Synthesis of disaccharide modified berberine derivatives and their anti-diabetic investigation in zebrafish using a fluorescence-based technology

Article abstract of DOI:10.1039/d0ob00327a

Berberine is a naturally occurring isoquinoline alkaloid and has been used as an important functional food additive in China due to its various pharmacological activities. Berberine exhibits great potential for developing anti-diabetic agents against type

Full text of DOI:10.1039/d0ob00327a

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Synthesis of disaccharide modified berberine derivatives and their anti-diabetic
investigation in zebrafish using a fluorescence-based technology
Lizhen Wang,a,§, Haotian Kong,a,§ Meng Jin,a Xiaobin Li,a,b Rostyslav Stoyka,c Houwen Lin,d
Kechun Liua,b,
Abstract
Berberine is a naturally occurring isoquinoline alkaloid and has been used as important
functional food additive in China due to its various pharmacological activities. Berberine exhibits
great potential for developing anti-diabetic agents against type 2 diabetes mellitus (T2DM), as it can
reduce the blood glucose level in many animal models. However, the low anti-diabetic activity and
poor bioavailability of berberine (below 5%) by oral administration significantly limit its practical
applications. To solve these problems, this article focuses on the structural modification of berberine
using some disaccharide groups, because the carbohydrate moiety has been proved to improve the
bioavailability and enhance the receptor-binding affinity of drugs. Anti-diabetic investigation of the
synthesized compounds was performed in zebrafish model using a fluorescently labeled glucose
analog 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose (2-NBDG) as glucose
tracker. The results indicated that modification of berberine with carbohydrate groups could give
derivatives with improved anti-diabetic activity, in particular the diglucose modified berberine
derivative 1 which could dramatically promote the uptake of 2-NBDG both in zebrafish larvae and
their eyes even at very low concentrations. Furthermore, the fluorescence-based anti-diabetic
investigation method in zebrafish shows great potential for anti-diabetic drug screening.
Keywords: Berberine, Disaccharide, Anti-diabatic, Zebrafish, 2-NBDG
aBiology Institute, Qilu University of Technology (Shandong Academy of Sciences), Jinan 250014, Shandong Province, China.
Corresponding author: Lizhen Wang, e-mail: wlzh1106@126.com; Kechun Liu, e-mail: liukechun2000@163.com
bBioengineering Technology Innovation Center of Shandong Province, Heze 274000, Shandong Province, China
cDepartment of Regulation of Cell Proliferation and Apoptosis, Institute of Cell Biology, National Academy of Sciences of Ukraine,
Lviv 79005, Ukraine
dResearch Center for Marine Drugs, State Key Laboratory of Oncogenes and Related Genes, Shanghai Jiao Tong University,
Shanghai 200127, China
§These authors contributed equally to this paper
†Electronic supplementary information (ESI) available. See DOI:
1

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Introduction
Diabetes is a chronic metabolic disorder disease that threatens human health all over the world.
The number of people having diabetes is increasing every year, and it is estimated that over 422
million people worldwide have diabetes in 2019. Type 2 diabetes mellitus (T2DM), which occupies
90-95% of diabetes, is characterized as abnormal hyperglycaemic state caused by insulin resistance
and impaired insulin secretion.1 Treatment of T2DM can be achieved by oral hypoglycemic drugs
including metformin, pioglitazone, exenatide, pramlintide, thiazolidinediones, and sulphonylureas.2, 3
However, currently used drugs always suffer from many adverse effects, such as hypoglycemia,
lactic acidosis, peripheral edema, and serious hepatotoxicity and gastrointestinal reaction caused by
chronic administration.4, 5 Therefore, it is of great importance to develop novel anti-diabetic agents
with good hypoglycemic activity but low side effects.
Berberine is a naturally occurring isoquinoline alkaloid isolated from the well-known Chinese
herb Coptischinensis.6-9 In China and India, berberine is widely used for treating gastrointestinal
diseases caused by bacteria infection, such as gastroenteritis and diarrhea. Recent studies have
11
revealed that berberine exhibits various pharmacological activities, such as anti-diabetic,10,
anti-leishmanial,12 anti-inflammation,13-16 anti-oxidative,17, 18 anti-cancer,19-23 anti-cardiovascular24
and anti-alzheimer’s diseases.15, 25, 26 Due to the positive effect on human health described above,
berberine has been employed as important functional food added to tea and beverages in China.27
The anti-diabetic effect of berberine attracts increasing interests recently, because it can obviously
increase the glucose consumption in cells and reduce the blood glucose level in animal models.11, 28-30
Molecular mechanism investigation reveals that berberine shows good inhibition activity on
α-glucosidase, thereby restraining the hydrolyzation of polysaccharides to result in low glucose level
in vivo.31 Berberine also affects glucose level through some other signal pathways, such as activating
AMP-activated protein kinase (AMPK), protecting the function of pancreatic β-cells,
down-regulating the expression of phosphoenolpyruvate carboxykinase (PEPCK) and
glucose-6-phosphatase (G6Pase) gene, and inhibiting the function of mitochondria.32-37 Human
clinical study has proved that berberine can reduce the fasting and post-load plasma glucose level in
T2DM patients.38, 39 All these data indicate that berberine should be a promising lead compound for
developing high efficient anti-diabetic drugs with low side effects.
However, the major problem for developing berberine-based drugs against T2DM is the poor
bioavailability (below 5% by oral administration).30 The poor water solubility of berberine caused by
the rigid planer structure and quaternary ammonium unit (Scheme 1) always leads to very low
absorption efficiency in gastrointestinal tract.40 In previous research, the pharmacokinetic study of
berberine in human was carried out on a liquid chromatography–electrospray ionization–mass
2

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spectrometry (LC–ESI–MS), which revealed that the Cmax of berberine in human plasma was as low
as 0.4 ng/mL after oral administration of 400 mg of berberine.41, 42 Another problem is the moderate
anti-diabetic activity of berberine compared with the clinically used anti-diabetic drugs.27 To
overcome these problems, many structurally modified berberine derivatives have been prepared, and
some
of
them
exhibit
improved
anti-diabetic
effect.43-51
For
example,
the
8,8-dimethyldihydroberberine45 and 9/10-OH pseudoberberines51 exhibit higher anti-diabetic activity
than berberine. Structure-activity relationship investigation reveals that the methylenedioxyl group
and quaternary ammonium unit (Scheme 1) in berberine are two key structural components related to
its anti-diabetic activity.50 Removal of the methylenedioxyl group or reduction of the quaternary
ammonium to a tertiary amine will result in significant decrease of the anti-diabetic effect of
berberine. Among all the references reported previously, modification of berberine on its
9-O-position is the most attracting work, as it can obviously enhance its anti-diabetic activity.43, 44, 46
Furthermore, the 9-O-modified berberine derivatives can be easily synthesized by constructing a key
intermediate 9-OH pseudoberberine (Scheme 1), obtained by pyrolysis of berberine with high yield
and excellent selectivity.52, 53 In contrast, the 8-C-, 13-C- and 10-O-modified berberine derivatives
are always achieved with very low overall yields, due to the poor leaving activity of hydrogen atom
and the low selectivity of 10-O-demethylation reaction.
Scheme 1. Construction of 9-OH pseudoberberine and 9-O-modified berberine derivatives
Glycosylation has been proved as an efficient strategy for drug modification, because it can
improve the bioavailability and enhance the receptor-binding affinity of drugs.54-57 Therefore, many
glycosylated drugs or natural products have been developed in the past several years.58-61 In 2012,
Chen and coworkers43 synthesized a 9-O-glucosylated berberine derivative, a major metabolite of
berberine in vivo.62, 63 This derivative exhibited improved bioavailability and anti-diabetic effect, but
3

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could be easily degraded into 9-OH pseudoberberine under mild conditions due to the presence of a
O-glycosidic bond.43 Later, Han and coworkers48 synthesized some 9-O-monosaccharide modified
berberine derivatives with good water solubility and high stability under acidic condition, and some
of them exhibited improved but moderate anti-diabetic activity compared with berberine. Compared
with monosaccharide moiety, disaccharide should have excellent water solubility due to the presence
of more hydroxyl groups. Furthermore, previous reports have revealed that conjugation of drugs with
disaccharide moiety can improve their biological activity, because carbohydrate plays critical role in
mediating the cellular uptake of disaccharide-drug conjugates.64-68 For example, bleomycin bearing a
disaccharide group exhibits higher tumor cell targetability than monosaccharide modified
bleomycin.64 Therefore, this article focuses on the synthesis and biological investigation of a set of
9-O-disaccharide modified berberine derivatives. The disaccharide building blocks are prepared by a
highly efficient procedure from different types of water-soluble monosaccharides, such as glucose,
mannose and ribose. Conjugation of disaccharides with berberine is accomplished using a stable
triazole group as linker, which can avoid the degradation reaction occurred in 9-O-glycosylated
berberine derivative. All the synthesized compounds were subjected to anti-diabetic investigation in
zebrafish model using a fluorescence-based technology. In order to conveniently visualize the
glucose
uptake
in
zebrafish,
a
fluorescently
labeled
glucose
mimetic
2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl)amino]-D-glucose69 (2-NBDG, Figure 1) with low
cytotoxicity is used as bioprobe for zebrafish maintenance. 2-NBDG has been proved to exhibit
higher binding ability towards glucose transporters in cells than glucose, and can be taken in by
zebrafish as glucose.70, 71
OH
O
HO
OH
HO
NH
N
O
N
NO2
Figure 1. Structure of 2-NBDG
Results and discussion
Synthesis of disaccharide modified berberine derivatives
Disaccharide building blocks 6–10 were synthesized from previously reported monosaccharide
β-D-glucopyranosyl azide 1148 as shown in Scheme 2. Selective protection of the 6-OH group in 11
with a tert-butyldimethylsilyl (TBS) group followed by benzoylation of the remaining hydroxyl
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groups (OH) afforded compound 12 with 52% total yield. Subsequent selective removal of the TBS
group in 12 promoted by tetrabutylammonium fluoride (TBAF) provided glycosyl acceptor 13 with
89% isolated yield. On the other hand, reaction of fully acetylated sugars 14–18 with p-thiocresol
(p-STol) under the influence of stannic chloride (SnCl4) gave glycosyl donors 19–23, which were
subjected to standard glycosylation procedure described previously72, 73 with acceptor 13 in the
presence of N-iodosuccinimide (NIS) and silver trifluoromethanesulfonate (AgOTf) to provide fully
protected disaccharides 24–28. Cleavage of all the acetyl (Ac) and benzoyl (Bz) groups in 24–28
using sodium methoxide (CH3ONa) in methanol smoothly furnished the disaccharide building blocks
6–10 with high total yields.
Scheme 2. Synthesis of carbohydrate building blocks 6–10
1. TBSCl
pyridine
2. BzCl, DMAP
OH
OTBS
O
OH
TBAF
THF
O
O
BzO
BzO
BzO
BzO
HO
HO
N3
N3
N3
89%
52%
OBz
OBz
OH
11
13
12
13
OAc
OH
O
OAc
O
OAc
AcO
p-STol, SnCl4
CH2Cl2
O
NIS, AgOTf
CH3ONa
CH3OH
O
HO
HO
AcO
O
CH2Cl2
O
OAc
AcO
AcO
AcO
AcO
OH
HO
HO
STol
OAc
O
90%
N3
82%
81%
BzO
BzO
O
OAc
OAc
N3
19
14
OBz
OH
24
6
OH
OAc
HO
HO
AcO
AcO
13
NIS, AgOTf
O
O
OAc
OAc
CH3ONa
CH3OH
AcO
AcO
p-STol, SnCl4 AcO
CH2Cl2
O
O
CH2Cl2
O
OAc
O
OH
STol
OAc
AcO
86%
86%
N3
63%
O
O
HO
HO
BzO
BzO
OAc
N3
OAc
15
20
OH
OBz
7
25
OAc
OH
13
NIS, AgOTf
AcO
AcO
HO
HO
OAc
STol
O
O
AcO
AcO
O
O
CH3ONa
CH3OH
p-STol, SnCl4
CH2Cl2
O
O
CH2Cl2
89%
O
79%
77%
O
BzO
BzO
HO
HO
AcO OAc
AcO
OAc
N3
N3
OBz
16
OH
21
26
OAc
8
OH
OH
O
OAc
O
13
HO
HO
OAc
AcO
AcO
OAc
OAc
NIS, AgOTf
CH3ONa
CH3OH
OAc
p-STol, SnCl4
O
CH2Cl2
O
O
O
CH2Cl2
AcO
AcO
AcO
AcO
92%
69%
O
68%
O
HO
HO
OAc
BzO
BzO
STol
N3
N3
17
22
OH
OBz
9
27
OH
OAc
HO
HO
AcO
AcO
13
NIS, AgOTf
STol
OAc
O
CH3ONa
CH3OH
O
p-STol, SnCl4
CH2Cl2
CH2Cl2
O
O
O
O
AcO
AcO
88%
84%
80%
AcO
AcO
O
OH
OAc
O
HO
HO
OAc
BzO
BzO
N3
N3
18
23
OBz
10
28
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Conjugation of alkynylated berberine derivative 2952 with disaccharide building blocks 6–10
was achieved by the classcial “click” chemistry74 in the presence of CuSO4ꞏ5H2O and sodium
ascorbate (Scheme 3), which afforded the desired disaccharide modified berberine derivatives 1–5.
Purification of the target compounds on silica gel column chromatography resulted in very low
isolated yields (<10%), possibly due to the high polarity of disaccharide modified berberine
derivatives. To solve this problem, all the compounds were subjected to a neutral alumina column
chromatography, which gave derivatives 1–5 with moderate isolated yields (46%–61%).
Scheme 3. Synthesis of disaccharide modified berberine derivatives 1–5
Uptake of 2-NBDG in zebrafish
Biological investigations are performed in zebrafish model, because drug screening can be
conveniently carried out using 24/48-well plate format compared with the currently used animal
models.75 More importantly, zebrafish owns highly similar genome (~80%) and glucose
transportation mechanisms as humans,76-79 which indicates its great potential for anti-diabetic
investigation in vivo. Therefore, the zebrafish model had been used by Williams group for studying
the anti-diabetic activity of various isolated natural products, and 2-NBDG with green fluorescent
emission was employed for conveniently visualizing the glucose uptake in zebrafish.70
In our work, the 3-day old zebrafish larvae are employed for drug screening because of the
well-worked glucose transportation and metabolism at this stage,79, 80 and the transparent body of
larvae makes 2-NBDG uptake being easily visualized on a fluorescent microscopy.70 Therefore, we
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firstly investigated the 2-NBDG uptake in zebrafish by treating 3-day old larvae with different
concentrations (0, 0.1 0.2, 0.6, 1, and 2 mM) of 2-NBDG for 3 h. The results were depicted in Figure
2A, zebrafish larvae without any treatment gave very weak background interference in abdominal
cavity, due to the presence of interfering biological species, such as ascorbic acid (AA) and
dehydroascorbic acid (DHA).81 In contrast, addition of 2-NBDG led to obvious fluorescence
enhancement with a dose-dependent dependence on the 2-NBDG concentration (Figure 2B), thus
indicating the uptake of 2-NBDG in zebrafish. This result was also proved by the enhanced
fluorescent emission in zebrafish eyes (Figure 2C), an organ that could express a large amount of
glucose transporters80 for 2-NBDG transportation. To achieve the anti-diabetic drug screening, the
optimized concentration for zebrafish incubation was 0.6 mM, as low or high concentration gave too
weak or strong fluorescence which was not suitable for discriminating the fluorescence improvement.
In parallel, the time-dependent uptake of 2-NBDG in zebrafish was carried out by treating zebrafish
larvae with 0.6 mM of 2-NBDG for 0, 1, 2, 3, 6, and 12 h as shown in Figure 2D, 2E and 2F. The
results indicated that 2-NBDG could be taken in zebrafish after 0.5 h of incubation, and the
fluorescent signal became steady in 3 h. Consequently, the optimized incubation time was 3 h. To
confirm the uptake of 2-NBDG was promoted by glucose transporter, zebrasfish larvae were further
competitively coincubated with glucose (0.6 mM) and 2-NBDG (0.6 mM) for 3 h. As a result, the
fluorescent signal in zebrafish obviously decreased (Figure S1), which revealed that the uptake of
2-NBDG was significantly reduced due to the competitive uptake of glucose. These results gave
solid evidence that uptake of 2-NBDG was mainly caused by glucose transporter, which could
promote the uptake of glucose. In our experiments, the Image J software was used for calculating the
integrated optical density (IOD). The 2-NBDG in abdominal cavity that was not transported to
tissues of zebrafish might give interfering fluorescent signals, therefore, the fluorescence in
abdominal cavity was deducted.
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Figure 2. Time and dose-dependent uptake of 2-NBDG in zebrafish. (A) Treatment of 3-day old
zebrafish with different concentrations (0, 0.1, 0.2, 0.6, 1, and 2 mM) of 2-NBDG for 3 h could
induce increased fluorescent emission; (B) Integrated optical density (IOD) in zebrafish after
incubating with different concentrations of 2-NBDG; (C) IOD in zebrafish eyes after incubating with
different concentrations of 2-NBDG; (D) Treatment of 3-day old zebrafish with 0.6 mM of 2-NBDG
for 0, 1, 2, 3, 6 and 12 h could induce increased fluorescent emission; (E) IOD in zebrafish after
incubating with 0.6 mM of 2-NBDG for 0, 1, 2, 3, 6 and 12 h; (F) IOD in zebrafish eyes after
incubating with 0.6 mM of 2-NBDG for 0, 1, 2, 3, 6 and 12 h. *P <0.05, **P <0.01, ***P <0.001 vs
zebrafish treated with 0 mM 2-NBDG. The fluorescence in abdominal cavity was deducted to
remove the interfering fluorescent signals.
Anti-diabetic investigations
To study the anti-diabetic activity of the synthesized compounds, 3-day old zebrafish larvae
were treated with compounds 1–5 and berberine (10 μM) for 1 h, which was followed by incubating
with 2-NBDG (0.6 mM) for 3 h. According to the literature, treating larvae with tested compounds
for 1 h could induce 2-NBDG uptake accompanied with dramatic fluorescent emission.70 Images of
larvae were captured on a fluorescent microscopy, and the results were depicted in Figure 3A and 3B.
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Compared with larvae treated with 2-NBDG only, incubation of larvae with berberine and 2-NBDG
could result in obvious fluorescent enhancement, thereby proving the improvement of 2-NBDG
uptake in zebrafish and the moderate anti-diabetic activity of berberine. In contrast, treatment of
larvae with 2-NBDG and berberine derivative 1 could cause significant fluorescence improvement
and high 2-NBDG uptake, and other berberine derivatives 2–5 also induced detectable fluorescence
enhancement. These results indicated that introducing disaccharide group to berberine could enhance
its anti-diabetic activity, in particular the diglucose modified berberine derivative 1 which exhibited
the highest anti-diabetic effect. This result was confirmed by the dramatic fluorescence improvement
in zebrafish eyes (Figure 3C), because the overexpression of glucose transporters promoted by
compounds 1–5 could enhance the uptake of 2-NBDG. As a comparison, the anti-diabetic activity of
emodin (a natural product with high anti-diabetic activity) and monoglucosylated berberine 30
(Figure 3D) was investigated in zebrafish through the procedure described above. The results
indicated that diglucose modified berberine 1 could induce significant fluorescence improvement as
emodin, thereby proving again the good anti-diabetic activity of compound 1. In addition, incubation
of zebrafish larvae with glucose and diglucose 6 could induce no detectable fluorescence
improvement (Figure S1), which indicated that the fluorescence signal was not induced by glucose
hydrolysis.
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Figure 3. Berberine derivative induced 2-NBDG uptake in zebrafish. (A) Treatment of 3-day old
zebrafish with 10 μM of tested compounds for 1 h and 0.6 mM of 2-NBDG for 3 h could induce
obvious fluorescent emission; (B) IOD in zebrafish after incubating with tested compounds and
2-NBDG; (C) IOD in zebrafish eyes after incubating with tested compounds and 2-NBDG. Control
group (Ctr) was incubated with E3 water for 1 h, and then treated with 0.6 mM of 2-NBDG for 3 h.
*P <0.05, **P <0.01, ***P <0.001 vs Ctr. The fluorescence in abdominal cavity was deducted to
remove the interfering fluorescent signals.
Diglucose modified berberine derivative 1 with high anti-diabetic activity was further subjected
to a dose-dependent investigation in zebrafish. The 3-day old larvae were incubated with different
concentrations (0, 2.5, 5, 10, 20, and 40 μM) of 1 for 1 h, and then treated with 2-NBDG (0.6 mM)
for 3 h. As shown in Figure 4, fluorescent images of larvae revealed that 10 μM of compound 1
could significantly promote the uptake of 2-NBDG in body and eyes of zebrafish, while increasing
the concentration induced inapparent fluorescence improvement. These results indicated that
compound 1 showed excellent anti-diabetic effect even at low concentrations.
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Figure 4. (A) 2-NBDG uptake in zebrafish incubated with different concentrations (0, 2.5, 5, 10, 20,
and 40 μM) of 1 for 1 h, and then treated with 2-NBDG (0.6 mM) for 3 h; (B) IOD in zebrafish after
incubating with 1 and 2-NBDG; (C) IOD in zebrafish eyes after incubating with 1 and 2-NBDG. **P
<0.01, ***P <0.001 vs zebrafish treated with 0 mM 2-NBDG. The fluorescence in abdominal cavity
was deducted to remove the interfering fluorescent signals.
Conclusions
In this paper, we synthesized some novel disaccharide modified berberine derivatives 1–5.
Attachment of disaccharide moiety with berberine was accomplished by the classical “click”
chemistry, which afforded the target compounds with high total yields. Anti-diabetic investigation of
the synthesized derivatives was performed in 3-day old zebrafish larvae, and 2-NBDG with strong
fluorescent emission was used for tracking the glucose transportation on a fluorescent microscope.
As a result, zebrafish larvae incubated with berberine derivative 1 and 2-NBDG could result in
significant fluorescence improvement, which indicated the huge uptake of 2-NBDG in zebrafish.
Furthermore, the zebrafish eyes also emitted strong fluorescent signals, thereby proving the
overexpression of various glucose transporters that could promote the uptake of 2-NBDG.
Dose-dependent investigation revealed that treatment of zebrafish with low concentration (10 μM) of
compound 1 could promote the uptake of 2-NBDG both in zebrafish larvae and their eyes. These
results indicated that the diglucose modified berberine derivative 1 should be an interesting lead
compound for developing novel anti-diabetic drugs, and the 2-NBDG based fluorescent technology
could be widely applied for anti-diabetic drug screening in zebrafish.
Experimental section
Compounds and reagents
Chemicals and reagents were obtained commercially and used without any further purification.
1H NMR and 13C NMR spectra were measured on a Bruker AV 400 spectrometer using CDCl3, D2O,
or DMSO as solvent. HRMS spectra were recorded on an Agilent QTOF 6520 mass spectrometer.
Thin layer chromatography (TLC) was carried out on silica gel HF254 plates with detection using a
UV detector or by charring with a solution of H2SO4 in MeOH (1/5, v/v).
2,3,4-Tri-O-benzoyl-β-D-glucopyranosyl azide 13
To a solution of azido sugar 11 (2.00 g, 9.76 mmol) in 50 mL of pyridine was slowly added
tert-butyldimethylsilyl chloride (TBSCl, 1.77 g, 11.71 mmol). The mixture was stirred at room
temperature overnight, and TLC (dichloromethane/methanol, 4/1, v/v) showed the complete
conversion of the starting material. Then, to the mixture was slowly added benzoyl chloride (BzCl,
6.83 g, 48.80 mmol) and a catalytic amount of 4-dimethylaminopyridine (DMAP, 122 mg, 1.00
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mmol). The reaction was stirred at room temperature for 6 h, diluted with 150 mL of ethyl acetate
and washed with water (50 mL×3). The organic layer was evaporated, and the resulting residue was
subjected to column chromatography (petroleum ether/ethyl acetate, 2/1, v/v) to give 12 as a white
solid. To the mixture of compound 12 (3.20 g, 5.08 mmol) in 50 mL of tetrahydrofuran was added
tetrabutylammonium fluoride (TBAF, 1.0 M solution in THF, 6.1 mL, 6.10 mmol), and the reaction
was stirred at room temperature for 8 h. After TLC (petroleum ether/ethyl acetate 2/1) showed the
complete conversion of the starting material, the solvent was removed under reduced pressure. The
resulting residue was purified by column chromatography (petroleum ether/ethyl acetate, 1/1, v/v) to
give 13 as a white solid (2.34 g, 46% for 2 steps). 1H NMR (400 MHz, CDCl3): δ 7.95 (t, J = 6.8 Hz,
4 H, ArH), 7.82 (d, J = 7.6 Hz, 2 H, ArH), 7.56–7.51 (m, 2 H, ArH), 7.45–7.35 (m, 5 H, ArH), 7.28
(t, J = 8.0 Hz, 2 H, ArH), 5.96 (t, J = 9.6 Hz, 1 H), 5.54 (t, J = 10.0 Hz, 1 H), 5.47 (t, J = 9.6 Hz, 1
13
H), 4.96 (d, J = 8.8 Hz, 1 H), 3.93–3.87 (m, 2 H), 3.79 (dd, J = 4.4, 12.8 Hz, 1 H); C NMR (100
MHz, CDCl3): δ 166.01, 165.73, 164.98, 133.82, 133.53, 133.40, 129.97, 129.91, 129.74, 128.75,
128.63, 128.56, 128.46, 128.39, 128.36, 88.40, 72.62, 71.27, 69.04, 61.18; ESI-TOF HRMS (m/z):
calcd for C27H24N3O8 , [M + H]+, 518.1558; found, 518.1560.
+
General procedure for the synthesis of fully protected disaccharide 24–28
To a solution of acetylated sugar 14–18 (3.08 mmol) and p-thiocresol (p-STol, 764 mg, 6.16 mmol)
o
in 100 mL of dry dichloromethane was added stannic chloride (SnCl4, 801 mg, 3.08 mmol) at 0 C.
The reaction was slowly warmed to room temperature and stirred for 4 h. Thereafter, the reaction
was neutralized with saturated NaHCO3 aqueous solution and extracted with dichloromethane (60
mL×3). The organic layer was evaporated and the resulting residue was purified by column
chromatography (petroleum ether/ethyl acetate, 1/1, v/v) to give glycosyl donor 19–23 as colorless
oil. To the mixture of donor 19–23 (1.10 mmol) and acceptor 13 (569 mg, 1.10 mmol) in 10 mL of
dry dichloromethane was added N-iodosuccinimide (NIS, 297 mg, 1.32 mmol) followed by a
o
catalytic amount of silver trifluoromethanesulfonate (AgOTf, 26 mg, 0.10 mmol) at –20 C. The
reaction was slowly warmed to room temperature and stirred for 2 h. Then, the reaction was
neutralized with three drops of triethylamine, and the solvent was evaporated. The resulting residue
was purified by column chromatography (hexane/ethyl acetate, 1/1, v/v) to give fully protected
disaccharide 24–28 as colorless oil.
2,3,4,6-Tetra-O-acetyl-β-D-glucopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl azide 24
Compound 24 was obtained as colorless oil (755 mg, 66% for 2 steps) from 14 by the general
procedure described above. 1H NMR (400 MHz, CDCl3): δ 7.96–7.90 (m, 4 H, ArH), 7.80 (d, J = 7.6
Hz, 2 H, ArH), 7.53 (t, J = 7.6 Hz, 2 H, ArH), 7.45–7.37 (m, 5 H, ArH), 7.29–7.26 (m, 2 H, ArH),
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5.87 (t, J = 9.6 Hz, 1 H), 5.41 (dt, J = 3.6, 10.0 Hz, 2 H), 5.20 (t, J = 9.6 Hz, 1 H), 5.08–4.98 (m, 2
H), 4.91 (d, J = 8.8 Hz, 1 H), 4.62 (d, J = 8.0 Hz, 1 H), 4.23 (dd, J = 4.8, 12.0 Hz, 1 H), 4.15–4.04 (m,
3 H), 3.78 (dd, J = 7.6, 11.2 Hz, 1 H), 3.70–3.66 (m, 1 H), 2.10 (s, 3 H), 2.01 (s, 6 H), 2.00 (s, 3 H);
13C NMR (100 MHz, CDCl3): δ 170.58, 170.19, 169.43, 169.38, 165.62, 165.24, 164.98, 133.71,
133.54, 133.36, 129.90, 129.84, 129.72, 128.71, 128.59, 128.53, 128.50, 128.45, 128.32, 101.04,
88.16, 76.04, 72.75, 71.92, 71.24, 71.11, 69.19, 68.42, 68.29, 61.80, 20.65, 20.61, 20.60, 20.58;
ESI-TOF HRMS (m/z): calcd for C41H42N3O17+, [M + H]+, 848.2509; found, 848.2505.
2,3,4,6-Tetra-O-acetyl-β-D-galactopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl azide
25
Compound 25 was obtained as colorless oil (587 mg, 54% for 2 steps) from 15 by the general
procedure described above. 1H NMR (400 MHz, CDCl3): δ 7.97–7.90 (m, 4 H, ArH), 7.81 (d, J = 7.2
Hz, 2 H, ArH), 7.53 (t, J = 7.2 Hz, 2 H, ArH), 7.45–7.37 (m, 5 H, ArH), 7.30–7.26 (m, 2 H, ArH),
5.87 (t, J = 9.6 Hz, 1 H), 5.45–5.40 (m, 2 H), 5.38 (d, J = 3.2 Hz, 1 H), 5.35 (s, 1 H), 5.24 (dd, J =
8.0, 10.4 Hz, 1 H), 5.00 (dd, J = 3.6, 10.8 Hz, 1 H), 4.93 (d, J = 8.8 Hz, 1 H), 4.58 (d, J = 8.0 Hz, 1
H), 4.15–4.04 (m, 4 H), 3.89 (t, J = 6.4 Hz, 1 H), 3.76 (dd, J = 7.2, 11.2 Hz, 1 H), 2.16 (s, 3 H), 2.12
(s, 3 H), 2.11 (s, 3 H), 1.98 (s, 6 H); 13C NMR (100 MHz, CDCl3): δ 170.33, 170.30, 170.17, 170.10,
170.08, 169.85, 169.54, 165.61, 165.24, 164.98, 133.71, 133.53, 133.36, 129.89, 129.82, 129.70,
128.71, 128.59, 128.53, 128.45, 128.32, 101.35, 89.73, 88.16, 76.06, 72.77, 71.24, 70.86, 70.80,
69.15, 68.77, 68.62, 68.42, 67.44, 67.38, 66.99, 66.46, 61.25, 61.19, 20.69, 20.61, 20.56, 20.53;
ESI-TOF HRMS (m/z): calcd for C41H42N3O17+, [M + H]+, 848.2509; found, 848.2503.
2,3,5-Tri-O-acetyl-β-D-ribofuranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl azide 26
Compound 26 was obtained as colorless oil (656 mg, 61% for 2 steps) from 16 by the general
procedure described above. 1H NMR (400 MHz, CDCl3): δ 7.97–7.91 (m, 4 H, ArH), 7.81 (d, J = 7.6
Hz, 2 H, ArH), 7.53 (t, J = 7.2 Hz, 2 H, ArH), 7.45–7.36 (m, 5 H, ArH), 7.30–7.26 (m, 2 H, ArH),
5.88 (t, J = 9.6 Hz, 1 H), 5.50 (t, J = 10.0 Hz, 1 H), 5.44 (t, J = 9.6 Hz, 1 H), 5.34–5.29 (m, 2 H),
5.08 (s, 1 H), 4.94 (d, J = 8.8 Hz, 1 H), 4.34–4.26 (m, 2 H), 4.13–4.05 (m, 2 H), 3.92 (dd, J = 1.6,
11.6 Hz, 1 H), 3.73 (dd, J = 6.8, 12.0 Hz, 1 H), 2.10 (s, 3 H), 2.05 (s, 3 H), 2.02 (s, 3 H); 13C NMR
(100 MHz, CDCl3): δ 170.53, 169.57, 169.47, 165.67, 165.09, 164.96, 133.58, 133.48, 133.32,
129.91, 129.83, 129.75, 128.78, 128.64, 128.49, 128.43, 128.31, 106.10, 88.14, 78.94, 76.08, 74.66,
72.81, 71.40, 71.27, 69.11, 67.01, 64.43, 20.68, 20.53, 20.50; ESI-TOF HRMS (m/z): calcd for
C38H38N3O15+, [M + H]+, 776.2297; found, 776.2290.
2,3,4,6-Tetra-O-acetyl-α-D-mannopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl azide
27
Compound 27 was obtained as colorless oil (643 mg, 47% for 2 steps) from 17 by the general
13

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procedure described above. 1H NMR (400 MHz, CDCl3): δ 7.98–7.93 (m, 4 H, ArH), 7.81 (d, J = 7.2
Hz, 2 H, ArH), 7.54 (t, J = 7.6 Hz, 2 H, ArH), 7.45–7.38 (m, 5 H, ArH), 7.30–7.26 (m, 2 H, ArH),
5.90 (t, J = 9.6 Hz, 1 H), 5.53 (t, J = 9.6 Hz, 1 H), 5.46 (t, J = 9.6 Hz, 1 H), 5.37 (dd, J = 3.6, 10.0 Hz,
1 H), 5.30–5.25 (m, 2 H), 5.02 (d, J = 8.8 Hz, 1 H), 4.84 (s, 1 H), 4.24 (dd, J = 5.2, 12.4 Hz, 1 H),
4.13 (t, J = 8.4 Hz, 1 H), 4.07–4.02 (m, 2 H), 3.94 (dd, J = 6.8, 11.2 Hz, 1 H), 3.73 (d, J = 8.8 Hz, 1
H), 2.14 (s, 3 H), 2.08 (s, 3 H), 2.03 (s, 3 H), 2.00 (s, 3 H); 13C NMR (100 MHz, CDCl3): δ 170.59,
169.94, 169.81, 169.77, 165.67, 165.16, 164.93, 133.73, 133.52, 133.37, 129.91, 129.88, 129.76,
128.74, 128.55, 128.45, 128.34, 97.44, 88.14, 75.33, 72.72, 71.22, 69.26, 69.10, 69.01, 68.68, 66.53,
65.92, 62.23, 20.84, 20.69; ESI-TOF HRMS (m/z): calcd for C41H42N3O17+, [M + H]+, 848.2509;
found, 848.2504.
2,3,4-Tri-O-acetyl-α-L-rhamnopyranosyl-(1→6)-2,3,4-tri-O-benzoyl-β-D-glucopyranosyl azide 28
Compound 28 was obtained as colorless oil (729 mg, 67% for 2 steps) from 18 by the general
procedure described above. 1H NMR (400 MHz, CDCl3): δ 7.90–7.85 (m, 4 H, ArH), 7.75 (d, J = 7.6
Hz, 2 H, ArH), 7.46 (t, J = 7.2 Hz, 2 H, ArH), 7.37–7.29 (m, 5 H, ArH), 7.21 (t, J = 7.0 Hz, 2 H,
ArH), 5.82 (t, J = 9.6 Hz, 1 H), 5.45–5.35 (m, 2 H), 5.25–5.24 (m, 1 H), 5.21 (dd, J = 3.6, 10.0 Hz, 1
H), 4.97 (t, J = 10.0 Hz, 1 H), 4.87 (d, J = 8.8 Hz, 1 H), 4.76 (s, 1 H), 4.07–4.03 (m, 1 H), 3.83–3.78
(m, 2 H), 3.73 (dd, J = 6.4, 12.0 Hz, 1 H), 2.06 (s, 3 H), 1.96 (s, 3 H), 1.92 (s, 3 H), 1.08 (d, J = 6.4
Hz, 3 H); 13C NMR (100 MHz, CDCl3): δ 168.97, 168.90, 168.85, 164.65, 164.06, 163.94, 132.59,
132.84, 132.32, 128.90, 128.85, 128.75, 127.74, 127.57, 127.53, 127.48, 127.42, 127.30, 97.22,
87.15, 75.28, 71.70, 70.23, 69.96, 68.47, 68.16, 67.97, 65.65, 65.58, 19.83, 19.77, 19.69, 16.31;
ESI-TOF HRMS (m/z): calcd for C39H40N3O15+, [M + H]+, 790.2454; found, 790.2457.
General procedure for the synthesis of disaccharide 6–10
Disaccharide 24–28 (0.70 mmol) was dissolved in 15 mL of a dichloromethane/methanol solution
(1:2, v/v). To the mixture was added sodium methoxide (CH3ONa, 38 mg, 0.70 mmol), and the
reaction was stirred at room temperature for 2 h. Then, the reaction was neutralized with acetic acid,
and the solvent was evaporated. The resulting residue was purified by column chromatography using
methanol as eluent to give disaccharide 6–10 as colorless oil.
β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl azide 6
Compound 6 was obtained as white solid (231 mg, 90%) from 24 (593 mg, 0.70 mmol) by the
general procedure described above. 1H NMR (400 MHz, D2O): δ 4.69 (s, 1 H), 4.67 (s, 1 H), 4.40 (d,
J = 8.0 Hz, 1 H), 4.11 (d, J = 10.8 Hz, 2 H), 3.84–3.76 (m, 4 H), 3.65–3.59 (m, 4 H), 3.45–3.15 (m,
14 H); 13C NMR (100 MHz, D2O): δ 102.76, 90.24, 76.87, 75.93, 75.65, 73.09, 72.76, 69.63, 69.05,
68.58, 60.74; ESI-TOF HRMS (m/z): calcd for C12H22N3O10+, [M + H]+, 368.1300; found, 368.1304.
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β-D-galactopyranosyl-(1→6)-β-D-glucopyranosyl azide 7
Compound 7 was obtained as white solid (221 mg, 86%) from 25 (593 mg, 0.70 mmol) by the
general procedure described above. 1H NMR (400 MHz, D2O): δ 4.66 (d, J = 8.8 Hz, 1 H), 4.34 (d, J
= 8.0 Hz, 1 H), 4.11 (d, J = 11.6 Hz, 1 H), 3.80 (d, J = 2.8 Hz, 1 H), 3.76 (dd, J = 6.4, 11.6 Hz, 1 H),
13
3.69–3.52 (m, 6 H), 3.46–3.36 (m, 3 H), 3.17–3.13 (m, 1 H); C NMR (100 MHz, D2O): δ 103.36,
90.27, 76.89, 75.66, 75.19, 72.77, 70.77, 69.05, 68.67, 68.47, 61.01; ESI-TOF HRMS (m/z): calcd
for C12H22N3O10+, [M + H]+, 368.1300; found, 368.1306.
β-D-ribofuranosyl-(1→6)-β-D-glucopyranosyl azide 8
Compound 8 was obtained as white solid (210 mg, 89%) from 26 (543 mg, 0.70 mmol) by the
general procedure described above. 1H NMR (400 MHz, D2O): δ 4.93 (s, 1 H), 4.63 (d, J = 8.8 Hz, 1
H), 4.13 (dd, J = 4.8, 6.4 Hz, 1 H), 4.01–3.91 (m, 3 H), 3.71 (dd, J = 2.8, 12.4 Hz, 1 H), 3.62–3.52
(m, 3 H), 3.41 (t, J = 9.2 Hz, 1 H), 3.32 (t, J = 9.6 Hz, 1 H), 3.15 (t, J = 9.6 Hz, 1 H); 13C NMR (100
MHz, D2O): δ 107.47, 90.16, 82.97, 76.60, 75.65, 74.27, 72.76, 70.72, 69.22, 66.83, 62.78; ESI-TOF
+
HRMS (m/z): calcd for C11H20N3O9 , [M + H]+, 338.1194; found, 338.1198.
α-D-mannopyranosyl-(1→6)-β-D-glucopyranosyl azide 9
Compound 9 was obtained as white solid (236 mg, 92%) from 27 (593 mg, 0.70 mmol) by the
general procedure described above. 1H NMR (400 MHz, D2O): δ 4.80 (s, 1 H), 4.68 (s, 1 H), 4.66 (s,
1 H), 3.89–3.84 (m, 2 H), 3.77 (d, J = 12.0 Hz, 1 H), 3.74–3.64 (m, 3 H), 3.61–3.53 (m, 3 H),
3.43–3.38 (m, 2 H), 3.17–3.13 (m, 1 H); 13C NMR (100 MHz, D2O): δ 99.69, 90.26, 76.14, 75.93,
72.77, 70.57, 69.89, 60.70, 66.69, 65.38, 60.89; ESI-TOF HRMS (m/z): calcd for C12H22N3O10+, [M
+ H]+, 368.1300; found, 368.1305.
α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl azide 10
Compound 10 was obtained as white solid (216 mg, 88%) from 28 (552 mg, 0.70 mmol) by the
general procedure described above. 1H NMR (400 MHz, D2O): δ 4.72 (s, 1 H), 4.65 (d, J = 8.8 Hz, 1
H), 3.93–3.88 (m, 2 H), 3.70–3.55 (m, 4 H), 3.41 (t, J = 8.8 Hz, 1 H), 3.33 (t, J = 9.2 Hz, 1 H), 3.17
13
(t, J = 9.2 Hz, 1 H), 1.20 (d, J = 6.0 Hz, 1 H); C NMR (100 MHz, D2O): δ 100.57, 90.19, 76.77,
75.67, 72.75, 72.04, 70.20, 70.00, 69.31, 68.76, 66.73, 16.65; ESI-TOF HRMS (m/z): calcd for
+
C12H22N3O9 , [M + H]+, 352.1351; found, 352.1357.
General procedure for the synthesis of disaccharide modified berberine derivative 1–5
Disaccharide 6–10 (0.447 mmol) and 9-O-(propargyl) berberine chloride (177 mg, 0.447 mmol)
were dissolved in 23 mL of a methanol/acetonitrile/H2O solution (10:10:3, v/v) at room temperature.
To the mixture was added CuSO4ꞏ5H2O (67 mg, 0.268 mmol) and sodium ascorbate (53 mg, 0.268
mmol), the reaction was heated to 60 °C and stirred for
6
h. After TLC
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(dichloromethane/methanol/water, 5/5/1, v/v) showed the conversion of the starting materials, the
solvent was evaporated and the resulting residue was subjected to neutral alumina column
chromatography (acetonitrile/methanol/water, 3/3/1, v/v) to give the desired berberine derivative 1–5
as yellow solid.
9-O-(1-(β-D-glucopyranosyl-(1→6)-β-D-glucopyranosyl)-4-methylene-1H-1,2,3-triazole)berberine
chloride
1
Compound 1 was synthesized from 6 (164 mg, 0.447 mmol) and 29 (177 mg, 0.447 mmol) by the
1
general procedure described above (184 mg, 54%). H NMR (400 MHz, DMSO-d6): δ 9.62 (s, 1 H,
CH=N), 8.93 (s, 1 H, ArH), 8.57 (s, 1 H, ArH), 8.22 (d, J = 9.2 Hz, 1 H, ArH), 8.01 (d, J = 8.8 Hz, 1
H, ArH), 7.79 (s, 1 H, ArH), 7.09 (s, 1 H, ArH), 6.17 (s, 2 H), 5.54 (d, J = 9.2 Hz, 1 H), 5.46 (s, 2 H,
CH2), 5.39 (d, J = 4.4 Hz, 1 H), 5.35 (d, J = 5.6 Hz, 1 H), 5.28 (d, J = 5.6 Hz, 1 H), 4.93 (d, J = 4.8
Hz, 1 H), 4.90–4.84 (m, 3 H), 4.48 (t, J = 5.6 Hz, 1 H), 4.11–4.04 (m, 4 H), 3.76 (q, J = 6.4 Hz, 1 H),
3.68–3.64 (m, 2 H), 3.50 (q, J = 4.4 Hz, 1 H), 3.45–3.37 (m, 2 H), 3.29–3.25 (m, 1 H), 3.24–3.16 (m,
2 H), 3.12–2.99 (m, 3 H), 2.95–2.90 (m, 1 H); 13C NMR (100 MHz, DMSO-d6): δ 151.43, 150.33,
148.16, 145.60, 142.07, 137.94, 133.41, 131.16, 127.04, 124.55, 122.40, 120.87, 120.78, 87.84,
78.70, 77.37, 77.25, 77.22, 73.82, 72.57, 70.42, 70.04, 68.96, 67.08, 61.44, 57.58, 55.90, 26.82.
ESI-TOF HRMS (m/z): calcd for C34H39N4O14+, [M – Cl]+, 727.2457; found, 727.2460.
9-O-(1-(β-D-galactopyranosyl-(1→6)-β-D-glucopyranosyl)-4-methylene-1H-1,2,3-triazole)berberin
e chloride
2
Compound 2 was synthesized from 7 (164 mg, 0.447 mmol) and 29 (177 mg, 0.447 mmol) by the
1
general procedure described above (171 mg, 50%). H NMR (400 MHz, DMSO-d6): δ 9.66 (s, 1 H,
CH=N), 8.98 (s, 1 H, ArH), 8.63 (s, 1 H, ArH), 8.22 (d, J = 9.6 Hz, 1 H, ArH), 8.03 (d, J = 8.8 Hz, 1
H, ArH), 7.81 (s, 1 H, ArH), 7.09 (s, 1 H, ArH), 6.18 (s, 2 H), 5.53 (d, J = 9.2 Hz, 1 H), 5.46 (s, 2 H,
CH2), 4.92 (t, J = 5.6 Hz, 2 H), 4.83–4.81 (m, 1 H), 4.68–4.63 (m, 1 H), 4.52–4.46 (m, 1 H), 4.09 (s,
3 H), 4.06–4.01 (m, 2 H), 3.75 (t, J = 8.8 Hz, 1 H), 3.66–3.60 (m, 3 H), 3.54–3.40 (m, 8 H),
3.32–3.16 (m, 8 H); 13C NMR (100 MHz, DMSO-d6): δ 175.66, 151.43, 150.25, 148.09, 145.58,
142.85, 141.95, 137.83, 133.38, 131.15, 126.93, 125.04, 124.60, 122.39, 120.86, 120.77, 108.88,
105.95, 104.21, 102.52, 87.90, 78.77, 77.19, 75.68, 74.01, 72.67, 71.05, 69.91, 68.85, 68.32, 66.97,
60.66, 57.56, 55.87, 26.81, 25.21. ESI-TOF HRMS (m/z): calcd for C34H39N4O14+, [M – Cl]+,
727.2457; found, 727.2462.
9-O-(1-(β-D-ribofuranosyl-(1→6)-β-D-glucopyranosyl)-4-methylene-1H-1,2,3-triazole)berberine
chloride
3
Compound 3 was synthesized from 8 (151 mg, 0.447 mmol) and 29 (177 mg, 0.447 mmol) by the
1
general procedure described above (200 mg, 61%). H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1 H,
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CH=N), 8.98 (s, 1 H, ArH), 8.62 (s, 1 H, ArH), 8.22 (d, J = 9.2 Hz, 1 H, ArH), 8.03 (d, J = 9.2 Hz, 1
H, ArH), 7.80 (s, 1 H, ArH), 7.09 (s, 1 H, ArH), 6.17 (s, 2 H), 5.56–5.41 (m, 6 H), 5.05 (s, 1 H), 4.92
(s, 3 H), 4.68–4.63 (m, 2 H), 4.09 (s, 3 H), 3.90 (d, J = 10.8 Hz, 1 H), 3.79–3.63 (m, 5 H), 3.49–3.28
(m, 6 H), 3.24–3.13 (m, 3 H); 13C NMR (100 MHz, DMSO-d6): δ 151.42, 150.30, 148.14, 145.64,
142.86, 142.06, 137.91, 133.43, 131.15, 127.00, 125.05, 124.56, 122.40, 120.89, 120.79, 108.89,
108.12, 105.97, 102.54, 87.69, 84.11, 78.63, 77.22, 74.70, 72.59, 71.46, 70.44, 67.79, 67.02, 63.67,
57.56, 55.86, 26.83. ESI-TOF HRMS (m/z): calcd for C33H37N4O13+, [M – Cl]+, 697.2352; found,
697.2356.
9-O-(1-(α-D-mannopyranosyl-(1→6)-β-D-glucopyranosyl)-4-methylene-1H-1,2,3-triazole)berberine
chloride
4
Compound 4 was synthesized from 9 (164 mg, 0.447 mmol) and 29 (177 mg, 0.447 mmol) by the
1
general procedure described above (157 mg, 46%). H NMR (400 MHz, DMSO-d6): δ 9.67 (s, 1 H,
CH=N), 8.98 (s, 1 H, ArH), 8.66 (s, 1 H, ArH), 8.21 (d, J = 9.2 Hz, 1 H, ArH), 8.03 (d, J = 9.2 Hz, 1
H, ArH), 7.80 (s, 1 H, ArH), 7.09 (s, 1 H, ArH), 6.17 (s, 2 H), 5.62–5.39 (m, 6 H), 4.93–4.71 (m, 5
H), 4.60 (s, 1 H), 4.49 (s, 1 H), 4.09 (s, 3 H), 3.77–3.55 (m, 6 H), 3.45–3.29 (m, 6 H), 3.21–3.16 (m,
2 H); 13C NMR (100 MHz, DMSO-d6): δ 151.41, 150.28, 148.12, 145.64, 142.92, 142.04, 137.88,
133.43, 131.17, 126.98, 124.93, 124.57, 122.37, 120.88, 120.78, 108.89, 105.97, 102.53, 100.31,
87.84, 78.25, 77.36, 74.22, 72.66, 71.37, 70.62, 69.84, 67.29, 67.00, 65.89, 61.48, 57.57, 55.88,
26.83. ESI-TOF HRMS (m/z): calcd for C34H39N4O14+, [M – Cl]+, 727.2457; found, 727.2454.
9-O-(1-(α-L-rhamnopyranosyl-(1→6)-β-D-glucopyranosyl)-4-methylene-1H-1,2,3-triazole)berberine
chloride
5
Compound 5 was synthesized from 10 (157 mg, 0.447 mmol) and 29 (177 mg, 0.447 mmol) by the
1
general procedure described above (197 mg, 59%). H NMR (400 MHz, DMSO-d6): δ 9.68 (s, 1 H,
CH=N), 8.98 (s, 1 H, ArH), 8.63 (s, 1 H, ArH), 8.21 (d, J = 9.2 Hz, 1 H, ArH), 8.03 (d, J = 8.8 Hz, 1
H, ArH), 7.80 (s, 1 H, ArH), 7.09 (s, 1 H, ArH), 6.17 (s, 2 H), 5.58 (d, J = 9.2 Hz, 1 H), 5.49–5.42 (m,
5 H), 4.93 (t, J = 5.2 Hz, 2 H), 4.81–4.77 (m, 2 H), 4.69–4.66 (m, 1 H), 4.50 (s, 1 H), 4.09 (s, 3 H),
3.83 (d, J = 8.8 Hz, 1 H), 3.78–3.72 (m, 1 H), 3.63 (t, J = 7.2 Hz, 1 H), 3.56 (s, 1 H), 3.45–3.23 (m, 6
H), 3.24–3.13 (m, 4 H); 13C NMR (100 MHz, DMSO-d6): δ 151.41, 150.29, 148.13, 145.65, 142.91,
142.05, 137.90, 133.44, 131.15, 126.98, 124.92, 124.56, 122.37, 120.89, 120.78, 108.89, 105.97,
102.54, 100.94, 87.77, 78.72, 77.22, 72.61, 72.44, 71.14, 70.73, 70.18, 68.84, 67.02, 66.96, 57.56,
55.87, 26.83, 18.33. ESI-TOF HRMS (m/z): calcd for C34H39N4O13+, [M – Cl]+, 711.2508; found,
711.2504.
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DOI: 10.1039/D0OB00327A
Zebrafish maintenance
The light (14 h) and dark (10 h) alternate culture condition was adopted for zebrafish maintenance ,
o
and the cycle temperature was controlled at 28 ± 0.5 C by the air conditioner system to ensure
normal spawning. Before experiment, female and male zebrafishes were placed in the breeding tank
in proportion to 1/1 or 1/2, and were separated by the partition board. In the next day, the female and
male zebrafishes chased each other after contacting and spawning. Then, zebrafish eggs were
maintained with E3 water containing 5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl2, and 0.33 mM
MgSO4). 2-Phenylthiourea (0.2 mM) was added to the water for increasing transparency of zebrafish
larvae. Finally, the larvae were incubated in light incubator at 28 ± 0.5 oC.
Anti-diabetic investigation
The 3-day old zebrafish larvae were placed into a 24-well plate with 10 larvae and 2 mL of E3
water in each well. Larvae were treated with tested compounds (10 μM) for 1 h, and the E3 water
was removed. Then, larvae were incubated with E3 water containing 2-NBDG (0.6 mM) for 3 h, and
the E3 water removed. The resultant larvae were washed with E3 water for 3 times, and anesthetized
using E3 water containing 0.02% tricaine. Fluorescent images of 2-NBDG uptake in zebrafish larvae
were captured on a fluorescent microscopy, and the 2-NBDG uptake was quantified using an Image J
software.
Conflict of Interest
The authors declare no competing financial interests.
Acknowledgments
This work was supported by the Natural Science Foundation of Shandong Province (No.
ZR2018PB007), Young Science Foundation of Shandong Academy of Sciences (No. 2020QN0018),
the Taishan Scholar Project from Shandong Province to Houwen Lin, High-end Talents Program of
Jinan (No. 2019GXRC044), Jinan High-end Foreign Experts Recruitment Program (No. 2019T10),
and National Natural Science Foundation of China (No. 81802629). Furthermore, we thank Mrs.
Ximin Wang for zebrafish maintenance.
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Graphical abstract
DOI: 10.1039/D0OB00327A
Synthesis of disaccharide modified berberine derivatives and their anti-diabetic
investigation in zebrafish using a fluorescence-based technology
Lizhen Wang, Haotian Kong, Meng Jin, Xiaobin Li, Rostyslav Stoyka, Houwen Lin, Kechun Liu
Diglucose modified berberine derivative can dramatically promote the uptake of 2-NBDG both in
zebrafish larvae and their eyes.