Tuning the optical properties of BODIPY dye through Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction

Article abstract of DOI:10.1007/s11426-011-4452-2

Borondipyrromethenes (BODIPY) are a class of fluorescent dyes whose fluorescence quantum yields are generally high and independent of the solvent. In this paper, we report the synthesis of a new type of BODIPY compound that carries an azido group on the 3-position of the pyrrole core. The azido group quenches the fluorescence of the dye due to its weak electron-donating effect. The fluorescence of the BODIPY dye can be switched on after reacting with alkynes via a Cu(I) catalyzed azide-alkyne cycloaddition (CuAAC) reaction. We further demonstrate that this azido-BODIPY compound can be used in the cell imaging applications.

Full text of DOI:10.1007/s11426-011-4452-2

SCIENCE CHINA
Chemistry
January 2012 Vol.55 No.1: 125–130
doi: 10.1007/s11426-011-4452-2
• ARTICLES •
· SPECIAL TOPIC · The Frontiers of Chemical Biology and Synthesis
Tuning the optical properties of BODIPY dye through Cu(I)
catalyzed azide-alkyne cycloaddition (CuAAC) reaction
WANG Chao1, XIE Fang2, SUTHIWANGCHAROEN Nisaraporn2, SUN Jian1* & WANG Qian2*
1Natural Products Center, Chengdu Institute of Biology, Chinese Academy of Sciences, Chengdu 610041, China
2Department of Chemistry and Biochemistry, University of South Carolina, Columbia, SC 29208, USA
Received September 17, 2011; accepted October 22, 2011; published online November 29, 2011
Borondipyrromethenes (BODIPY) are a class of fluorescent dyes whose fluorescence quantum yields are generally high and
independent of the solvent. In this paper, we report the synthesis of a new type of BODIPY compound that carries an azido
group on the 3-position of the pyrrole core. The azido group quenches the fluorescence of the dye due to its weak electron-
donating effect. The fluorescence of the BODIPY dye can be switched on after reacting with alkynes via a Cu(I) catalyzed
azide-alkyne cycloaddition (CuAAC) reaction. We further demonstrate that this azido-BODIPY compound can be used in the
cell imaging applications.
CuAAC reaction, fluorogenic, BODIPY dye, cell imaging, fluorescent probe
1 Introduction
It has been found that Cu(I) catalyzed 1,3-dipolar az-
ide-alkyne cycloaddition (CuAAC) reaction [1, 2], a proto-
typical click chemistry, could activate the fluorescence of
Figure 1 Structure of the BODIPY core.
some pro-fluorophores with either azido or alkynyl groups
BODIPY (borondipyrromethene) dyes are a type of
by the formation of triazole rings [3–6]. This fluorogenic
interesting fluorophores which have numerous advantages
process has shown widespread applications, especially in
including great chemical and photo stability, relatively high
the emerging field of cell biology and functional proteomics
absorption coefficients, high quantum yields (ca. фf = 0.5
[7], due to the high reaction efficiency of the CuAAC reac-
0.8), visible excitation wavelength (ca. 500 nm), narrow
tion under mild reaction conditions and the distinct fluores-
emission bands and high emission intensity. They have been
cence properties of the products [8]. So far only a few types
used as laser dyes [9–20], markers [21, 22], sensors [23–28]
of fluorophores, including coumarin, anthracene, and naph-
in modern chemistry and biology [29]. It is well known that
thalimide, have been explored [8], and all of them can only
the functionalities on 3-, 4-, 5- and 8-positions of the
be irradiated by UV light. In biological systems, irradiation
BODIPY core (Figure 1) strongly impact its optical proper-
at UV range may cause high background noise and cell
ties [30–33], which led to many efforts to design
damage. Therefore, fluorophores with longer excitation and
BODIPY-based sensor molecules [34–39]. In particular, an
emission wavelengths, which can be activated in situ, are
internal charge transfer (ICT) process has often been em-
highly demanded to develop.
ployed by introducing an electron donating amino group at
the 3-position of the pyrrole ring of the BODIPY. For
*Corresponding authors (email: sunjian@cib.ac.cn; wang@mail.chem.sc.edu)
example, it has been shown that mono- or di-substitution of
© Science China Press and Springer-Verlag Berlin Heidelberg 2011
chem.scichina.com www.springerlink.com

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Wang C, et al. Sci China Chem January (2012) Vol.55 No.1
3,5-dichloro BODIPY caused the products to have quite
different absorption and emission spectra [32, 40].
(trifluoromethyl)benzene (9 mg, 0.05 mmol) were suspend-
ed in a 7:3 mixture (V/V) of ethanol and water (8 mL).
Copper sulfate (3 mg, 0.015 mmol), sodium ascorbate (6 mg,
0.03 mmol) and tris[(N-benzyltriazolyl)-methyl]amine lig-
and (8 mg, 0.015 mmol) were added. The mixture was then
stirred at room temperature for 12–16 h, and monitored by
TLC. EtOAc (20 mL  2) were added to extract the product.
The combined organic layers were washed with brine and
dried over anhydrous Na2SO4. The solvent was removed
under vacuum and the resulting residue was purified with
flash chromatography on silica gel to get the triazole com-
pound as a yellow solid (22 mg, 89%).
Based on our previous study, we postulated that when an
azido group is introduced to the 3-position of the BODIPY
core, the electron donating effect of the -nitrogen of the
azido group will quench the fluorescence via an ICT pro-
cess. Upon CuAAC reaction with a terminal alkyne, the
formation of triazole ring will lower the electron donating
ability of this nitrogen, release the fluorescence quenching,
and lead to a fluorogenic phenomenon [3].
1
2 Experimental
6: H NMR (300 MHz, CDCl3): δ (ppm) 9.12 (s, 1 H),
7.94 (s, 1 H), 7.73–7.69 (m, 2 H), 7.57 (d, J = 8.1 Hz, 2 H),
7.45–7.39 (m, 2 H), 7.15 (s, 1 H), 7.17–7.01 (m, 4 H), 6.62
(s, 1 H), 3.92 (s, 3 H); 13C NMR (100 MHz, CDCl3): δ (ppm)
146.1, 135.4, 130.5, 130.2, 129.6, 128.7, 127.8, 126.6,
125.9, 125.6, 125.5, 125.1, 124.4, 120.9, 55.5, 45.9. HRMS
calculated for C24H17BF3N5O (M+): m/z 459.1593, found
m/z 459.1587.
2.1 General experimental section
Chemical reagents and solvents were purchased from VWR
Scientific, and were used without further purification. H
1
NMR spectra were recorded on Varian 300 NMR spectrom-
eter and the chemical shifts were reported relative to TMS.
13C NMR spectra were recorded either on Varian 300 NMR
or Varian 400 NMR spectrometer. Analytical thin-layer
chromatography (TLC) was performed on silica 60F-254
plates. Flash column chromatography was carried out on
silica gel. The UV-vis absorption spectra were measured on
Agilent 8453 spectrometer. Emission spectra were meas-
ured on Varian Cary Eclipse fluorescence spectrophotome-
ter. Fluorescent microcopy was performed on Olympus IX
86. RPMI 1640 without folic acid and Dulbecco’s Modified
Eagle’s Medium (DMEM) were purchased from Invitrogen
(Carlsbad, CA). KB cells and Human Mesenchymal Stem
Cell (HMSCs) were purchased from ATCC.
1
7: H NMR (300 MHz, CDCl3): δ (ppm) 9.08 (s, 1 H),
7.91 (s, 1 H), 7.87 (d, J = 8.1 Hz, 2 H), 7.55 (d, J = 8.1 Hz,
2 H), 7.28 (d, J = 8.1 Hz, 2 H), 7.18 (d, J = 4.2 Hz, 1 H),
7.09–7.06 (m, 3 H), 6.99 (d, J = 2.1 Hz, 1 H), 6.63 (s, 1 H),
3.92 (s, 3 H), 2.65 (t, J = 7.2 Hz, 2 H), 1.67–1.63 (m, 2 H),
1.37–1.34 (m, 4 H), 0.92–0.82 (m, 3 H); 13C NMR (75 MHz,
CDCl3): δ (ppm) 162.3, 148.4, 147.1, 143.6, 143.3, 139.3,
134.3, 133.4, 132.5, 132.4, 131.1, 128.9, 127.2, 126.1,
125.8, 120.3, 120.2, 120.1, 119.0, 114.1, 113.0, 55.6, 35.8,
33.2, 31.9, 22.6, 14.0. HRMS calculated for C29H28BF2N5O
(M+): m/z 511.2469, found m/z 511.2483.
8: 1H NMR (300 MHz, CDCl3): δ (ppm) 9.11 (s, 1 H),
7.64 (s, 1 H),7.51–7.46 (m, 2H), 7.22 (d, J = 3.6 Hz, 1 H),
7.03–7.00 (m, 3 H), 6.76–6.75 (m, 1 H), 6.45–6.43 (m, 1 H),
5.23 (s, 1 H), 5.13 (s, 1 H), 3.89 (s, 3 H), 3.26 (s, 3 H); 13C
NMR (75 MHz, CDCl3): δ (ppm) 167.2, 160.5, 151.0, 141.5,
137.4, 136.4, 133.8, 132.5, 131.0, 130.0, 125.8, 125.2,
116.9, 115.0, 113.0, 112.0, 54.5, 21.3.
2.2 Synthesis of azido BODIPY 5 and sequential Cu-
AAC reaction
2.2.1 Synthesis of azido BODIPY 5
To 30 mL acetonitrile solution of monochloride BODIPY 4
[41–43] (240 mg, 0.69 mmol), 3 equivalents of sodium
azide (134 mg, 2.07 mmol) were added. The mixture was
stirred in dark at room temperature for 3 h, monitored with
TLC. The reaction was concentrated under vacuum and the
resulting residue was purified by chromatography to get
1
9: H NMR (400 MHz, CDCl3): δ (ppm) 8.63 (s, 1 H),
7.88 (s, 1 H), 7.54 (d, J = 9.0 Hz, 2 H), 7.17–7.05 (m, 3 H),
6.98–6.97 (m, 1 H), 6.60–6.59 (m, 1 H), 3.91 (s, 3 H), 3.71
(t, J = 4.5 Hz, 2 H), 2.88 (t, J = 5.7 Hz, 2 H), 1.89–1.84 (m,
2 H), 1.75 - 1.67 (m, 2 H); 13C NMR (100 MHz, CDCl3): δ
(ppm) 162.2, 148.5, 147.3, 147.2, 143.2, 134.2, 133.3, 132.4,
131.1, 125.8, 122.3, 122.2, 122.1, 118.9, 114.2, 112.9, 62.6,
55.6, 32.2, 30.0, 25.3. HRMS calculated for C22H22BF2N5O2
(M+): m/z 437.1949, found m/z 437.1957.
1
BODIPY 5 as a red solid (210 mg, 86%). H NMR (400
MHz, CDCl3): δ (ppm) 7.79 (s, 1 H), 7.48 (d, J = 8.4 Hz, 2
H), 7.04 (d, J = 8.4 Hz, 2 H), 6.99 (d, J = 4.8 Hz, 1 H), 6.84
(d, J = 4.2 Hz, 1 H), 6.50 (m, 1 H), 6.35 (d, J = 4.2 Hz, 1 H),
3.91 (s, 3 H); 13C NMR (100 MHz, CDCl3): δ (ppm) 161.8,
152.4, 144.0, 140.8, 134.0, 133.3, 132.9, 132.2, 128.8,
125.9, 117.4, 114.1, 109.9, 55.5; HRMS calculated for
(C16H12BF2N5O + Na)+: m/z 345.1607, found m/z 345.1613.
1
10: H NMR (300 MHz, CDCl3): δ (ppm) 9.17 (s, 1 H),
8.07 (d, J = 6.6 Hz, 2 H), 7.94 (s, 1 H), 7.74–7.65 (m, 4 H),
7.58 (d, J = 6.6 Hz, 2 H), 7.50–7.45 (m, 2 H), 7.40–7.35 (m,
1 H), 7.21 (d, J = 4.5 Hz, 1 H), 7.11–7.07 (m, 3 H), 7.02 (s,
1 H), 6.64 (s, 1 H), 3.94 (s, 3 H); HRMS calculated for
C30H22BF2N5O (M+): m/z 517.2000, found m/z 517.2005.
11: 1H NMR (300 MHz, DMSO): δ (ppm) 8.98 (s, 1 H),
7.90 (s, 1 H), 7.81 (d, J = 8.7 Hz, 2 H), 7.56 (d, J = 8.4 Hz,
2.2.2 Typical experimental procedure for CuAAC reac-
tions
Azido BODIPY 5 (17 mg, 0.05 mmol), 1-ethynyl-3-

Wang C, et al. Sci China Chem January (2012) Vol.55 No.1
127
2 H), 7.21 (d, J = 4.8 Hz, 1 H), 7.09–7.07 (m, 3 H), 6.97 (d,
J = 3.9 Hz, 1 H), 6.75 (d, J = 8.7 Hz, 2 H), 6.61 (s, 1 H),
3.93 (s, 3 H), 3.44 (q, J = 6.6 Hz, 4 H), 1.19 (t, J = 6.6 Hz, 6
H). HRMS calculated for C28H27BF2N6O (M+): 512.2422,
found m/z 512.2439.
ro-BODIPY precursor 4 [43]. The nucleophilic reaction was
tested in several different conditions. The monochloro
BODIPY 4 disappeared, and no desired azido compound
was formed at room temperature in the most often used
solvent such as DMSO and DMF. Finally with acetonitrile
as the solvent, 4 was smoothly converted to the final azido
BODIPY compound 5 in three hours at room temperature.
The product was purified by choromatography, and con-
firmed by NMR, IR, and HRMS analysis. As we predicted,
5 showed very weak fluorescence under the irradiation of
520 nm while it had a strong absorption peak around this
range.
The CuAAC reactions between BODIPY azide 5 and al-
kynes were first conducted in DMF/H2O (4:1) following the
common reaction conditions. However, the yield of the de-
sired triazole products was very low while some unknown
impurities were observed. Upon optimization, moderate to
high yields of the CuAAC reaction was obtained when
EtOH/H2O (7:3) was used as solvent. Eight different al-
kynes with different electron density and structure diversity
were selected to react with BODIPY azide 5, and the yields
of the designed triazole products were listed in Scheme 1.
The optical properties of the resulting triazole com-
pounds were analyzed by UV-vis and fluorescence spec-
troscopy, which revealed that the CuAAC reaction could
indeed trigger the fluorogenic process. The maximum ab-
sorption and emission wavelengths of triazole products in
12:1H NMR (300 MHz, CDCl3): δ (ppm) 9.17 (s, 1 H),
8.24 (s, 1 H), 8.15 (d, J = 5.7 Hz, 1 H), 7.95 (s, 1 H), 7.65–
7.57 (m, 4 H), 7.18 (d, J = 3.6 Hz, 1 H), 7.10–7.07 (m, 3 H),
7.04 ( s, 1 H), 6.65 (s, 1 H), 3.93 (s, 3H); 13C NMR (100
MHz, CDCl3): δ (ppm) 162.4, 147.6, 146.8, 146.3, 144.0,
134.5, 133.4, 132.5, 131.9, 131.7, 130.9, 130.7, 129.4,
125.7, 125.4, 125.2, 125.1, 123.0, 122.9, 122.7, 121. 2,
119.4, 114.3, 112.8, 55.6. HRMS calculated for
C26H17BF5N5O (M+): m/z 509.1561, found m/z 509.1569.
13:1H NMR (300 MHz, DMSO): δ (ppm) 8.98 (s, 1 H),
7.90 (s, 1 H), 7.81 (d, J = 8.7 Hz, 2 H), 7.55 (d, J = 8.7 Hz,
2 H), 7.20 (s, 1H), 7.10–7.07 (m, 3 H), 6.98 (s, 1 H), 6.70 (d,
J = 8.7 Hz, 2 H), 6.60 (s, 1 H), 3.93 (s, 3 H), 3.23 (q, J = 7.2
Hz, 2 H), 1.30 (t, J = 7.2 Hz, 3 H). HRMS calculated for
C26H23BF2N6O (M+): m/z 484.2109, found m/z 484.2119.
2.3 Cell labeling and detection by fluorescence mi-
croscopy
KB cells were cultured in RPMI 1640 medium without folic
acid for 2 weeks prior to the experiment, adjusted to contain
1% penicillin/streptomycin and 10% fetal bovine serum
(FBS). HMSCs were grown in DMEM supplemented with
10% FBS and 1% penicillin/streptomycin. Both cells were
o
maintained in humid 5% CO2 atmosphere at 37 C. Cells
were plated into a 12-well plate at a density of 1 × 105
cells/well 24 h prior to the experiment. Then the medium
was replaced with 1 mL of fresh medium containing 5 µg
triazole folate compound 15 which was obtained by the
congjuation of folate ligand 14 and BODIPY 5. After 2 h,
the cells were washed 3 times with PBS, and then fixed with
4% paraformaldehyde in PBS for 20 min at room tempera-
ture. The nucleus was labeled with DAPI for 10 min fol-
lowed by several washes with PBS, and then visualized us-
ing a confocal microscope.
3 Results and discussion
As shown in Scheme 1, the monoazide derivatized BODIPY
dyes were synthesized to test our hypothesis. The starting
compound 1 was obtained according to the reported method
by condensation of p-anisaldehyde with neat excess pyrrole
under the catalysis of trifluoroacetic acid at room tempera-
ture [41]. It was purified by chromatography and recrystal-
lization, and then chlorinated with one equivalent of
N-chlorosuccinimide in tetrahydrofuran at 78 °C [42]. The
monochlorinated compound 2 was oxidized with p-chloranil
in dichloromethane to give 3, which was then refluxed with
triethylamine and BF3-OEt2 in toluene to afford the chlo-
Scheme 1 Synthesis of the azido-BODIPY dye and sequential CuAAC
reactioin.

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Wang C, et al. Sci China Chem January (2012) Vol.55 No.1
different solvent systems were summarized in Table 1. The
results showed that alkynes with different structure and
electron density could only slightly tune the maximum ab-
sorption and emission wavelength of the triazole products.
The fluorescence intensity comparison between the azido
compound 5 and the triazole compounds were conducted in
chloroform. For most products, a significant intensity in-
crease was observed when comparing the fluorescence of
the trazole products with the azido compound 5. Generally,
the electron withdrawing groups on the benzene ring (e.g. 6
and 12) increased the fluorescence intensities of the triazole
products, whereas the electron donating groups (e.g. 11 and
13) significantly decreased the fluorescence signals as
shown in Figure 2.
With all the results mentioned above, it clearly shows
that the fluorescence of the azido compound 5 can be effi-
ciently switched on through a CuAAC reaction. In addition
to the preparation of the azido compound, its application in
cancer cell imaging was also studied. The propargyl amine
modified folate 14 was prepared according to reported
method [44, 45], and then it was used as the targeting ligand
to test whether the BODIPY azide 5 can be used in cell im-
aging study through in situ click reaction. In this study, KB
cells were chosen as the targeting cells because they over-
express the folate receptors [46, 47] while human mesen-
chymal stem cells (HMSCs), which do not overexpress fo-
late receptors, were used as a negative control. The cells
were exposed to compound 14, and then compound 5. Un-
fortunately, a strong fluorescent signal was also detected
from the negative control which only has compound 5. We
postulated that the problem could be resulted from the in-
stability of the azido compound 5 under the experimental
conditions.
In order to increase the stability of the BODIPY dye, the
folate ligand 14 was conjugated to the compound 5 via
CuAAC reaction to generate a stable triazole compound 15,
before it was used for the cell studies (Scheme 2). Follow-
ing the aforementioned procedure, the KB cells and HMSCs
were exposed to the mixture of the triazole compound 15
for 2 hours before the cells were washed and fixed with 4%
paraformaldehyde in PBS for 20 min at room temperature.
Then the cellular uptake was examined with confocal mi-
croscope. As expected, the KB cells could efficiently uptake
the triazole compound and show clear red fluorescence in
cytoplasm when visualized under the Cy5 channel. As
comparison, the very weak fluorescence signals of HMSCs
after incubating with compound 15 is likely due to the lack
of folate receptors (Figure 3). In both situations, no obvious
cytotoxicity has been observed. The results demonstrated
that our fluorogenic BODIPY dye can potentially be used in
cell imaging studies.
Table 1 Optical properties of azide 5 and triazole products 6–13 in vari-
ous solvents
4 Conclusions
Absorption (λ, nm)
hexane MeOH CHCl3
Emission (λ, nm)
Entry
hexane
MeOH
530
534
536
529
532
538
536
534
533
CHCl3
In summary, we have synthesized a new type of BODIPY
dye that the azido group on the 3-position of the pyrrole
core could quench the fluorescence of the dye. The fluores-
cent emission could be significantly switched on after re-
acting with alkynes. It could be used to selectively target
human KB cells when it was conjugated with the folate lig-
and, which demonstrated the potential applications of this
dye in bioimaging. Further detailed study about the photo-
physical mechanism of switch on/off of fluorescence and
5
6
522
528
533
516
523
532
529
527
533
515
516
518
514
514
518
518
515
522
521
528
532
521
523
532
531
528
530
529
538
544
532
534
544
542
538
539
533
541
546
534
536
546
541
541
541
7
8
9
10
11
12
13
Figure 2 The comparison between the maximum emission intensity of
BODIPY triazole products 6–13 and azide 5 in chloroform (optical density
kept constant at 0.1 mM at their excitation wavelength).
Scheme 2 Synthesis of BODIPY-folate conjugates.

Wang C, et al. Sci China Chem January (2012) Vol.55 No.1
129
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This work was partially supported by the Alfred P. Sloan Scholarship,
Camille Dreyfus Teacher Scholar Award, the W. M. Keck Foundation, and
the Colon Cancer Center of University of South Carolina. We are also
grateful for financial support from the National Natural Science Founda-
tion of China (91013006).
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