Novel Liposomal Azido Mannosamine Lipids on Metabolic Cell Labeling and Imaging via Cu-Free Click Chemistry

Article abstract of DOI:10.1021/acs.bioconjchem.9b00509

In comparison with the popular Ac₄ManNAz applied as cell labels via Cu-free click chemistry, two novel azido mannosamine lipids with C6 and C12 esters on anomeric hydroxyl groups were prepared and encapsulated in a liposome delivery system, which enhanced chemical stabilities and showed good cell-metabolizable labeling efficiency on MDA-MB-231 cells with strong fluorescence after the treatment of DBCO-Cy5 by triazole formation via click chemistry.

Full text of DOI:10.1021/acs.bioconjchem.9b00509

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Communication
Novel Liposomal Azido Mannosamine Lipids on Metabolic
Cell Labelling and Imaging via Cu-free Click Chemistry
Li Shen, Kaimin Cai, Jin Yu, and Jianjun Cheng
Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/
acs.bioconjchem.9b00509 • Publication Date (Web): 12 Aug 2019
Downloaded from pubs.acs.org on August 13, 2019
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O
N3
Derivative Process
HN
AcO
AcO
AcO
O
OAc
Ac4ManNAz
Liposomal Formation
Release
Hydrolysis
ManNAz
Sialic acid
Metabolism
Biosynthesis
Click
SiaNAz
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Novel Liposomal Azido Mannosamine Lipids on Metabolic Cell Labelling and
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Imaging via Cu-free Click Chemistry
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,
,
‡
†
Li Shen,* Kaimin Cai, ‡ Jin Yu, ‡ and Jianjun Cheng *
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† Ocean College, Zhejiang University, Zhoushan 316021, China. E-mail: shenli@zju.edu.cn
‡ Department of Materials Science and Engineering, University of Illinois at Urbana-Champaign
Urbana, IL 61801, USA. E-mail: jianjunc@illinois.edu
ABSTRACT:
Comparing with the popular Ac4ManNAz applied on cell labelling via Cu-free click chemistry,
two novel azido mannosamine lipids with C6 and C12 ester on anomeric hydroxyl group were
prepared and encapsulated in liposome delivery system, which enhanced chemical stabilities and
showed good cell-metabolizable labelling efficiency on MDA-MB-231 cells with strong
fluorescene after the treatment of DBCO-Cy5 by triazole formation via click chemistry.
INTRODUCTION:
Metabolic oligosaccharide engineering of unnatural carbohydras which created by Bertozzi group
has become a powerful tool to introduce chemical receptors onto the cell surface. [1-3] Based on the
successfully employment of modified N-azidoacetyl mannosamine (ManNAz) which can also be
metabolized through sialic acid biosynthetic pathway and incorporated onto cell surface
[4, 5]
sialoglycoconjugates as normal sialic acids,
such biosynthetic precursor of ManNAc
derivative containing an azido group as a chemical probe can be used as metabolic tracers for
[6, 7]
labelling sialoglycans through different types of click chemistry on the azido group.
Since
then the application of the click chemistry in vitro and in vivo was widely applied on the research
of cell labelling due to their favorable features especially on biocompatibility and high specificity
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[8]
with good reaction rate. This type of cell labelling strategy has been developed as an efficient
5
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7
method of incorporating selectively reactive chemical groups into cell-surface glycoproteins by
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[9, 10]
[11-14]
[15, 16]
novel molecular,
nanoparticle
and liposomal strategies
of deliver systems.
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Tetraacetyl-N-azidoacetylmannosamine (Ac4ManNAz, AAM) is one of the most extensively
studied metabolic cell-labelling reagents which can be typically metabolized in the cell through
sialic acid biosynthesis to azido sialic acid on the cell membrane and then labelled the cell through
click chemistry after treatment of dibenzocyclooctyne (DBCO)-bearing reagents. [3, 6]
Scheme 1. Design of liposomal azido mannosamine lipids on metabolic cell labelling and imaging
In order to improve the chemical stability and cell labelling efficiency of Ac4ManNAz (AAM), we
prepared derivatives of AAM with different carbon chain length ester at the C-1-OH of
Ac3ManNAzOH for hydrophobic lipids (Scheme 1). Herein, two novel liposomal azido
mannosamine lipids loaded with hexanoic (C6) or dodecanoic (C12) ester tail instead of acetyl
(C2) ester on anomeric hydroxyl group of Ac3ManNAzOH were designed and prepared to test the
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cell labelling efficiency via Cu-free click chemistry. To overcome the low water solubility of the
lipids with long ester tail (Scheme 2), we took the advantage of liposome system and all the
azido-lipids were encapsulated by liposome system with good loading efficiencies.
RESULTS AND DISCUSSION
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H N
HCl
O
2
O
HO
HO
HO
O
N3
O
OH
HN
NaN3
H2O
HO
HO
HO
Ac2O
N3
Br
O
HO
HO
OH
HOBt,EDC,TEA
NaOAc
1
O
O
O
O
O
N3
N3
N3
(NH4)2CO3
MeOH
HN
HN
HN
R
O
O
R
AcO
AcO
AcO
AcO
AcO
AcO
AcO
O
O
O
AcO
AcO
or
OH
OAc
O
O
R
Cl
R
3
2
4
R =
5
Scheme 2. Synthesis of Ac4ManAz and its derivative lipids
[15, 17, 18]
Starting with AAM from previous work,
triacetyl-N-azidoacetylmannosamine lipid
(Ac3ManNAzOH-lipid) was successfully synthesized through selective exposure of hydroxyl
[19]
[20]
group on anomeric carbon
and acylation
with varying carbon chain length lipid tail of
saturated C6 or C12 fatty acids in concise routes (Scheme 2). In the assessment of the hydrolytic
degradation by HPLC, C6 and C12 lipids (Figure 1A) showed much more chemical stable than
AAM in the 37oC incubation in the PBS (Figure S2 in the supporting information).
Ac3ManNAzOH lipid-loaded liposomes were continually prepared with highly efficient process [15,
21]
by extrusion of the lipid mixture with 1,2-distearoyl-sn-glycero-3- phosphocholine (DSPC),
cholesterol and 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [amino (polyethylene
glycol)-2000] (DSPE-PEG2000, Figure 1B). The prepared Ac3ManNAzOH lipid-loaded
liposomes had effective diameters of around 200 nm by dynamic light scattering (DLS) and sugar
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loading efficiency of 72% (Figure S3 in the supporting information) on C2 lipid (AAM), 41%
(Figure S4 in the supporting information) on C6 lipid and 50% (Figure S5 in the supporting
information) on C12 lipid (Figure 1C). The C2 lipid (AAM) had the highest loading efficiency
might be affiliate with its higher water solvability in the four types of lipids (Figure S1 in the
supporting information), so AAM might not only distribute into the liposome membrane as other
hydrophilic lipids but also partially dissolve in the aqueous fluid part of liposome.
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Figure 1 (A). The structure of Ac3ManNAzOH lipids with C2, C6 or C12 ester tail on anomeric
hydroxyl group; (B). Preparation of Ac3ManNAzOH lipid-loaded liposomes; (C). Diameter and
diameter distribution of Ac3ManNAzOH lipid-loaded liposomes; (D). Azido carbohydrate release
of Ac3ManNAzOH lipid-loaded liposomes by reverse-titrate method of DBCO-Cy3.
Sugar release test of Ac3ManNAzOH lipid-loaded liposomes by reverse-titrate method of
DBCO-Cy3 (Figure S6 in the supporting information) indicated that the C2 and C6 lipid-loaded
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liposomes had the faster release rate, while C12 lipid-loaded liposomes had significant lower
release rates under the same physiological conditions (Figure 1D).
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We next tested the cell-labelling efficiency of Ac3ManNAzOH lipids on MDA-MB-231 breast
cancer cells. The cells were incubated with 10 μM Ac3ManNAzOH lipid-loaded liposomes for 2 h
or 12 h, and then treated with DBCO-Cy5 independently for another 1 h to detect the expression
of azido groups via Cu-free click chemistry. The cells without liposomes treatment were used as
negative control in PBS. Flow cytometry measurements of Cy5 fluorescence intensity (Figure 2A)
showed that all the Ac3ManNAzOH lipid-loaded liposomes could label MDA-MB-231 cells
efficiently in the contrast of PBS as the negative control after 12 h incubation. In contrast of the
result of 12 h incubation group, fluorescence intensities of 2 h incubation showed no significant
differences between the cells treated with Ac3ManNAzOH lipid-loaded liposomes and PBS. There
were also no significant differences of fluorescence intensities between the cells treated with free
AAM as the positive control comparing with AAM liposome (Figure 2C), which attributed that
the difference of the 2 h and 12 h incubation groups was probably due to the time needed for the
biosynthesis and metabolism pathway.
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Furthermore, the cell labelling results indicated that all the Ac3ManNAzOH lipids could be
metabolized onto the cell membrane glycoprotein through sialic acid biosynthesis by SDS-PAGE
comparing with coomassie blue staining (Figure 2B). Fluorescence intensity could be observed
directly on the SDS-PAGE gel under 365 nm ultraviolet because of the highly effective formation
of a stable triazole from azido-glycoprotein binding with the fluorescent dye DBCO-Cy5 through
click chemistry.
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Figure 2. (A). Flow cytometry analysis of the cellular internalization of the liposomes after 2 h
and 12 h incubation with the liposome and free C2 lipid (AAM). (B). SDS-PAGE analysis of
azide groups in glycoprotein of MDA-MB-231 cells in vitro after 2 h and 12 h incubations with
the liposomes and free C2 lipid (AAM). The gel was detected under 365 nm ultraviolet directly
after DBCO-Cy5 incubation for another 1 h. The coomassie stain shown the total amount of
protein; (C). SDS-PAGE analysis of azide groups in glycoprotein of MDA-MB-231 cells in vitro
after 2 h and 12 h incubations with free C2 lipid and C2 liposome. The gel was detected under 365
nm ultraviolet directly after DBCO-Cy5 incubation for another 1 h. The coomassie stain shown
the total amount of protein; (D). Confocal microscopy images of MDA-MB-231 cells incubated
with 10 μM of Ac3ManNAzOH-lipid-loaded liposomes and free AAM for 12 h at 37oC, followed
by labelling with DBCO-Cy5 for another 1 h. Cells treated with PBS and labelled with
DBCO-Cy5 were used as control. The cell nucleus was stained with hoechst and membrane was
stained with cell mask orange.
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The results of confocal microscopy (Figure 2D) showed that all the cells treated with
Ac3ManNAzOH lipid-loaded liposomes could generated fluorescence on the cell membrane after
treatment of DBCO-Cy5 comparing with the PBS as the negative control and the free AAM as the
positive control. Cell labelling efficiencies were significantly strengthened by the long ester tail
lipid loaded liposome. The C6 and C12 lipid-loaded liposomes showed better intensities of
fluorescence than C2 lipid-loaded liposomes, which indicated that long ester tails on C-1-OH
could not only improve chemical stability of the sugar part but also enhance labelling efficiency.
SUMMARY AND CONCLUSION
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In summary, we developed an enhanced metabolic cell labelling strategy of liposomal azido
carbohydrates carrier by Ac3ManNAzOH lipids with C6 and C12 ester tail on anomeric hydroxyl
group. All the lipids with different length of ester tail could be delivered in liposome systems in
good loading efficiency and kept metabolic cell labelling efficiencies on MDA-MB-231 cells as
AAM. The labelling efficiency seemed better after prolonged the length of ester on anomeric
carbon. This work may provide a promising cell labelling system based on of metabolic
oligosaccharide engineering via Cu-free click chemistry.
EXPERIMENTAL SECTION
General Experimental Details
All other reagents were used as obtained from commercial sources. Room temperature refers to
ambient temperature. Temperatures of 0oC were maintained using an ice-water bath. Thin-layer
chromatography was performed using silica gel 60 with F254 indicator on glass plates (Merck).
Flash chromatography was performed using Merck 40-63 μm silica gel. Solvent ratios for the
purification of compounds by flash chromatography are reported as percent volume (v/v).
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Reactions were performed using oven-dried glassware apparatus under an atmosphere of nitrogen
or argon with anhydrous solvents. Anhydrous dichloromethane (DCM), hexane, and ethyl acetate
were purified by passing them through alumina columns and kept anhydrous in molecular sieves.
NMR spectroscopy was performed in deuterated solvents at 20°C. Chemical shifts (δ) are given in
parts per million (ppm) relative to TMS, and the solvent residual signal is used as a reference.
Abbreviations for peak multiplicities are s (singlet), d (doublet), t (triplet), dt (doublet of triplets),
m (multiplet), and br (broad). Mass spectra were measured using an electrospray ionization (ESI)
mass spectrometer. Details for the spectrometers and other methods are given in the Supporting
Information.
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Synthesis of Ac4ManNAz (2, AAM). Azidoacetic acid was prepared by adding sodium azide (NaN3,
2.6 g, 40.0 mmol) to bromoacetic acid (2.78 g, 20.0 mmol) in deionized water (30 mL) and
stirring at room temperature for 48 h. Thin-layer chromatography (TLC) analysis using detection
by bromocresol green stain indicated the reaction was completed. The solution was extracted by
ethyl acetate and washed with 1 M HCl aq., the organic layer was washed with saturated NaCl aq.,
and dried over Na2SO4. The solvent was removed to obtain yellow oil (1.4 g, 69%). The crude
product could be used directly for the next step. Compound 1 was synthesized as follows,
D-Mannosamine hydrochloride (1.5 g, 7.0 mmol) was added to azidoacetic acid (0.98 g, 9.7 mmol)
from the previous step. Triethylamine (TEA, 2.5 ml, 17.0 mmol) was added and the reaction
mixture was stirred for 5 min at room temperature. Then the solution was cooled to 0oC and
N-hydroxybenzotriazole (HOBt, 0.95 g, 7.1 mmol) was added, followed by addition of
1-[3-(dimethylamino) propyl]-3-ethylcarbodiimide hydrochloride (EDC, 2.68 g, 14.0 mmol). The
reaction was allowed to warm to room temperature overnight, at which point TLC analysis with
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cerric ammonium molybdate (CAM) stain indicates that the reaction is complete. The solution was
concentrated to obtain crude 1 and then purified by silica gel chromatography with
DCM-methanol (9:1, v/v) to colorless semisolid (3.1 g, 84%) which was acetylated directly. To a
suspension of 1 (0.9 g, 3.4 mmol) in acetic anhydride (15 mL) was added anhydrous NaOAc (0.31
g, 3.8 mmol) and stirred at 140 oC for 10 h. The solution was resuspended in DCM and washed
with saturated NaHCO3 aq. saturated NaCl aq. The organic phase was dried over Na2SO4, filtered,
and concentrated. The crude material was then purified by silica gel chromatography, eluting with
hexane–ethyl acetate (2:1, v/v) to obtain pain white solid (0.6 g, 41.0%, 1/1 α/β isomers). 1H NMR
(500 MHz, CDCl3) δ 6.67 (d, J = 9.0 Hz, 1H), 6.60 (d, J = 9.3 Hz, 1H), 6.06 (s, 1H), 5.91 (s, 1H),
5.35 (dd, J = 10.2, 4.3 Hz, 1H), 5.23 (d, J = 10.1 Hz, 1H), 5.18 (t, J = 9.8 Hz, 1H), 5.07 (dd, J =
9.9, 3.9 Hz, 1H), 4.75 (ddd, J = 9.1, 4.0, 1.7 Hz, 1H), 4.63 (ddd, J = 9.4, 4.3, 2.0 Hz, 1H), 4.26
(ddd, J = 12.0, 7.4, 4.4 Hz, 2H), 4.16 (dd, J = 12.5, 2.4 Hz, 1H), 4.13 (d, J = 2.5 Hz, 2H), 4.10 –
4.09 (m, 2H), 4.06 (d, J = 16.7 Hz, 2H), 3.84 (ddd, J = 9.7, 4.6, 2.5 Hz, 1H), 2.20 (s, 3H), 2.13 (d,
J = 2.1 Hz, 9H), 2.08 (s, 6H), 2.02 (d, J = 2.3 Hz, 6H).13C NMR (126 MHz, CDCl3) δ 170.48,
170.12, 170.07, 169.55, 169.53, 168.32, 168.08, 167.29, 166.71, 91.31, 90.26, 73.38, 71.42, 70.26,
68.85, 65.13, 64.96, 61.78, 61.69, 52.60, 52.42, 49.72, 49.27, 20.85, 20.77, 20.75, 20.70, 20.66,
20.63, 20.59. ESI-HRMS (m/z) C16H22N4O10: calculated 430.1336; found: 453.1224 [M+Na]+.
Synthesis of Ac3ManNAzOH (3). Ac4ManNAz (2, 0.3 g, 0.7 mmol) and ammonium carbonate
(0.074 g, 0.77 mmol) were dissolved in methanol (10 mL) and stirred at room temperature for 8 h.
TLC analysis using detection by CAM indicated the reaction was finished, the solvent was
removed and the crude product was purified by silica gel column chromatography with
DCM–ethyl acetate (2:1, v/v) to yield a white solid (0.2 g, 73.6%, 5/1 α/β isomers). 1H NMR (500
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MHz, CDCl3) δ 6.60 (d, J = 9.1 Hz, 1H), 5.39 (dd, J = 10.1, 4.2 Hz, 1H), 5.20 – 5.14 (m, 2H),
4.58 (ddd, J = 9.2, 4.3, 1.9 Hz, 1H), 4.47 (s, 1H), 4.26 – 4.23 (m, 1H), 4.19 (d, J = 4.2 Hz, 1H),
4.16 (d, J = 2.6 Hz, 1H), 4.06 (d, J = 3.6 Hz, 1H), 4.03 (s, 1H), 2.11 (s, 3H), 2.05 (s, 3H), 1.98 (s,
3H). 13C NMR (126 MHz, CDCl3) δ 170.82, 170.35, 169.90, 167.16, 93.06, 69.16, 68.03, 65.78,
62.31, 60.48, 52.40, 50.86, 20.84, 20.71, 20.68.
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Synthesis of C6 ester of Ac3ManNAzOH (4) Ac3ManNAzOH (43 mg, 0.11 mmol) and hexanoic
anhydride (0.1 mL, 0.42 mmol) were dissolved in anhydrous acetone (5 mL) at 0oC.
4-Dimethylaminopyridine (DMAP, 5 mg, 0.41 μmol) was added. The mixture was stirred
overnight at room temperature and then concentrated. The residue was dissolved in DCM and then
washed with saturated NaHCO3 aq. saturated NaCl aq. The organic phase was dried over Na2SO4,
filtered, and concentrated. The crude material was then purified by silica gel chromatography,
eluting with hexane–ethyl acetate (1:1, v/v) to obtain colorless semisolid (45 mg, 84%, 5/1 α/β
isomers). 1H NMR (500 MHz, CDCl3) δ 6.56 (d, J = 9.3 Hz, 1H), 6.05 (s, 1H), 5.33 (dd, J = 10.1,
4.1 Hz, 1H), 5.21 (t, J = 10.1 Hz, 1H), 4.61 (dd, J = 9.2, 4.1 Hz, 1H), 4.22 (dd, J = 12.2, 4.3 Hz,
1H), 4.13 – 4.10 (m, 1H), 4.09 (s, 1H), 4.06 (s, 1H), 4.02 (d, J = 6.7 Hz, 1H), 2.42 (t, J = 7.4 Hz,
2H), 2.11 (s, 3H), 2.06 (s, 3H), 2.00 (s, 3H), 1.68 – 1.65 (m, 2H), 1.33 (dt, J = 6.9, 3.6 Hz, 4H),
0.90 (t, J = 6.6 Hz, 3H). 13C NMR (126 MHz, CDCl3) δ 170.90, 170.47, 170.05, 169.53, 166.66,
91.11, 77.29, 77.04, 76.78, 70.28, 68.92, 65.15, 61.80, 52.44, 49.31, 33.99, 31.12, 24.34, 22.25,
20.75, 20.64, 20.58, 13.86. ESI-HRMS (m/z) C20H30N4O10: calculated 486.1962; found: 509.1862
[M+Na]+.
Synthesis of C12 ester of Ac3ManNAzOH (5). Ac3ManNAzOH (20 mg, 51.5 μmol) and TEA (10
μL, 70 μmol) were dissolved in anhydrous DCM (3 mL). Dodecanoicacid chloride (12.8 μL, 55.8
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μmol) or octadecanoicacid chloride (23.7 mg, 78 μmol) was added at 0oC. The mixture was
warmed to room temperature and stirred overnight. The reaction solution was washed with
saturated NaHCO3 aq. saturated NaCl aq. The organic phase was dried over Na2SO4, filtered, and
concentrated. The crude material was then purified by silica gel chromatography, eluting with
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hexane–ethyl acetate (1:1, v/v) to obtain colorless semisolid (20.0 mg, 68%, 5/1 α/β isomers). H
NMR (500 MHz, CDCl3) δ 6.56 (d, J = 9.2 Hz, 1H), 6.06 (s, 1H), 5.34 (dd, J = 10.2, 4.2 Hz, 1H),
5.22 (t, J = 10.1 Hz, 1H), 4.61 (dd, J = 7.6, 4.1 Hz, 1H), 4.24 (dd, J = 12.1, 3.9 Hz, 1H), 4.13 (d, J
= 8.0 Hz, 1H), 4.10 (s, 1H), 4.07 (s, 1H), 4.03 (d, J = 10.1 Hz, 2H), 2.42 (t, J = 7.5 Hz, 2H), 2.12
(s, 3H), 2.07 (s, 3H), 2.00 (s, 3H), 1.68 – 1.63 (m, 3H), 1.25 (d, J = 3.6 Hz, 16H), 0.88 (s, 4H). 13
C
NMR (126 MHz, CDCl3) δ 170.91, 170.47, 170.06, 169.53, 166.63, 91.11, 77.27, 77.02, 76.76,
70.27, 68.93, 65.14, 61.77, 52.46, 49.31, 34.03, 31.91, 29.60, 24.66, 22.69, 20.59, 14.12.
ESI-HRMS (m/z) C26H42N4O10: calculated 570.2901; found: 593.2782 [M+Na]+.
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ACKNOWLEDGMENTS
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This work was supported by National Institute of Health (R21CA198684) and National Natural
Science Foundation of China (No. 81402845).
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Supporting Information
The Supporting Information is available free of charge on the ACS Publications website.
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Derivative Process
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Ac4ManNAz
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Liposomal Formation
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Release
Hydrolysis
ManNAz
Sialic acid
Biosynthesis
Metabolism
Click
SiaNAz
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( A )
( B )
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C2 lipid（AAM）
C12 lipid
C6 lipid
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(D)
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DSPC
DSPE-PEG2000
C2 liposome
C6 liposome
C12 liposome
Cholesterol
Sugar lipid
In PBS solution
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C2 liposome
C12 liposome
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PDI:
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efficiency: 72 %
Diameter: 200 nm
PDI:
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efficiency: 50 %
Diameter: 198 nm
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( A )
12 h
PBS
( D )
PBS
2 h
Free C2 lipid
C 2 liposome
C 6 liposome
C12 liposome
Free C2 lipid
C 2 liposome
C 6 liposome
C12 liposome
Membrane
(Cell Mask
Orange)
Nucleus
(Hoechst)
DBCO Cy‐5
Merge
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Cy5 Fluorescence
Cy5 Fluorescence
( C )
( B )
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