1.0, H2O);
1H NMR (CD3OD): d 1.94 (m, 2H, 6-H), 2.85 (dd, 1H, J = 17.2 Hz,
J = 7.3 Hz, CH2, 4-H), 3.27 (dd, 1H, CH2, J = 17.2 Hz, J = 10.2 Hz, 4-
H), 3.43 (s, 3H, OCH3), 3.49 (dd, 1H, J = 9.3 Hz, J = 3.7 Hz, 10-H),
3.68–3.74 (m, 5H, CH, 8-H, 40-H, 7-H, 12-H), 3.83 (m, 1H, 20-H),
3.94 (m, 2H, 9-H, 30-H), 4.23 (m, 1H, 11-H), 4.35 (d, 1H,
J = 10.0 Hz, 50-H), 4.73 (d, 1H, J = 3.5 Hz, 10-H), 4.78 (m, 1H, 5-H);
13C NMR (CD3OD): d 30.7 (CH2, 6-C), 38.7 (CH2, 4-C), 54.9 (OCH3),
61.4 (CH2, 12-C), 67.7 (CH, 8-C), 68.9 (CH, 40-C), 69.3 (CH, 20-C),
70.8 (CH, 50-C), 71.9 (9-CH,), 72.0 (CH, 7-C), 72.2 (CH, 30-C), 73.2
(CH, 10-C), 73.6 (CH, 11-C), 78.2 (CH, 5-C), 100.6 (CH, 10-C), 158.3
(C@N); HRMS(ESI): calcd for C16H27NNaO11 ([M+Na]+), 432.1482,
found: 432.1478.
3.4.2. In vitro colorimetric reverse transcriptase assay
The HIV-RT inhibition assay was performed by using a colori-
metric reverse transcriptase assay (Roche), and the procedure for
assaying RT inhibition was performed as described in the kit proto-
col. Briefly, the reaction mixture consists of template/primer com-
plex, 20-deoxy-nucleotide-50-triphosphates (dNTPs) and reverse
transcriptase (RT) enzyme in the lysis buffer with or without inhib-
itors. After 1 h incubation at 37 °C the reaction mixture was trans-
ferred to streptavidine-coated microtiter plate (MTP). The biotins
labeled dNTPs that are incorporated in the template due to activity
of RT were bound to streptavidine. The unbound dNTPs were
washed using wash buffer and antidigoxigenin-peroxidase (DIG-
POD) was added in MTP. The DIG-labeled dNTPs incorporated in
the template was bound to anti-DIG-POD antibody. The unbound
anti-DIG-POD was washed and the peroxide substrate (ABST)
was added to the MTP. A colored reaction product was produced
during the cleavage of the substrate catalyses by a peroxide en-
zyme. The absorbance of the sample was determined at OD
405 nM using microtiter plate ELISA reader. The resulting color
intensity is directly proportional to the actual RT activity. The per-
centage inhibitory activity of RT inhibitors was calculated by com-
paring to a sample that does not contain an inhibitor. The
percentage inhibition was calculated by formula as given below:
% Inhibition = 100 ꢁ [(OD 405 nm with inhibitor/OD 405 nm with-
out inhibitor) ꢂ 100].
3.3.18. ((5R)-3-(Methyl-6-deoxy-a-D-glucopyranoside-5-
yl)isoxazoline-5-yl) methyl a-C-D-galactopyranoside (17c)
White solid, mp: 161 °C, decomposed; Yield: 92%; ½a D25
ꢀ
+47.0 (c
1.0, H2O); 1H NMR (CD3OD): d 1.92 (m, 1H, 6-H), 2.18 (m, 1H, 6-H),
2.96 (dd, 1H, J = 17.1 Hz, J = 8.4 Hz, CH2, 4-H), 3.20 (dd, 1H, CH2,
J = 17.0 Hz, J = 10.3 Hz, 4-H), 3.45 (s, 3H, OCH3), 3.49 (dd, 2H,
J = 9.6 Hz, J = 3.7 Hz, 10-H, 8-H), 3.56–3.73 (m, 3H, CH, 40-H, 7-H,
12-H), 3.83 (m, 2H, 20-H, 12-H), 3.94 (m, 2H, 9-H, 30-H), 4.23 (m,
1H, 11-H), 4.35 (d, 1H, J = 9.9 Hz, 50-H), 4.73 (d, 1H, J = 3.7 Hz, 10-
H), 4.83 (m, 1H, 5-H); 13C NMR (CD3OD): d 29.9 (CH2, 6-C), 37.8
(CH2, 4-C), 54.9 (OCH3), 61.4 (CH2, 12-C), 67.8 (CH, 8-C), 68.8
(CH, 40-C), 69.3 (CH, 20-C), 70.8 (CH, 50-C), 71.7 (9-CH), 72.2 (CH,
7-C), 72.4 (CH, 30-C), 73.4 (CH, 10-C), 73.6 (CH, 11-C), 79.2 (CH,
5-C), 100.6 (CH, 10-C), 158.1 (C@N); HRMS(ESI): calcd for
C
16H27NNaO11 ([M+Na]+), 432.1482, found: 432.1476.
3.4. Biological activity assays
3.4.1. Inhibition of glycosidases
The inhibitory activity of the synthesized compounds against a-
amylase,
(yeast) and b-glucosidase (almonds) were obtained from Fluka;
-amylase (Bacillaceae, 4000 U/mg) from Sanland-chen Interna-
tional Inc., Xiamen, China. Two substrates, p-nitrophenyl -gluco-
-salicin, were purchased from Sigma
a-glucosidase, and b-glucosidase: The a-glucosidase
3.4.3. Antitumor activity
a
The cytotoxicity of the compounds against breast cancer cell
line was examined by the modified Mosmann’s protocol as follows:
Briefly, cells (104 cells per well) were plated in 96-well culture
plates and cultured overnight at 37 °C in a 5% CO2 humidified incu-
bator. Compounds were added to the wells at final concentrations
a
pyranoside (PNPG) and (ꢁ)-
D
Chemical Co. All other commercial reagents were used as received.
Each enzyme assay was measured as follows.
The
a-amylase assay was performed using 1% starch as sub-
of 1, 10, and 100 lmol/L. Control wells were prepared by addition
strate in phosphate buffer, pH 6.0, at 50 °C. The enzyme solution
(0.1 mL, 5 mg of solid enzyme in 50 mL of pH 6.0 phosphate buf-
fer), 0.1 mL of inhibitor (1 mg/mL) and 1 mL of buffer were incu-
bated for 10 min, and then 1 mL of substrate was added. After
10 min, 2 mL of 3,5-dinitrosalicylic acid was added, and then the
reaction was heated in boiling water for 5 min. The solution was fi-
nally diluted to 20 mL after cooling down. Absorbance readings
were taken on a TU-1901 UV–vis spectrophotometer at 540 nm
using distilled deionized water as a blank control and acarbose
as a positive control.
of DMEM. Wells containing DMEM without cells were used as
blanks. The plates were incubated at 37 °C in a 5% CO2 incubator
for 48 h. Upon completion of the incubation, stock MTT dye solu-
tion (10
lL, 5 mg/mL) was added to each well. After 4 h of incuba-
tion, the supernatant was removed and DMSO (100
lL) was added
to dissolve the MTT. The optical density of each well was measured
on a microplate spectrophotometer at a wavelength of 570 nm. The
cytotoxicity effect was calculated according to the formula:
(ODcontrol ꢁ ODtreated)/ODcontrol ꢂ 100%.
The b-glucosidase assay was performed using (ꢁ)-
D
-salicin
Acknowledgments
(2 mg/mL) as substrate in phosphate buffer, pH 4.8, at 35 °C. The
enzyme solution (0.1 mL, 10 mg of solid enzyme in 10 mL of pH
4.8 acetate buffer), 0.1 mL of inhibitor (1 mg/mL), and 0.9 mL of
buffer were incubated for 10 min, and then 0.8 mL of substrate
The financial supports from the 973 Program (2010CB534913),
the National Natural Science Foundation of China (20972039,
21172051), the Natural Science Foundations of the Department

16
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Supplementary data
Supplementary data (1H and 13C NMR spectra for compounds
5–10 and 12–17, table for the chemical shifts of H-4 and coupling
constants of J4a/4b and J4a,4b/5 of compounds and tables of glycosidase
inhibitory activities and cytotoxicity) associated with this article
can be found, in the online version, at doi:10.1016/j.carres.2011.
11.025.
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