An inexpensive fluorescent labeling protocol for bioactive natural products utilizing Cu(I)-catalyzed Huisgen reaction

Article abstract of DOI:10.1016/j.tet.2007.04.069

Labeling of bioactive small molecules with organic dyes for various applications in cell biology has been emerging as an attractive research field. Using an easily prepared and inexpensive fluorescein derivative 1 and a Cu(I)-catalyzed Huisgen reaction, a

Full text of DOI:10.1016/j.tet.2007.04.069

Tetrahedron 63 (2007) 6813–6821
An inexpensive fluorescent labeling protocol for bioactive natural
products utilizing Cu(I)-catalyzed Huisgen reaction
Yan-Hong Zhang,a,b Zheng-Xi Gao,a Chun-Long Zhong,a,c Hai-Bin Zhou,a Lei Chen,a
Wen-Min Wu,a Xin-Jun Pengb and Zhu-Jun Yaoa,c,
*
aState Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry,
Chinese Academy of Sciences, 354 Fenglin Road, Shanghai 200032, China
bDepartment of Analysis Chemistry, Hunan Traditional Chinese Medicine University, 113 Middle Shaoshan Road,
Changsha, Hunan 410007, China
cDepartment of Chemistry, University of Science and Technology of China, Hefei, Anhui 230026, China
Received 1 March 2007; revised 20 April 2007; accepted 23 April 2007
Available online 25 April 2007
Abstract—Labeling of bioactive small molecules with organic dyes for various applications in cell biology has been emerging as an attractive
research field. Using an easily prepared and inexpensive fluorescein derivative 1 and a Cu(I)-catalyzed Huisgen reaction, an efficient fluores-
cent labeling strategy is developed generally for bioactive natural products. Essentials of a successful labeling include the personalized
introduction of an azido functionality to specific targets by a selective and efficient manner, and the strategic adjustment of reaction sequence
to avoid possible side reactions under the ‘click’ reaction conditions. Such a protocol has been successfully applied to the fluorescent labeling
of four bioactive small molecules in different chemical categories in this study. Advantages of this labeling protocol include the use of
inexpensive reagents, ease of operation, free-of-protections at the ‘click’ step, and suiting a wide range of bioactive molecules bearing the
reactive functionalities.
Ó 2007 Elsevier Ltd. All rights reserved.
1. Introduction
between an azide and a terminal alkyne (to afford the
1,2,3-triazole regioselectively, Fig. 1). Cu(I)-based catalysts
dramatically improve the regioselectivity and reaction rate
of Huisgen 1,3-dipolar cycloaddition and thus eliminate
the need for elevated temperatures. This high-yielding reac-
tion also tolerates a variety of functional groups and affords
the 1,2,3-triazole product with minimal work-up and purifi-
cation. These significances make Huisgen reaction to be an
ideal research tool in many applications, including the fluo-
rescent labeling of biomolecules.3a,6,7 Another focus in-
volved in the fluorescent labeling is fluorescent reagents.
Though many choices are commercially available today
and ready to use in most experiments, most fluorescent
reagents are relatively expensive and required for feasible
examinations in individual cases. Up to date, fluorescent
Fluorescence allows qualitative and quantitative determina-
tions of cellular events at molecular level.1 Such visualiza-
tions are usually performed easily and reliably by rapid
and economic methods with improved sensitivity and selec-
tivity. In various chemical biology studies, proper labeling of
the bioactive compounds (including natural products and
biochemical probes) with organic dyes has been recognized
as a very useful technique in recent years.2,3 Therefore, de-
velopment of convenient labeling protocols and acquirement
of suitably labeled bioactive molecules are of great impor-
tance and are becoming an attractive area in organic chem-
istry. In recent years, Sharpless and co-workers have
identified a number of reactions called ‘click chemistry’.4
The foundation of ‘click chemistry’ is termed by generation
of chemical substances by joining small units together
through a convenient heterocycle-formation reaction in
high chemical yield and selectivity in a wide range of sub-
strates. Among these, the most powerful example is to utilize
the Cu(I)-catalyzed Huisgen 1,3-dipolar cycloaddition4,5
Ο
Ο
OH
N
R1
R1
N
N
N
cat. CuSO4
N
N
Na ascorbate
Ο
R2
R2
Ο
1
* Corresponding author. Tel.: +86 21 54925133; fax: +86 21 64166128;
e-mail: yaoz@mail.sioc.ac.cn
Figure 1. Cu(I)-catalyzed Huisgen reaction and the inexpensive fluorescein
derivative 1.
0040–4020/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tet.2007.04.069

6814
Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
labeling of the bioactive small molecules is still a high-cost
and time-consuming work, especially for those bioactive
compounds naturally bearing multiple reactive functional-
ities. Herein, we report our explorations and results in devel-
oping a versatile and economic ‘click’ fluorescent labeling
protocol, which is suitable for a wide range of bioactive
compounds and natural products in different chemical cate-
gories. An easily prepared and inexpensive fluorescein
derivative 1 bearing a terminal alkyne functionality8,9 is suc-
cessfully used as the fluorescent reagent.
interactions between naringenin and nod-D protein. The
above-mentioned ‘click-labeling’ concept was successfully
employed in our recent study on naringenin, in which a quick
synthesis of a fluorescent labeled naringenin derivative 3
was accomplished in a site-selective and protection-free
fashion.8 In naringenin, three hydroxyl groups and one
ketone carbonyl group exist in the different rings of one mol-
ecule, bringing great complexity to the site-selective azido-
group introduction by a direct manner (without protection
manipulations). However, fine structural analysis indicates
that the A ring of naringenin, especially C-6 and C-8 posi-
tions, might be more reactive toward the electrophiles than
the C ring. Relatively mild Mannich reaction11 was thus con-
sidered as a direct derivation method for naringenin. After
careful optimizations of reaction conditions, a direct and
regioselective Mannich reaction of naringenin with a variety
of secondary amines and formaldehyde was successfully
developed.8
2. Results and discussions
For genetic reasons, all original natural products have no
azido group, which is essentially required for the ‘click’
reaction. Therefore, selective and efficient introduction of
an azido group to a proper position of the bioactive targets
becomes a critical work for this ‘click’ labeling strategy.
Usually, it is a difficult and time-consuming work because
most bioactive targets contain a variety of reactive functional
groups. Also, they present similar reactivity in many cases.
Such contradictory prevents people from convenient ac-
quirement of corresponding azido derivatives starting from
their parents. However, existence of complex but slightly
different functionalities in the bioactive targets also provides
us the chance to find a selective azido-group introduction di-
rectly or indirectly. Undoubtedly, such operations (to intro-
duce an azido group selectively into a specific target) have
to be done in a ‘personalized’ fashion because of the differ-
ent chemical environment of individual targets.
From the above successful two-step labeling of naringenin,
several conclusions were drawn in general. First, convenient
preparation of an ideal derivative bearing an azido group is
the most important issue for this ‘click’ labeling protocol
with fluorescein derivative 1. Unambiguously, direct and
selective introduction of an azido functionality to a specific
target (a natural product or a bioactive small molecule) can
be achieved, of course, using the ‘personalized’ innovative
chemistry. Second, the ‘click’ labeling in a ‘free-of-protec-
tion’ fashion proves to be a versatile and efficient method,
especially for those targets bearing multiple hydrophilic and
reactive functionalities. Encouraged by the above results,
further applications of this ‘click’ labeling protocol was
examined to the representative bioactive natural products,
including muramyl dipeptide (MDP, 6, a bioactive unit of
peptidoglycan),12 sinomenine (7, an immunosuppressive al-
kaloid from traditional Chinese medicine),13 and artemisinin
(qinghaosu, 8, an anti-malarial terpenoid from traditional
Chinese medicine)14 (Fig. 2). These three bioactive natural
The first natural product we examined is naringenin (2 in
Scheme 1), a representative flavonone involved in plant root
nodulation and nitrogen fixation.10 Of course, a fluorescent
probe based on naringenin will be much more helpful
to those biochemical visualization studies, such as the
Step 1
regioselective
Mannich reaction
Personalized
selective bond formation
Fluorescein
OH
10
O
HO
formaldehyde,
5
4
Bioactive small
flexible
6
3
N3
amine 5 ZnCl2 (0.1 eq),
2' EtOH 65 °C, 3h, 87%
O
O
OH
N
molecules
spacer
A
B
3'
C
2'
2
HO7
O
9
4'
3'
8
1
1'
= devised functional groups
Protection-free
"click" reaction
N3
OH
OH
naringenin (2)
4
5
HN
.
CuSO4 5H2O (cat.)
F
ascorbic acid, 1
BuOH-H2O, 67%
t
Step 2
N3
Bioactive small
molecules
Flu-labeled
Cu(I)-catalyzed
click reaction
compounds
OH
O
MeO
HO
HO
N
O
H
O
HO
OH
N
N
N
O
HO
O
O
NHAc
OH
O
NH
O
O
H
OH
O
O
OH
NMe
O
O
O
HN
O
O
O
OMe
H2N
MDP (6)
O
sinomenine (7)
artemisinin (8)
3
Scheme 1. ‘Click’ fluorescent labeling of naringenin (2) using a direct and
regioselective Mannich reaction.
Figure 2. General concepts for ‘click’ fluorescent labeling, and three
selected bioactive targets 6–8 for further studies.

Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
6815
Step 1
C-1 glycosidation
HO
HO
O
HO
OH
OH
NHAc
O
O
O
O
OH
NH
O
O
N3
HN
O
O
H2N
O
Step 2
Cu(I)-catalyzed click reaction
1
6
Figure 3. A personalized protocol for ‘click’ fluorescent labeling of MDP (6).
products belong to different chemical categories, presenting
different chemical and biological properties. Of course, they
represent the substrate generality to some extent.
cells in the study of immuno-adjuvant cellular interactions in
experimental model system. According to Chirva’s previous
work,15 the alkyl b-glycosides of muramyl dipeptide (MDP)
having different hydrocarbon-chain lengths exerted no
substantial effect on the corresponding immunostimulating
activity. Thus, its labeling attachment point to an azido func-
tionality was decided to fix at C-1 position via glycosylation
of muramyl dipeptide (Fig. 3).
Muramyl dipeptide (MDP, 6), the minimal bioactive unit
of peptidoglycan of Gram-positive bacterial cell walls, is
known to have pleiotropic stimulatory effects on host
immune systems and to elicit defensive responses. MDP is
also known to have many effects on both the humoral- and
cell-mediated immune systems, and has been implicated in
the stimulation of macrophage.14 A fluorescent-labeled MDP
would be useful to visualize the binding of MDP to various
The synthesis of MDP derivative 15 bearing an internal
azido-spacer is shown in Scheme 2. Both the dipeptide
amine 10 and sugar precursor 11 were conveniently prepared
t-BuO
t-BuO
COOH
H2N
O
O
CbzHN
O
O
H2/Pd-C
MeOH, 100%
HN
HN
CbzHN
O
O
O
H2N
10
H2N
H2N
N-Cbz-L-Glu-NH2
9
N3
)
FeCl3
HO(CH2)10N3 (neat)
pTsOH, 4A MS
rt, 60%
dry acetone
reflux, 20min
71%
OH
H
O
(
8
H
O
HO
O
O
O
OH
O
OH
O
O
HO
H
O
NHAc
HO
H
NHAc
N
12
11
N3
N3
10
O
O
O
NaH, THF
O
(
O
)
O
O
10
(
)
O
(S)-ClCH(CH3)COOH
O
O
NHAc
t
80%
NHAc
BuO
EDCI, HOBt
DIPEA, 10
DCM, 80%
O
NH
O
OH
O
O
HN
13
14
O
H2N
N3
HO
O
O
HO
(
)
O
10
1, DIPEA, CuI
BuOH-H2O (1:1)
25% TFA
in DCM, rt
t
NHAc
HO
O
NH
O
40% after HPLC
O
HN
O
15
H2N
N
N
O
OH
O
N
HO
O
O
HO
O
O
NHAc
OH
O
NH
O
O
O
MDP-flu (16)
HN
O
H2N
Scheme 2. Synthesis of MDP-flu 16 by click chemistry.

6816
Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
Step 2
for keeping its bioactivities. Considering this, a personalized
‘click’ labeling plan for sinomenine was designed (Fig. 4).
By this strategy, the C–C bond formation was fixed at the
para-position of its phenol ring through a selective halogen
introduction. Such a strategy might lead to the accomplish-
ment of a very short and highly efficient route.
Step 1
C-C bond formation
Cu(I)-catalyzed click reaction
2FG
N3
(
)
n
MeO
HO
FG1
18
O
O
OH
NMe
O
Treatment of natural sinomenine (7) with N-iodosuccin-
imide at room temperature afforded the iodide 17 in 85%
yield, regioselectively (Scheme 3). Heck reaction of aryl
iodide 17 with pre-prepared acrylamide 18 was smoothly
performed under Pd(OAc)2-catalyzed conditions to give
the azido-containing derivative 19 in 81% yield. Final ‘click’
labeling was successfully carried out again in a mixture of
tert-butanol and water (1:1), affording sinomenine-flu (20)
in 65% yield after purification by a silica gel column chro-
matography. Using this personalized protocol, the fluores-
cent labeling of sinomenine was efficiently achieved in
three steps.
O
OMe
O
sinomenine (7)
1
Figure 4. A personalized ‘click’ fluorescent-labeling protocol for sino-
menine (8).
in good yield.16 Treatment of the protected sugar derivative
11 with 10-azidodecanol8 afforded exclusively unprotected
b-glucopyranosides 12 in the presence of p-TsOH and 4A
MS. O-Alkylation of free alcohol 12 with (S)-2-chloropro-
pionic acid gave the carboxylic acid 13. Coupling of 13
with dipeptide 10 using EDCI/HOBt gave the protected
MDP derivative 14. Final global deprotection of 14 was car-
ried out in a DCM solution containing 25% of TFA at room
temperature. This highly polar and water-soluble crude
product 15 was successfully used for the last ‘click’ reaction
without any further purification. The 1,3-dipolar Huisgen
cycloaddition between MDP derivative 15 and fluorescein
derivative 1 was finally accomplished in tBuOH/H2O (v/v¼
1:1) in the presence of catalytic amount of CuI and excess of
DIPEA (partially to neutralize the remaining TFA in crude
15). The reaction smoothly proceeded at room temperature,
and the crude product was isolated in high yield by simple
filtration of solid CuI. A pure sample of the labeled MDP de-
rivative 16 was collected in 40% yield after a reverse-phase
HPLC purification.
Artemisinin (Qinghaosu, 8) is a unique sesquiterpene 1,2,4-
trioxane isolated from the Chinese medicinal herb Qinghao
(Artemisia annua L.).14 Artemisinin and many of its deriva-
tives are highly effective against multidrug-resistant malaria
caused by Plasmodium falciparum. The detailed mecha-
nisms of these drugs remain unclear yet. However, many
studies have shown that the activity of artemisinin is a conse-
quence of the cleavage of the endoperoxide group by intra-
parasitic heme of associated heme alkylation, and of
inhibition of hemin polymerization to hemozoin.19 In order
to demonstrate the mechanism of artemisinin compounds
through real-time imaging study, acquirement of suitable
artemisinin derivatives containing a fluorescent group is of
great value. However, this class of peroxides are rather reac-
tive when they are exposed to the Cu(I) species, leading to
cleavage of their endoperoxide bridge and loss of bioactivity.
Therefore, a strategic consideration has to be required in
order to successfully employ this ‘click’ labeling protocol to
the artemisinin derivatives. After various comparisons, a
protocol by simply reversing the order of labeling sequence
was finally adopted. According to this protocol, coupling of
Sinomenine (7) is a naturally occurring alkaloid from
Chinese medicinal plant Sinomenium acutum, exhibiting
immunomodulating and anti-inflammatory activities with
unambiguous clinical effects.17 Guided by our previous re-
sults on modification of sinomenine,18 maintenance of orig-
inally existing functional groups would be more favorable
MeO
HO
I
MeO
HO
H
N
( )5
18
N3
NIS
DCM, rt
85%
O
N
N
Pd(OAc)2 (5 mol%)
PPh3 (10 mol%),
DMF-Et3N (1:1), 80 o
81%
O
O
C
OMe
17
OMe
sinomenine (7)
O
N3
O
MeO
( )7
N
H
MeO
HO
( )7
N
H
N
HO
O
N
1, CuI, DIPEA
N
t-BuOH-H2O (1:1)
N
N
HO
OMe
O
O
64%
O
OMe
O
19
20
O
Scheme 3. Click chemistry-based short synthesis of sinomenine-flu (20).

Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
6817
Step 1
catalyst (added in three portions) afforded the labeled com-
pound 25 (E-isomer only) in 33% yield after purification by
a preparative TLC.
Cu(I)-catalyzed click reaction
H
O
O
N3
O
O
O
OH
H
(
)
6
O
3. Conclusion
O
O
In summary, a flexible and efficient ‘click’ fluorescent label-
ing strategy is described. Personalized introduction of an
azido functionality to specific targets by selective reactions
and strategic order adjustment of labeling sequence are two
essentials for a successful labeling. Applications to naringe-
nin, MDP, sinomenine, and artemisinin were successfully
achieved in short routes. Advantages of this labeling proto-
col include ease of operation, use of an inexpensive fluores-
cein reagent, and a catalyzed Huisgen cycloaddition in a
free-of-protection fashion, as well as high efficiency
achieved. It thus provides a practical and useful tool for
developing bioactive small molecule-based probes and the
corresponding studies on biological mechanisms. Further
applications of these labeled compounds to related biologi-
cal studies are underway in this laboratory and will be
reported in due course.
21
22
1
Step 2
cross metathesis
Figure 5. A reverse sequence for ‘click’ labeling of artesiminin derivatives
using cross metathesis and Huisgen reaction.
fluorescein derivative 1 with an azido-containing spacer was
executed at first by a click reaction in the presence of Cu(I)
catalyst, so that the final attachment to the peroxide target
could be done under Cu(I)-free conditions. Such a strategic
modification can avoid the exposure of artemisinin endoper-
oxides to the Cu(I) species in the same reaction media, and
does not require additional steps. Under these consider-
ations, an easily prepared and known artemisinin derivative,
10b-allyldeoxoartemisinin 21 was chosen as the labeling
precursor, in which its terminal olefin functionality was de-
vised as the final C–C bond formation position by a Ru-cat-
alyzed cross metathesis.20 In addition, a hydrocarbon spacer
22 bearing a terminal olefin and azido group was designed to
join both the artemisinin derivative 21 and fluorescein deriv-
ative 1 in this personalized protocol (Fig. 5).
4. Experimental
4.1. General
Optical rotations were measured at room temperature. Infra-
red spectra were recorded on a Perkin–Elmer 983 FTIR
spectrophotometer, with all samples examined as films on
a KBr disk. NMR spectra were recorded at Bruker Avance
instruments (300 MHz or 500 MHz for 1H NMR, and
75 MHz or 125 MHz for 13C NMR) and are reported in parts
per million (d). HRMS spectra were recorded on Kratos
Concept, Q-Tof Micro or APEXIII 7.0 TESLA FTMS. Ele-
mental analyses were preformed on VARIO EL Elemental
Starting from the commercially available 10-undecen-1-ol,
a linear spacer 22 was prepared (Scheme 4). ‘Click’ linkage
of fluorescein derivative 1 was efficiently accomplished with
azido-containing spacer 22 under the Cu(I)-catalyzed condi-
tions. Final cross metathesis of newly produced fluorescein
derivative 24 with 5 equiv of artemisinin derivative 21 in
the presence of 20 mol % of the second generation Grubbs
MsCl, Et3N
CH2Cl2, 98%
R
23 R = OMs
NaN3, DMF
70oC, 92%
OH
22 R = N3
( )7
N
N
22, CuSO4·5H2O (cat.)
N
HO
O
O
ascorbic acid
t
BuOH, H2O, 80%
HO
O
O
O
O
O
24
O
1
Cl2(Cy3P)(sIMes)Ru=CHPh (20 mol%)
21, CH2Cl2, 45 °C, 33%
O
O
H
N
N
N
O
O
O
O
O
OH
(
)
6
O
H
25
Scheme 4. A two-step synthesis of artemisinin-flu (25) using cross metathesis and click reaction.

6818
Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
˚
4 mmol) were added molecular sieves (4 A, 300 mg) and an-
Apparatus. All melting points were uncorrected. Flash
column chromatography was performed on silica gel
(10w40 mm) using a mixture of petroleum ether and ethyl
acetate as the eluant.
hydrous p-TsOH (172 mg, 1 mmol). The mixture was stirred
at room temperature overnight. The reaction was quenched
by adding K2CO3 slowly until the pH reached 7, when
TLC indicated that reactant disappeared. After removal of
the solid by filtration, the filtrate was concentrated under re-
duced pressure. The residue was washed several times with
CH2Cl2 to afford a brown syrup. This syrup was then sus-
pended in a mixture of acetone (8 mL) and 2,2-dimethoxy-
propane (5 mL). p-Toluenesulfonic acid (17 mg, 0.1 mmol)
was added, and the mixture was stirred for 3 h at room tem-
perature. The solvent was removed under reduced pressure,
and the residue was then taken up into dichloromethane.
This solution was washed with dilute sodium bicarbonate,
water, and brine successively, dried over magnesium sulfate,
filtered, and concentrated. The resulting light brown oil was
purified by chromatography on silica gel (2% methanol/
DCM) to yield 12 as a yellow syrup (265 mg, 60%); [a]D20
ꢂ39.8 (c 1.83, CHCl3). 1H NMR (CDCl3, 300 MHz):
d 5.77 (br s, 1H), 4.61 (d, 1H, J¼8.1 Hz), 4.31 (br s, 1H),
3.95–3.80 (m, 5H), 3.59–3.46 (m, 3H), 3.27 (t, 3H,
J¼6.6 Hz), 2.05 (s, 3H), 1.59–1.25 (m, 25H). 13C NMR
(CDCl3, 75 MHz): d 172.1, 101.2, 99.8, 74.3, 71.6, 70.0,
67.1, 62.0, 57.9, 51.4, 32.7, 29.5, 29.5, 29.4, 29.4, 29.3,
29.1, 29.0, 28.8, 26.7, 25.9, 23.3, 19.1. HRMS (ESI, m/z)
calcd for [C21H38N4O6+Na] (M+Na)+: 465.2683, found:
465.2678.
4.1.1. Compound 4. To a solution of naringenin (272 mg,
1 mmol), paraformaldehyde (60 mg, 2 mmol), and ZnCl2
(14 mg, 0.1 mmol) in ethanol (4 mL) was added the second-
ary amine 5 (234 mg, 1.5 mmol) at room temperature. The
mixture was then heated to 65 ꢀC, and the reaction process
was judged by TLC monitoring. After the reactants were
consumed, EtOAc (30 mL) and acidic water (30 mL, pH
3) were added to the mixture. The pH of aqueous phase
was then adjusted to 7. The aqueous phase was extracted
with EtOAc (15 mLꢁ3). The combined extracts were dried
over anhydrous Na2SO4. The crude product was purified by
silica gel chromatography (CH2Cl2/MeOH¼50:1) to afford
pure 4 (383 mg, 87%). IR (KBr): nmax 3025, 2938, 2862,
2096, 1646, 1614, 1540, 1519, 1459, 1367, 1184, 1168,
1
1082, 835 cmꢂ1. H NMR (acetone-d6, 300 MHz): d 7.34
(2H, d, J¼8.4 Hz), 6.88 (2H, d, J¼8.4 Hz), 5.78 (1H, s),
5.35 (1H, dd, J¼12.6, 3.0 Hz), 3.83 (2H, s), 3.30 (2H, t,
J¼6.9 Hz), 3.09 (1H, dd, J¼17.1, 12.6 Hz), 2.70 (2H, t,
J¼7.4 Hz), 2.66 (1H, dd, J¼17.1, 3.0 Hz), 2.44 (3H, s),
1.69–1.56 (4H, m), 1.39–1.37 (4H, m). 13C NMR (ace-
tone-d6, 75 MHz): d 195.49, 171.13, 162.70, 161.25,
157.94, 129.90, 128.03, 115.32, 100.67, 100.01, 95.85,
78.84, 56.05, 52.65, 51.02, 42.57, 40.05, 28.49, 26.33,
26.23, 25.87. HRMS (ESI, m/z) calcd for C23H29N4O5
(M+H)+: 441.2133, found: 441.2139. Anal. Calcd for
C23H28N4O5: C, 62.71; H, 6.41; N, 12.72. Found: C,
62.49; H, 6.40; N, 12.58.
4.1.4. Compound 13. Sodium hydride (100 mg, 2.52 mmol,
60% dispersion in oil) was placed in a 25-mL round-bottom
flask, washed with hexane three times, and finally suspended
in dry THF (3 mL). A solution of compound 12 (0.35 g,
0.80 mmol) in dry THF (2 mL) was added dropwise over
10 min under nitrogen. To this mixture was added dropwise
over 10 min a solution of (S)-(ꢂ)-2-chloropropionic acid
(77 mg, 0.72 mmol) in dry THF (1 mL). The mixture was
heated at reflux for 18 h, cooled, quenched with ethanol, di-
luted with water, and washed with ether. The aqueous layer
was acidified with 1 M phosphoric acid and extracted with
dichloromethane three times. The organic solution was
again extracted with dilute sodium bicarbonate solution.
The aqueous layer was then re-acidified with 1 M phospho-
ric acid and extracted with ethyl acetate. The ethyl acetate
solution was washed with brine, dried over magnesium sul-
fate, filtered, and concentrated to yield 13 as an amorphous
solid (298 mg, 80%); [a]D20 18.1 (c 0.75, CHCl3). IR (KBr):
nmax 3338, 2993, 2933, 2858, 2098, 1722, 1624, 1561, 1459,
4.1.2. Naringenin-flu (3). To a mixture of compounds 4
(400 mg, 0.91 mmol) and 1 (504 mg, 1.36 mmol) in t-
BuOH/H2O (20 mL, v/v¼1:1) were added CuSO4$5H2O
(2 mg, 0.008 mmol) and ascorbic acid (5 mg, 0.028 mmol).
The mixture was stirred at room temperature for 24 h.
EtOAc (200 mL) was added to dilute the reaction. The or-
ganic phase was washed by saturated brine, and dried over
anhydrous Na2SO4. The crude product was purified by silica
gel chromatography (CH2Cl2/MeOH¼20:1), affording pure
naringenin-flu 3 (493 mg, 67%). IR (KBr): nmax 2937, 1759,
1637, 1616, 1505, 1459, 1364, 1250, 1174, 1110, 1085,
834 cmꢂ1. 1H NMR (DMSO-d6, 300 MHz): d 8.24 (1H, s),
8.00 (1H, d, J¼7.5 Hz), 7.79–7.69 (2H, m), 7.32 (1H, s),
7.28 (2H, d, J¼8.4 Hz), 7.09 (1H, d, J¼2.7 Hz), 6.81–6.74
(4H, m), 6.65 (1H, d, J¼9.3 Hz), 6.59 (2H, d, J¼1.2 Hz),
5.66 (1H, s), 5.35 (1H, dd, J¼12.6, 2.4 Hz), 5.22 (2H, s),
4.36 (2H, t, J¼6.6 Hz), 3.83 (2H, s), 3.15 (1H, dd, J¼17.1,
12.6 Hz), 2.69 (2H, t, J¼7.2 Hz), 2.61 (1H, dd, J¼17.1,
2.4 Hz), 2.40 (3H, s), 1.82 (2H, t, J¼6.6 Hz), 1.56 (2H, br
m), 1.27 (4H, br m). 13C NMR (DMSO-d6, 75 MHz):
d 194.8, 173.4, 169.1, 162.6, 161.7, 160.2, 158.1, 152.8,
152.3, 142.6, 136.1, 130.6, 129.7, 129.5, 129.4, 128.7,
126.6, 125.1, 125.0, 124.5, 115.6, 113.4, 112.9, 111.9,
109.8, 102.7, 102.2, 100.0, 99.8, 96.6, 83.4, 78.5, 62.1,
55.7, 52.1, 49.8, 42.3, 40.4, 29.9, 26.1, 25.9, 25.3. HRMS
(ESI, m/z) calcd for C46H43N4O10 (M+H)+: 811.2974, found:
811.2993.
1
1376, 1110 cmꢂ1. H NMR (MeOH-d4, 500 MHz): d 4.45
(d, 1H, J¼6.7 Hz), 4.39 (d, 1H, J¼8.3 Hz), 3.84 (m, 2H),
3.67–3.40 (m, 5H), 3.30–3.25 (m, 3H), 1.94 (s, 3H), 1.59–
1.51 (m, 5H), 1.38–1.30 (m, 17H). 13C NMR (CDCl3,
125 MHz): d 187.9, 177.9, 174.0, 103.0, 83.5, 78.1, 77.1,
72.5, 70.9, 63.0, 56.5, 52.8, 33.3, 31.0, 30. 9, 30.8, 30.6,
30.2, 28.1, 27.4, 23.5, 19.9. HRMS (ESI, m/z) calcd for
[C24H42N4O8+Na] (M+Na)+: 537.2894, found: 537.2914.
4.1.5. Compound 14. To a solution of 13 (925 mg,
1.8 mmol) and 10 (410 mg, 1.5 mmol) in dry dichloro-
methane (20 mL) were added DIPEA (232 mg, 1.8 mmol),
N-ethyl-N-(3-dimethylaminopropyl)carbodiimide (EDCI)
(350 mg, 1.8 mmol), and N-hydroxybenzotriazole hydrate
(HOBt) (275 mg, 1.8 mmol). The reaction was stirred for
24 h at room temperature. The mixture was diluted with
4.1.3. Compound 12. To a mixture of oxazoline 1116b
(240 mg, 1 mmol) and 10-azidodecan-1-ol (813 mg,

Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
6819
dichloromethane, washed successively with saturated
NaHCO3, water and brine, dried over magnesium sulfate, fil-
tered, and concentrated. The residue was purified by column
chromatography on silica gel (2% methanol in DCM) to
yield 14 as an amorphous solid (922 mg, 80%); [a]2D0 5.59
(c 1.69, CHCl3). IR (KBr): nmax 3367, 2934, 2859, 2098,
145.8, 145.1, 131.9, 124.3, 119.2, 114.5, 87.8, 57.0, 56.0,
54.8, 48.7, 46.9, 45.5, 42.7, 41.1, 35.4, 31.8. HRMS (ESI,
m/z) calcd for C19H23INO4 (M+H)+: 456.0666, found:
456.0661.
4.1.8. Compound 19. To a mixture of iodo-sinomenine de-
rivative 17 (282 mg, 0.62 mmol), N-(10-azidodecyl)acryl-
amide 18 (156 mg, 0.62 mmol), PPh3 (16 mg, 0.062 mmol),
and Pd(OAc)2 (7 mg, 0.031 mmol) were added degassed
DMF (15 mL) and Et3N (3 mL) under nitrogen atmosphere.
The resulting mixture was heated to 80 ꢀC for 2 h, cooled
down to room temperature and filtered over a pad of Celite.
The pad was washed with ethyl acetate (20 mL) and the
filtrate was washed by H2O and brine, dried over Na2SO4, fil-
tered, and concentrated. The residue was purified by flash
chromatography (CH2Cl2/CH3OH¼50:1 to 20:1) to give
19 (290 mg, 81%) as a pale yellow solid; [a]2D0 28.9 (c
0.52, CHCl3); mp: 125–127 ꢀC (decomp.). IR (KBr): nmax
2930, 2854, 2096, 1692, 1654, 1617, 1465, 1422, 1290,
1
1736, 1660, 1561, 1458, 1370, 1267, 1203, 1158 cmꢂ1. H
NMR (CDCl3, 500 MHz): d 7.58–7.45 (m, 2H), 6.93 (s,
1H), 6.64 (d, 1H, J¼7.6 Hz), 6.00 (s, 1H), 4.82 (d, 1H,
J¼8.2 Hz), 4.45–4.40 (m, 1H), 4.32–4.27 (m, 1H), 4.17–
4.14 (m, 1H), 3.97–3.90 (m, 2H), 3.82–3.75 (m, 2H), 3.59
(t, 1H, J¼9.2 Hz), 3.47–3.33 (m, 2H), 3.32–3.28 (m, 1H),
3.25 (t, 2H, J¼6.9 Hz), 2.44–2.34 (m, 2H), 2.17–2.11 (m,
1H), 2.01–1.93 (m, 4H), 1.61–1.27 (m, 37H). 13C NMR
(CDCl3, 75 MHz): d 174.4, 174.2, 172.9, 172.7, 171.3,
100.7, 99.3, 80.9, 79.9, 78.0, 74.6, 69.9, 66.6, 62.1, 57.0,
52.6, 51.4, 49.4, 31.9, 29.5, 29.4, 29.4, 29.3, 29.1, 28.8,
28.1 (3C), 28.0, 27.0, 26.7, 25.8, 23.6, 19.5, 19.0, 18.0.
HRMS (ESI, m/z) calcd for [C36H63N7O11+Na] (M+Na)+:
792.4478, found: 792.4506.
1203, 1148, 1109, 1073 cmꢂ1
.
1H NMR (CDCl3,
300 MHz): d 7.92 (1H, d, J¼15.3 Hz), 6.86 (1H, s), 6.39
(1H, br s), 6.15 (1H, d, J¼14.7 Hz), 5.69 (1H, t,
J¼5.1 Hz), 5.45 (1H, s), 4.33 (1H, d, J¼15.6 Hz), 3.82
(3H, s), 3.47 (3H, s), 3.38 (2H, q, J¼6.6 Hz), 3.28–3.24
(3H, m), 3.12 (1H, d, J¼18.0 Hz), 3.02 (1H, s), 2.68 (1H,
dd, J¼5.1, 18.6 Hz), 2.41 (3H, s), 2.02–1.88 (3H, m),
1.62–1.55 (4H, m), 1.30–1.26 (12H, m). 13C NMR
(CDCl3, 75 MHz): d 194.0, 166.2, 152.3, 146.4, 145.2,
137.8, 130.7, 123.6, 123.2, 119.9, 115.0, 106.5, 56.1, 55.8,
54.7, 51.4, 49.1, 47.0, 45.4, 42.8, 40.6, 39.8, 35.5, 29.7,
29.4 (2C), 29.3, 29.1, 28.8, 26.9, 26.7, 22.0. HRMS (ESI,
m/z) calcd for C32H46N5O5 (M+H)+: 580.3494, found:
580.3489.
4.1.6. MDP-flu (16). Treatment of 14 (40 mg, 0.05 mmol)
with TFA (0.25 mL) in methylene chloride (0.75 mL) at
room temperature for 4 h afforded crude 15 (37 mg,
100%). To a mixture of crude 15 (37 mg, 0.05 mmol) and
fluorescein derivative 1 (22 mg, 0.06 mmol) in t-BuOH/
H2O (1 mL/1 mL) were added CuI (1 mg, 0.005 mmol)
and DIPEA (7 mg, 0.05 mmol). The mixture was stirred at
room temperature for 24 h. The whole mixture was evapo-
rated to dryness under reduced pressure. The residue was
re-dissolved into MeOH and purified by reverse-phase
HPLC (conditions: Vydac C18 column (monomeric 120A,
250ꢁ10 mm); 40% acetonitrile in water to 60% acetonitrile
in water in 20 min, and then to 100% acetonitrile in 15 min;
flow rate 1 mL/min; UV 224 nm; retention time of product
is 16.8 min), affording pure 16 (21 mg, 40%) as a light
4.1.9. Sinomenine-flu (20). To a solution of azido-sinome-
nine derivative 19 (92 mg, 0.16 mmol), acetylene-fluores-
cein derivative 1 (59 mg, 0.16 mmol), and CuI (3 mg,
0.016 mmol) in t-BuOH/H2O (5 mL, v/v¼1:1) was added
DIPEA (83 mL, 0.48 mmol) dropwise under nitrogen atmo-
sphere. The resulting mixture was stirred at room tempera-
ture for 4 h. The mixture was extracted with 5% MeOH/
CH2Cl2 for three times. The combined organic phases
were washed with brine, dried over Na2SO4, filtered, and
concentrated. The residue was purified by flash chromato-
graphy on silica gel (CH2Cl2/CH3OH¼30:1 to 10:1) to af-
ford 20 (96 mg, 64%) as a yellow solid; [a]2D0 22.8 (c 0.52,
CHCl3); mp: 138–140 ꢀC (decomp.). IR (KBr): nmax 2928,
2854, 1764, 1687, 1611, 1465, 1427, 1288, 1249, 1180,
1
yellow syrup. H NMR (MeOH-d4, 300 MHz): d 8.11 (s,
1H), 8.05 (d, 1H, J¼8.1 Hz), 7.82–7.70 (m, 2H), 7.22
(d, 1H, J¼7.5 Hz), 7.05 (s, 1H), 6.82–6.74 (m, 3H), 6.68–
6.60 (m, 2H), 5.27 (s, 2H), 4.93 (d, 1H, J¼8.2 Hz), 4.44–
4.35 (m, 4H), 4.29–4.20 (m, 2H), 4.08–3.95 (m, 1H),
3.90–3.68 (m, 3H), 3.46–3.42 (m, 2H), 2.40 (t, 2H,
J¼7.5 Hz), 2.24–2.22 (m, 1H), 1.96–1.94 (m, 2H), 1.93 (s,
3H), 1.52–1.26 (m, 22H). HRMS (MALDI, m/z) calcd
for [C52H65N7O16+Na] (M+Na)+: 1066.4380, found:
1066.4390.
4.1.7. Compound 17. To a solution of sinomenine (7, 3.29 g,
10.0 mmol) in CH2Cl2 (80 mL) was added NIS (2.36 g,
10.5 mmol) at room temperature. After 2 h, the reaction
mixture was quenched by adding aqueous Na2S2O3 solution.
The organic layer was separated and washed with H2O and
then brine, dried over Na2SO4, filtered, and concentrated.
The residue was purified by silica gel column chromato-
graphy (CH2Cl2/CH3OH¼50:1 to 20:1) to give 17 (3.87 g,
85%) as a pale yellow solid; [a]2D0 5.8 (c 0.46, CHCl3);
mp: 108–110 ꢀC. IR (KBr): nmax 2934, 2837, 1689, 1628,
1
757 cmꢂ1. H NMR (DMSO, 500 MHz): d 10.15 (1H, br
s), 8.93 (1H, br s), 8.24 (1H, s), 8.00 (1H, d, J¼7.7 Hz),
7.97 (1H, t, J¼5.5 Hz), 7.79 (1H, t, J¼7.3 Hz), 7.71 (1H,
t, J¼7.3 Hz), 7.66 (1H, d, J¼15.4 Hz), 7.27 (1H, d,
J¼7.7 Hz), 7.09 (1H, d, J¼2.3 Hz), 6.96 (1H, s), 6.77 (1H,
dd, J¼2.4, 8.8 Hz), 6.73 (1H, s), 6.66 (1H, d, J¼8.8 Hz),
6.59 (2H, s), 6.41 (1H, d, J¼15.4 Hz), 5.69 (1H, s), 5.22
(2H, s), 4.34 (2H, t, J¼7.0 Hz), 4.16 (1H, d, J¼15.4 Hz),
3.78 (3H, s), 3.34 (3H, s), 3.23 (1H, s), 3.15 (2H, q,
J¼6.4 Hz), 2.92–2.88 (2H, m), 2.73–2.69 (1H, m), 2.44
(2H, d, J¼15.3 Hz), 2.30 (3H, s), 1.91–1.74 (5H, m),
1.44–1.42 (2H, m), 1.24–1.22 (12H, m). 13C NMR
(DMSO, 125 MHz): d 192.5, 168.6, 165.2, 159.7, 159.6,
152.4, 151.8, 151.7, 151.4, 146.5, 145.5, 142.1, 135.58,
135.55, 130.1, 130.1, 129.0, 128.9, 126.0, 124.6, 124.5,
1
1471, 1435, 1278, 1202, 1148, 1052, 885, 749 cmꢂ1. H
NMR (CDCl3, 300 MHz): d 7.12 (1H, s), 6.67 (1H, br s),
5.42 (1H, s), 4.32 (1H, d, J¼15.6 Hz), 3.75 (3H, s), 3.46
(1H, s), 3.21 (1H, d, J¼3.9 Hz), 2.97 (1H, s), 2.86 (1H, d,
J¼18.9 Hz), 2.53–2.43 (3H, m), 2.39 (3H, s), 2.00–1.85
(3H, m). 13C NMR (CDCl3, 125 MHz): d 193.7, 152.2,

6820
Y.-H. Zhang et al. / Tetrahedron 63 (2007) 6813–6821
123.9, 122.8, 120.9, 115.9, 112.8, 112.4, 111.3, 109.4, 106.9,
102.2, 101.6, 82.6, 62.8, 61.5, 55.5, 55.2, 54.2, 49.3, 46.6,
44.4, 42.3, 40.1, 38.6, 34.6, 29.6, 29.1, 28.82, 28.75,
28.65, 28.3, 26.4, 25.8, 21.6. HRMS (ESI, m/z) calcd for
C55H59N5O10Na (M+Na)+: 927.4154, found: 927.4115.
Acknowledgements
This work is financially supported by the Ministry of Science
and Technology of China (2002CB0713808), National
Natural Science Foundation of China (Nos. 20425205 and
20621062), Chinese Academy of Sciences (KGCX2-SW-
213, KSCX2-YW-R-23, KJCX-YW-H08), and Shanghai
Municipal Commission of Science and Technology
(04DZ14901, 06QH14016).
4.1.10. Compound 24. To a suspension of 11-azido-undec-
1-ene 22 (918 mg, 4.7 mmol) and acetylene-fluorescein de-
rivative 1 (1.446 g, 3.9 mmol) in a 1:1 mixture of water and
tert-butyl alcohol (60 mL) were added cupric sulfate
(17 mg, 0.04 mmol) and ascorbic acid (43 mg, 0.14 mmol)
successively. The mixture was stirred vigorously at room
temperature for 24 h. The reaction mixture was diluted
with ethyl acetate (400 mL), washed with water (10 mLꢁ3),
saturated brine solution (10 mL), dried over anhydrous so-
dium sulfate, and concentrated in vacuo. The residue was
chromatographed on silica gel to afford 24 as a yellow solid
(1.03 g, 80%); mp 78–80 ꢀC. IR (KBr): nmax 2927, 1765,
1634, 1615, 1504, 1465, 1430, 1249, 1183, 1108, 760,
Supplementary data
Supplementary data associated with this article can be found
in the online version, at doi:10.1016/j.tet.2007.04.069.
References and notes
1
692 cmꢂ1. H NMR (300 MHz, CDCl3): d 8.00 (d, 1H,
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J¼7.2 Hz), 7.70–7.58 (m, 3H), 7.15 (d, 1H, J¼7.2 Hz),
6.86 (d, 1H, J¼1.5 Hz), 6.77 (s, 1H), 6.69–6.56 (m, 4H),
5.84–5.73 (m, 1H), 5.22 (s, 2H), 5.01 (dd, 1H, J¼3.9,
1.5 Hz), 4.96–4.90 (m, 1H), 4.36 (t, 1H, J¼6.9 Hz), 2.06–
1.99 (m, 2H), 1.92 (t, 2H, J¼6.6 Hz), 1.32–1.20 (m, 12H).
MS (ESI, m/z): 566 (M+H)+, 588 (M+Na)+. Anal. Calcd
for C34H35N3O5: C, 72.19; H, 6.24; N, 7.43. Found: C,
71.96; H, 6.50; N, 7.43.
4.1.11. Fluorescein-labeled artemisinin derivative 25. A
suspension of 24 (48 mg, 0.085 mmol) and 12b-allyldeoxo-
artemisinin 21 (131 mg, 0.425 mmol) in anhydrous CH2Cl2
(0.2 mL) was added the second generation Grubbs catalyst
(7 mg, 0.0085 mmol) over 2 h via a syringe pump under
N2 at 45 ꢀC. After 12 h, the second portion of Grubbs cata-
lyst (3.5 mg, 0.0042 mmol) in dichloromethane (0.1 mL)
was added. This mixture was stirred for 24 h, and then the
third portion of Grubbs catalyst (3.5 mg, 0.0042 mmol) in
dichloromethane (0.1 mL) was added. After additional 12 h,
the reaction was allowed to cool down to room temperature
and filtered through a pad of Celite. The filtrate was concen-
trated in vacuo. The residue was purified by a preparative
thin layer chromatography to afford 25 as a yellow solid
(24 mg, 33%); mp 68–69 ꢀC; [a]D23 ꢂ59.5 (c 0.073, CHCl3).
IR (KBr): nmax 2926, 1769, 1637, 1563, 1504, 1374, 1248,
1179, 1108, 1038, 840, 764, 668, 420 cmꢂ1 1H NMR
.
(500 MHz, CDCl3): d 8.01 (d, 1H, J¼7.6 Hz), 7.66 (t, 1H,
J¼7.5 Hz), 7.62–7.59 (m, 2H), 7.15 (d, 1H, J¼7.6 Hz),
6.86 (s, 1H), 6.75 (d, 1H, J¼2.2 Hz), 6.69–6.55 (m, 4H),
5.50 (dt, 1H, J¼15.5, 6.8 Hz), 5.44 (dt, 1H, J¼14.7,
6.0 Hz), 5.24 (s, 2H), 5.23 (s, 1H), 4.35 (t, 2H, J¼7.2 Hz),
4.15–4.11 (m, 1H), 3.56 (s, 1H), 2.25–2.14 (m, 3H),
2.00–1.91 (m, 6H), 1.81–1.70 (m, 3H), 1.56 (s, 3H),
1.30–1.19 (m, 18H), 0.91–0.87 (m, 6H). 13C NMR
(75 MHz, CDCl3): d 169.8, 159.6, 158.8, 152.5, 152.3,
143.2, 135.1, 132.1, 129.6, 129.5, 129.1, 127.1, 126.7,
124.9, 124.0, 122.9, 115.3, 112.7, 111.8, 110.2, 107.0,
103.1, 102.0, 96.8, 83.0, 69.8, 69.0, 61.9, 53.7, 50.6, 41.2,
39.9, 35.2, 34.4, 34.3, 32.6, 31.7, 30.4, 30.1, 29.7, 29.4,
29.3, 29.2, 28.9, 26.4, 25.1, 20.3, 18.5, 14.1, 12.0. MS
(MALDI, m/z): 846 (M+H)+, 868 (M+Na)+. HRMS
(MALDI, m/z) calcd for C50H59N3O9 (M)+: 845.4251,
found: 845.4246.
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