Monosaccharide Analogues of Anticancer Peptide R-Lycosin-I: Role of Monosaccharide Conjugation in Complexation and the Potential of Lung Cancer Targeting and Therapy

Article abstract of DOI:10.1021/acs.jmedchem.9b00634

Glycoconjugation is a promising modification strategy for the optimization of peptide drugs. In this study, five different monosaccharide derivatives (7a-e) were covalently linked to the N-terminal of R-lycosin-I, which yielded five glycopeptides (8a-e). They demonstrated increased or reduced cytotoxicity depending on monosaccharide types, which might be explained by the changes of physicochemical properties. Among all synthesized glycopeptides, only 8a exhibited increased cytotoxicity (IC₅₀ = 9.6 ± 0.3 μM) and selectivity (IC₅₀ = 37.4 ± 5.9 μM). The glucose transporter 1 (GLUT1) with high expression in cancer cells was approved to be involved in the cytotoxicity and selectivity enhancement of 8a. Furthermore, 8a but not R-lycosin-I inhibited tumor growth in the nude mice xenograft model without generating side effects intraperitoneally. Taken together, this study reveals the different monosaccharide roles in peptide modification and also provides an optimized anticancer peptide with high activity and selectivity, that is, 8a might be a promising lead for developing anticancer drugs.

Full text of DOI:10.1021/acs.jmedchem.9b00634

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Article
Monosaccharide Analogues of Anticancer Peptide R-lycosin-
I: Role of Monosaccharide Conjugation in Complexation
and Potential of Lung Cancer Targeting and Therapy
peng zhang, jing ma, qianqian zhang, shandong jian, xiaoliang sun, bobo
liu, liqin nie, meiyan liu, Songping Liang, Youlin Zeng, and Zhonghua Liu
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00634 • Publication Date (Web): 05 Jul 2019
Downloaded from http://pubs.acs.org on July 6, 2019
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Monosaccharide Analogues of Anticancer Peptide R-lycosin-I: Role of Monosaccharide
Conjugation in Complexation and Potential of Lung Cancer Targeting and Therapy
Peng Zhang1,2†, Jing Ma , Qianqian Zhang , Shandong Jian , Xiaoliang Sun , Bobo Liu , Liqin
2†
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Nie , Meiyan Liu , Songping Liang , Youlin Zeng * and Zhonghua Liu *
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1
. The National &Local Joint Engineering Laboratory of Animal Peptide Drug Development,
College of Life Sciences, Hunan Normal University, Changsha Hunan 410081, China. E-mail:
liuzh@hunnu.edu.cn
2. The National and Local Joint Engineering Laboratory for New Petrochemical Materials and
Fine Utilization of Resources, Hunan Normal University, Changsha Hunan 410081, China.
E-mail: youlinzengcn@gmail.com
3. State Key Laboratory of Developmental Biology of Freshwater Fish, College of Life Sciences,
Hunan Normal University, Changsha Hunan 410081, China
*
Corresponding author
†
These authors contribute equally to this paper.
Abstract
Glycoconjugation is a promising modification strategy for the optimization of peptide drugs. In
this study, five different monosaccharides derivatives (7a-e) were covalently linked to N-terminal
of R-lycosin-I, which yielded five glycopeptides (8a-e). They demonstrated increased or reduced
cytotoxicity depending on monosaccharide types, which might be explained by the changes of
physicochemical properties. Among all synthesized glycopeptides, only 8a exhibited both
increased cytotoxicity (IC =9.6±0.3 μM) and selectivity (IC =37.4±5.9μM). The glucose
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transporter 1(GLUT1) with high expression in cancer cells was approved to involve in the
cytotoxicity and selectivity enhancement of 8a. Furthermore, 8a but not R-lycosin-I inhibited
tumor growth in nude mice xenograft model without generating side effects by intraperitoneally.
Taken together, this study revealed the different monosaccharide roles in peptide modification and
also provides an optimized anticancer peptide with high activity and selectivity, that is, 8a might
be a promising lead for developing anticancer drugs.
Keyword: Glycoconjugation, anticancer peptides, R-lycosin-I, monosaccharide, xenograft model.
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Introduction
Although there are a growing number of progress have been made in reducing incidence and
mortality rates of cancers, cancers remain
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major cause of mortality worldwide and further strategies for cancer treatment are needed1-3.
Small cell lung cancer (SCLC) is an exceptionally lethal malignancy for which more effective
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therapies are urgently needed . Cation anticancer peptides (ACPs) have been considered as novel
therapeutic candidates due to their ability to kill target cells rapidly and low immunogenicity5, 6
.
However, their development as novel anticancer agents has been hampered by their lower
cytotoxic activity especially in solid tumor, poor serum half-lives, and cytotoxicity to host7, 8. To
address these problems, modification strategies have been applied to develop novel synthetic
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ACPs. There are two principally approaches used for ACP optimization . (1) One approach is
template modification. This strategy includes the systematic sequence truncation, amino acid
substitution, hybridization and/or cyclization, which is characterized by using an active naturally
occurring sequence as a starting “template”9, 10. Some studies demonstrated that amino acid
substitutions and/or sequence truncations at peptide termini can enhance salt and serum
stability11-13, as well as reduce toxicities . Our previous study showed that lysine substitution by
arginine in the sequence of lycosin-I endows this peptide higher anticancer activity in solid
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tumors in vitro and ex vivo . (2) The other one is conjugation of peptides with other materials
including carbohydrate, PEG, fatty acid, photothermal materials etc16-20. Many studies show that
this peptide modification strategy is promising in anticancer drug development. For example,
photothermal material conjugation of ACPs has been found to improve anticancer activity and
selectivity in vitro and in vivo21.
Glycosylation is a naturally occurred process which has many functions, including affecting the
fold, stability, localization, immunogenicity, activity of proteins and other macromolecules.
Therefore, glycosylation is also an important parameter in the optimization of many protein and
peptide drugs. For example, the approved cyclic peptide antifungal drug caspofungin conjugated
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with β-D-glucopyranose shows stronger and broader antifungal activity ; monosaccharide
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modification can also improve the antifungal activity of tunicyclin D ; the addition of sialyl
LacNAc to GLP-1 greatly improves stability against DPP-IV and NEP 24.11 and extends the
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blood glucose-lowering activity in vivo as compared to the native type . Furthermore, the
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“
Warburg effect” explains why there are over expression of glucose-transporters in cancer cells
and drastically increased glucose intake, which provides clinically validated targets for cancer
treatment26-28. Therefore, researches regarding glycoconjugates for specific delivery of various
radiotracers, fluorophores, and organic anticancer drugs have attracted many attentions. Studies
indicate that multifarious anticancer drugs, for example, azomycin, adriamycin, paclitaxel, can be
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delivered specifically to cancer cells by covalently conjugating with carbohydrates . Stephen J.
Lippard group synthesized and characterized six positional isomers of glucose-platinum
conjugates which exhibit different cellular uptake and cytotoxicity30, 31. This study also well
reveals specific cancer targeting through glucose-transporter mediated uptake in vitro and in vivo.
In addition to, by covalently conjugated with maltose, the chemical and biological activity of
Eugenol were improved 32.
Our previous study demonstrated that R-lycosin-I, an analogue derived from the anticancer
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peptide lycosin-I, is a promise lead from the development of anticancer peptide drugs . However，
this peptide displays some drawbacks including low activity, selectivity and stability in serum.
Considering the advantages of carbohydrate modification mentioned above, we made an attempt
to optimize R-lycosin-I, that is, to improve its anticancer potential by conjugate monosaccharides
to R-lycosin-I. In this study, five glycopeptides were synthesized and characterized by coupling
monosaccharide derivatives 7a, 7b, 7c, 7d and 7e to the N-terminal of R-lycosin-I named as 8a,
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b, 8c, 8d and 8e, respectively. Compared with R-lycosin-I, they displayed stronger or weaker
cytotoxic activities depending on monosaccharide types, which might be explained by the
alteration of the physiochemical properties including size, zeta potential, secondary structure of
the glycopeptides. Interestingly, 8a showed not only higher cytotoxic activity but also higher
selectivity against cancer cells. The glucose transporter GLUT1 which is high expressed in cancer
cells might involve in this process. Furthermore, 8a inhibited the tumor growth in ex vivo and in
vivo experiments. Taken together, our study developed a physicochemical properties-activity
relationship to describe the roles of different monosaccharides in the anticancer process, and
provided clues and leads for the development of anticancer drugs with high efficacy and safety.
Results
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Synthesis of R- lycosin-I-monosaccharide conjugates
Five monosaccharide derivatives, 3’-aminopropyl -D-glucopyranoside (7a), 3’-aminopropyl
-D-galactopyranoside (7b), 3'-aminopropyl -D-mannopyranoside (7c), 3'-aminopropyl

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-L-arabinopyranoside (7d), 3'-aminopropyl 2-deoxy-2- acetamido--D-glucopyranoside (7e),
were synthesized from monosaccharide by acetylation, 1-O-selective deacetylation,
trichloroacetimidation, glycosylation, deprotection, and staudinger reaction (see Experimental
Section, Scheme 1-5). The structures of target compounds and some synthetic intermediates were
characterized by H NMR and 13C NMR. The NMR spectrums of monosaccharide derivatives
were included in the Supporting Information Page S3-S8. The peptide R-lycosin-I was synthesized
using the standard 9-fluorenyl-methoxycarbonyl (Fmoc) solid-phase peptide synthesis method.
The monosaccharide derivative was conjugated to the N-terminal of R-lycosin-I with glutaric
anhydride as the linker between the monosaccharide derivative and the peptide. The five
monosaccharide derivatives (7a, 7b, 7c, 7d and 7e) were used to synthesize five glycopeptides,
which were named as 8a, 8b, 8c, 8d and 8e, respectively. As shown in Figure 1A-B, the synthetic
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a and 8b were purified by using prepared C18 RP-HPLC and analytical C18 RP-HPLC analysis
indicated that the two synthetic glycopeptides all had high purity (Figure 1A-B, inset). So did the
other three glycopeptides 8c, 8d and 8e (Figure S1A-C). As the determined by MALDI-TOF MS,
the average molecular masses were 3263.47 Da for 8a, 3263.39 Da for 8b, 3264.20 Da for 8c,
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229.23 Da for 8d and 3304.49 Da for 8e (Figure 1C-D; Figure S1D-F), which were same as those
of calculated ones based on the sequences, respectively. The data confirmed the successful
synthesis of the five glycopepitdes. The fluorescein isothiocyanate (FITC) labeled 8a was also
synthesized by coupling FITC to the side chain amide group of the N-terminal lysine residue of
R-lycosin-I (see Supporting Information Page S2).
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Figure 1. The purification and molecular mass determination of 8a and 8b. The purification of 8a
(A) and 8b (B) by using RP-HPLC (column, Welch, C18, 300 Å, 10 × 250 mm). The synthetic
glycopeptides were eluted using a linear acetonitrile gradient (0-80% acetonitrile/0.1% TFA) at a
-1
flow rate of 3.0 ml min . The elution of the peptides was monitored at 280 nm. Inset, the purity of
purified glycopeptides was analyzed by analytical RP-HPLC. The molecular masses of 8a (C) and
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b (D) were determined by MALDI-TOF MS.
Cytotoxicity profile of R-lycosin-I and their conjugates
The cytotoxic activity of the five glycopeptides was evaluated on human lung carcinoma
cells (A549) and non-cancer cells (HEK-293T). The IC50 values were calculated to evaluate the
potency of glycopeptides, which represent the concentrations of the glycopeptides that confer 50%
growth inhibition of cells. As shown in Figure 2A, for A549 cells, the cytotoxic activities of the
peptides were ranked as 8b ≈ 8d ≈ 8a > R-lycosin-I > 8e > 8c; while for HEK-293T cells, those
were ranked as 8b > 8d > R-lycosin-I > 8e > 8a > 8c. These data indicated that 7b and 7d
modification enhanced the cytotoxic activity of R-lycosin-I on both cell lines, while 7c and 7e
modification resulted in reduction or loss of activity. It was interesting that 7a coupling to the
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N-terminal of R-lycosin-I had increased inhibition on A549 cancer cells but decreased effect on
HEK-293T non-cancer cells, suggesting improved selectivity. In addition to CCK-8 assay, another
cell viability assay also examined. Trypan blue is a cell-active dye that is commonly used to detect
cell membrane integrity and is often used to detect cell survival. Live cells are not stained blue,
and dead cells are dyed light blue. Therefore, according to the CCK-8 assay, we choose the IC50
value of R-lycosin-I and performed the trypan blue staining. As shown in Figure 2B and S2, when
treated with 15 μM R-lycosin-I, about half of cells turned blue; cells treated with 8a, 8b and 8d
were basically stained blue and a few blues were observed to the 8c, 8e and control. Next, 8a and
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b were further applied in the expanded cytotoxicity tests. As shown in Table 1, 8b exhibited high
potency and inhibited cancer cell growth with IC50 values of 4-9 µM in cancer cell lines
MDA-MB-231, H460, H1437, HGC-27 and PC-3) and non-cancer cell lines (HEK-293T and
(
L-02).The cytotoxic activity of 8a against those cancer and non-cancer cell lines was 6.89-13.05
µM and 25.91-37.44 µM, respectively, unlike 7b, further confirming that 7a conjugation enhanced
not only the cytotoxic activity but also the selectivity on cancer cells over non-cancer cells. The
A549 cells and 8a were chosen as the study subject in follow-up experiments. We further verified
the effects of 8a on cell proliferation by colony formation assay. Compared with control, the
treatment group caused a strong inhibition of colony formation at the concentration of 4 μM 8a
(Figure 2C-D). The data indicated that 8a could also inhibit cell growth at low concentration less
than IC values. On the other hand, we measured if the glycopeptides triggered apoptosis of A549
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cells using flow cytometry (Figure 2E). The percentage of apoptotic cells increased from 4.59 %
in the control to about 40.16 % in 8a-treated A549 cells at the concentration of 8 μM, respectively.
However, the 8 μM R-lycosin-I treatment did not cause obvious apoptotic effect on A549 cells.
These results indicated monosaccharide conjugation to the N-terminal of R-lycosin I could
incearse or reduce the cytototoxic activity of the peptide depending on the monosaccharide
types.Note that damage to the integrity of the cell membrane also causes Annexin V to be colored.
So, in order to accurately exam the apoptosis, the caspase-3 activity was performed as described
blow. For the 8a, in low concentration, its cytotoxic activity may be derived from the proliferation
inhibition and apoptosis inducement. These results indicated monosaccharide conjugation to the
N-terminal of R-lycosin-I could incearse or reduce the cytotoxic activity of the peptide depending
on the monosaccharide types.
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Figure 2. The cytotoxic activities of R-lycosin-I and five glycopeptides. (A) The IC values of the
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six peptides on A549 and HEK-293T cells as determined by CCK-8 assay. IC50 value was
determined by averaging three repeated experiments. Each experiment consisted of 3 repetitions.
(B) Trypan blue staining of 5glycopeptides and R-lycosin-I. (C) Colony formation viability of
A549 cells treated with 4 μM R-lycosin-I and 8a were evaluated by clonogenic assay. (D)
Quantitative results are illustrated for clonogenic assay, (n=3). (E) Cell apoptosis in A549 cells
treated with 8 μM R-lycosin-I and 8a determined by flow cytometry. The circular dichroism
spectra of R-lycosin-I and glycopeptides. (E) In the presence of 50% TFE at 25 °C. (F) In benign
medium (PBS) at 25 °C. (G) The morphology of R-lycosin-I and glycopeptides analyzed using
TEM.
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Table 1 Cytotoxic activity (IC50) activities of peptides against non-cancer cells and cancer cells.
IC50 (μM)
Peptides
HEK-293T
25.8±3.7
37.4±5.9
8.3±0.4
L-02
MDA-MB-231
10.5±0.5
A549
H460
H1437
HGC-27
PC-3
R-lycosin-I
12.6±1.1
25.9±3.8
4.8±0.3
14.3±1.3 12.9±1.3 16.5±1.3 12.5±1.2 15.6±0.6
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8a
8b
10.1±0.2
9.6±0.3
7.0±0.3
8.5±0.4 13.0±0.2 12.6±0.6 6.9±0.4
4.9±0.9 5.3±0.2 5.3±0.2 4.9±0.2
6.0±0.2
IC50 value (μM) represents the concentration of a certain peptide at which cell viability was inhibited by 50% in
comparison with the untreated cells. IC50 value was determined by averaging three repeated experiments. Each
experiment consists of 3 repetitions.
Physicochemical characteristics of glycopeptides
Some physicochemical parameters such as shape, size, surface charge, hydrophobicity, secondary
18
structure, are known to play important roles in cytotoxic activity of ACPs . Therefore, we
assumed that the varied cytotoxic activities of the glycopeptides might in part be related to the
parameter changes which were resulted from the conjugation of different monosaccharides. The
TEM analysis showed that all of the six peptides were spherical in shape with a diameter of
~80-150 nm (Figure S3). As shown in Table 2, all the peptides had similar hydrophobicity as
represented by retention time in RP-HPLC analysis, except that 8e had slightly higher
hydrophobicity; the sizes, surface charges and helicities of R-lycosin-I were all affected by the
monosaccharide modification. The zeta potentials are +26.13±3.45 mV for R-lycosin-I,
+
25.9±1.24 mV for 8a, +50.43±2.94 mV for 8b, +3.81±1.02 mV for 8c, +35.73±7.24 mV for 8d,
and+9.66±2.03 mV for 8e. The hydrodynamic sizes as determined by DLS are 252.77±28.25 nm
Polymer dispersity index (PDI) = 0.601±0.033) for R-lycosin-I, 471.67±74.28 nm (PDI = 0.689
0.028) for 8a, 232.30±14.23 nm (PDI = 0.259±0.016) for 8b, 1101.00±80.89 nm (PDI = 1.000)
(
±
for 8c, 222.57±9.73 nm (PDI = 0.458±0.071) for 8d, and 696.70±45.01 nm (PDI=0.919±0.062)
for 8e. Same as R-lycosin-I, the five glycopeptides adopted random coil conformation in PBS
buffer and alpha-helical conformation in 50% TFE as determined by CD (Figure 2 F -G). As
compared with R-lycosin-I, 8d displayed a perfect -helical structure, 8a and 8b had similar
helicities, 8e had reduced helicities, whereas 7c modification drastically disrupted the stabilization
of the α-helix conformation of R-lycosin-I (Table 2).
It has been accepted that the cytotoxic activity of ACPs was affected by multi-factors. Most
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ACPs are positively charged, which may facilitate the electrostatic attraction between ACPs and
the negatively charged components of the membrane of cancer cells. Because cancer cell surface
contains more negatively charged components than that of non-cancer cells, the positive charges
of ACPs are believed to be important to their selective action on cancer cells over non-cancer
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cells . The zeta potential which is considered strongly cationic when the value is greater than +30
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mV . The strongly cationic particles generally displaying stronger toxicity associated with cell
membrane disruption, which may cause peptide loss of selectivity. The size effect proposed by
Zheng et al. indicated that relatively smaller particle size is more likely to be taken up by cells35.
The increased hydrodynamic size may result from peptide self-association in aqueous solutions36,
37,
which may decrease the ability of the peptide to dissociate, and penetrate into the cytoplasmic
38
membrane to kill cells . In addition，amphipathic helical conformation is often required to the
cytotoxic activity of ACPs.
With these determined parameters, we attempted to establish the relationship between the
cytotoxicity potency and these parameters of the peptides. Accordingly, the five glycopeptides
could be classified into three groups. (1) 8b and 8d. Their increased activity might closely
correlated with their sharply increased net positive charges ， reduced hydrodynamic sizes and
high helicities, which might even cause their loss of selectivity on cancer and non-cancer cells. (2)
8
c and 8e. It was evident that their low activity might be derived from their reduced net charges,
greatly increased hydrodynamic sizes and most importantly low helicity. These parameters might
function in a combination way. The large sizes of 8c and 8e might be resulted from their
self-association in aqueous because of small net charges and low helicities. (3) 8a. It displayed
unchanged net charges, increased hydrodynamic size and high helicity. It seems that these changes
could not explain the increased activity and selectivity of 8a. Therefore, beside the
physicochemical characteristics, some biological properties would be involved, which would be
described as below.
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Table 2 Physicochemical parameters of R-lycosin-I and glycopeptides
Benign
50% TFE
Zeta potential Average sizes
Hydrophobicitya
Peptides
PDI
(mv)
(nm)
t
R (min)
[
θ]222 % Helixc [θ]222b % Helixc
b
R-lycosin-I +26.13±3.45
252.77±28.25
471.67±74.28
232.30±14.23
1101.00±80.89
222.57±9.73
0.601±0.033
0.689±0.028
0.259±0.016
1.000
15.14
15.87
15.96
15.58
15.43
17.80
-1724.82 12.48 -12911.59
-1830.29 13.50 -12143.39
-2186.47 15.53 -12373.36
93.54
89.64
87.92
29.60
0
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0
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8a
+25.90±1.24
+50.43±2.94
+3.81±1.02
+35.73±7.24
+9.66±2.03
8
b
8
c
-1380.73 9.94
-4090.38
8d
0.458±0.071
0.919±0.062
-2057.99 14.97 -13807.07 100.00
-1455.69 10.57 -10083.70 73.00
8
e
696.70±45.01
a
The overall hydrophobicity was represented by retention time (t
The mean 222 values (in deg.cm .dmol ) at wavelength 222 nm were measured at 25°C by circular dichroism
R
) by RP-HPLC at room temperature.
b
2
-1
spectroscopy.
c
The helical content (in percent) of a peptide is relative to the molar ellipticity value of the peptide.
Intracellular distribution and cellular uptake analysis
Our previous study indicated that R-lycosin-I can interact with cell membrane and penetrate cell
membrane to locate in cytoplasm. We wonder whether 7a modification would affect such an effect
of R-lycosin-I. In order to precisely locate 8a in A549 cells, the fluorescence distribution of
FITC-modified 8a in cells was visualized using laser confocal microscopy after 2 h incubation at
the concentration of 15 μM. As shown in Figure 3A, the two peptides were observed inside A549
cells, but there were differences regarding their distribution in cells. First, FITC-R-lycosin-I
exhibited granular-like distribution in the cytoplasm, may be because some R-lycosin-I molecules
still accumulated in granular structures in cytoplasm when entering inside cells. Compared to
R-lycosin-I, 8a relatively dispersed and equally distributed in the cytoplasm, as observed for
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histidine-rich peptides Ctry2459-H2 and Ctry2459-H3 , indicating that they could escape from
vesicles more easily than R-lycosin-I, which might account for their enhanced cytotoxic activity.
Second, the fluorescent signals of 8a but not R-lycosin-I were found to be aggregated in the cell
nucleus, indicating that monosaccharide modification could significantly enhance the ability of the
peptide to penetrate membrane including the nuclear envelope (NE). It was reported that after
translocation into cell nucleus, the peptide, Azurin, would interact with nucleus targets, such as
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P53, triggering cell apoptosis . In this respect, 8a may enhance the NE penetration to improve the
cytotoxic activity.
Next, we determined that the effect of 7a on the cellular uptake of R-lycosin-I. As shown in Figure
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B-C, for R-lycosin-I at low concentration of 6.25 μM, long time treatment could not lead to the
increment of fluorescent cells. When the concentration of the peptides was increased from 6.25
μM to 12.5 μM, the number of fluorescent cells would increase by 2-3 times. At the same
concentration (6.25 μM or 12.5 μM) or time point, 8a demonstrated significantly higher cell
uptake capacity than R-lycosin-I, consistent with their higher cytotoxic activity on A549 cells than
R-lycosin-I. These data implied that their cytotoxicity potency might be positively correlated with
their cell uptake capacity.
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Figure 3. Cellular distribution and cell uptake ratio of R-lycosin-I and 8a. (A) Peptides
distribution in A549 cells examined by laser scanning confocal microscopy. (C and D)
Quantitative comparison of cellular uptake of R-lycosin-I and 8a at the concentration of 6.25 μM
(C) and 12.5 μM (D), respectively, (n=3). The number of fluorescence-labeled cells was counted
using Cellometer K2. The scale bar represents 50 μm.
Glucose-transporter mediated cytotoxic activity of 8a
It was interesting that 8a demonstrated much higher selectivity on cancer cells over
non-cancer cells than R-lycosin-I and 8b. However, this seem cannot be explained by chemical
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and biological properties mentioned above. It has been accepted that glucose transporter 1
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(
GLUT1) is broadly over-expressed in many different cancers , which provides a good surface
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receptor for selectively targeting cancer cells . Therefore, we wondered if the high expression of
GLUT1 on cancer cells contributed to the enhanced selectivity of 8a through 7a and GLUT1
interaction. Firstly, the cytotoxic activity of the 8a was determined in the absence and presence of
the GLUT1 inhibitor STF-31 on A549 cells. As shown in Figure 4A, the cell viability of A549
cells treated with 12.5 μM 8a was proximately 54% and 40% (P<0.05) in the absence and
presence of STF-31 treatment, respectively. However, there was no obvious difference when A549
cells treated with 12.5 μM R-lycosin-I, 8b and 8d (Figure S4A) in the presence and absence of
STF-31. In addition, LDH assay performed to exam the selectively penetration of cancer cell and
non-cancer cell membrane when treated with 12.5 μM 8a, 8b and 8d which showed enhanced
cytotoxicity compared to R-lycosin-I. As shown in Figure S4B, this result demonstrated that only
the 8a had selectivity.
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Next, when GLUT1 expression in A549 cells was knocked down by RNAi, the cytotoxic
activity of 8a on A549 cells was accordingly reduced. Compared with control oligos, GLUT1
RNAi oligos led to approximately 50 % knockdown of GLUT1 expression in A549 cells (Figure
4
B; Table S1), which made the IC50 value of 8a increased by 1.4 folds (13.4 μM in A549 cells
with low GLUT1 expression vs 9.9 μM in cells with normal GLUT1 expression) (Figure 4D).
Accordingly, the cellular uptake of FITC-8a was also significantly reduced in response to knock
down of GLUT1 expression in A549 cells (Figure 5E). In addition, HEK293T cells exhibited
lower GLUT1 expression than A549 cells (Figure 4C). These data indicated that the high
expression level of GLUT1 in cancer cells might be related to the selective cytotoxic activity of
8
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Figure 4. Glucose-transporter mediated cytotoxic activity of 8a
(
A) Effect of GLUT1 inhibitor STF-31 on the cytotoxic activity of R-lycosin-I, 8a and 8b. A549
cells were pre-incubated with 1 μM STF-31 for 4 hours and then treated with 12.5 μM peptides for
h. (B and C) Western blot analysis of GLUT1 expression in A549cells and HEK293T cells. (D)
2
Representative dose-response curves for A549 and A549-GLUT1 RNAi cells treated with a series
of concentrations of 8a. (E) The cellular uptake of FITC-8a in A549 and A549-GLUT1 RNAi
cells, respectively at the concentration of 12.5 µM for the exposure time of 2 h, (n=3).
Mechanism underlying the cytotoxic activity of 8a
As mentioned above, the two peptides, R-lycosin-I and 8a, could bind to cell surface and
enter into the interior of A549 cells, which could induce cell death. The binding of the peptides to
cell surface result in cell membrane disruption. Actually, this is a common mechanism shared by
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most ACPs . SEM examination was applied to observe the effect of 8a on cell morphology.
Untreated A549 cells showed plump spindle cell morphology (Figure 5A). In contrast, A549 cells
treated with 15 μM R-lycosin-I (Figure S5A) and 8a (Figure 5B) for 24 h showed marked
alterations in cell morphology. Mostly shrunken cells with membrane disruption and lysis were
observed. The release of lactate dehydrogenase (LDH) was widely used to measure membrane
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integrity. The peptide treatment indeed led to LDH release from A549 cells. An obvious release
occurred at the peptide concentration near IC50 value of the corresponding peptide. For example,
considering 1% TritonX-100 caused 100% release of LDH from A549 cells, the incubation of 12.5
μM 8a or R-lycosin-I for 2 hours resulted in more than 30 % LDH release, respectively, indicating
the rapid permeabilizing effect on the cancer cell membranes (Figure 5D). With the increasing of
peptide concentration, more LDH release was observed, indicating more membrane
permeabilization. These data suggested that rapid membrane disruption or membrane-lytic activity
should be one of the mechanisms underlying these peptides inducing cell death.
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In addition, some cells seem presented apoptosis bodies and severe surface bubbling (Figure 5C
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and Figure S5B), a characteristic event during the execution phase of cell apoptosis . Considering
mitochondrion plays a central part in cell apoptosis and many crucial factors and events in
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apoptotic death are regulated by it , the interaction of 8a with mitochondrion was determined by
co-localization assays. Expectably, the merge images showed overlapping between signals of
FITC-labeled peptides and Mito-Tracker red (Figure 5E), which indicated that 8a could be
localized to mitochondria and implied that mitochondria-mediated cell apoptosis pathway might
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be involved . Mitochondrial membrane potential (MMP) measurement demonstrated that the
treatment with 15 μM peptides caused a significant loss of MMP in A549 cells (Figure 5F-G).
Accordingly, MMP depolarization is often accompanied with increasing mitochondrial membrane
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permeability and Cytochrome C (CytC) release . Therefore, immuno-fluorescence imaging was
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performed according to the method reported by chen C et.al . As shown in Figure 5H, CytC in
A549 cells displayed clear, grainy staining distribution in control cells, in contrast to diffuse
staining in the cells treated with 15 μM peptides, indicating the transportation of mitochondria
Cyt-C to cytoplasm. The released CytC further activates caspase cascade reaction and triggers cell
apoptosis.50 The caspase 3 activity was therefore examined using the specific protease-peptide
substrate chromomeric reaction due to the caspase 3 activation was known as an effector of
apoptotic pathways.51 When treated with peptides, respectively, the caspase 3 activities in A549
cells were elevated by approximately 1.5-fold compared with the control (Figure 5I). These data
indicated that 8a brought about apoptosis of A549 cells through mitochondria-mediated pathway.
These results mentioned above characteristically demonstrated that 8a could initiate cell
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membrane disruption and mitochondria-dependent apoptotic pathway, then synergistically
achieving maximized cytotoxicity efficacy.
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Figure 5. 8a caused direct cell membrane disruption and cell apoptosis. Representative SEM
micrograph of A549 cells in control (A) or treated with 15 μM 8a (B and C) for 24 h. (D) LDH
leakage in A549 cells after treatment with R-lycosin-I and 8a for 2h, respectively, (n=3). (E)
Co-localization with mitochondria after treatment with FITC-labeled peptides for 24 h. Nuclei
(blue color) and mitochondria (red color) were stained with DAPI and Mito-Tracker Red,
respectively. (F) Fluorescent images of MMP staining with JC-1 in A549 cells before and after
incubation with 15 μM R-lycosin-I and 8a for 24 h, respectively. (G) Quantitative analysis of
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MMP in A549 cells treated with R-lycosin-I and 8a respectively, (n=3). (H) Immuno-fluorescence
staining of CytC in A549 cells before and after treatment with 15 μM R-lycosin-I and 8a for 24 h,
respectively. (I) Caspase-3 activity in A549 cells before and after treatment with 15 μM
R-lycosin-I and 8a for 24 h, respectively, (n=3). The scale bar represents 100 μm.
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Ex vivo antitumor performance
Overwhelming evidences approve that ex vivo 3D tumor model more accurately reflects the
morphology, growth kinetics, and protein expression profile of human tumors than simple
two-dimensional cell monolayer.52 Therefore, we performed a series of experiments to evaluate
the activity of R-lycosin-I and 8a on 3D tumor spheroids of A549 cells. Studies showed that
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penetration into tumor spheroids is required for the inhibitory activity of anticancer drugs. First,
we examined the penetration ability of the two peptides. A549 3D tumor spheroids were cultured
with 30 μM FITC-modified peptides for 2 h and the FITC-fluorescence was determined by laser
confocal microscopy using Z scan model (Figure 6A). In the 24 μm layer of the tumor spheroids
treated with R-lycosin-I, no green fluorescence was detected in the center of the tumor spheroids,
indicating that it might penetrate into the spheroids less than 16 μm in distance. However, when
treated tumor spheroids with 30 μM 8a, green fluorescence was detected even in the 48 μm layer,
showing that monosaccharide modification markedly improved the peptide penetration ability into
3
D tumor spheroids.
Next, in order to evaluate the activity of the two peptides on the tumor spheroids, A549 3D
tumor spheroids were treated with the peptides at the concentration of 30 μM for six days, which
were added three times, at day 0, day2 and Day 4. As shown in Figure 6B, in the control group,
spheroids were gradually growing and generated tight spheroids, but looser morphology, physical
shrinking and dissociation of the three-dimensional structure were observed in the peptide
treatment groups. Note more evident changes were observed in 8a groups than these of
R-lycosin-I group, which was consistent with the tumor spheroid volume measurement and
comparison (Figure 6C).
Finally, the cell viability of tumor spheroids in the 8a groups was significantly lower than
those observed for the control and R-lycosin-I groups, as revealed by the CCK-8 cytotoxicity
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assay (Figure 6D). The increased activity of 8a might be due to the improved cytotoxicity in
two-dimensional cell monolayer, higher stability in serum (described below), and more effective
penetration into the tumor spheroids. Given the relatively superior performance on the 3D tumor
spheroids, we further performed in vivo animal model test.
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Figure 6. The effect of R-lycosin-I and 8a on the 3D tumor spheroids of A549 cells. (A) The
Z-stack confocal microscopy images of the A549 3D tumor spheroids treated with FITC-modified
R-lycosin-I and 8a at a concentration of 30 μM for 2 h, respectively. The spheroids were imaged
using an Olympus FV1000 confocal microscope, preceding every 8 μm. Scale bar: 100 μm. The
tumor spheroids size images(B), volume (C), and cell viability (D), were obtained in the presence
30 μM R-lycosin-I and 8a, respectively, (n=3). The scale bar represents 100 μm.
Effect of 8a on inhibiting tumor growth in nude xenograft mice
The in vivo antitumor activities of R-lycosin-I and 8a were investigated using the
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A549-luciferase xenograft model of lung cancer in nude mice . R-lycosin-I and 8a had the same
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cytotoxic activity on A549-luciferase cells as on A549 cells used above (Figure S6). The lung
tumor xenograft models of mice injected with 8a showed strong fluorescent signals around tumor
with 6 h post-injection (Fig. 7A). Compared with FITC treatment which had an extensive
distribution in kidney and lung, the fluorescence of peptides treatment was detected in the liver,
with minimal in the spleen and no detectable activity in the heart. Two weeks after implantation of
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A549-luciferase cells, the average tumor volume reached 150-180 mm . R-lycosin-I and 8a were
intraperitoneally injected every two days at the dose of 9 mg/kg. At day 14, significant inhibition
of tumor growth was found in the 8a treatment group as revealed by bioluminescent imaging, as
compared with R-lycosin-I and PBS control groups (Figure 7B, upper). Furthermore, as shown in
Figure 7C, a certain degree of inhibition of tumor growth was observed in R-lycosin-I treatment
group compared to the control group, but only 8a had a significant tumor volume reduction. On
the other hand, if the two peptides were administrated by intratumor injection, a significant
inhibition in tumor growth was observed during the course of experiments in mice treated with the
two peptides at the dose of 3 mg/kg (Figure 7B, lower and 7D). In the way of intratumor
administration, due to the drug can reach the tumor directly without blood circulation and there are
only 1.6 times of cytotoxicity between R-lycosin-I and 8a on A549 cells, R-lycosin-I and 8a
showed similar activity to inhibit A549-luciferase tumor xenograft growth in vivo. It was
noteworthy that no significant body weight changes were observed whether in intraperitoneal or
intratumor treatments, indicating that these peptides did not diminish their overall health (Figure
7
E-F). H&E staining showed no obvious changes in the primary organs (heart, liver and kidney)
after treatment with the two peptides, indicating no potential toxicity of the two peptides to these
organs (Figure 7G). In addition, our data indicated that 8a is stable in serum and retained
approximately 80% of its inhibitory activity in the exist of 10% serum within 48 hours, but
R-lycosin-I was lack of most activity within 24 hours (Figure S7). This result indicated that the 8a
have higher serum stability than R-lycosin-I that will facilitate the enrichment of more 8a at the
tumor. Furthermore, TUNEL assays demonstrated much more apoptotic cells in the tumor tissues
of 8a group compared with those of groups (Figure 7H). All data above confirmed the in vivo
anticancer activity of the two peptides, but only 8a possessed efficacy in both intratumor and
intraperitoneal injections.
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Figure 7. R-lycosin-I and 8a inhibited A549-luciferase tumor xenograft growth in vivo. (A)
Representative ex vivo fluorescence image of organs isolated from the animals after 6 hours
administration of peptides; R-L represents the anticancer peptide R-lycosin-I. (B)bio-luminescent
imaging on A549-luciferase tumor-bearing mice at the final day after 14 days for intraperitoneal
(
upper) and 12 days for intratumor injection (lower). (C)Tumor inhibition curves for
intraperitoneal injection (n=5). (D) Tumor inhibition curves for intratumor injection (n=5).
E)Toxicity analysis by monitoring body weight after the peptide administration for intraperitoneal
(
injection. (F) Toxicity analysis by monitoring body weight after the peptide administration for
intratumor injection. (G) H&E assay for mice heart, liver and kidney tissues. The treatments were
administrated via the intraperitoneal every other day for seven total times. (H)TUNEL assay for
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mice tumor tissues. The treatments were administrated via the intraperitoneal every other day for
seven total times.
Discussion and conclusions
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An unprecedented number of marketing approvals (6 peptides) have witnessed a resurgence
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of peptide drug development which has greatly inspired the researchers . The anticancer peptides
(ACPs) have been described as promising molecules to treat cancer cells due to their kill target
cells rapidly, broad spectrum activity and hardly to develop resistance56-58. Except those
extraordinary properties, several obstacles have also appeared in development of peptide-based
drugs for example poor pharmacokinetics, low cytotoxic activity to solid tumor and lack of
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selectivity . Some strategies have been attempted to develop peptide molecules with optimal
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therapeutic index in the anticancer field. Strategies such as combination therapy , peptide
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template modification and covalent coupling modification can immensely improve the peptide
performance. Among them, glycoconjugation is one of effective modification method for the
optimization of anticancer drugs including serum stability enhancement, tumor targeting
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improvement and cytotoxicity increased . Here, five glycopeptides were produced by covalently
linked different monosaccharides (7a-e) to the N-terminal of R-lycosin-I. This article is mainly
from two aspects to expound the research of glycoconjugation.
One aspect is significance of glycosylation as a strategy to enhance the selectivity and
cytotoxicity of ACPs. Cytotoxicity assay including CCK-8 and trypan blue staining were used to
measure the cytotoxic activity or selectively of 5 glycopeptides in cancer cell lines. In the paper,
we used some cell lines in our laboratory as cytotoxicity test subject to make the conclusion of the
change of cytotoxicity profile. Actually, there are many kinds of cell lines, and it is more difficult
to test all of them with 5 glycopeptides which indicated that other cells may not have the equal
behavior as the A549 and HEK293T. The result in Table 2 also demonstrated glycopeptides
exhibit different cytotoxic activities against various cell lines. The primary target of most ACPs is
cell membrane, and the interactions between cells and ACPs or the inherent membrane
constitutions between cancer cells and non-cancer cells provide a valuable information and an
opportunity to optimize the ACPs. It is well accepted that the cytotoxicity and selectively of ACPs
are determined by numerous factors such as shape, size, surface charge, hydrophobicity, and
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secondary structure63 and the main mechanism of ACPs induce the cell death is destabilizing the
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membrane through multistep process . Systematic combined physicochemical and biological
properties to explain various cytotoxicity after monosaccharide conjugation provides a better
understanding of the relationship between these factors and it also provides an effectively
information to modify such peptide. Glucose transporter 1 (GLUT1) is broadly over-expressed in
many different cancers, which provides a good surface receptor for selectively targeting cancer
cells. The improvement selectively of 8a closely related to the expression of GLUT1 cancer cell
and non-cancer cell (Figure 4C). The glucose transporter uptake (Figure 4A) and internalization
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(data not shown) synergistically increased cell uptake of 8a. The way 8a was taken up by cells is
diverse due to it contains both monosaccharides and peptide elements. The former may not only
facilitate 8a binding to GLUT1 to enhance its enrichment on the membrane and then
internalization by the cells but also increase the glucose transporter uptake. Due to ACPs exert the
cytotoxicity via a membrane disruption mechanisms only when its binds to the membrane to a
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‘critical’ concentration , the enhanced cytotoxicity and selectivity might be explained as follows:
the higher expression level of GLUT1 might attract more 8a molecules to accumulate on the
surface of cancer cells; consequently, 8a might form a relatively higher concentration on cancer
cell surface and then for cancer cells tend to induce cell death at a relatively lower concentration in
solution than that for non-cancer cells.
Another aspect is efforts in the identification of 8a as potential lead for anti-cancer drug
development. Our previous data showed R-lycosin-I could inhibit cancer cells growth though
15
mitochondria-mediated cell death pathway . Accordingly, a series experiments including MMP
measurement, cyt C release and caspase-3 activity detection (Figure 5F-I) verified that the
mitochondria-dependent apoptotic pathway was also involved in 8a treatment. Notably, rapid
membrane disruption or membrane-lytic activity were appeared (Figure 5A-D) which is the main
mode of action in 8a brought about cell death and this mechanism was confirmed in majority
ACPs. Due to 8a exhibited relatively superior performance on the 3D tumor spheroids which more
accurately reflects the real status of tumors than cell monolayer, in vivo animal model was
performed to assess the anticancer potential of the molecule. Significant tumor volume reduction
was observed in 8a not in R-lycosin-I treatment group by intraperitoneally. The highest
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bio-stability in serum is crucial for the in vivo therapeutic effects of drugs. It has been reported
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that glycosylation modification can enhance the bio-stability of peptides . The data could explain
the low efficacy of R-lycosin-I in vivo. It has been reported that particles with zeta potentials
outside of the range of -30 mV to +30 mV are prone to opsonization, leading to recognition and
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clearance by mononuclear phagocyte system , it seemed that 8a did not probably undergo such
clearance because of its moderate zeta potential. Furthermore, 8a was able to target GLUT1 whose
expression is high in cancer cells, making 8a tend to accumulate in cancer tissue, uptake by tumor
cells and then suppress tumor growth efficiently. However, the fact that these tumors by
intratumor treatments were not completely eradicated and these tumors volume by
intraperitoneal treatments were also constantly growing although smaller volume than the control
can be explained in difficulties to diffuse through the necrotic area and relatively fast clearance of
66
the peptides from the tumor site . Therefore, further modification the 8a with more therapeutic
index to make it clinically applicable is our research focus in the next step.
In summary, the goal of this study is to improve the anticancer potential of R-lycosin-I. Five
peptide-monosaccharide conjugates were synthesized and characterized. Fortunately, it was found
that 7a conjugation rendered R-lycosin-I higher cytotoxic activity and selectivity, two important
parameters for therapeutic development of ACPs. The anticancer potential of 8a was derived from
the data at three levels, cell assay, 3D tumor spheroids and animal tumor models. Compared with
R-lycosin-I, 8a displayed more potent cell penetrating ability, which makes more 8a molecules
distributed inside cancer cells and subsequently causes more cell death. Direct cell membrane
disruption and mitochondria-mediated cell apoptosis were involved in the cell death induced by
8
a. We also found that glucose transporter GLUT1 played important role in the cancer
cell-selective killing of 8a, which provides new evidence for the targeted anticancer drug
development based on GLUT1. 8a had superior performance on the A549 3D tumor spheroids,
that is, it could potently penetrate into and suppress the growth of tumor spheroids. Furthermore,
because of the optimized properties of 8a, intraperitoneal injection of 8a demonstrated good
therapeutic effect on A549 tumor models of mice without evident side effects, which provides
direct evidence confirming the potential application of 8a in anticancer drug development. In
addition, our study established the relationship between cytotoxic activity and physiochemical
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properties of monosaccharide-conjugated R-lycosin-I, which provides references for ACP
optimization by glycosylation modification.
Experimental Section
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General Experimental Procedure. Chemicals used were reagent grade as supplied except
where noted. Analytical thin-layer chromatography was performed using silica gel 60 HF254 glass
plates; Compound spots were visualized by UV light (254 nm), or in some cases by staining with a
yellow solution containing ninhydrin (1.5 g) in butanol (100 mL)-HAc (3 mL), or by charring with
3
0% sulfuric acid-alcohol, or by staining with iodine in silica gel. Flash column chromatography
was performed on columns (16×240 mm, 18×300 mm, 35×400 mm) of silica gel 60 (200-300
Mesh) with EtOAc-petroleum ether (60-90°C) as the eluent. Solutions were concentrated at <60°C
under reduced pressure. NMR spectra were referenced using Me Si (0 ppm), residual CHCl3
4
(1
H-NMR 7.26 ppm, 13C NMR 77.3 ppm) for CDCl , using residual DOH ( H-NMR 4.79 ppm)
1
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for D O, or using residual CD OH ( H-NMR 4.87 ppm, C NMR 49.0 ppm) for CD OD. Peak
2
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assignments are based on H NMR, and 13C NMR experiments. NMR experiments were
1
conducted at 500, and 125 MHz for H, 13C, respectively, using Bruker Avance 500 MHz NMR
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Spectrometer equipped with a switchable QNP ( H, C) probe enabling back-to-back data
acquisition for the different nuclei without the need to remove sample or tune the probe. The
glycopeptides were purified using preparing RP-HPLC (Milford, MA, USA) (column, C18, 300
Å, 10 × 250 mm; Welch Materials, Inc.) and determined using MALDI-TOF MS. All
glycopeptide analogues evaluated in the biological assays were greater than 95% pure based on
the HPLC methods. Purity, 8a: 96.4 %, 8b: 95.1 %, 8c: 95.0 %, 8d: 96.2 %, 8e: 95.4 %.
Synthesis of 1,2,3,4,6-penta-O-acetyl-,-D-glucopyranose (1a)
o
In a 500 mL round bottom flask, the acetic anhydride (200 mL) was cooled to 0 C under
stirring. Then perchloric acid (1.0 mL) was added dropwise. The glucose (50.0 g, 277.8 mmol)
o
was added in partition under the temperature was not excess 20 C. The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 3 : 1) showed the starting material was
disappeared, during which time the temperature was gradually raised to ambient temperature.
DCM (200 mL) was added, and then washed with water and CH Cl for three times. The organic
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layer was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated
on a rotavapor to give crude 1a (106.6 g, 273.1 mmol, 98.3%) as a brown yellow syrup.
Synthesis of 1,2,3,4,6-penta-O-acetyl-, -D-galactopyranose (1b)
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In a 1L round bottom flask, the acetic anhydride (400 mL) was cooled to 0 C under stirring.
Then perchloric acid (0.4 mL) was added dropwise. The galactose (100.0 g, 0.56 mol) was added
o
in partition under the temperature was not excess 10 C. The reaction mixture was stirring until
TLC (petroleum ether: ethyl acetate = 3 : 1) showed the starting material was disappeared, during
which time the temperature was gradually raised to ambient temperature. DCM (200 mL) was
added to it, and then washed with water and CH Cl for three times. The organic layer was
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collected, dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate was
evaporated on a rotavapor to give crude product 1b (199.5 g, 0.51 mol, 92% yield) as a brown
yellow syrup.
Synthesis of 1,2,3,4,6-penta-O-acetyl-α,β-D-mannopyranose (1c)
o
In a 500 mL round bottom flask, the acetic anhydride (100 mL) was cooled to 0 C under
stirring. Then perchloric acid (1.0 mL) was added dropwise. The mannose (25.0 g, 138.9 mmol)
o
was added in partition under the temperature was not excess 20 C. The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 3 : 1) showed the starting material was
disappeared, during which time the temperature was gradually raised to ambient temperature.
DCM (200 mL) was added to it, and then washed with water and CH Cl for three times. The
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organic layer was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was
evaporated on a rotavapor to give crude product 1c (43.5 g, 111.4 mmol, 80.3%) as a brown
yellow syrup.
Synthesis of 1,2,3,4-tetra-O-acetyl-,-L-arabinopyranose (1d)
o
In a 500 mL round bottom flask, the acetic anhydride (100.0 mL) was cooled to 0 C under stirring.
Then perchloric acid (0.2 mL) was added dropwise. The L-arabinose (25.0 g, 166.7 mmol) was
o
added in partition under the temperature was not excess 20 C. The reaction mixture was stirring
until TLC (petroleum ether : ethyl acetate = 1 : 1) showed the starting material was disappeared,
during which time the temperature was gradually raised to ambient temperature. DCM (100.0 mL)
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was added to it, and then washed with water and CH Cl for three times. The organic layer was
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collected, dried over anhydrous sodium sulfate, filtered and the solvent from the filtrate was
evaporated on a rotavapor to give crude product 1d (50.0 g, 157.2 mmol, 94.3%).
2
-Acetamido-1,3,4,6-tetra-O-acetyl-2-deoxy-D-glucopyranose (1e)
A solution of D-glucosamine hydrochloride (6.0 g, 27.8 mmol) in a mixture of anhydrous pyridine
60 mL) and acetic anhydride (30 mL) was stirred overnight at RT. The mixture was diluted with
00 mL of chloroform and washed successively with cold water, saturated sodium bicarbonate
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solution, and finally with water. Then the solvent was removed to give compound 1e (7.8 g, 20.0
mmol, 72%) as a white solid.
Synthesis of 2,3,4,6-tetra-O-acetyl-,-D-glucopyranose (2a)
In a 1 L round bottom flask, piperazine (25.0 g, 293.6 mmol) was added to the solution of
compound 1a (104.5 g, 267.7 mmol) in tetrahydrofuran (300 mL). The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed the starting material was almost
disappeared, filtered and the filtrate was evaporated on a rotavapor. The residue was washed with
water and CH Cl .The organic layer was collected, dried over anhydrous sodium sulfate, filtered
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and concentrated, then purified by silica gel column chromatography with petroleum ether : ethyl
acetate = 3 : 1 as the eluent to give compound 2a (75.7 g, 217.3 mmol, 81.2%)as a pale-yellow
syrup.
Synthesis of 2,3,4,6-tetra-O-acetyl-,-D-galactopyranose (2b)
In a 1 L round bottom flask, piperazine (14.6 g, 171.5 mmol) was added to the solution of
compound 1b (60.0 g, 153.7 mmol) in tetrahydrofuran (150 mL). The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed the starting material was almost
disappeared. The mixture was filtered and the filtrate was evaporated on a rotavapor. The residue
was dissolved with CH Cl and washed with water. The organic layer was collected, dried over
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anhydrous sodium sulfate, filtered and concentrated, then purified by silica gel column
chromatography with petroleum ether : ethyl acetate = 3 : 1 as the eluent to give compound 2b
(42.0 g, 120.6 mmol, 78.5%) as a pale-yellow syrup.
Synthesis of 2,3,4,6-tetra-O-acetyl-α,β-D-mannopyranose (2c)
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In a 1.0 L round bottom flask, piperazine (12.9 g, 151.5 mmol) was added to the solution of
compound 1c (29.0 g, 74.3 mmol) in tetrahydrofuran (150 mL). The reaction mixture was stirring
until TLC (petroleum ether: ethyl acetate = 2: 1) showed the starting material was almost
disappeared, filtered and the filtrate was evaporated on a rotavapor. The residue was washed with
water and CH Cl . The organic layer was collected, dried over anhydrous sodium sulfate, filtered
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and concentrated, then purified by silica gel column chromatography with petroleum ether : ethyl
acetate = 3 : 1 as the eluent to give compound 2c (21.0 g, 60.3 mmol, 81.1%)as a pale-yellow
syrup.
Synthesis of 2,3,4-tri-O-acetyl-,-L-arabinopyranose (2d)
In a 1000mL round bottom flask, piperazine (14.9 g, 172.8 mmol) was added to the solution of
compound 1d (50.0 g, 157.2 mmol) in tetrahydrofuran (100 mL). The reaction mixture was
stirring until TLC (petroleum ether : ethyl acetate = 2 : 1) showed the starting material was almost
disappeared, filtered and the filtrate was evaporated on a rotavapor. The residue was washed with
water and CH Cl .The organic layer was collected, dried over anhydrous sodium sulfate, filtered
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and concentrated, then purified by silica gel column chromatography with petroleum ether : ethyl
acetate = 3 : 2 as the eluent to give compound 2d (20.8 g, 75.3 mmol, 47.9%).
2
-Acetamido-3,4,6-tri-O-acetyl-2-deoxy-D-glucopyranose (2e)
To a stirred solution of 1e (3.0 g, 7.7 mmol) in dry methanol (10 mL) and THF (40 mL) were
introduced ammonia gas. The reaction mixture was stirred for 40 min at RT. The mixture was
concentrated in vacuo and the residue was purified by flash column chromatography on silica gel
(ethyl acetate : petroleum ether = 1 : 1) to give compound 2e (1.75 g, 5.0 mmol, 65.2%) as a
colorless oil.
Synthesis of 2,3,4,6-tetra-O-acetyl--D-glucopyranosetrichloroacetimine (3a)
In a 1 L round bottom flask, trichloroacetonitrile (58.4 mL, 582.4mmol) was added to a
mixture of the intermediate 2a (74.3 g, 213.3 mmol), K CO (53.6 g, 388.4 mmol) in anhydrous
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DCM (400 mL). The mixture was stirring at room temperature until TLC (petroleum ether : ethyl
acetate = 2 : 1) showed the starting material was almost disappeared, filtered and the solvent from
the filtrate was evaporated on a rotavapor. The residue was purified by silica gel column
chromatography with petroleum ether : ethyl acetate = 3 : 1 as the eluent to give compound 3a
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(
80.0 g, 162.4 mmol, 76.1%) as a white solid.
Synthesis of 2,3,4,6-tetra-O-acetyl--D-galactopyranoyltrichloroacetimidate (3b)
In a 500 mL round bottom flask, trichloroacetonitrile (43 mL, 428.8 mmol) was added to a
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mixture of the intermediate 2b (33.1 g, 95.0 mmol), K CO (30.5 g, 220.3 mmol) in anhydrous
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DCM (400 mL). The mixture was stirring at room temperature until TLC (petroleum ether : ethyl
acetate = 2 : 1) showed the starting material was almost disappeared, filtered and the filtrate was
evaporated on a rotavapor. The residues were purified by silica gel column chromatography with
petroleum ether : ethyl acetate = 3 : 1 as the eluent to give compound 3b (40.3 g, 81.8 mmol,
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6.1%) as a white solid.
Synthesis of 2,3,4,6-tetra-O-acetyl--D-mannopyranosyltrichloroacetimine (3c)
In a 250 mL round bottom flask, trichloroacetonitrile (22.6 mL, 120.0 mmol) was added to a
mixture of the intermediate 2c (20.0 g, 57.4 mmol), K CO (25.0 g, 180.9 mmol) in anhydrous
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DCM (200 mL). The mixture was stirring at room temperature until TLC (petroleum ether : ethyl
acetate = 2 : 1) showed the starting material was almost disappeared, filtered and the solvent from
the filtrate was evaporated on a rotavapor. The residue was purified by silica gel column
chromatography with petroleum ether : ethyl acetate = 3 : 1 as the eluent to give compound 3c
(24.7 g, 50.1 mmol, 87.3 %) as a white solid.
Synthesis of 2,3,4-tri-O-acetyl--L-arabinopyranosyltrichloroacetimidate (3d)
In a 500 mL round bottom flask, trichloroacetonitrile (22.5 mL, 225.5 mmol) was added to a
mixture of the intermediate 2d (20.0 g, 72.4 mmol), K CO (20.8 g, 150.7 mmol) in anhydrous
2
3
DCM (150 mL). The mixture was stirring at room temperature until TLC (petroleum ether : ethyl
acetate = 2 : 1) showed the starting material was almost disappeared, filtered and the solvent from
the filtrate was evaporated on a rotavapor. These residues were purified by silica gel column
chromatography with petroleum ether : ethyl acetate = 4 : 1 as the eluent to give compound 3d
(19.7 g, 45.3 mmol, 62.5%).
2
-Acetamido-3,4,6-tri-O-acetyl-2-deoxy--D-glucopyranosyl trichloroacetimidate (3e)
To a stirred solution of 2e (1.2 g, 3.5 mmol) in dry dichloromethane (25 mL) were added
trichloroacetonitrile (1.36 mL, 7.2 mmol) and anhydrous potassium carbonate (2.5 g, 18.1 mmol).
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The reaction mixture was stirred for 3 h at RT. The crude product was filtered, concentrated and
purified by flash column chromatography on silica gel (ethyl acetate : petroleum ether = 1 : 1) to
give 3e (1.1 g, 2.3 mmol, 67.5%) as a light yellow solid.
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Synthesis of 3’-chloropropyl 2,3,4,6-tetra-O-acetyl--D-glucopyranoside (4a)
The compound 3a (41.0 g, 83.2 mmol) and 4Å MS (3.0 g) were dried under vacuum for 2 h,
then 3-chloro-1-propanol (25.0 mL, 179.4 mmol) and dry DCM (150 mL) were added. The
o
mixture was stirred and cooled to -20 C, and then TMSOTf (260.0 L, 1.5 mmol) was added
under nitrogen atmosphere. The reaction mixture was stirring until TLC (petroleum ether: ethyl
acetate = 3 : 1) showed the starting material was disappeared, during which time the temperature
was gradually raised to ambient temperature. The mixture was washed with water and CH Cl .
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The organic layer was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was
evaporated on a rotavapor. The residue was purified by silica gel column chromatography with
petroleum ether : ethyl acetate = 4 : 1 as the eluent to give compound 4a (28.4 g, 66.8 mmol,
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0.3%)as a pale-yellow syrup.
Synthesis of 3’-chloropropyl 2,3,4,6-tetra-O-acetyl--D-galactopyranoside (4b)
The compound 3b (38.3 g,77.7 mmol) and 4Å MS (3.0 g) were dried under vacuum for 2 h,
then 3-chloro-1-propanol (19.5 mL, 233.4 mmol) and dry DCM (150 mL) were added. The
o
mixture was stirred and cooled to -20 C, and then TMSOTf (296.1 L, 1.6 mmol) was added
under nitrogen atmosphere. The reaction mixture was stirring until TLC (petroleum ether: ethyl
acetate = 3 : 1) showed the starting material was disappeared, during which time the temperature
was gradually raised to ambient temperature. The mixture was washed with water and CH Cl .The
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organic layer was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was
evaporated on a rotavapor. The residues was purified by silica gel column chromatography with
petroleum ether: ethyl acetate = 4 : 1 as the eluent to give compound 4b (29.1 g, 68.5 mmol,
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8.2%) as a pale yellow syrup.
Synthesis of 3’-chloropropyl 2,3,4,6-tetra-O-acetyl--D-mannopyranoside (4c)
The compound 3c (23.1 g, 46.9 mmol) and 4Å MS (3.0 g) were dried under vacuum for 2 h,
then 3-chloro-1-propanol (8.5 mL, 101.7 mmol) and dry DCM (250 mL) were added. The mixture
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was stirred and cooled to -20 C, and then TMSOTf (100.0 µL) was added under nitrogen
atmosphere. The reaction mixture was stirring until TLC (petroleum ether : ethyl acetate = 3 : 1)
showed the starting material was disappeared, during which time the temperature was gradually
raised to ambient temperature. The mixture was washed with water and CH Cl . The organic layer
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was collected, dried over anhydrous sodium sulfate, filtered and the filtrate was evaporated on a
rotavapor. The residue was purified by silica gel column chromatography with petroleum ether :
ethyl acetate = 4 : 1 as the eluent to give compound 4c (13.4 g, 31.5 mmol, 67.1%) as a
pale-yellow syrup.
Synthesis of 3’-Chloropropyl 2,3,4-tri-O-acetyl--L-arabinopyranoside (4d)
The compound 3d (19.0 g, 45.2 mmol) and 4Å MS (3 g) were dried under vacuum for 2 h, then
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-chloro-1-propanol (11.0 mL, 131.6 mmol) and dry DCM (200 mL) were added. The mixture
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was stirred and cooled to -20 C, and then TMSOTf (220.0 L, 0.8 mmol) was added under
nitrogen atmosphere. The reaction mixture was stirring until TLC (petroleum ether: ethyl acetate =
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: 1) showed the starting material was disappeared, during which time the temperature was
gradually raised to ambient temperature. The mixture was washed with water and CH Cl .The
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organic layer was collected, dried over anhydrous sodium sulfate, filtered and the solvent from the
filtrate was evaporated on a rotavapor. These residues were purified by silica gel column
chromatography with petroleum ether : ethyl acetate = 4 : 1 as the eluent to give compound 4d
(11.0 g, 31.1 mmol, 68.8%).
3
’-Chloropropyl3,4,6-tri-O-acetyl-2-deoxy-2-acetamido--D-glucopyranoside (4e)
To a stirred solution of 3-chloropropanol (1.8 mL, 20.2 mmol) and 3e (3.3 g, 6.71 mmol) in
dichloromethane (30 mL) containing freshly activated 4 AMS (0.5 g) was slowly added TMSOTf
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75 L, 410.0 mol) at -20 C. The reaction mixture was stirred for 30 min at RT. The crude
product was filtered. After removal of solvent, the residue was purified by flash column
chromatography on silica gel (ethyl acetate : petroleum ether = 1 : 2 to 1 : 1) to give compound 4e
(1.2 g, 2.8 mmol, 42.1%) as a white solid.
Synthesis of 3’-Azidopropyl 2,3,4,6-tetra-O-acetyl- -D-glucopyranoside (5a)
Sodium azide (8.5 g, 130.8 mmol) was added slowly to a stirred solution of compound 4a
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27.6 g, 64.0 mmol) in DMF (150 mL). The reaction mixture was stirring at 75 C for 12 h until
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