Synthesis of a novel fluorescent ruthenium complex with an appended Ac4GlcNAc moiety by click reaction

Article abstract of DOI:10.3390/molecules23071649

The O-linked β-N-acetylglucosamine (O-GlcNAc) modification is an abundant post-translational modification in eukaryotic cells, which plays a fundamental role in the activity of many cells and is associated with pathologies like type II diabetes, Alzheimer’s disease or some cancers. However, the precise connexion between O-GlcNAc-modified proteins and their function in cells is largely undefined for most cases. Confocal microscopy is a powerful and effective tool for in-cell elucidation of the function of biological molecules. Chemical labeling of non-ultraviolet or non-fluorescent carbohydrates with fluorescent tag is an essential step that makes intra-cellular microscopic inspection possible. Here we report a strategy based on the 1,3-dipolar cycloaddition, called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues Ac₄GlcNAc (substituted with an azido group) and the corresponding fluorescent tag Ru(bpy)₂(Phen-alkyne)Cl₂ (4) to synthesize the fluorescent dye Ru(bpy)₂(Phen-Ac₄GlcNAc)Cl₂ (5) under mild and neutral reaction conditions. Moreover, 5 showed good stability, desirable fluorescence characteristics, and exhibited rather low levels of cytotoxicity against sensitive MCF-7 cells. Additionally, we have achieved successful fluorescent imaging of 5 transported in living MCF-7 cells. Cell images displayed that proteins are potentially labelled with 5 in the cytoplasm.

Full text of DOI:10.3390/molecules23071649

molecules
Article
Synthesis of a Novel Fluorescent Ruthenium
Complex with an Appended Ac GlcNAc Moiety
4
by Click Reaction
Qi Cheng , Yalu Cui , Nao Xiao * , Jishun Lu 2 and Chen-Jie Fang 1
1
2
1, ID
1
Beijing Area Major Laboratory of Peptide and Small Molecular Drugs, Engineering Research Center of
Endogenous Prophylactic of Ministry of Education of China, School of Pharmaceutical Sciences,
Capital Medical University, Beijing 100069, China; qicheng@ccmu.edu.cn (Q.C.); cjfang@ccmu.edu.cn (C.-J.F.)
Key Laboratory of Systems Biomedicine (Ministry of Education) and Collaborative Innovation Center of
2
Systems Biomedicine, Shanghai Center for Systems Biomedicine (SCSB), Shanghai Jiao Tong University,
8
00 Dongchuan Road, Shanghai 200240, China; cuiyalu2011@126.com (Y.C.); lujishun@sjtu.edu.cn (J.L.)
*
Correspondence: xiaonao@ccmu.edu.cn; Tel.: +86-10-8391-1524
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 9 June 2018; Accepted: 2 July 2018; Published: 6 July 2018
Abstract: The O-linked
β-N-acetylglucosamine (O-GlcNAc) modification is an abundant
post-translational modification in eukaryotic cells, which plays a fundamental role in the activity
of many cells and is associated with pathologies like type II diabetes, Alzheimer’s disease or some
cancers. However, the precise connexion between O-GlcNAc-modified proteins and their function
in cells is largely undefined for most cases. Confocal microscopy is a powerful and effective tool
for in-cell elucidation of the function of biological molecules. Chemical labeling of non-ultraviolet
or non-fluorescent carbohydrates with fluorescent tag is an essential step that makes intra-cellular
microscopic inspection possible. Here we report a strategy based on the 1,3-dipolar cycloaddition,
called click chemistry, between unnatural N-acetylglucosamine (GlcNAc) analogues Ac GlcNAc
4
(
substituted with an azido group) and the corresponding fluorescent tag Ru(bpy) (Phen-alkyne)Cl (4)
2 2
to synthesize the fluorescent dye Ru(bpy) (Phen-Ac GlcNAc)Cl (
5
) under mild and neutral reaction
showed good stability, desirable fluorescence characteristics, and exhibited
rather low levels of cytotoxicity against sensitive MCF-7 cells. Additionally, we have achieved
successful fluorescent imaging of transported in living MCF-7 cells. Cell images displayed that
2
4
2
conditions. Moreover,
5
5
proteins are potentially labelled with 5 in the cytoplasm.
Keywords: O-GlcNAc; click chemistry; cell imaging; fluorescent labeling; living cells
1
. Introduction
O-Linked
diverse cellular processes including transcription, cell division, and glucose homeostasis [
Therefore, understanding the glycosylation of proteins is critical to elucidate the functional roles of
proteins within the dynamic environment of cells in physiological and diseased states [ ]. Currently,
β-N-acetylglucosamine (O-GlcNAc) glycosylation is an important regulation for the
1–5].
6–9
glycosylation levels are typically monitored by immunoblotting with a general O-GlcNAc antibody.
Due to the lack of specificity and selectivity of the antibody used, only a limited number of O-GlcNAc
proteins can be detected [10]. With this regard, great challenge remains to develop simple but efficient
methods to monitor non-ultraviolet or non-fluorescent O-GlcNAc glycosylation in response to cellular
stimuli [11].
Microscopic inspection is a powerful and widely used tool for high-resolution and non-invasive
imaging of target molecules in their native environments [12]. With spectroscopic probes such as
alkyne-functionalized rhodamine or 1,8-naphthalimide derivatives, imaging of O-GlcNAc proteins in
Molecules 2018, 23, 1649; doi:10.3390/molecules23071649
www.mdpi.com/journal/molecules

Molecules 2018, 23, 1649
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their native environment is feasible [13
–
16]. This method definitely facilitates and advances proteomic
studies by helping monitor the expression and dynamics of O-GlcNAc-glycosylated proteins in cells
and tissues [17]. Herein, we report an alternative fluorescent dye (Scheme 1) as a potential labeling
reagent, which utilizes the intrinsic reactivity of the Ac GlcNAc moiety at end of a ruthenium complex
5
4
to specifically address the target protein by either chemical or enzymatic reactions [18,19]. This is the
first example of the use of a ruthenium complex appended with the glycosylation reagent Ac GlcNAc to
4
image proteins within cells. We anticipate that this fluorescent dye will be a powerful tool for facilitating
our understanding of the physiological functions and dynamic regulation of O-GlcNAc glycosylation
1
13
within cells. The monoglucosylated complex
fluorimetry, and mass spectrometry. Further the use of complex
with MCF-7 cells, which suggested the accumulation of 5 in the cytoplasm.
5
was characterized with IR, UV-vis, H-NMR, C-NMR,
5
for intracellular imaging was tested
Scheme 1. Synthesis of Ac GlcNAz (2), Ru(bpy) (Phen-alkyne)Cl (4) and Ru(bpy) (Phen-Ac GlcNAc)Cl (5).
2 2 2 4 2
4
2
. Results
The synthetic routes to the intermediates and the target ruthenium complex
Scheme 1. Compound was synthesized by copper-catalyzed azide-alkyne cycloaddition (CuAAC)
reaction of alkyne-dye and azide-based O-GlcNAc glycosylation metabolite Ac GlcNAz ( ).
M) against living cells under mild and neutral
was mostly located in the cytoplasm
5
are summarized in
5
4
2
4
Compound
5
has trivial cytotoxicity (IC50 = 549
µ
◦
conditions (37 C, 1 h). According to microscope imaging,
5
and not the nucleus of living MCF-7 cells, which provides direct imaging and visualization of
5
transported in living cells, showing its potential as a tool for biological function elucidation of
O-GlcNAc modified proteins.

Molecules 2018, 23, 1649
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3
. Discussion
3
.1. Synthesis of 2 and 3
Peracetylated N-azidoacetylglucosamine
2
was synthesized starting from glucosamine
hydrochloride (Scheme 1). The monosaccharide was deprotonated with sodium methoxide, followed
by acetylation with chloroacetic anhydride in methanol. Displacement of the halide with sodium azide
in dry DMF provided the compound
with acetic anhydrate in pyridine. Ligand
the reagents 1,10-phenanthrolin-5-amine and 6-heptynoic acid in a DCM solution of EDCI/DMAP.
1. The acetyl protected target
2 was obtained by acetylation of
1
3
appended with an alkynyl chain was obtained with
1
13
GlcNACl,
Figures S2, S4 and S6) and mass spectroscopy (Figures S11–S14, S16 and S17).
In the FTIR spectrum (Figure S18), a characteristic stretching band at around 2100 cm appeared,
indicating the introduction of the azido group together with the glucosyl group to form , which
1, 2, and 3 were fully characterized by FTIR, H-NMR (Figures S1, S3 and S5), C-NMR
(
−
1
2
completely disappeared after the click reaction with terminal alkyne
complex unit was successful conjugated with the glucosyl unit.
4
. It suggested the ruthenium
To provide further insight into the structure of GlcNACl,
1,
2
and
3, ESI-MS measurements were
carried out. For GlcNACl, the main peak at 277.64 (m/z) (Figure S11) is in good agreement with
the theoretical molecular-ion of the sodium-cationized GlcNACl (C H ClNNaO : 278.04), the peaks
8
14
6
nearby main peak at 279.57, 280.17, and 280.50 are assigned to isotope forms of sodium-cationized
GlcNACl. For , the peaks at 243.25, and 258.75 (m/z) (Figure S12) are in a good agreement with the
theoretical molecular-ions of [M-N ] (C H NNaO : 243.07), and [M-N ] (C H N NaO : 257.07),
1
+
+
3
8
14
6
2
8
14
2
6
receptivity. For
2, the peaks at 452.82, and 883.04 (m/z) (Figure S13) are in a good agreement with the
+
+
theoretical molecular-ions of [M + Na] (C H N NaO : 453.12) and [2M + Na] (C H N NaO :
16
22
4
10
32 44
8
20
8
83.26) without any unreacted material peaks or byproducts. For
3
, the peak at 303.81 (m/z) (Figure
+
S14) is in a good agreement with the theoretical molecular-ions of [M + H] (C H N O: 304.14)
19
17
3
without any unreacted material peaks or byproducts.
Overall, FTIR, ESI-MS, and NMR results (see Supplementary Information) clearly evidenced
that the and were successfully synthesized by acetylation or nucleophilic substitution reactions.
2
3
1
13
The detailed characteristic H-NMR and C-NMR spectra of
Figures S1–S6.
1, 2 and 3, are provided in Supplementary
3
.2. Synthesis of Ruthenium Complexes
The ruthenium complexes of and
shown in Scheme 1) were synthesized by efficient methods. As shown in Scheme 1, the alkynyl chain
with a bidentate phenanthroline ligand reacted with ruthenium precursor complex cis-Ru(bpy) Cl
in CH Cl :MeOH (1:1) under reflux to produce the heteroleptic complex 4.
0
5 (where bpy = 2,2 -bipyridine) complexes (cationic forms are
4
3
2
2
2
2
CuAAC is a popular strategy in biological applications such as labeling proteins and nucleic
acids in situ with minimal undesired side reactions [20 22]. Both azide and alkyne groups are stable,
and it is easy to introduce them into biomolecules or probe molecules without dramatically altering
their function or metabolic processes [23 26]. Through the CuAAC method, the terminal alkyne
fluorescent tag is further reacted with peracetylated glucosyl moieties bearing the azido-ethyl linker
overnight. The catalytically active Cu was obtained with reducing Cu (CuSO ) by ascorbic acid in
–
–
4
I
II
2
4
DMF solution. After all the azides were converted into the triazole-substituted adduct
5, the solvent
was removed by evaporation, and the desired was obtained as the sole product in nearly quantitative
5
yields. Both designed ruthenium complexes were achieved by only a very simple, rapid, and efficient
one step reaction. The purity of prepared ruthenium complexes residue was examined by thin-layer
chromatography (TLC), and the raw materials and byproducts in the residue were then removed by
column chromatography on silica with MeCN:H O:KNO (100:7.5:0.5) as eluent.
fully characterized with HPLC, FTIR, H-NMR, C-NMR, and mass spectroscopy.
4 and 5 were further
2
13
3
1

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Typically, the synthesized target ruthenium complex
5
was determined by HPLC, which
showed a single peak with no impurities detected. The HPLC traces confirmed the formation
of , as shown in Figure S15. The pure products were later ascertained to be by mass
spectroscopy and NMR spectroscopy. The calculated and experimental results of the mass
spectra of and are given in Figures S16 and S17, respectively. The calculated molar masses is
17.18 g/mol for Ru(bpy) (Phen-alkyne) and 1147.31 g/mol for Ru(bpy) (Phen-Ac GlcNAc) .
5
5
4
5
2+
2+
7
2
2
4
Each ruthenium complex has an overall charge of +2, so the calculated molar masses
2
+
of m/z (mass/charge) are 358.59 g/mol and 573.655 g/mol for Ru(bpy) (Phen-alkyne) and
2
2+
Ru(bpy) (Phen-Ac GlcNAc) , respectively. The measured molecular-ion peaks appear at 358.07
2
4
(
Figure S16, Calcd. for C H N ORu/2: 358.59) and 573.06 (Figure S17, Calcd. for C H N O Ru/2:
39 33 7 55 55 11 11
573.655) are exactly consistent with the calculated molar masses, confirming the identity and the
integrity of the complexes. As expected, no other byproducts were observed in the MS profile,
suggesting a clean reaction.
1
Furthermore, the structural information of
4
and
5
were investigated by H-NMR measurements.
In the analysis of Figure S19 (upper), the proton signals of a’–e’ due to the methyne and alkynyl on
4
were clearly observed in the range of 1.63–2.76 ppm, and the signals of the aromatic part assignable
to the bpy and phenanthroline segments were simultaneously observed. The click reaction was also
1
1
further verified by H-NMR measurements. The H-NMR spectrum of
5
is shown in Figure S19
(
lower), the new proton signals due to the glucosyl and methyl group were observed in the range of
4
.02–6.13 ppm and 1.81–2.27 ppm, respectively. Additionally, the signals of the methylene protons of
were fully shifted from 2.84 ppm to 2.69 ppm after the click reaction.
a’ and b’ on the alkyl chain of
4
The observed protons are consistent with the presence of protons on ruthenium complexes structures
by integration for each signal. These results indicated the formation of the Ru(II) complex 4 and 5.
1
13
Data from ESI-MS, HPLC, H-NMR, and C-NMR indicated that the
4 could be quantitatively
conjugated with to afford via click reactions with no byproducts, implying its potential in fluorescent
2
5
1
13
labeling of proteins modified with O-GlcNAc. Detailed characteristic H-NMR and C-NMR spectra
of 4 and 5, are presented in Supplementary Figures S7–S10.
3
.3. UV-Vis and Fluorescence Properties Studies of 4 and 5
To explore the optical properties of and , the electronic absorption and emission spectra were
recorded, as shown in Figure 1. Compounds and displayed maxima excitation and emission
4
5
4
5
wavelength at 450 nm and 590 nm, respectively, indicating that the inherently favorable spectroscopic
and photophysical properties of the parent complex are not substantially altered by the ligand
substitution. The absorbance peak appeared around 450 nm is caused by the metal-to-ligand charge
transfer (MLCT) effect in the ruthenium complex core, which resulted in the slightly red color of the
aqueous solution. Both complexes exhibit a single emission band. The observed emission spectra of the
complexes are very close to that of the ruthenium complex previously reported by Yuan’s group [27].
Figure 1. UV-vis absorption and fluorescence spectra of 4 and 5 in MeOH. λex = 450 nm.

Molecules 2018, 23, 1649
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As shown in Figure S20 and Table S1, both ruthenium complexes
4
and
5
underwent
a signal-exponential decay with almost the same lifetime of around 190 ns. The good fluorescence
properties implied the potential to imaging of 2 transported within living cells.
3
.4. Cytotoxicity of 5 against Living Cells
MCF-7 cells were adopted since they were used for studying the labeling O-GlcNAc-modified
proteins in the past [28
should be friendly to living cells. To ascertain whether the imaging reagent might alter the
cells’ biological function, the survival rates of cells administered with was determined by
a standard 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) analysis (Figure 2).
Cells incubated with medium only were used as control. At concentrations ranging in 0.001 to 100 M,
the survival rates of the cells was higher than 80%, suggesting has hardly any negative effect on the
M (Figure S21), which also suggested a trivial
,29]. In terms of fluorescent imaging in living cells, the fluorescent tags
5
µ
5
proliferation of living cells. The IC50 value of 5 is 549 µ
cytotoxicity of 5 against MCF-7 cells.
Figure 2. Cytotoxicity of 5 against MCF-7 cells.
3
.5. Imaging of 5 against Living MCF-7 Cells
Imaging of transported in living MCF-7 cells, which were cultured with
and 5% CO , followed by stained with Hoechst 33342 in PBS (10 mg/mL) for 10 min was performed.
◦
5 in PBS for 24 h at 37 C
5
2
To track the sub-cellular location of the target molecule
MCF-7 cells were imaged under confocal microscopy at 20
Figure 3, compound -tagged proteins appeared as red fluorescent spots that were predominantly
located not in the nucleus but inside the cytoplasm of MCF-7 cells (left panel).
5
transported within living cells, these treated
×
by two-channel scanning. As shown in
5
Figure 3. Confocal microscopy images of MCF-7 cells subjected to ligation with 5, followed by staining
with Hoechst 33342 (blue, nucleus-specific dye). The overlay indicates the colocalization of ruthenium
complex 5 and the Hoechst 33342.

Molecules 2018, 23, 1649
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Hoechst 33342 (nucleus specific dye, middle panel) dyed proteins appreared as blue fluorescent
spots that were dominantly located in the nucleus of MCF-7 cells. Overlapping of the fluorescent
image profiles (right panel) deduced from co-localization with 5 and Hoechst 33342 imaging further
demonstrated that label 5 was basically located in cytoplasm of the MCF-7 cell line, indicating
its potential utility in sub-cellular localizations of the O-GlcNAc-glycosylation within living cells.
Apparently, the imaging profiles demonstrated that the 5 could be desirably used as a fluorescent tags,
which enables further fluorescence microscopy studies of O-GlcNAc-glycosylation modified proteins
such as visualization of imaging, locating or tracking of target molecules within living cells
4
. Materials and Methods
4
.1. Materials
All chemicals and reagents were of analytical grade or of the highest purity available
and were used without further purification. Ultrapure water was used throughout the
experiments. D(+)-Glucosamine hydrochloride, sodium methylate (MeONa), chloroacetic anhydride,
N,N-dimethylformamide (DMF), methanol (MeOH), triethylamine, pyridine, dichloromethane
(
(
(
(
DCM), ethanol (EtOH), 1,10-phenanthrolin-5-amine (Phen-NH ), 4-dimethylaminopyridine
DMAP), 6-heptynoic acid, the complex (cis-bis-(2,2 -bipyridine)dichlororuthenium dihydrate,
2
0
cis-Ru(bpy) Cl2
·
2H O), copper sulfate pentahydrate (CuSO4 5H O), ascorbic acid, potassium nitrate
·
2
2
2
KNO ), and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride (EDC HCl, or EDC) were
·
3
purchased from Aladdin Reagent (Shanghai, China), fetal bovine serum from ExCell Biology, Inc.
Shanghai, China), and 1% antibiotics from Solarbio (Beijing, China). TLC analyses were performed on
(
silica gel plates and column chromatography was conducted over silica gel (mesh 200–300), both of
which were obtained from the Qingdao Haiyang Chemicals (Qingdao, China). A semi-preparative
column (Dikma Tech. Inc., Beijing, China, Luster C18, 10 mm, 21.2
×
250 mm) was used to purify
ruthenium complexes. The purities of ruthenium complex (>98%) and the intermediates (>95%) were
5
measured by TLC analysis (Qingdao silica gel plates of GF254, 0.25 mm layer thickness) and HPLC
®
analysis (XTerra MS C18, 5
µm, 2.1
×
150 mm, Waters, Milford, MA, USA). UV-vis absorption
spectra were recorded on a UV-2550 spectrophotometer (Shimadzu, Kyoto, Japan). Photoluminescence
was recorded with a F-2500 fluorescence spectrometer (Hitachi, Tokyo, Japan) with the excitation
and emission slit widths at 5.0 nm and the excitation wavelength at 450 nm. Fluorescence lifetimes
were detected by a FLS980 fluorescence spectrometer from Edinburgh Instruments. The monitored
wavelength was 590 nm. The fitting parameters (decay times and pre-exponential factors) were decided
2
by minimizing the reduced chi square
χ
. IR spectra were recorded using a FT-IR spectrometer
(
Thermo Nicolet IS5, Thermo Fisher Scientific, Waltham, MA, USA). Approximately 1 mg of sample
1
13
was measured using the KBr pellet method at room temperature. H-NMR (300 MHz) and C-NMR
75 MHz) spectra were recorded on an INOVA-300 MHz spectrometer (Varian, Palo Alto, CA, USA)
(
with MeOD as the solvent and tetramethylsilane as internal standard. ESI/MS spectra were recorded
on ZQ 2000 (Waters) and solariX FT-ICR mass spectrometer (Bruker Daltonics, Breman, Germany)
with an ESI/MALDI dual ion source and 9.4 T superconductive magnet.
4
.2. Synthesis of 2-Azidoacetamido-2-deoxy-β-D-glucopyranose (GlcNAz, 1)
Compound was synthesized through a modification of a previously reported procedure [30].
1
Glucosamine hydrochloride (800 mg, 3.71 mmol) was dissolved in MeOH (10 mL), to which
an equivalent amount of sodium methoxide (200 mg, 3.71 mmol) had been added. The reaction mixture
was stirred at room temperature for 4 h, and then triethylamine (0.57 mL, 4.11 mmol) and chloroacetic
anhydride (1.500 g, 8.77 mmol) were added. The reaction mixture was stirred for another 6 h.
Removal of the solvent provided the corresponding chloro intermediate, which was partially purified
by silica gel chromatography eluting with a gradient of CH Cl :MeOH (6:1 to 4:1). The resulting oil
2
2
was dissolved in DMF (20 mL), NaN (2.0 g, 33.33 mmol) and 15-crown-5 (100 µL) were added and
3

Molecules 2018, 23, 1649
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◦
the reaction mixture was heated to 80 C for 2 h. After cooling to room temperature, the solvent was
removed under reduced pressure and the residue was purified by silica gel column chromatography
(
CH Cl :MeOH 5:1) to give the title compound as a light yellow oil (600 mg, 2.29 mmol, 62% over
2 2
two steps).
4
.3. Synthesis of 1,3,4,6-Tetra-O-acetyl-2-N-azidoacetyl-2-deoxy-
β-D-glucopyranose (Ac GlcNAz, Mixture of
4
Anomers, 2)
Compound
Compound (524 mg, 2 mmol) was dissolved in pyridine (16 mL) and Ac O (8 mL). After stirring
for 12 h at room temperature, the solvent was removed under reduced pressure and the residue
was dissolved in CH Cl (50 mL). The organic solution was washed with H O (3 100 mL),
dried over anhydrous Na SO , concentrated, and purified by silica gel chromatography eluting
2 was synthesized through a modification of a procedure reported in the literature [31].
1
2
×
2
2
2
2
4
with CH Cl :MeOH (5:1) to afford 2 as a lear thick oil (632 mg, 1.47 mmol, 73.5%).
2
2
4
.4. Synthesis of N-(1,10-phenanthrolin-5-yl)hept-6-ynamide (Phen-alkyne, 3)
Compound was synthesized through a modification of a procedure reported in the literature [32].
-Amino-1,10-phenanthroline (488 mg, 2.50 mmol, 1 eq.) was dissolved in DCM (20 mL) at
3
5
room temperature. 6-Heptynoic acid (315 mg, 2.50 mmol, 1 eq.) was added, followed by
-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 959 mg, 5.00 mmol, 2.0 eq.)
and finally 4-dimethylaminopyridine (DMAP, 305 mg, 2.50 mmol, 1 eq.). The mixture was allowed to
1
stir for 24 h. The solvent was removed under reduced pressure before H O (50 mL) was added causing
2
precipitation of a white solid which was isolated by filtration. The resulting solid was redispersed in
MeCN (10 mL) and again filtered before being collected by filtration under vacuum and dried under
high vacuum to give a beige/white solid (637 mg, 2.10 mmol, 84.0%).
4
.5. Synthesis of Ru(bpy) (Phen-alkyne)Cl (4)
2 2
Ligand
3
(152 mg, 0.5 mmol, 1 eq.) and cis-Ru(bpy) Cl2 2H O (260 mg, 0.5 mmol, 1 eq.) were
·
2
2
suspended in EtOH:H O (8:2), and the suspension was degassed by bubbling with argon for 15 min
and heated at 160 C for 8 h. After cooling to room temperature, the reaction mixture was filtered
2
◦
through a funnel, the solvent was removed under reduced pressure, and the residue was purified
by silica gel column chromatography eluting with MeCN:H O:KNO (100:7.5:0.5). The fractions
2
3
containing compound
4
was collected, and the solvent was evaporated. The resulting solid was
as a red solid
dissolved in CH CN to remove the excess KNO by filtration to give compound
4
3
3
(262 mg, 0.37 mmol, 73.18%).
4
.6. Synthesis of Ru(bpy) (Phen-Ac GlcNAc)Cl (5)
2 4 2
DMF (20 mL) was added into a round bottomed flask containing
72.0 mg 0.1 mmol), copper (II) sulfate pentahydrate (7.5 mg, 0.03 mmol) and ascorbic acid (17.6 mg,
.1 mmol). The mixture was stirred at room temperature overnight. The solvent was removed under
2 (52.0 mg, 0.12 mmol),
4
0
(
,
reduced pressure, and the residue was purified by silica gel chromatography eluting with a 100:7.5:0.5
MeCN:H O:KNO ratio. The fractions containing were collected, and the solvent was evaporated.
5
2
3
The resulting solid was dissolved in CH CN to remove the excess KNO by filtration to give compound
3
3
5
as a red solid (103.25 mg, 0.09 mmol, 90.00%).
4
.7. Evaluation of the Cell Viability and Metabolic Activity
The survival rates of cells administered with free
5
was determined by a standard
3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) analysis. In brief, the MCF-7
cells were seeded in a 96-well microplate for 18 h and cultured with
for 36 h, supplemented with 10% (v/v) fetal bovine serum, 1% antibiotics, and cultured at 37 C in
5
at various concentrations
◦
an atmosphere of 5% CO . Meanwhile, the cells treated with medium only were used as control.
2

Molecules 2018, 23, 1649
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Then, the 20
µL of 0.5 mg/mL MTT solution was added into each well for 4 h. The incubation was
suspended by removing the medium and adding 100
µL of DMSO. After a 15-min gentle vortex,
the light absorption value of each well was recorded at 490 nm on an Infinite M 200 Pro Multilabel
Plate Reader (Tecan, Mannedorf, Switzerland).
4
.8. Imaging Cellular Uptake of 5
Cellular uptake and distribution of
5 was observed by TCS SP5 confocal microscopy
(
Leica Microsystems, Wetzlar, Germany). MCF-7 cells were incubated on cell culture dishes at a density
5
of 1 × 10 cells per dish for 16 h with
5
(100
µ
mol/mL, 2 mL), supplemented with 10% (v/v) fetal
◦
bovine serum, 1% antibiotics, and cultured at 37 C in an atmosphere of 5% CO . After removing
2
drugs and washing the dishes twice with PBS, the cell nuclei were then stained with Hoechst 33342 in
PBS (10 mg/mL) for 10 min. The following wavelengths were used: excitation at 346 nm and detection
through a 460 nm filter for Hoechst 33342, and excitation at 450 nm and detection through a 593 nm
filter for 5.
5
. Conclusions
We demonstrate here a robust and general method to synthesize a novel water-soluble ruthenium
bearing an Ac GlcNAc moiety by a click reaction, which could be used in living cell imaging.
dye
Our results indicated that the sugar-derived
, and could be delivered into the inside of cells through the fat-soluble cell membrane. The IC50
of against living MCF-7 cells is 549 M. By microscope imaging, compound was mostly located
in the cytoplasm and not the nucleus of living MCF-7 cells, which provides direct insight into the
imaging and visualization of transported in living cells, showing its potential for biological function
elucidation of O-GlcNAc-modified proteins. According to the results, the dye-Ac GlcNAc reagent
5
4
5
displayed similar fluorescence properties to its precursor
4
5
µ
5
5
5
4
might has potential applications as a metabolic labeling reagent. The bioconjugation reactions between
and relevant proteins are underlined within living cells.
5
1
Supplementary Materials: The following are available online. Figure S1: H-NMR spectrum of
1
in MeOD,
1
3
1
13
Figure S2: C-NMR spectrum of
1
in MeOD, Figure S3: H-NMR spectrum of
2
in CDCl , Figure S4: C-NMR
3
1
13
spectrum of
2
in CDCl , Figure S5: H-NMR spectrum of
3
in CDCl , Figure S6
in MeOD, Figure S8: C-NMR spectrum of
:
C-NMR spectrum of
3
in
3
3
1
13
CDCl , Figure S7: H-NMR spectrum of
4
4
in MeOD, Figure S9:
3
1
13
H-NMR spectrum of
5
in MeOD, Figure S10: C-NMR spectrum of
5
in MeOD, Figure S11: ESI-MS spectrum of
GlcNACl in CH OH, Figure S12: ESI-MS spectrum of
1
in CH OH, Figure S13: ESI-MS spectrum of in CH OH,
2
3
3
3
Figure S14: ESI-MS spectrum of
60:36:4). λ = 450 nm, Figure S16: ESI-MS spectrum of
Figure S18: IR spectra of glucosamine hydrochloride,
Figure S20: The fluorescent decay curves (collected at 590 nm) of
cell viability after incubation of MCF-7 cells with
in MeOH solution at room temperature.
3 in CH OH, Figure S15: HPLC trace of 5 determined in CH CN/H O/HAc
3
3
2
(
4
in CH OH. Figure S17: ESI-MS spectrum of
5
in CH OH.
3
3
1
1,
2
and
5.
Figure S19: H-NMR spectra of
4
and
5
in MeOD.
4
and
5
in MeOH solution. Figure S21: In vitro
and were recorded
5
. Table S1: Photophysical properties of
4
5
Author Contributions: Conceptualization, N.X. and J.L.; Data curation, Q.C.; Formal analysis, N.X.; Funding
acquisition, N.X.; Investigation, N.X.; Methodology, N.X.; Project administration, N.X.; Resources, N.X.; Software,
Q.C. and Y.C.; Supervision, N.X.; Validation, J.L. and C.-J.F.; Visualization, N.X.; Writing—original draft,
N.X.; Writing—review & editing, N.X. and C.-J.F.
Funding: This research was funded by the Beijing Natural Science Foundation (2174069) and Scientific Research
Common Program of Beijing Municipal Commission of Education (KM201510025007).
Acknowledgments: We would like to thank Jun Deng, Yuanjie Fan, Xueying Song, Shurui Zhao, and Meiqing
Zheng of the Core Facilities Center of Capital Medical University, Beijing, China.
Conflicts of Interest: The authors declare no conflict of interest. The funders had no role in the design of the
study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, and in the decision to
publish the results.

Molecules 2018, 23, 1649
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