Click and Fluoresce: A Bioorthogonally Activated Smart Probe for Wash-Free Fluorescent Labeling of Biomolecules

Article abstract of DOI:10.1021/acs.joc.6b02654

Bioorthogonally activated smart probes greatly facilitate the selective labeling of biomolecules in living system. Herein, we described a novel type of smart probes with tunable reaction rates, high fluorescence turn-on ratio, and easy access. The practic

Full text of DOI:10.1021/acs.joc.6b02654

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Article
Click and Fluoresce: A Bioorthogonally Activated Smart
Probe for Wash-free Fluorescent Labeling of Biomolecules
Xingyue Ji, Kaili Ji, Vayou Chittavong, Robert E. Aghoghovbia, Mengyuan Zhu, and Binghe Wang
J. Org. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.joc.6b02654 • Publication Date (Web): 09 Jan 2017
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Click and Fluoresce: A Bioorthogonally Activated Smart Probe for Washꢀfree
Fluorescent Labeling of Biomolecules
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Xingyue Ji, Kaili Ji, Vayou Chittavong, Robert E. Aghoghovbia, Mengyuan Zhu, and
Binghe Wang*
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Department of Chemistry and Center for Diagnostics and Therapeutics, Georgia State
University, Atlanta, Georgia 30303 USA.
* To whom correspondence should be addressed: Email: wang@gsu.edu
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Abstract: Bioorthogonally activated smart probes greatly facilitate the selective
labeling of biomolecules in living system. Herein, we described a novel type of smart
probes with tunable reaction rates, high fluorescence turnꢀon ratio, and easy access.
The practicality of such probes was demonstrated by selective labeling of lipid and
hCAII in Hela cells.
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INTRODUCTION
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The ability to selectively label biomolecules greatly facilitates the
understanding of their dynamic roles under physiological and pathological
conditions. Such understanding could help the identification of specific
biomarkers and development of therapeutic agents against human diseases.1ꢀ5
Bioorthogonal chemistry holds great promise in selective labeling of
biomolecules, which normally involves two steps: tagging biomolecules of
interest with a reporter group and then “clicking’’ with a secondary reagent
normally bearing a fluorescent probe.6ꢀ10 In most cases, an excess of the
secondary reagent is employed to guarantee an adequate labeling efficiency. As
a result, an intensive washing step is required to remove the excessive
unreacted fluorescent reagent in order to eliminate the background fluorescence,
which can also reduce the specific labeling when a nonꢀcovalent tagging
strategy is employed.11 Additionally, such florescent reagents cannot be applied
to situations, where a washing step is impractical, such as realꢀtime monitoring
of biomolecules’ dynamic processes and inꢀvivo labeling.
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In order to address aforementioned issues, there have been a high level of
interest in developing bioorthogonally activated smart probes, where the
florescence is “turned on” upon the click reaction between the two reagents
used, and hence the tedious washing steps become unnecessary.11 For optimal
performance, the smart probes should ideally possess the following
characteristics: 1) tunable reaction kinetics; 2) fast reaction without the need of
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additional catalysis; and 3) high fluorescence turnꢀon ratio (a 100ꢀfold turned
on ratio was considered to be preferable for robust labeling).12 There have been
several elegant types of smart probes developed, where the fluorescence of the
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fluorophore is quenched by azido, alkyne,13ꢀ17 phosphorus (Staudinger
12,22ꢀ28
reaction),18ꢀ21 or tetrazine,
and the fluorescence is turned on after the
click reaction. However, few of them meet all the aforementioned
requirements.12 The fast and tunable reaction rates for tetrazine ligation (with
transꢀcyclooctene) stand out among others; however, only a moderate turnꢀon
ratio (10ꢀ20 fold) was achieved in most cases.22,23,29 Consequently, there is still
need for further improvement, especially in terms of reaction rate tunability,
reaction rate enhancement, and fluorescence “turn on” ratio.
One way to design a labeling system with a high fluorescence turn on ratio is
the formation of a fluorophoreas the result of the labeling reaction, in
contrast to fluorescence quenching strategies. In such a case, only one
fluorescent species is involved, and hence a high fluorescence turnꢀon ratio can
be expected. Very recently, Guo and coꢀworkers described a new probe based
on such a strategy employing the reaction between tetrazine and a terminal
alkene for selective protein labeling. Although the stability of the terminal
alkene is superior to strained alkenes (e.g. transꢀcyclooctene), the reaction rate
is compromised (k2 = 0.078 Mꢀ1sꢀ1).30 Herein, we described a novel
bioorthogonal turnꢀon probe based on the fluorophore generation strategy,
which does not involve any catalyst, has tunable reaction rates, becomes
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significantly fluorescent after the labeling reaction, and can be easily
synthesized in just one step from readily available starting materials.
Previously, we have described the click reaction between
tetraphenylcyclopentadienone and a strained alkyne (e.g. endoꢀBCN) as a
prodrug for carbon monoxide.31 We envisioned that attachment of an
appropriate chromophore to the cyclopentadienone system could lead to the
formation of a fluorophore upon the click reaction. After examining various
possibilities, we found that fusing a naphthalene group at positions 3 and 4 of
the cyclopentadienone moiety allowed for the synthesis of a series of
cyclopentadienones (Figure 1), which are nonꢀfluorescent, and yet are readily
converted to highly fluorescent compounds after click reaction with a strained
alkyne. Therefore, by tagging the biomolecules of interest with a strained
alkyne, such smart probes could be employed for selective washꢀfree labeling
of biomolecules.
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Figure 1. The selective labeling of biomolecules of interest with smart probes
RESULTS AND DISCUSSION
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The synthesis of the smart probes is quite straightforward, and only
involves one step, as shown in Scheme 1. Specifically, commercially ketone
7
8
9
compounds 1a/c and diketone
2 were reacted to afford probes 3a/cby following
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literature procedures.32,33 By using a similar method used for the synthesis of 3c
3b was also synthesized in high yield using 1b as starting material.
,
O
O
O
R1
R2
O
KOH, EtOH, reflux
4
3
+
R1
R2
Or THF, MeOH, Et3N,rt
1a: R1 = Ph, R2 = Ph;
1b: R1 = Ph, R2 = COOMe;
2
3a: R1 = Ph, R2 = Ph, Yield: 75%;
3b: R1 = Ph, R2 = COOMe, Yield: 60%;
1c: R1 = COOMe, R2 = COOMe;
3c: R1 = COOMe, R2 = COOMe, Yield: 65%;
Scheme 1. The synthesis of the smart probes
With these compounds in hand, we next tested their spectroscopic profiles.
The solution of compounds 3a , and showed a blue, purple, or red color
Figure 2a), respectively, but no fluorescence. Click reaction with endoꢀBCN
,
b
c
(
(
o
4
) in DMSO/PBS (10:1) at 37 C led to timeꢀdependent appearance of strong
blue fluorescence with the concomitant disappearance of the color of the
dienone compounds (Figure 2b and ).
3
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Figure 2. Colorimetric (a) and fluorescent changes (b) after the reaction
between 3a (300 µM, 6 h), 3b (300 µM, 1 h), or 3c (300 µM, 5 min) and
4 (1
mM) (DMSO/PBS (pH = 7.4) = 10:1, 37 °C).
Figure 3. Timeꢀdependent fluorescence changes of the reaction of 3a-c
(DMSO/PBS (pH = 7.4) = 10:1, 37 °C): The initial concentrations for
and were a: 80 ꢁM (3a)/1 mM ( ); b: 50 ꢁM (3b)/500 ꢁM ( ) c: 16
ꢁM(3c)/160 ꢁM ( ).
with
3a
4
ꢀ
c
4
4
4
4
The fluorescent products were confirmed by NMR and HRMS as the
cycloaddition products 5a (Table 1.). As shown in Figure 3, the λem for
ꢀ
c
compounds 5a
compounds 5a
ꢀ
c
is 461, 462 and 465 nm, respectively. The quantum yields for
were determined by a relative comparison method using a well
ꢀ
c
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characterized standard, quinine sulfate34 and were found to be in the range of
0.13±0.01ꢀ0.17±0.02 with compound 5a having the highest quantum yield of
0.17±0.02.
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Table 1. The click reactions between
4 and 3aꢀc
k (Mꢀ1sꢀ1)a
λex /λem (nm)b
370/461
Фc
BR (Mꢀ1cmꢀ1) e
Compounds
3a: R1 = R2 = Ph
0.11±0.01
1.30± 0.11
12.2± 0.6 d
0.17± 0.02
0.13±0.01
0.13±0.01
2.7
1.4
1.7
3b: R1 = Ph, R2 = COOMe
3c: R1 = R2 = COOMe
370/462
368/465
a: The second order reaction rate constants; b: The excitation/emission
wavelength for the clicked products 5a ; c: The quantum yield of the clicked
products 5a in methanol; d: Reaction rate constant was determined at room
ꢀc
ꢀ
c
temperature; e: The brightness of compounds 5aꢀc was calculated by extinction
coefficient × quantum yield / 1000.
The significant difference in the fluorescence properties between the
dienone compounds 3a and the cycloaddition products 5a-c allowed for the
easy determination of the second order rate constant (DMSO/PBS, 10:1,
pH=7.4). Since the reaction between 3c and is very fast (e.g. the reaction is
ꢀc
4
finished within 16 min with an initial concentration of 160 ꢁM), the second
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order reaction rate constant was determined at room temperature instead of 37
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°C, which was the condition used for determination of rate constants for 3a
, b
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with . The reaction rate constant between compound 3c and (12.2± 0.6
4
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Mꢀ1sꢀ1) is the highest because of the two strong electron withdrawing ester
groups (Table 1). This is expected because such electronꢀwithdrawing groups
decrease the gap between the LUMO of the diene and the HOMO of the
dienophile in this DAinv reaction. The reaction rate constant for compound 3b
with endoꢀBCN (1.30 ± 0.11 Mꢀ1sꢀ1) is higher than that of compound 3a (0.11 ±
0.01 Mꢀ1sꢀ1), presumably because the ester group is more electronꢀwithdrawing
than the phenyl group in 3a. We also calculated the HOMO energy of
4 and
LUMO energies of 3a
,
3b and 3c by following published procedures.35 The
HOMO energy level for
energy level for compounds 3a
respectively. Such results are qualitatively consistent with the trend of the
observed second order rate constants for the reaction between and dienone
compounds 3a
Having confirmed that compounds 3aꢀc could be turned on by clicking with
4
was calculated to be ꢀ144.4 kcal/mol, and the LUMO
,
3b, and 3c was ꢀ62.9, ꢀ68.4 or ꢀ74.1 kcal/mol,
4
ꢀc.
a strained alkyne, we next tested whether these probes could be applied to a real
biological milieu for labeling purpose. Lipids have been shown to play vital
roles in regulating many signaling pathways, and their abnormality are
associated with many human diseases.36 Selective labeling of lipids facilitates
the study of lipid metabolism, protein lipidation and other lipid biological
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roles.37,38 Some elegant labeling strategies have been successfully employed to
visualize lipids in living cells, including bioorthogonally activated probes.24,39
In order to demonstrate the practicality of our probes, compound 3b was chosen
for the labeling of lipids in Hela cells. For this purpose, an endoꢀBCN modified
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phospholipid
7
6
was synthesized by conjugating DOPE with the activated ester
(Scheme 2).
of endoꢀBCN
O
O2N
O
O
O
H
DOPE-NH2, Et3N, DMF
Yield: 60%
OH
H
O
O
O
O
O
P
N
H
O
H
O
O
H
7
6
Scheme 2. The synthesis of lipid with endoꢀBCN conjugation
Hela cells were then preꢀincubated with compound
7 for 1h. After washing
with medium, the cells were subsequently incubated with probe 3b for another
4 h. Then the cells were fixed and imaged using a fluorescent microscope,
under DAPI imaging channel (excitation: 358 nm, emission: 461 nm). As
shown in Figure 4a, the cells incubated with both compounds
7 and 3b showed
strong blue fluorescence, yet the control cells treated with compound 3b only
did not exhibit any fluorescence (Figure 4c), which highlighted the benefit of
the bioorthogonally activated smart probes. The cells treated with
4 and 3b also
showed blue fluorescence, yet the fluorescence primarily located in the
cytoplasm (Figure 4b), and is different from the one shown in Figure 4a, which
was presumably wellꢀdistributed on the cell surface.
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Figure 5. The twoꢀstep labeling of lipids in Hela cells. a) Cells pretreated with
7
(100 ꢁM) for 1h, and followed by treatment of 3b (50 ꢁM) for 4h; b) Cells
pretreated with (100 ꢁM, without phospholipid conjugation) for 1h, and
4
followed by treatment of 3b (50 ꢁM) for 4h; c) Cells treated with dienone
compound 3b (50 ꢁM) only. The image was taken under DAPI channel.
By installing a clickable handle on smallꢀmolecule inhibitors without
disturbing their binding affinity to the target proteins, bioorthogonal smart
probes have been applied to the investigation of the distribution of such
inhibitors, and thus the expression level and subꢀlocation of the protein of
interest in cells.22,24 Human carbonic anhydrase II (hCAII) is one of fourteen
forms of human alpha carbonic anhydrase, which regulate numerous
physiological processes, and play pivotal roles in a variety of diseases,
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including glaucoma, epilepsy, and cancer.40 It has been reported that
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4ꢀ(aminomethyl)benzenesulfonamide (8, scheme 3) showed potent inhibitory
8
9
activity to hCAII, and chemical modifications to the benzylamino group impose
little impact on the binding to hCAII.41,42 Therefore, compound
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9
with
endoꢀBCN conjugation was synthesized (Scheme 3) for twoꢀstep labeling of
hCAII protein in Hela cells.
Scheme 3. The synthesis of endoꢀBCN conjugated hCAII inhibitor.
Hela cells were preꢀtreated with compound
9 for 1 h. After washing with
medium, the cells were subsequently incubated with probe 3b for another 4h.
Then the cells were fixed and imaged under DAPI channel. As shown in Figure
6a, the cells treated with compound
yet the cells treated with compound
fluorescence (data not shown). In order to confirm that the blue florescence was
mainly the result of binding by to hCaII, the cells were preꢀtreated with 100
M of the inhibitor for 1 h. After washing with medium, the cells were
further incubated with compound for another 1h. The cells were washed again
to remove the unbound compound , followed by the incubation with probe 3b
9
and 3b showed strong blue fluorescence,
9
or 3b only did not present any
9
ꢀ
8
9
9
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for another 4h. Then the cells were fixed for imaging study under DAPI
channel. As shown in Figure 5b, the intensity of the blue fluorescence was
significantly weaker as compared to the one in Figure 5a, which presumably
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was due to the competition between compound
9 and the inhibitor 8 in binding
to the target protein. Such a strategy showed potential as a highꢀthroughout
screening assay for the discovery of new inhibitors for the target protein in
living cells.
Figure 5. The twoꢀstep labeling of hCAII with biorthogonal activated probe. a)
Cells preꢀtreated with compound
treatment of probe 3b (50 M) for another 4 h; b) Cells treated with inhibitor
(100 M) for 1h, and followed the treatment of (20 M, 1 h) and 3b (50 M,
9 (20 ꢀM) for 1h, then followed by the
ꢀ
8
ꢀ
9
ꢀ
ꢀ
4 h) separately. The image was taken under DAPI channel.
CONCLUSIONS
In conclusion, several cyclopentadienones were designed as a novel type of
smart probes. These probes are nonꢀfluorescent, and generate a highly
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fluorescent fluorophore after labeling reaction with a strained alkyne (e.g.
endoꢀBCN) under near physiological conditions. The second order reaction rate
is tunable with the rate constants ranging from 0.11± 0.01 to 12.2± 0.6 Mꢀ1sꢀ1
with the three cyclopentadienones studied. This represents a 100ꢀfold change in
reaction rate constants. The application of these probes was demonstrated by
the washꢀfree labeling of lipids and hCAII protein in Hela cells using BCN
modified probes and compound 3b. Therefore, we believe that the probes
reported herein hold great promise in the washꢀfree labeling of other
biomolecules.
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Experimental sections
General: All reagents and solvents were of reagent grade. Column
chromatography was carried out using flash silica gel (230–400 mesh) and Pꢀ2
Gel (particle size range 45ꢀ90 ꢁm). TLC analysis was conducted on silica gel
1
plates (Silica G UV254). NMR spectra were recorded at 400 MHz for H and
13
100 MHz for C. Chemical shifts (δ values) and coupling constants (J values)
are given in ppm and hertz, respectively, using the respective solvent (1H NMR,
13C NMR) as the internal reference. IR spectra were recorded on a FTꢀIR
system, and were reported in frequency of absorption (cm−1). Mass
spectrometric results were obtained by the GSU Mass Spectrometry Facilities
with a TOF analyzer.
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7, 9-Diphenyl-8H-cyclopenta[a]acenaphthylen-8-one (3a). Compound 3a was
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8
synthesized by following previously published procedures (yield: 75%).33 1H
NMR (CDCl3): 8.08 (d, = 7.2 Hz, 2H), 7.89ꢀ7.86 (m, 6H), 7.61 (t, = 7.6 Hz,
2H), 7.55 (t, = 7.6 Hz, 4H), and 7.43 (t, = 7.2 Hz, 2H). HRMS (ESI) m/z:
J
J
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J
J
[M+H]+ Calcd for C27H17O 357.1279; Found 357.1291.
Methyl 8-oxo-9-phenyl-8H-cyclopenta[a]acenaphthylene-7-carboxylate (3b). A
solution of compound 1b (1.0 g, 5.2 mmol), which was synthesized following
literature procedures,32 and acenaphthyleneꢀ1, 2ꢀdione (0.95 g, 1 equiv.) in
THF/MeOH (30/10 ml) was treated with Et3N (0.79 g, 1.5 equiv.), and the reaction
mixture was stirred overnight at room temperature. The formed dark green precipitate
was filtered, and washed with methanol to give compound 3b as dark green solid (1.0
g, yield: 60%). 1H NMR (CDCl3): 8.77 (d,
= 8.0 Hz, 1H), 7.84 (d, = 7.2 Hz, 2H), 7.80 (t,
1H), 7.54 (t, = 8.0 Hz, 2H), 7.47 (t, = 7.6 Hz, 1H), and 4.03 (s, 3H). HRMS (ESI)
m/z: [M+H]+ Calcd for C23H15O3 339.1021; Found 339.1036.
J
= 7.2 Hz, 1H), 8.09ꢀ8.05 (m, 2H), 7.94 (d,
J
J
J
= 7.6 Hz, 1H), 7.63 (t, = 8.0 Hz,
J
J
J
43
Dimethyl 8-oxo-8H-cyclopenta[a]acenaphthylene-7, 9-dicarboxylate (3c). By a
similar method used to synthesize 3b, 3c was obtained as a red solid (210mg, yield:
65%). 1H NMR (CDCl3): 8.67 (d,
J = 7.2 Hz, 2H), 8.09 (d, J = 8.0 Hz, 2H), 7.80 (t, J
= 7.2Hz, 2H), and 4.01 (s, 6H). HRMS (ESI) m/z: [M+H]+ Calcd for C19H13O5
321.0763; Found 321.0755.
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General procedure for the click reaction between compounds 3a-c and 4
A solution of 4 (10 mg, 0.067 mmol) and compound 3a, 3b or 3c (0.5 equiv.) in
DMSO/PBS (pH = 7.4, 10:1, 11 ml) was incubated at 37 oC till the featured color for
3aꢀc disappeared. Then water (30 ml) was added, and product was extracted with
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ethyl acetate (3
× 30 ml). The combined organic layer was dried over anhydrous
Na2SO4. After filtration and concentration, the obtained residue was purified over
silica gel column using CH2Cl2/ethyl acetate (20:1) to obtain compound 5aꢀc.
1
o
5a, pale yellow solid (13mg, yield: 85%). H NMR (CDCl3, 60 C) δ 7.67 – 7.50 (m,
8H), 7.46 (t,
(d,
J
= 7.6 Hz, 4H), 7.24 (t,
J
= 7.6 Hz, 2H), 6.37 (d,
J
= 7.2 Hz, 2H), 3.75
13
J
= 7.2 Hz, 2H), 2.97 – 2.69 (m, 4H), 2.06 (br, 2H), 1.31ꢀ1.10 (m, 5H). C NMR
(CDCl3): 141.1, 136.9, 135.2, 132.8, 129.6, 129.3, 129.1, 128.9, 127.6, 127.4, 125.9,
122.5, 65.8, 59.8, 29.7, 17.2, and 15.4. IR (
, cm−1): 3356, 2922, 1425, 1237, 1019, 825,
ν
770, 700. HRMS (ESI) m/z: [M+Na]+ Calcd for C36H30ONa 501.2194; Found
501.2197.
5b, pale yellow solid (13mg, yield: 87%). 1H NMR (CD3CN, 60 oC) δ 7.90 (d,
J
= 8.0
= 8.0 Hz, 2H), 7.69ꢀ7.62 (m, 4H), 7.44ꢀ7.39 (m, 2H), 7.35 – 7.21
= 8.0 Hz, 1H), 4.15 (s, 3H), 3.62 (br, 2H), 3.08 (br, 1H), 2.99 –
Hz, 1H), 7.79 (d,
(m, 1H), 6.34 (d,
J
J
2.79 (m, 2H), 2.72 (br, 1H), 2.30 (br, 1H), 2.19ꢀ2.18 (m, 1H), 1.71 – 1.38 (m, 2H),
1.02ꢀ0.93 (m, 3H). 13C NMR (CDCl3): 171.2, 136.2, 136.0, 134.7, 132.8, 132.7, 129.8,
129.7, 129.1, 129.0, 127.8, 127.7, 127.6, 127.0, 126.4, 122.8, 121.4, 60.4, 59.8, 52.5,
34.8, 29.7, 28.8, 27.9, and 14.1. IR (ν
, cm−1): 3410, 2929, 1719, 1429, 1225, 1284,
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1141, 1018, 824, 772, 704. HRMS (ESI) m/z: [M+Na]+ Calcd for C32H28O3Na
483.1936; Found 483.1913.
7
8
9
5c, pale yellow solid (13mg, yield: 88%). 1H NMR (CD3CN, 60 oC) δ 7.98 (d,
J = 8.0
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Hz, 2H), 7.80 (d, = 8.0 Hz, 2H), 7.71(t, = 8.0 Hz, 2H), 4.14 (s, 6H), 3.62 (br, 2H),
J
J
3.09ꢀ3.03 (m, 2H), 2.92ꢀ2.85 (m, 2H), 2.35 – 2.13 (m, 3H), 1.62 (br, 2H), 1.02 (br,
1H), 0.91 (br, 2H). 13C NMR (CDCl3): 170.2, 134.1, 133.8, 132.6, 130.1, 129.9, 127.9,
127.4, 121.8, 59.7, 52.6, 29.7, 22.7, 21.1, and 14.2. IR (ν
, cm−1): 2923, 1715, 1430,
1224, 1284, 1139, 1014, 822, 769. HRMS (ESI) m/z: [M+Na]+ Calcd for C28H26O5Na
465.1678; Found 465.1663.
The synthesis of lipid with endo-BCN conjugation (7)
The activated ester of endoꢀBCN 6 (60 mg, 0.19 mmol) was dissolved in DMF/THF
(3 mL/1.5 mL), and then DOPEꢀNH2 (100 mg, 0.14 mmol) and Et3N (23 mg, 0.23
mmol) were added in one portion. The obtained solution was stirred at 60 oC for 6 h.
Then the reaction mixture was dried under vacuum. The obtained residue was purified
over silica gel using DCM/MeOH (10:1) to afford compound 7 as a colorless oil
(77mg, Yield: 60%). 1H NMR (CDCl3): 9.35 (s, 1H), 5. 40ꢀ5.31 (m, 4H), 5.24 (br, 1H),
4.41ꢀ4.38 (m, 1H), 4.21ꢀ4.15 (m, 3H), 3.98 (br, 4H), 3.43ꢀ3.25 (m, 3H), 2.30ꢀ2.22 (m,
13
9H), 2.07ꢀ1.99 (m, 7H), 1.51ꢀ1.21 (m, 49H), 0.96ꢀ0.88 (m, 8H). C NMR (CDCl3):
173.4, 173.0, 130.0, 129.7, 98.8, 46.5, 34.2, 34.1, 31.9, 29.8, 29.54, 29.4, 29.3, 29.2,
29.2, 29.1, 27.2, 27.1, 24.9, 24.8, 22.7, 21.4, 20.3, 20.2, 18.9, 14.1. IR (ν
, cm−1): 3360,
2923, 2853, 1738, 1719, 1460, 1233, 1061. HRMS (ESI) m/z: [MꢀH]ꢀ Calcd for
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C52H89NO10P 918.6224; Found 918.6258.
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8
9
The synthesis of hCAII inhibitor with endo-BCN conjugation (9)
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The activated ester of endoꢀBCN 6 (50 mg, 0.16 mmol) was dissolved in DMF (4
mL), and then 8 (44mg, 0.24mmol) and Et3N (24mg, 0.24mmol) were added in one
portion. The obtained solution was stirred at room temperature for 6 h. Then the
reaction mixture was dried under vacuum. The obtained residue was purified over
silica gel (hexane : ethyl acetate = 2:1) to afford compound 9 as a white solid (40mg,
1
Yield: 70%). H NMR (DMSOꢀ
d
6): δ 7.77 (d,
= 6.0 Hz, 2H), 4.08 (d,
6H), 1.54ꢀ1.52 (m, 2H), 1.38 – 1.22 (m, 1H), 0.90ꢀ0.85 (m, 2H). 13C NMR
(DMSOꢀ 6): δ 157.2, 144.5, 143.1, 127.8, 126.1, 99.5, 62.2, 43.9, 29.1, 21.3, 20.1,
18.1. IR (
, cm−1): 3383, 3216, 3092, 1689, 1537, 1317, 1267, 1151, 1138, 910, 808,
J
= 8.0 Hz, 3H), 7.42 (d,
J = 8.0 Hz,
2H), 7.31 (s, 2H), 4.24 (d,
J
J
= 8.0 Hz, 2H), 2.24ꢀ2.09 (m,
d
ν
670. HRMS (ESI) m/z: [M+H]+ Calcd for C18H23N2O4S 363.1373; Found 363.1366.
The determination of the quantum yield for compound 5a-c
The quantum yields of compounds 5a-c were determined by a comparative
method using a well characterized standard, quinine sulfate (Φ = 0.54 in 0.05 M of
H2SO4). Briefly, a series of solutions of compounds 5aꢀc and standard with different
concentrations (4, 8, 12, 16 and 20 ꢁM) were prepared, and the UV absorbance at
their excitation wavelength was taken. Then the fluorescence of the same solution was
recorded. The integrated fluorescence was plotted against the absorbance, and the
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quantum yield for the compounds was calculated according to the following equation:
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6
7
2
2
Φx
=
Φ
ST(Gradx/GradST)( ηx /ηST
)
(1)
8
9
Where the subscripts ST and x denote standard and compound respectively;
Φ
is the
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fluorescence quantum yield; Grad is the gradient from the plot of integrated
fluorescence intensity against absorbance; and η is the refractive index of the solvent.
The determination of the second order reaction rate constants between 3a-c and
4
The fluorescent property of the cycloaddition product 5aꢀc greatly facilitates the
determination of the second order reaction rate constants between 3aꢀc and 4
(DMSO/PBS = 10:1, 37 oC). The reaction rate constant for 3c was determined at room
temperature. Briefly, the second order reaction was treated as pseudo first order
reaction by using an excessive amount of 4 (>10 fold). The reaction process was
monitored by the increase of the fluorescence intensity of the click product at different
time intervals. The fluorescence intensity was then plotted against time, and the
resulting curve was fitted using Sigmaplot to get the pseudo first reaction rate
constant k’. The obtained k’ was then plotted against the concentration of 4 used, and
the slope is the second order reaction rate constant between 3aꢀc and 4.
Cell imaging
HeLa cells (ATCC® CCLꢀ2™) were maintained in DMEM (Dulbecco’s Modified
Eagle’s Medium) supplemented with 10% fetal bovine serum (MidSci; S01520HI)
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and 1% penicillinꢀstreptomycin (SigmaꢀAldrich; P4333) at 37 °C with 5% CO2. The
media was changed every other day. All the experiments were done within 10
passages of HeLa cells.
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1). Lipid imaging
The HeLa cells were seeded on coverslips in the 6ꢀwell plate one day before the
compound treatment. The compounds were dissolved in DMSO as stock solution. The
cells were incubated with 7 (100
ꢀ
M) for 1 hour. The cells were then washed with
PBS followed by incubation with 3b (50
ꢀM) for 4 hours under 37 °C. The cells
treated with 7 or 3b only were used as negative controls.
2). Carbonic anhydrase II imaging
The HeLa cells were seeded on coverslips in the 6ꢀwell plate one day before the
compound treatment. The compounds were dissolved in DMSO as stock solution. The
cells were incubated with 9 (20
followed by incubation with 3b (50
binding assay, the cells were first incubated with 100
ꢀ
M) for 1 hour. The cells were then washed with PBS
M) for 4 hours at 37 °C. For the competitive
M inhibitor 8 for 1 hour, and
ꢀ
ꢀ
then followed the incubation steps stated above. After that, the cells were washed with
PBS twice and fixed with 4% paraformaldehyde in PBS for 30 minutes at room
temperature. Then, the cell samples were immersed in 300 ꢀM glycine in PBS for 1
hour at room temperature to quench the auto fluorescence. The cells were then
washed with PBS twice and the coverslips with cells were immersed in deionized
water. The coverslips were mounted onto glass slides using the mounting media
without DAPI (ProLong® Live Antifade Reagent; P36974). The fluorescent imaging
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was performed on a Zeiss fluorescent microscope, using DAPI imaging channel
(excitation: 358 nm, emission: 461 nm).
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ASSOCIATED CONTENT
The data for the reaction rate constant and quantum yield of the click product, the
tables of atom coordinates, along with copies of 1H and 13C NMR spectra of products.
This material is available free of charge via the Internet at http://pubs.acs.org.
Acknowledgement: Partial financial support from the National Institutes of Health
(CA180519) is gratefully acknowledged.
NOTES
The authors declare no competing financial interest.
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