Expanding the application scope of glycosidases using click chemistry

Article abstract of DOI:10.1016/j.tet.2009.11.044

Glycosidase-mediated glycosylation of alkynyl alcohols and azide-containing alcohols was followed by a click reaction, affording various types of triazole glycosides. The activities of triazole glycosides detected in subsequent bioassays show that this procedure is a feasible approach to the development of anti-fungal drugs.

Full text of DOI:10.1016/j.tet.2009.11.044

Tetrahedron 66 (2010) 750–757
Contents lists available at ScienceDirect
Tetrahedron
journal homepage: www.elsevier.com/locate/tet
Expanding the application scope of glycosidases using click chemistry
,
Wen-Ya Lu a, Xing-Wen Sun b, Chen Zhu a, Jian-He Xu c, Guo-Qiang Lin a
*
a CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, Shanghai 200032, China
b Department of Chemistry, Fudan University, Shanghai 200433, China
c State Key Laboratory of Bioreactor Engineering, East China University of Science and Technology, Shanghai 200237, China
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycosidase-mediated glycosylation of alkynyl alcohols and azide-containing alcohols was followed by
a click reaction, affording various types of triazole glycosides. The activities of triazole glycosides
detected in subsequent bioassays show that this procedure is a feasible approach to the development of
anti-fungal drugs.
Received 18 September 2009
Received in revised form 10 November 2009
Accepted 10 November 2009
Available online 17 November 2009
Ó 2009 Elsevier Ltd. All rights reserved.
1. Introduction
In our previous reports, we demonstrated that crude meal of
some cyanogenic plant seeds, such as almond, peach kernel, apple
The enzymatic approach, by virtue of its mildness, high selec-
tivity, and acceptance of unprotected sugars as substrates, is of
increasing importance in the synthesis of glycosides. The glyco-
syltransferase,1 and recent glycosynthases2 show remarkable
application potential. The glycosidase, mediating the cleavage of
glycosidic bonds in vivo, can be used for glycoside synthesis via
reverse hydrolysis (Fig. 1A, thermodynamic control) or trans-
glycosylation (Fig. 1B, kinetic control), is also attractive due to its
ability of synthesis of glycosides from unprotected and unactivated
sugars in one step.3
seed, and apricot kernel, are robust and cheap alternatives to
purified or immobilized -glucosidases for glycoside synthesis via
b
reverse hydrolysis.4,5 This procedure was successfully applied to
synthesize some naturally occurring glycosides, such as salidroside
and rosvin (Fig. 2).5 Furthermore, a reactor integrating with product
adsorption column was developed for salidroside production. This
process shows a higher efficiency than the chemical or glycosyl-
transferase approach.6 Recently, the synthesis of salidroside was
scaled up to kilogram in a pilot reactor.
Reversed hydrolysis, though elegant in its simplicity, has its
inherited problems, the yield of product will decrease with the
increase of molecular weight of alcohols. To overcome this problem,
glucosidation of allylic alcohol followed by Mizoroki–Heck (MH)
type reaction approach was developed for the synthesis of Rosvin
and its analogs.7 However, the Mizoroki–Heck (MH) type reaction
glycosidase
reverse hydrolysis
+
+
(A)
Glycosyl-OH
Glycosyl-OR
H2O
ROH
glycosidase
transglycosylation
(B)
+
Glycosyl-OR'
Glycosyl-OR
+
ROH
R'OH
between the aryl boron reagents and unprotected allyl
pyranoside afforded only a 20% yield and protection–deprotection
b-gluco-
Figure 1. Enzymatic synthesis of glycosides based on reverse hydrolysis and
transglycosylation.
OH
OH
HO
apple seed meal
glucose +
O
HO
+ H2O
O
HO
OH
OH
salidroside
OH
O
HO
HO
apple seed meal
glucose +
O
+ H2O
HO
OH
rosvin
Figure 2. Enzymatic synthesis of salidroside and rosvin.
steps are still required. In the meanwhile, to develop an efficient
and seamless chemoenzymatic approach to various glycosides, the
copper(I) catalyzed azide–alkyne cycloaddition (CuAAC), or ‘click
* Corresponding author. Tel.: þ86 21 54925169; fax: þ86 21 64166263.
E-mail address: lingq@mail.sioc.ac.cn (G.-Q. Lin).
0040-4020/$ – see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2009.11.044

W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
751
reaction’8 was introduced for the ligation of simple glycosides and
other molecules.
accepting terminal alkynyl alcohols, internal alkynes alcohols, and
N3-containing alcohols. The glycosylation of 2-azidoethanol (1j)
and 3-azidopropan-1-ol (1k) achieved a higher yield (Table 1,
entries 10 and 11) than the alkynes containing alcohols. It seems
that N3-containing alkyl alcohols have no toxicity to apple/peach
CuAAC has gained great popularity because of its unique ‘click’
nature: namely, the reaction proceeds with high yields and no
byproducts and exhibits functional group orthogonality. CuAAC not
only is an effective ligation tool but also creates a 1,2,3-triazole
structure, which provides a facile route to mimic triazole fungal-
cides.9 Interestingly, it was also reported that alkyl glycosides
containing C8 to C12 alkyl chains showed a broad spectrum of
antimicrobial activity.10 Therefore, we envisaged that the glycoside
with a triazole moiety could be a good candidate for fungalcide.
b-glucosidase. However, due to the instability of aromatic azide,
a yield for 4-azidobenzyl
1, entry 12).
b-glucoside (2l) cannot be obtained (Table
Table 1
Synthesis of
b-glucosides using fruit kernel meal
OH
peach kernel meal
reverse hydrolysis
O
HO
HO
2. Results and discussion
HO
R
glucose +
+ H2O
O
R
OH
1a-1l
2a-2l
Propargyl, a highly reactive functional group, is sensitive to heat
and light. Unsurprisingly, there is no product isolated from the
system and the glucosidase was deactivated when propargyl
alcohol was applied both as substrate and organic phase (Fig. 1A).
Therefore, a co-solvent was introduced to reduce the concentration
of propargyl alcohol. Based on our early result,11 seven water-
miscible solvents, including tert-butyl alcohol, tert-amyl alcohol,
acetonitrile, acetone, 1,4-dioxane, 1,2-dimethoxyethane, and 1,2-
diacetoxyethane were employed as media for the enzymatic
glycosidation of propargyl alcohol. The results were summarized in
Figure 3. The content of propargyl alcohol should not be higher
than 50% (v/v) to maintain the activity of the enzyme (Fig. 3A).
When 50% (v/v) of solvent was added, acetonitrile can achieve the
best result (yield, 35%, Fig. 3B).
( )2
( )3
( )4
HO
HO
HO
HO
1a
1b
1c
1d
C3H7
Ph
HO
HO
HO
OH
HO
1e
1f
1g
1h
HO
C2H3
HO
( )2
HO
N3
N3
HO
N3
1j
1i
1k
1l
Entry
Substrate
Solvent
2 Yield (%)
Peach kernela
Apple seedb
35
30
25
20
15
10
5
A
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
CH3CN
CH3CN
CH3CN
CH3CN
tert-Butyl alcohol
CH3CN
CH3CN
CH3CN
CH3CN
CH3CN
32
30
17
15
31
12
5
35
31
18
16
27
12
12
14
16
45
51
N.R.
0
0
20
40
60
80
100
Propargyl alcohol (v/v)
9
9
15
39
53
N.R.
10
11
12
1j
1k
1l
40
35
30
25
20
15
10
5
B
CH3CN
Various
a
Isolated yield, using 60 mg (1.8 U) of home-made acetone powder of peach
kernel meal per mL of reaction mixture.
b
Isolated yield, using 60 mg (1.9 U) of home-made acetone powder of apple seed
meal per mL of reaction mixture.
0
l
e
l
e
ol
oh
ne
n
ne
ho
ri
it
o
t
xa
ha
co
lc
o
i
on
et
ac
ce
et
a
d
a
l al
my
xy
yl
ut
-b
rt
te
Generally, reversed hydrolysis (Fig. 1A), a reaction of a mono-
saccharide with an alcohol, is simpler than transglycosylation
(Fig. 1B), a reaction of a reactive glycosyl donor with an alcohol
(acceptor). In contrast to the synthesis of glucosides, trans-
ho
t
a
-
rt
te
me
di
solvent
-glucosides synthesis on volume fraction of
Figure 3. (A) Dependence of propargyl
b
glycosylation is preferred for the synthesis of
b-galactosides, be-
the propargyl alcohol in tert-butyl alcohol/water system; The reaction was carried out
by shaking at 50 ꢀC a mixture of 0.30 g P. persica kernel meal, 0.5 ml water containing
0.25 mmol glucose, 4.5 mL propargyl alcohol/tert-butyl alcohol with various ratio. (B)
Screen of solvents for the synthesis of propargyl b-glucosides. The reaction was carried
out by shaking at 50 ꢀC a mixture of 0.30 g P. persica kernel meal, 0.5 ml water con-
cause lactose, glycosyl donor, is abundant and cheap. The synthesis
of
b-galactosides catalyzed by b-galactosidase has been docu-
mented.13 However, little investigation had been focused on the
galactosidation of alkynes alcohols or N3-containing alcohols.14 The
enzymatic galactosidation were then carried out as trans-
glycosylation with lactose as the glycosyl donor. Gratifyingly, all the
taining 0.25 mmol glucose, 4.5 mL 50% (v/v) propargyl alcohol/mentioned solvent.
With the optimized conditions established, we began to evalu-
ate the reaction scope. Four terminal alkynyl alcohols (1a–1d) and
three N3-contained alcohols (1j–1l) were employed by applying
two different types of enzyme preparation, from peach kernel meal
and apple seed meal. Additionally, to investigate the substrate
scope of the enzyme and the potential activity of alkynyl glyco-
sides12 several internal alkynyl alcohols (1e–1i) were also included
as substrates.
substrates (1a–1l) were accepted by
gillus oryzae and afforded corresponding
b
-galactosidase from Asper-
-galactosides in an
b
acceptable yield (Table 2). However, attempts to enhance the yield
by adding a water-miscible solvent, such as DMF, tert-butyl alcohol,
acetonitrile, or acetone, were not hopeful.
The [3þ2] cycloaddition of an alkynl between an azide ach-
ieved near complete conversion and high isolated yield (>85%)
regardless of which functional group the glycoside was linked to
(Table 3). An increased reaction rate could be achieved using
sodium ascorbate8b to reduce CuII to CuI (Table 3, entries 1 and
As indicated in Table 1, the peach and apple glucosidase
exhibited relatively broad substrate scope. They were capable of

752
W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
3). When a bulky organic azide (6) was used, copper powder8d
Table 2
Synthesis of
b
-galactosides based on transglycosidation
was found to be a better reductant (Table 3, entries 2 and 4).
Additionally, it is worth to note that the standard thermal cy-
cloaddition of and alkyne can also conduct smoothly (Table 3,
entry 8).
The obtained alkynl and triazole glycosides were tested for their
antifungal activities. The alkynl glycosides (2d, 2f) and triazole-
containing glycosides (13, 15, and 17) showed inhibitory activities
against Candida albicans, Candida tropicalis, Candida glabrata, and
Cryptococcus neoformans with minimum inhibitory concentration
OH
OH
β -galactosidase
O
HO
R
+ glucose
lactose +
O
R
HO
OH
1a-1l
3a-3l
Entry
Substrate
3 Yielda (%)
1
2
3
4
5
6
7
8
1a
1b
1c
1d
1e
1f
1g
1h
1i
36
18
17
11
21
5
14
16
15
12
16
6
(MIC) of 4 mg/mL. Despite the anti-candida activity of triazole gly-
coside is only one quarter of that of fluconazole, these results in-
dicate the potential and feasibility of this approach to the
development of antifungal drugs.
We next investigated the possibility of performing the glyco-
sidation and cycloaddition as a sequential one-pot-like reaction.
Because of high molar excess of the alcohols, filtration and evapo-
ration are needed to remove the enzyme and the excess alcohol.
The click reaction was then accomplished by dissolving the re-
sultant syrup and adding other reactants. Mostly, this sequential
one-pot-like reaction could achieve a similar yield to enzymatic
glycosylation (Fig. 4).
9
10
11
12
1j
1k
1l
a
Isolated yield. The reaction was conducted under 25 ꢀC for 4–24 h.
Table 3
Synthesis of triazole-containing glycosides
R1
N
N
Cu+
( )n
( )n
3. Conclusion
+
N3 R1
Glycosyl
O
Glycosyl
N3
O
N
N3
In conclusion, a series of sugar modules bearing two kinds of
reactive handles (alkynl and azide) were enzymatically synthesized
from unprotected sugars in one step. The efficient synthesis of
alkynl and azide containing glycosides provide a series of modules,
which can be directly installed to other molecules. Furthermore,
sequential one-pot procedures for enzymatic glycosylation and
azide–alkyne cycloaddition have been developed giving access to
triazole-containing glycosides commencing with alkynl alcohols
being commercially available in a great variety.
N3
R1 N3
=
( )10
4
5
6
Entry
Glycoside
Azide
Time (h)
Product
Yield (%)
1a
2b
3a
4b
2a
2a
3a
3a
4
6
5
6
4
24
6
11
12
13
14
96
89
90
85
24
4. Experimental section
4.1. Material and methods
NMR spectra were recorded on
a Varian Mercury 300
(300 MHz) or Bruker AM 300 spectrometer (300 MHz). The 1H
NMR chemical shifts are reported relative to
d
d
¼4.79 (D2O) and
¼4.87 (CD3OD), respectively. The 13C NMR chemical shifts are
reported as
standard) and
d
values relative to
d
¼0.00 (D2O, TMS as external
d
¼49.0 (CD3OD, the center of the solvent resonance),
Entry
Glycoside
Alkyne
Time (h)
Product
Yield (%)
5b
6b
7b
8c
2j
3j
3k
3l
7
7
8
9
8
8
12
12
15
16
17
18
95
92
95
86
respectively. The following abbreviations are used to indicate the
multiplicity: s-singlet; d-doublet; t-triplet; q-quartet; p-pentlet;
h-sixtet; m-multiplet. Mass spectra were recorded on a Bruker
APEXIII 7.0 TESLA FTMS using ESI mode. Optical rotations were
measured using Jasco P-1030 polarimeter. Analytical TLC was
performed with silica gel 60 F254 plates, and the products were
visualized with H2SO4 (10%) solution in MeOH followed by
a
CuSO4, sodium ascorbate.
CuSO4, copper powder.
Methanol, reflux.
b
c
1) β-galactosidase
1) fruit kernel meal
OH
+
+
nC12H25
OH
OH
O
N
N
HO
O
N
N
HO
HO
HO
lactose
glucose
O
O
N
2) Cu+
+
HO
N
2) Cu+
+
R1 N3
OH
R1 N3
OH
11
34% for two steps
35% for two steps
13
β
1) -galactosidase
1) fruit kernel meal
+
HO
OH
OH
+
OH
O
O
O
N3
N3
O
N
HO
2) Cu+
10% for two steps
HO
HO
N
O
N
O
HO
N
O
N
OH
N
+
R2
2) Cu+
44% for two steps
+
R2
OH
16
17
Figure 4. Sequential one-pot-like reaction.

W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
753
heating. Column chromatography was carried out using silica gel
4.2.2.3. Pent-4-ynyl
b
-glucoside (2c).
(300–400 mesh).
OH
O
Prunus persica L. var. scleropersica (Reich) Yu¨ et Lu (Peach) and
Malus pumila Mill (apple) was purchased from the supermarket in
Shanghai as fresh fruits. All other chemicals were of the highest
purity commercially available and used without further purification
HO
HO
( )
3
O
OH
23
White solid; [
CD3OD)
a
]
ꢁ32.6 (c 0.75, CH3OH); 1H NMR (300 MHz,
D
if not mentioned.
from Sigma (Lot 91K1450). Standardized with dextrin 1 U will hy-
drolyze 1
mol of indicated substrate per min at pH 4.5 at 30 ꢀC.
b-Galactosidase from A. oryzae was purchased
d
1.75 (p, J¼6.9 Hz, 2H), 2.16 (t, J¼2.4 Hz, 1H), 2.25 (dt,
J¼9.3, 2.4 Hz, 2H), 3.11 (t, J¼8.4 Hz, 1H), 3.22–3.28 (m, 3H), 3.55–
3.64 (m, 2H), 3.81 (dd, J¼18.0, 1.5 Hz, 1H), 3.88–3.95 (m, 1H), 4.20
m
Activity with lactose 9.0 U/mg solid; activity with o-nitrophenyl-b-
galactoside 10.4 U/mg solid.
(J¼8.1 Hz, 1H); 13C NMR (75 MHz, CD3OD)
d 16.1, 30.3, 63.0, 69.6,
69.9, 71.9, 75.4, 78.1, 75.4, 78.1, 78.3, 85.0, 104.7; HRMS (ESI): calcd
The
release of p-nitrophenol from pNPG, and 1 U of
activity (U) is defined as the amount of enzyme that release 1
b
-glucosidase activity was determined by measuring the
-glucosidase
mol
for C11H18O6Naþ (MþNaþ) 269.0996, found 269.0998.
b
m
4.2.2.4. Hex-5-ynyl
b
-glucoside (2d).
of p-nitrophenol per min. All samples were assayed in potassium
phosphate buffer (50 mM, pH 7.0) at 50 ꢀC under conditions that
activity was proportional to enzyme concentration. A control test
without enzyme was included.
OH
O
HO
HO
( )4
O
OH
P. persica L. (Peach) stones were cracked with a hammer to
release soft kernels inside. The kernels were collected, peeled, and
then powdered in cold ethyl acetate (0 ꢀC) with a homogenizer. The
powder was defatted by further three washes with ethyl acetate,
two washes with acetone, and dried in vacuum and then stored at
4 ꢀC. The apple seeds were treated in the same manner. P. persica
kernel meal (28.7 U/g) and M. pumila Mill seed meal (32.8 U/g)
were obtained as a white powder and stored at 4 ꢀC.
White solid; [a]
D
d
ꢁ32.9 (c 0.68, CH3OH); 1H NMR (300 MHz, D2O)
26
1.57–1.66 (m, 2H), 1.70–1.76 (m, 2H), 2.20–2.24 (m, 3H), 3.17 (t,
J¼8.1 Hz, 1H), 3.27–3.38 (m, 3H), 3.53–3.60 (m, 1H), 3.64–3.70 (m,
1H), 3.85–3.97 (m, 2H), 4.25 (d, J¼8.1 Hz, 1H); 13C NMR (75 MHz,
CD3OD)
d 18.8, 26.2, 29.7, 62.7, 69.2, 70.1, 70.6, 75.0, 77.8, 78.0,
84.9, 104.3; HRMS (ESI): calcd for C12H20O6Naþ (MþNaþ)
283.1152, found 283.1146.
4.2. Synthesis of
b-glucosides using peach kernel meal
4.2.2.5. 4-Hydroxybut-2-ynyl
b-glucoside (2e).
OH
4.2.1. General procedure. To
a
solution of glucose (0.27 g,
O
HO
1.5 mmol) in water (0.5 mL) was added a solution (4.5 mL) of
substrate (10–38 mmol) in mentioned solvent, and P. persica ker-
nel meal/apple seed meal (0.30 g) was then added. The mixture
was stirred for 72 h at 50 ꢀC, then filtered and concentrated under
vacuum. The resultant syrup was applied to flash column chro-
matography (eluent EtOAc/MeOH¼15–10/1 or CH2Cl2/MeOH¼5/1).
O
HO
OH
OH
24
White solid; [
d
a
]
ꢁ71.5 (c 1.55, CH3OH); 1H NMR (300 MHz, D2O)
D
3.29 (t, J¼8.1 Hz, 1H), 3.39 (t, J¼9.3 Hz, 1H), 3.45–3.54 (m, 2H),
3.72 (dd, J¼12.3, 6.0 Hz, 1H), 3.92 (dd, J¼12.3, 0.9 Hz, 1H), 4.29
(s, 2H), 4.52 (s, 2H), 4.64 (d, J¼8.1 Hz, 1H); 13C NMR (75 MHz,
The corresponding
b-D-glucopyranosides were collected as white
D2O)
d 49.4, 56.6, 60.6, 69.5, 72.8, 75.6, 75.9, 80.2, 85.2, 100.4;
solid or clear syrup.
HRMS (ESI): calcd for C10H16O7Naþ (MþNaþ) 271.0788, found
271.0785.
4.2.2. Spectral and analytical data for all b-glucosides.
4.2.2.6. 3-Phenylprop-2-ynyl
b-glucoside (2f).
4.2.2.1. Prop-2-ynyl
b-glucoside (2a).
OH
OH
O
O
HO
HO
HO
HO
O
O
OH
Ph
OH
23
White solid; [
a
]
ꢁ91.5 (c 0.90, CH3OH); 1H NMR (300 MHz,
D
26
White solid; [
d
a
]
ꢁ91.9 (c 0.66, CH3OH); 1H NMR (300 MHz, D2O)
D
CD3OD)
d
(dd, J¼9.0, 7.5 Hz, 1H), 3.62–3.67 (m, 1H), 3.85 (d,
: 2.92(t, J¼2.4 Hz, 1H), 3.29 (dd, J1¼8.1 Hz, J2¼9.0 Hz, 1H), 3.34–
J¼9.3 Hz, 1H), 4.49 (d, J¼7.8 Hz, 1H), 4.61 (dd, J¼16.9, 15.6 Hz, 1H),
3.53 (m, 4H), 3.71 (dd, J¼12.3, 5.7 Hz, 1H), 3.91 (dd, J¼12.3, 1.8 Hz,
7.26–7.31 (m, 3H), 7.36–7.40 (m, 2H); 13C NMR (75 MHz, CD3OD)
1H), 4.47 (t, J¼1.8 Hz, 2H), 4.50 (d, J¼8.1 Hz, 1H); 13C NMR
d
57.7, 63.0, 71.9, 75.2, 78.3, 85.8, 87.6, 102.7, 124.2, 129.7, 129.9,
(75 MHz, D2O)
d 56.6, 60.8, 69.6, 72.9, 75.7, 76.0, 76.4, 78.9, 100.6;
132.9; HRMS (ESI): calcd for C15H18O6Naþ (MþNaþ) 317.0091, found
MS(ESI) m/z: 241.0 [MþNaþ].
317.0096.
4.2.2.2. But-3-ynyl
b
-glucoside (2b).
4.2.2.7. But-2-ynyl
b-glucoside (2g).
OH
O
OH
O
HO
HO
HO
HO
O
O
OH
OH
26
23
White solid; [
d
a
]
D
ꢁ30.0 (c 1.35, CH3OH); 1H NMR (300 MHz, D2O)
White solid; [
d
a
]
D
ꢁ30.1 (c 0.73, CH3OH); 1H NMR (300 MHz, D2O)
2.39 (t, J¼2.4 Hz, 1H), 2.56 (dt, J¼6.1, 2.7 Hz, 2H), 3.29 (t, J¼8.4 Hz,
1.78 (s, 3H), 3.21 (t, J¼2.7 Hz, 1H), 3.28–3.46 (m, 3H), 3.64 (dd,
1H), 3.38–3.52 (m, 3H), 3.69–3.85 (m, 2H), 3.90–4.04 (m, 2H), 4.51
J¼16.9, 15.6 Hz, 1H), 3.85 (d, J¼12.0 Hz, 1H), 4.35 (s, 2H), 4.56 (d,
(d, J¼7.8 Hz, 1H); 13C NMR (75 MHz, D2O)
d
19.3, 60.9, 68.3, 69.8,
t, J¼8.1 Hz, 1H); 1H NMR (300 MHz, D2O)
d 2.5, 57.1, 60.6, 69.5,
70.6, 73.2, 75.9, 76.1, 82.5, 102.4; HRMS (ESI): calcd for C10H16O6Naþ
72.9, 73.8, 75.7, 75.9, 84.9,102.2; HRMS (ESI): calcd for C10H16O6Naþ
(MþNaþ) 255.0839, found 255.0839.
(MþNaþ) 255.0839, found 255.0839.

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W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
4.2.2.8. Hex-2-ynyl
b
-glucoside (2h).
4.2.2.13. 3-Azidopropyl b-glucoside (2k).
OH
O
OH
O
HO
HO
HO
HO
O
O
N3
OH
OH
C3H7
23
23
ꢁ26.7 (c 0.95, CH3OH); 1H NMR (300 MHz,
Clear syrup; [a]
D
d
ꢁ26.1 (c 1.20, CH3OH); 1H NMR (300 MHz, D2O)
White solid; [a]
D
1.89 (p, J¼6.6 Hz, 2H), 3.25 (dd, J¼8.4, 8.4 Hz, 1H), 3.33–3.50 (m,
CD3OD)
d
1.00 (t, J¼7.2 Hz, 3H), 1.47 (m, 2H), 2.18–1.24 (m, 2H),
6H), 3.66–3.77 (m, 2H), 3.88–4.00 (m, 2H), 4.43 (d, J¼8.1 Hz, 1H);
3.20 (dd, J¼9.0, 8.1 Hz, 1H), 3.30–3.40 (m, 3H), 3.64–3.70 (m, 1H),
3.87 (dd, J¼12.3, 1.8 Hz, 1H), 4.41 (dd, J¼4.8, 2.1 Hz, 2H), 4.47 (d,
13C NMR (75 MHz, D2O)
d 28.3, 47.9, 60.8, 67.3, 69.7, 73.2, 75.8,
75.9, 102.3; HRMS (ESI): calcd for C9H17N3O6Naþ (MþNaþ)
J¼7.5 Hz, 1H); 13C NMR (75 MHz, CD3OD)
d 13.8, 21.4, 23.1, 57.2,
286.1010, found 286.1008.
62.7, 71.6, 74.9, 78.00, 78.02, 87.9, 101.9; HRMS (ESI): calcd for
C12H20O6Naþ (MþNaþ) 283.1152, found 283.1148.
4.3. Synthesis of b-galactosides based on transglycosidation
4.2.2.9. Hex-3-ynyl
b
-glucoside (2i).
4.3.1. General procedure. A solution of 2.5 g lactose in 5 mL
sodium acetate buffer (20 mM, pH 4.5) was prepared and 0.5 mL
propargyl alcohol was added. The reaction was initiated by the
addition of 0.1 g (900 U) galactosidase from A. oryzae and the
mixture stirred at 100 rpm and 25 ꢀC. The reaction was stopped
by filtering off the residual solid enzyme particles and the filtrate
was dried under rotary vacuum to give yellow syrup. The
resultant syrup was applied to flash column chromatography
(eluent EtOAc/MeOH¼15–10/1 or CH2Cl2/MeOH¼5/1). The cor-
OH
O
C2H5
HO
HO
O
OH
23
White solid; [
CD3OD)
a
]
D
ꢁ21.2 (c 0.83, CH3OH); 1H NMR (300 MHz,
d
1.13 (t, J¼9.0 Hz, 3H), 2.14–2.20 (m, 2H), 2.48–2.54 (m,
2H), 3.23 (t, J¼8.4 Hz, 1H), 3.32–3.41 (m, 3H), 3.67–3.75 (m, 2H),
3.89–3.97 (m, 2H), 3.35 (d, J¼8.1 Hz, 1H); 13C NMR (75 MHz,
CD3OD)
d 13.0, 14.6, 20.8, 62.6, 69.5, 71.4, 74.9. 76.6, 77.80, 77.85,
responding
clear syrup.
b-D-glucopyranosides were collected as white solid or
83.5, 104.2; HRMS (ESI): calcd for C12H20O6Naþ (MþNaþ)
283.1152, found 283.1144.
4.3.2. Spectral and analytical data for all b-galactosides.
4.2.2.10. 2-Azidoethanol
(1j)15. 2-Chloroethanol
(25.2 mL,
375 mmol) was added to a solution of NaN3 (30 g, 461 mmol) and
NaOH (1.5 g, 37.5 mmol) in water (115 mL). The mixture was stirred
at rt for 3 days, and sodium sulfate (35 g) was added. After 10 min,
the mixture was extracted with CH2Cl2 (3ꢂ70 mL). The combined
extracts were dried in Na2SO4 and concentrated. The residue was
distilled (Caution! Explosion hazards) to give clear liquid (bp, 30 ꢀC,
4.3.2.1. Prop-2-ynyl b-galactoside (3a).
OH
OH
O
O
HO
OH
20 Pa). Yield: 86%. 1H NMR (300 MHz, CDCl3)
J¼5.1 Hz, 2H), 3.47–3.44 (t, J¼5.1 Hz, 2H), 2.02 (s, 1H).
d 3.79–3.76 (t,
26
White solid; [
a
]
D
ꢁ30.2 (c 0.71, CH3OH); 1H NMR (300 MHz, D2O)
d
2.91 (s, 1H), 3.53 (dd, J¼9.9, 8.1 Hz, 1H), 3.64–3.79 (m, 4H), 3.93 (d,
J¼2.7 Hz, 1H), 4.48 (s, 2H), 4.57 (d, J¼7.8 Hz, 1H); 13C NMR (75 MHz,
4.2.2.11. 2-Azidoethyl
b
-glucoside (2j).
D2O)
d 56.4, 60.8, 68.5, 70.4, 72.6, 75.2, 76.2, 78.9, 101.0; MS(ESI)
m/z: 241.0 [MþNaþ].
OH
O
HO
HO
N3
O
4.3.2.2. But-3-ynyl b-galactoside (3b).
OH
OH
OH
23
Clear syrup; [
d
a]
ꢁ38.9 (c 0.91, CH3OH); 1H NMR (300 MHz, D2O)
D
O
3.30 (dd, J¼9.0, 8.1 Hz, 1H), 3.39–3.53 (m, 3H), 3.56 (t, J¼5.1 Hz,
O
HO
OH
2H), 3.73 (dd, J¼12.3, 5.4 Hz, 1H), 3.84 (dt, J¼11.4, 5.1 Hz, 1H),
3.92 (dd, J¼12.3, 2.1 Hz, 1H), 4.06 (dt, J¼11.4, 5.1 Hz, 1H), 4.50 (d,
J¼8.1 Hz, 1H); 13C NMR (75 MHz, D2O) 50.5, 60.7, 68.5, 69.6, 73.1,
75.7, 75.9, 102.3; HRMS (ESI): calcd for C8H15N3O6Naþ (MþNaþ)
272.0853, found 272.0852.
26
White solid; [
D2O)
a
]
ꢁ24.6 (c 0.85, CH3OH); 1H NMR (300 MHz,
D
d
2.35 (t, J¼2.1 Hz, 1H), 2.53 (dt, J¼6.3, 2.1 Hz, 2H), 3.50
(dd, J¼9.6, 7.8 Hz, 1H), 3.60–3.82 (m, 5H), 3.90 (d, J¼3.0 Hz, 1H),
3.95–4.02 (m, 1H), 4.42 (d, J¼7.8 Hz, 1H); 13C NMR (75 MHz,
D2O)
d 21.79, 21.82, 63.6, 70.7, 71.3, 73.0, 75.4, 77.8, 105.5;
4.2.2.12. 3-Azidopropanol (1k)16. A solution of sodium azide
(29.3 g, 0.461 mol, in 113 mL of H2O) was added during 30 min to
a solution of acrolein (17.2 g, 0.307 mol, in 45 mL of HOAc) with
intermittent cooling (dry ice/acetone) to keep the temperature
below 5 ꢀC. Stirring was then continued for 30 min without cooling.
The solution was extracted with ether (2ꢂ250 mL), the extract was
washed with 100 mL of saturated Na2CO3 solution (Caution!
Foaming), dried (MgSO4), and concentrated at rt to ca. 250 mL. The
ether solution was added during 30 min to a solution of NaBH4 (5 g
in 30 mL of H2O) with cooling to keep the temperature below 20 ꢀC.
The mixture was stirred for 15 min and saturated with solid NaC1.
The organic layer was dried (MgSO4) and concentrated. The residue
was distilled (Caution! Explosion hazards) to give clear liquid (bp,
HRMS (ESI): calcd for C10H16O6Naþ (MþNaþ) 255.0839, found
255.0834.
4.3.2.3. Pent-4-ynyl b-galactoside (3c).
OH
OH
O
O
HO
OH
26
White solid; [a]
D
ꢁ24.6 (c 0.85, CH3OH); 1H NMR (300 MHz, D2O)
d
1.84 (p, J¼6.6 Hz, 2H), 2.31–2.37 (m, 3H), 3.50 (dd, J¼9.6, 7.8 Hz,
1H), 3.62–3.70 (m, 2H), 3.73–3.81 (m, 3H), 3.92 (d, J¼3.3 Hz, 1H),
3.97–4.05 (m, 1H), 4.39 (d, J¼7.8 Hz, 1H); 13C NMR (75 MHz, D2O)
35 ꢀC, 20 Pa). Yield: 86%. 1H NMR (300 MHz, CDCl3)
d
1.84 (p,
d 14.2, 27.7, 60.9, 68.6, 68.8, 69.6, 70.8, 72.8, 75.1, 85.1, 102.8;
J¼6.3 Hz, 2H), 2.12 (t, J¼1.5 Hz, 2H, 1H), 3.45 (t, J¼6.3 Hz, 2H), 3.75
HRMS (ESI): calcd for C11H18O6Naþ (MþNaþ) 269.0982, found
(dd, J¼10.5, 5.4 Hz, 2H).
269.0996.

W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
755
4.3.2.4. Hex-5-ynyl
b
-galactoside (3d).
4.3.2.9. Hex-3-ynyl
b
-galactoside (3i).
OH
OH
OH
O
OH
O
C2H5
O
O
HO
HO
23
OH
OH
26
ꢁ26.3 (c 0.59, CH3OH); 1H NMR (300 MHz,
White solid; [
CD3OD)
a]
D
ꢁ18.1 (c 0.98, CH3OH); 1H NMR (300 MHz,
White solid; [
CD3OD)
a
]
D
d
1.11 (t, J¼7.5 Hz, 3H), 2.12–2.20 (m, 2H), 2.46–2.52 (m,
d
1.54–1.63 (m, 2H), 1.67–1.73 (m, 2H), 2.16–2.21 (m, 3H),
2H), 3.51–3.57 (m, 3H), 3.63–3.71 (m, 1H), 3.76–3.79 (m, 2H), 3.86
(d, J¼2.4 Hz, 1H), 3.89–3.95 (m, 1H), 4.28 (d, J¼6.6 Hz, 1H); 13C
NMR (75 MHz, CD3OD) 13.0, 14.6, 20.8, 62.4, 69.5, 71.2, 72.4, 74.8,
76.59, 75.65, 83.5, 104.9; HRMS (ESI): calcd for C12H20O6Naþ
(MþNaþ) 283.1152, found 283.1142.
3.41–3.58 (m, 4H), 3.70 (d, J¼1.5 Hz, 1H), 3.76 (dd, J¼24.9, 3.0 Hz,
2H), 3.86–3,94 (m, 1H), 4.18 (d, J¼7.2 Hz, 1H); 13C NMR (75 MHz,
CD3OD)
d 19.0, 26.5, 30.0, 62.7, 69.9, 70.4, 70.5, 72.8, 75.3, 76.8,
105.2; HRMS (ESI): calcd for C12H20O6Naþ (MþNaþ) 283.1152,
found 283.1142.
4.3.2.10. 2-Azidoethyl b-galactoside (3j).
4.3.2.5. 4-Hydroxybut-2-ynyl b-galactoside (3e).
OH
OH
OH
OH
O
O
N3
O
HO
O
HO
OH
OH
OH
23
Clear syrup; [
d
a
]
D
ꢁ24.6 (c 0.14, CH3OH); 1H NMR (300 MHz, D2O)
22
White solid; [
d
a
]
D
ꢁ78.6 (c 0.63, CH3OH);1H NMR (300 MHz, D2O)
3.49–3.57 (m, 3H), 3.62–3.72 (m, 2H), 3.76 (d, 3.81–3.86 (m,
3.53 (dd, J1¼7.8 Hz, J2¼9.6 Hz, 1H), 3.65–3.80 (m, 4H), 3.93 (d,
2H)), 3.92 (d, J¼3.0 Hz, 1H), 4.02–4.09 (m, 1H), 4.42 (d, J¼7.8 Hz,
J¼3.3 Hz, 1H), 4.30 (s, 2H), 4.53 (s, 2H), 4.58 (d, J¼7.5 Hz, 1H); 13C
1H); 13C NMR (75 MHz, D2O)
d 50.5, 60.8, 68.3, 68.5, 70.6, 72.6,
NMR (75 MHz, D2O)
d 49.5, 56.7, 60.9, 68.6, 70.6, 72.7, 75.3, 80.4,
75.1, 102.8; HRMS (ESI): calcd for C8H15N3O6Naþ (MþNaþ)
85.2, 101.0; HRMS (ESI): calcd for C10H16O7Naþ (MþNaþ)
272.0853, found 272.0858.
271.0788, found 271.0786.
4.3.2.11. 3-Azidopropyl b-galactoside (3k).
4.3.2.6. 3-Phenylprop-2-ynyl b-galactoside (3f).
OH
OH
OH
OH
O
O
O
N3
HO
OH
O
HO
OH
Clear syrup; [
a
]
ꢁ15.9 (c 0.90, CH3OH); 1H NMR (300 MHz, D2O)
d:
23
D
23
White solid; [
D2O)
a
]
ꢁ104.3 (c 0.31, CH3OH);1H NMR (300 MHz,
1.93 (p, J¼6.6 Hz, 2H), 3.48 (t, J¼6.9 Hz, 2H), 3.52–3.55 (m, 1H),
3.63–3.80 (m, 5H), 3.94 (d, J¼3.3 Hz, 1H), 3.98–4.06 (m, 1H), 4.40
D
d
3.55 (dd, J¼9.6, 7.8 Hz, 1H), 3.63–3.80 (m, 4H), 3.92 (d,
(d, J¼7.8 Hz, 1H); 13C NMR (75 MHz, D2O)
d: 28.3, 47.9, 60.9, 67.2,
J¼3.3 Hz, 1H), 4.63 (d, J¼7.2 Hz, 1H), 4.68 (s, 2H), 7.39–7.43 (m,
68.6, 70.7, 72.8, 75.1, 102.9; HRMS (ESI): calcd for C9H17N3O6Naþ
3H), 7.51–7.54 (m, 2H); 13C NMR (75 MHz, D2O)
d
57.3, 60.9,
(MþNaþ) 286.1010, found 286.1005.
68.6, 70.6, 72.8, 75.3, 84.2, 86.9, 101.3, 121.6, 128.6, 129.1, 131.7;
HRMS (ESI): calcd for C15H18O6Naþ (MþNaþ) 317.0096, found
317.1010.
4.3.2.12. (4-Azidophenyl)methanol (1l).
HO
4.3.2.7. But-2-ynyl b-galactoside (3g).
N3
OH
OH
O
(4-Nitrophenyl)methanol (7.6 g, 5 mmol) in methanol. To a mixture
of aniline derivative (107 mmol) in AcOH (92 mL) and concd H2SO4
(43 mL) was added sodium nitrite (7.70 g, 111 mmol) in water
(50 mL) dropwise under vigorous stirring at 0–5 ꢀC. After 10 min
aqueous urea was added to the reaction mixture to remove excess
sodium nitrite. Then sodium azide (7.70 g, 118 mmol) in water
(50 mL) was added to the reaction mixture at 0–5 ꢀC for 3 h. The
reaction mixture was poured into ice water and the resulting
mixture was basified with sodium hydroxide and extracted with
ethyl acetate. The organic layer was washed with water, dried over
anhydrous sodium sulfate, and evaporated to give (4-azidophenyl)-
O
HO
OH
23
White solid; [
d
a
]
ꢁ68.3 (c 0.57, CH3OH); 1H NMR (300 MHz, D2O)
D
1.79 (t, J¼2.4 Hz, 3H), 3.45–3.49 (m, 3H), 3.68–3.79 (m, 3H), 4.32–
4.35 (m, 2H), 4.37 (d, J¼7.5 Hz,1H); 13C NMR (75 MHz, CD3OD)
d 3.6,
57.9, 62.3, 70.2, 74.7, 75.5, 76.7, 85.0, 102.6; HRMS (ESI): calcd for
C10H16O6Naþ (MþNaþ) 255.0839, found 255.0839.
4.3.2.8. Hex-2-ynyl b-galactoside (3h).
OH
methanol (1l). 1H NMR (300 MHz, CDCl3)
7.01 (d, J¼8.1 Hz, 2H), 7.33 (d, J¼8.1 Hz, 2H).
d
1.99 (s, 1H), 4.65 (s, 2H),
OH
O
O
HO
OH
C3H7
4.3.2.13. 4-Azidobenzyl b-galactoside (3l).
23
OH
White solid; [
CD3OD)
a
]
D
ꢁ43.5 (c 0.63, CH3OH); 1H NMR (300 MHz,
OH
O
d
0.98 (t, J¼7.2 Hz, 3H), 1.51 (h, J¼7.2 Hz, 2H), 2.18 (tt,
O
HO
J¼9.0, 2.1 Hz, 2H), 3.43–3.54 (m, 3H), 3.67–3.78 (m, 2H), 3.81 (d,
OH
J¼7.2 Hz, 1H), 4.38 (dd, J¼4.8, 2.7 Hz, 2H), 4.40 (d, J¼7.2 Hz, 1H);
N3
13C NMR (75 MHz, CD3OD)
d 14.0, 21.7, 23.4, 57.4, 62.8, 70.6, 72.6,
23
75.3, 76.9, 77.1, 88.1, 102.9; HRMS (ESI): calcd for C12H20O6Naþ
Yellow solid; [
CD3OD)
a]
ꢁ30.0 (c 1.30, CH3OH); 1H NMR (300 MHz,
D
(MþNaþ) 283.1152, found 283.1143.
d
3.43–3.58 (m, 3H), 3.74–3.83 (m, 3H), 4.29 (d, J¼7.5 Hz,

756
W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
1H), 4.76 (dd, J¼77.1, 12.0 Hz, 1H), 7.02 (d, J¼8.4 Hz, 2H), 7.44 (d,
(d, J¼8.1 Hz, 1H), 4.78 (d, J¼12.0 Hz, 1H), 4.96 (d, J¼12.0 Hz, 1H),
8.12 (s, 1H); 13C NMR (75 MHz, CD3OD)
29.6, 35.5, 42.5, 59.7,
J¼8.4 Hz, 2H); 13C NMR (75 MHz, CD3OD)
d
62.2, 70.1, 71.1, 72.3,
d
74.6, 76.5, 103.6, 119.8, 131.0, 135.7, 140.7; HRMS (ESI): calcd for
61.2, 61.8, 68.9, 71.0, 73.4, 75.4, 102.9, 120.6; HRMS (ESI): calcd for
C13H17N3O6Naþ (MþNaþ) 334.1010, found 334.1006.
C19H29N3O6Naþ (MþNaþ) 418.1949, found 418.1945.
4.4. Procedure for copper(I) catalyzed azide-alkyne
cycloaddition
4.4.5. Compound 15.
OH
N
N
N
O
HO
HO
Glycoside (0.5 mmol), substrate (0.6 mmol), copper (II) acetate
(9 mg, 0.05 mmol), and sodium ascorbate (20 mg, 0.1 mmol) or
copper powder (190 mg) were mixed in tert-butyl alcohol/water
(1:1; 4.0 mL) and stirred at rt overnight. The solution was directly
subjected to flash column chromatography (eluent EtOAc/MeOH/
H2O¼12–8/1/0.2).
O
OH
25
Clear syrup; [
a
]
ꢁ33.2 (c 1.10, CH3OH); 1H NMR (300 MHz,
D
CD3OD)
d
3.20 (t, 1H), 3.28–3.35 (m, 3H), 3.65 (dd, J¼12.0, 5.1 Hz,
1H), 3.86 (dd, J¼12.0, 1.2 Hz, 1H), 4.02–4.08 (m, 1H), 4.26–4.32
(m, 1H), 4.33 (d, J¼7.5 Hz, 1H), 4.69 (t, J¼5.1 Hz, 2H), 7.30–7.35
(m, 1H), 7.29–7.44 (m, 2H), 7.80–7.83 (m, 2H), 8.45 (s, 1H); 13C
4.4.1. Compound 11.
NMR (75 MHz, CD3OD)
d 50.6, 61.5, 67.9, 70.3, 73.8, 76.8, 76.9,
OH
103.4, 122.3, 125.5, 128.2, 128.8, 130.6, 147.5; HRMS (ESI): calcd
O
N
N
for C16H21N3O6Naþ (MþNaþ) 374.1323, found 374.1318.
HO
HO
O
N
OH
4.4.6. Compound 16.
25
Clear syrup; [
CD3OD)
a
]
D
ꢁ29.4 (c 2.30, CH3OH); 1H NMR (300 MHz,
OH
OH
d
3.15 (t, J¼7.8 Hz, 1H), 3.23–3.28 (m, 4H), 3.62 (dd,
O
O
O
N
J¼11.7, 4.8 Hz, 1H), 3.83 (d, J¼11.7, 1H), 4.32 (d, J¼8.1 Hz, 1H), 4.72
(d, J¼12.3 Hz, 1H), 4.92 (d, J¼12.3 Hz, 1H), 7.26–7.37 (m, 5H), 7.97
HO
O
N
O
N
OH
(s, 1H); 13C NMR (75 MHz, CD3OD)
d 54.9, 62.7, 63.0, 71.6, 75.0,
77.8, 78.0, 103.6, 125.3, 129.1, 129.6, 130.0, 136.7, 146.1; HRMS
25
Clear syrup; [
CD3OD)
a]
ꢁ26.3 (c 1.20, CH3OH); 1H NMR (300 MHz,
(ESI): calcd for C16H21N3O6Naþ (MþNaþ) 374.1323, found 374.1325.
D
d
3.47–3.59 (m, 3H), 3.70–3.82 (m, 2H), 3.85 (d, J¼3.0 Hz,
1H), 4.03–4.11 (m, 1H), 4.26–4.33 (m, 2H), 4.74 (t, J¼5.4 Hz, 2H),
5.44 (s, 2H), 6.07–6.08 (m, 1H), 7.35–7.38 (m, 2H), 7.61–7.67 (m,
1H), 7.83–7,88 (m, 1H), 8.42 (s, 1H); 13C NMR (75 MHz, DMSO)
4.4.2. Compound 12.
OH
nC12H25
OH
O
N
N
d
55.1, 65.6, 68.0, 72.4, 73.2, 73.2, 75.5, 78.3, 80.5, 96.4, 108.7,
O
HO
25
N
120.2, 121.5, 128.1, 129.4, 131.2, 137.9, 145.9, 157.9, 166.8, 169.6;
HRMS (ESI): calcd for C20H23N3O9Naþ (MþNaþ) 472.1327, found
472.1331.
OH
White solid; [
CD3OD)
a
]
D
ꢁ24.6 (c 1.20, CH3OH); 1H NMR (300 MHz,
d
0.85 (t, J¼6.6 Hz, 3H), 1.23–1.27 (m, 18H), 1.84 (p,
J¼6.6 Hz, 3H), 3.26 (t, J¼6.6 Hz, 2H), 3.41 (dd, J¼9.6, 3.0 Hz, 1H),
3.47–3.53 (m, 2H), 3.65–3.75 (m, 2H), 3.78 (d, J¼3.0 Hz, 1H), 4.28
(d, J¼7.5 Hz, 1H), 4.34 (t, J¼7.2 Hz, 2H), 4.82 (dd, J¼27.6, 12.6 Hz,
4.4.7. Compound 17.
OH
O
HO
HO
2H), 7.96 (s, 1H); 13C NMR (75 MHz, CD3OD)
d 14.7, 24.0, 27.7,
O
O
O
N
N
O
OH
30.4, 30.7, 30.8, 30.9, 31.0, 31.5, 33.3, 51.6, 62.8, 63.3, 70.6, 72.7,
75.1, 77.1, 104.5, 125.5, 146.0; HRMS (ESI): calcd for C21H39N3O6Naþ
(MþNaþ) 452.2731, found 452.2728.
N
Clear syrup; [
CD3OD)
a]
D
ꢁ30.8 (c 1.25, CH3OH); 1H NMR (300 MHz,
25
4.4.3. Compound 13.
d
2.24 (p, J¼6.3 Hz, 2H), 3.24–3.34 (m, 3H), 3.43 (t,
J¼9.0 Hz, 1H), 3.54–3.62 (m, 1H), 3.66–3.74 (m, 1H), 3.88–3.97 (m,
2H), 4.31 (d, J¼8.1 Hz, 1H), 4.65 (t, J¼6.6 Hz, 2H), 5.42 (s, 2H), 6.02
(s, 1H), 7.25–7.30 (m, 2H), 7.55–7.61 (m, 1H), 7.75 (dd, J¼7.8
OH
O
N
N
HO
HO
O
N
OH
1.5 Hz, 1H), 8.36 (s, 1H); 13C NMR (75 MHz, CD3OD)
d 31.4, 62.7,
25
Clear syrup; [
a
]
ꢁ35.6 (c 1.05, CH3OH); 1H NMR (300 MHz,
63.7, 66.8, 71.5, 75.0, 77.90, 77.95, 91.8, 104.3, 116.6, 117.5, 124.2,
125.5, 126.9, 133.9, 142.5, 154.4, 164.8, 166.8; HRMS (ESI): calcd
for C21H25N3O9Naþ (MþNaþ) 486.1483, found 486.1479.
D
CD3OD)
d
1.83 (s, 6H), 2.25 (s, 9H), 3.19–3.25 (m, 1H), 3.28–3.38
(m, 3H), 3.67 (dd, J¼12.0, 5.4 Hz, 1H), 3.90 (d, J¼11.4 Hz, 1H), 4.38
(d, J¼7.8 Hz, 1H), 4.77 (d, J¼12.3 Hz, 1H), 4.96 (d, J¼12.3 Hz, 1H),
8.12 (s, 1H); 13C NMR (75 MHz, CD3OD)
d 30.9, 36.9, 43.9, 61.1,
4.4.8. Compound 18.
62.8, 63.2, 71.6, 75.0, 77.9, 78.0, 103.6, 122.0, 144.9; HRMS (ESI):
OH
OH
calcd for C19H29N3O6Naþ (MþNaþ) 418.1949, found 418.1947.
O
O
COOMe
HO
4.4.4. Compound 14.
OH
N
N
COOMe
OH
OH
N
O
N
N
O
25
ꢁ41.3 (c 1.10, CH3OH); 1H NMR (300 MHz,
HO
N
White solid; [
CD3OD)
a
]
D
OH
d
3.46–3.65 (m, 3H), 3.75–3.81 (m, 2H), 3.85 (d, J¼3.3 Hz,
25
D
White solid; [
a
]
ꢁ18.6 (c 0.95, CH3OH); 1H NMR (300 MHz,
1H), 3.87 (s, 3H), 3.95 (s, 3H), 4.36 (d, J¼8.1 Hz, 1H), 4.79 (d,
CD3OD)
d
1.84 (s, 6H), 2.25 (s, 9H), 3.47 (dd, J¼9.6, 3.3 Hz, 1H),
J¼12.6 Hz, 1H), 5.04 (d, J¼12.6 Hz, 1H), 7.54 (d, J¼8.7 Hz, 2H), 7.67
3.52–3.58 (m, 2H), 3.73–3.80 (m, 2H), 3.83 (d, J¼3.0 Hz, 1H), 4.33
(d, J¼8.7 Hz, 2H); 13C NMR (75 MHz, CD3OD)
d 53.2, 54.4, 62.5,

W.-Y. Lu et al. / Tetrahedron 66 (2010) 750–757
757
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4.5. Procedure for sequential one-pot-like reaction
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wasconductedas generalprocedure. Totheresultant syrupwas then
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copper (II) acetate (9 mg, 0.05 mmol) and copper powder (190 mg,
3 mmol), and tert-butyl alcohol/water (1:1; 4.0 mL) and stirred at rt
for 4–24 h. The solution was filtered and concentrated under vac-
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(eluent EtOAc/MeOH/H2O¼12–8/1/0.2) to give the product.
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was conducted as general procedure, to the reaction mixture was
added 10 mL ethanol, and then filtered through Celite and concen-
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4.6. Antifungal susceptibility test
The antifungal activity was measured based on the recom-
mendations of NCCLS.17 The compounds were dissolved in water
and diluted in a twofold manner in RPMI 1640 (pH 7.0) in 96-
microwell plates. The MIC was the minimum concentration of the
agent that shows a full inhibition of the fungal growth in the well,
examined by naked eyes.
Acknowledgements
We would like to thank Sha-Sha Zou, Rong-Fei Cheng, and Shu-
Hua Zhang at Sichuan Industrial Institute of Antibiotics Co. Ltd.
(SIIA Co. Ltd.) for performing the antifungal susceptibility test. This
research was financial support by National Natural Science Foun-
dation of China (20672126 & 20672037) and the Major State Basic
Research Development Program (2006CB806106) and the Chinese
Academy of Sciences (KSCX2-YW-R-23).
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Supplementary data
Supplementary data associated with this article can be found in
the online version at doi:10.1016/j.tet.2009.11.044.
References and notes
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