A cell-permeable and triazole-forming fluorescent probe for glycoconjugate imaging in live cells

Article abstract of DOI:10.1039/c6cc08805h

A new fluorescence-forming probe, coumOCT, designed by fusing cyclooctyne with a coumarin fluorophore was successfully used for the imaging of azido-glycoconjugates in living HeLa cells. This probe is cell-permeable and generates fluorescence after triazole formation, thus minimizing the background signal and enabling the real-time intracellular imaging of glycoconjugate trafficking.

Full text of DOI:10.1039/c6cc08805h

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A Cell-Permeable and Triazole-Forming Fluorescence Probe for
Glycoconjugate Imaging in Live Cells†
Received 00th January 20xx,
Accepted 00th January 20xx
Jiun-Jie Shie,*a Ying-Chih Liu,b Jye-Chian Hsiao,a Jim-Min Fang,bc and Chi-Huey Wong*bd
DOI: 10.1039/x0xx00000x
www.rsc.org/
A new fluorescence-forming probe, coumOCT, designed by fusing
cyclooctyne with a coumarin fluorophore was successfully used
for the imaging of azido-glycoconjugates in living HeLa cells. This
probe is cell-permeable and generates fluorescence after triazole
formation, thus minimizing the background signal and enabling
the real-time intracellular imaging of glycoconjugate trafficking.
Figure 1. Structures of SPAAC-based fluorescence-forming
probe and two reported probes, CoumBARAC and Fl-DIBO.
1
Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC)1 is an
orthogonal reaction that has been widely utilized to label
specific biomolecules in complex mixtures, cells and tissues.2
Further studies have focused on using non- or weakly-
fluorescent azides and alkynes to generate highly fluorescent
triazole products upon cycloaddition reactions.3 The distinct
fluorescence enhancement induced by the highly efficient
the reactivity in cycloaddition reactions with azides.6i-j To date,
two cyclooctyne-based fluorogenic probes, CoumBARAC7 and
Fl-DIBO,8 have been reported by the Bertozzi and the Boons
groups, respectively (Fig. 1).
The low fluorescence 7-alkynylcoumarin derivative has been
shown to undergo a CuAAC reaction to give the corresponding
triazole product with enhanced fluorescence due to the
electron-donating property of the triazole ring.3c Thus,
CuAAC reaction has given
a potentially wide-range of
applications in the emerging field of cell biology and functional
proteomics.3a,3d,3j,4 However, the toxicity of Cu(I) has hindered
the use of CuAAC in living systems.
incorporation of a cyclooctyne to the coumarin moiety
1 may
decrease fluorescence, but the SPAAC reaction of 1 with azides
To circumvent the cytotoxicity associated with the metal
catalyst, ring strain-promoted azide-alkyne cycloadditions
(SPAAC) have been developed as an alternative strategy.5
Cyclooctyne moieties such as difluorinated cyclooctynes (DIFO)
and their derivatives are often incorporated as a stem
structure into SPAAC reagents to improve the reactivity.6a,6b In
addition, the cyclooctyne moiety can be fused with other rings
such as dibenzocyclooctyne (DIBO),6c-6f diarylazacyclooctynone
(BARAC),6g and bicyclononynes (BCN) to increase the ring
strain and reactivity.6h Tetramethylthiacycloheptyne (TMTH)
bearing a contracted seven-membered ring was also used to
increase
is expected to give highly fluorescent triazole products for
sensitive detection. This approach was reported by Bertozzi
and co-workers using the biarylazacyclooctynone ring fused
with a coumarin fluorophore (CoumBARAC).7 Although the
compound undergoes
azidoethanol to give
a
cycloaddition reaction with 2-
10-fold increase in fluorescence
a
intensity, the triazole product exhibited a low quantum yield
(Φf = 0.04) and requires relatively high energy excitation (~300
nm), making it difficult for imaging in living systems. Fl-DIBO
was not used in live cell imaging and its triazole adduct also
gave a relatively low quantum yield. In this study, we report a
SPAAC-based fluorescence-forming probe
1 (Fig. 1), namely
coumOCT that can be used for the real-time imaging of azido-
tagged glycans in living cells under no-wash and no-fixation
conditions.
Scheme 1 shows the synthesis of
1 using 1-benzosuberone
as the starting material. According to the previously reported
procedure,9 1-benzosuberone was subject to regioselective
nitration at the 8-position. The nitro group was then reduced,
followed by diazotization and hydroxylation under acid
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conditions to give the phenol product
the
2. After protection of observed the cycloaddition reaction between 1 and N-
azidoacetylmannosamine (ManNAz), proceeding similarly to
give the triazole 10 with a second-order rate constant of 0.010
o
M−1s−1 at 25 C in a solution of CD3OD–D2O (5:1, v/v) (Fig. S1
and S2, ESI
Table S1 (ESI
and 10 recorded under simulated physiological
conditions (PBS buffer containing 10% DMSO, pH 7.4). As
expected, formation of triazoles and 10 was accompanied by
†).
†) shows the absorption and fluorescence data
of
1, 9
9
a significant increase in fluorescence intensity with a large
Stokes shift into a standard range for coumarin emission (Fig.
S3a, ESI
weak emission band centered at 405 nm with a low quantum
yield (Φf = 0.011), whereas both triazoles and 10 exhibited a
†). Upon excitation at 330 nm, probe 1 produced a
9
strong fluorescence at 435 nm with a quantum yield of 0.23
and 0.21, respectively.
To probe the SPAAC reaction under conditions that would
Scheme 1. Synthesis of the SPAAC-based fluorescence-forming be more typical for biomolecule labeling, we investigated the
probe 1 and the corresponding triazoles 9 and 10. Reagents fluorescence response and time course for the reaction of 1
and conditions: (a) conc. H2SO4, KNO3, 0 oC, 1.5 h, 72%; (b) Sn, with ManNAz. The experiments indicated that more than 90%
conc. HCl, C2H5OH, reflux, 50 min, 82%; (c) 10% aq. H2SO4, of ManNAz was consumed in 40 min, and the fluorescence
o
NaNO2, 0 C to rt, 72 h, 76%; (d) BnBr, K2CO3, DMF, rt, 24 h, intensity reached a plateau in less than 1 h (Fig. S3b, ESI
†
).
98%; (e) TMSCHN2, BF3·OEt2, CH2Cl2, 0 oC, 12 h, 73%; (f) NaBH4,
Aberrant glycosylation on the surface of malignant cells is
o
CH3OH, 0 C, 1 h; (g) TIPSOTf, 2,6-lutidine, CH2Cl2, rt, 1 h, 96% often observed in pathological conditions, such as
for two steps; (h) H2, Pd/C, CH3OH, EtOAc,
1 h; (i) inflammation and cancer metastasis. HeLa cell is the most
paraformaldehyde, MgCl2, Et3N, CH3CN, reflux, 12 h, 87% for widely used model system for studying human cellular and
two steps; (j) Ph3P=C=C=O, toluene, 90 oC, 1.5 h, 83%; (k) TBAF, molecular biology as well as cancer biology.We sought to
THF, 0 oC to rt, 1 h; (l) (COCl)2, DMSO, Et3N, CH2Cl2, −78 oC to rt, evaluate the performance of
1
in live cell imaging.HeLa cells
1 h, 78% for two steps; (m) NaHMDS, Tf2NPh, −78 oC to rt, 2 h, were cultured in the presence of peracetylated N-
32%; (n) BnN3 (1.5 equiv.), CH3CN, rt, 2 h, 95%; (o) N- azidoacetylmannosamine (Ac4ManNAz) for days to
3
azidoacetylmannosamine (1.5 equiv.), MeOH, H2O, rt, 2 h, metabolically produce the azido-sialic acid experessing cells. As
92%.
(a)
hydroxyl group as a benzyl ether, the cyclic ketone
underwent ring expansion by treatment with TMS-
diazomethane in the presence of BF3·OEt2 to afford the
cyclooctanone product in 73% yield. Reduction of the
carbonyl group with NaBH4, followed by silylation, gave the
silyl ether in 96% yield. The benzyl group in was removed
3
a
4
5
5
by hydrogenation, and the phenol intermediate was treated
with excess of paraformaldehyde in the presence of Et3N and
MgCl2 to form the salicylaldehyde
was constructed by treatment of
ketenylidenetriphenylphosphosphorane. After desilylation and
oxidation, ketone was obtained in 78% yield. The carbonyl
group in was converted to enol triflate, which was
subsequently treated with a strong base NaHMDS to render an (b)
elimination reaction, giving the coumOCT probe
To evaluate the feasibility of probe as a reagent for the
6
. The coumarin scaffold in
7
6
with freshly prepared
8
8
1.
1
SPAAC-based fluorescence labeling, we first studied its
reaction scope and kinetics using benzyl azide (BnN3) as a
model substrate. The SPAAC reaction of
acetonitrile was completed in 2 h at room temperature to give
triazole in 95% yield (Scheme 1). The reactivity of with
1 with BnN3 in
9
1
benzyl azide (1:1) in CD3CN was determined by integration of
multiple chemical shifts in the 1H-NMR spectrum to yield a
o
second-order rate constant of 0.012 M−1s−1 at 25 C. We also
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HeLa cells were incubated in the presence of Ac4GalNAz and
Ac4GlcNAz for three days, respectively. Peracetylated N-
Figure 2. (a) Fluorescence images of sialylated glycoconjugate acetylgalactosamine (Ac4GalNAc) and N-acetylglucosamine
using coumOCT ( ) for SPAAC in live cells by confocal (Ac4GlcNAc) were
1
microscopy; (b) Localization of probe-labeled sialylated
glycoconjugates in HeLa cells under confocal microscopy. The
labeled cells were stained with anti-GM130 followed by Cy3-
conjugated antibody (for Golgi, red). (Scale bar: 10 μm)
a negative control, HeLa cells were grown in the presence of
peracetylated N-acetylmannosamine
(Ac4ManNAc). The cells were incubated with
1 for 30 min at
37 oC under no-wash conditions. The intense fluorescence
labeling was observed for Ac4ManNAz-treated cells relative to
the background with control (Ac4ManNAc) cells (Fig. 2a and
Fig. S4, ESI
glycoconjugates in living cells was visualized by confocal
microscopy. The cells labeled by probe were subsequently
†). Furthermore, the location of azido-containing
1
fixed and stained with anti-GM130 followed by Cy3-conjugated
antibody for marking Golgi apparatus.10 The blue fluorescent
signal derived from the coumarin probe was apparent in Figure 3. Time-lapse intracellular fluorescence imaging of
Ac4ManNAz-treated cells but not in Ac4ManNAc-treated cells. sialylated glycoconjugate trafficking using coumOCT (1) for
The images indicated that coumOCT was labeled to azido-sialic SPAAC in live cells by confocal microscopy. The labeled cells
acid bearing glycoconjugates inside the living cell as well as on were stained with DRAQ5TM for nucleus staining (red). (Scale
the cell surface, and significantly overlapped with the Golgi bar: 10 μm)
staining (red) (Fig. 2b and Fig. S5, ESI†).
Interestingly, the fluorescence signals were observed not employed as the corresponding control sugars. The cells
only on the cell surface but also inside the cells in the living treated with Ac4GlcNAz and Ac4GalNAz showed distinct
cell-imaging experiment. We therefore examined whether patterns in fluorescence channels (Fig. 4g, m), whereas those
coumOCT is a cell-permeable probe that could be utilized for treated with control sugar showed almost no labeling in the
direct intracellular labeling in live cells. In order to obtain corresponding channels (Fig. 4j, p). These images indicated
fluorescence images of azido-bearing glycoconjugates reacting that coumOCT can be used to specifically label azido-GalNAc
with coumOCT inside the living cell, HeLa cells were incubated and azido-GlcNAc containing glycoconjugates inside the living
with Ac4ManNAz for 1 h and subsequently the unbound cell and on the cell surface. Next, we compared the
Ac4ManNAz was removed. We expected that the cell- fluorescence cell imaging upon treatment with different azido-
permeable Ac4ManNAz was firstly taken up into the cytosol sugars using coumOCT. The coumOCT-labeled cells with
before incorporation into glycoconjugates in the Golgi different azido-sugars show cell surface staining and distinct
apparatus and the endoplasmic reticulum (ER).11 After labeling of intracellular Golgi apparatus. The phenomenon
treatment with natural or azido sugar in living cells and labeled suggested that these azido-sugar incorporated glycoconjugates
by coumOCT, we then performed imaging to monitor the were first produced in the Golgi apparatus through metabolic
trafficking of the azido-bearing glycoconjugates. The time processing, then transported to the cell surface. Moreover,
course experiment was conducted by exposing the cells at 30- although coumOCT also labels the cells treated with
min intervals under no-wash and no-fixation conditions (Fig. 3 Ac4GalNAz, the relatively weak fluorescence output is
and Fig. S6, ESI†). The Ac4ManNAz-treated cells showed a attributed to the low efficiency of Ac4GalNAz-mediated
time-dependent increase in fluorescence intensity inside the metabolic processes in HeLa cells.13 The experiments indicated
living cell and then reached saturation after 1.5 h incubation. that coumOCT can be employed as a fluorescence-forming
The punctate staining in Ac4ManNAz-treated cell may be probe for detection of azido-bearing glycoconjugates in living
related to transportation of sialic acid-containing cell by SPAAC-based triazole formation chemistry.
glycoconjugates during incubation with probe
1. These results
A potentially problematic side reaction of cyclooctynes is
indicate that coumOCT possesses good cell-membrane the addition reaction with the endogenous thiol group to form
permeability and specific for the azido-sialic acid the corresponding vinyl sulfide. For example, three
glycoconjugates labeling in live cells.
cyclooctynes (DIBO, DIBAC and BCN) are susceptible to thiol-
Peracetylated N-azidoacetylgalactosamine (Ac4GalNAz) and yne addition with peptidylcysteines.14 On the other hand, Fl-
N-azidoacetylglucosamine (Ac4GlcNAz) have been used for DIBO is a cyclooctyne-based fluorogenic probe, but the triazole
metabolic labeling of glycans in living cell.12 We further adduct will react with thiol-containing molecules such as
examined the use of coumOCT for glycan labeling with glutathione and DTT to quench fluorescence by addition of
different azido-sugars in living cells (Fig. 4 and Fig. S7, ESI
†
). thiol to the cyclopropenone moiety of triazole.8 To test
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Figure 4. A comparison of fluorescence images upon treatment
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fluorescence-forming probe coumOCT (
in living cells under no-wash and no-fixation conditions.
Without side-by-side comparison, probe and Fl-DIBO appear
to have a similar reactivity toward benzyl azide, but probe
given a higher quantum yield and more stable triazole product.
Furthermore, our study establishes that probe is
1) for real-time imaging
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a
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