Fluorescent Labeling of Protein Using Blue-Emitting 8-Amino-BODIPY Derivatives

Article abstract of DOI:10.1007/s10895-017-2164-5

8-Amino-BODIPY (boron-dipyrromethane) dyes show bright blue fluorescence. Disclosed here are synthesis and characterization of the photophysical properties of a series of functionalized 8-Amino-BODIPY (BP1–4) for protein labeling. The compact structure and solvent-insensitive absorption property of the dye are desirable features for protein labeling. For the model protein, bovine serum albumin (BSA), the labeling proceeds under mild condition via amide bond formation or thiol-ene conjugation with maintaining the bright blue fluorescence. The chromatography and mass spectroscopy analysis clearly support the labeling of the BODIPY dye on the BSA. The protein labeling with blue-emitting BODIPY would be applicable for studying protein dynamics and fluorescence resonance energy transfer (FRET) with intrinsic biomolecules.

Full text of DOI:10.1007/s10895-017-2164-5

J Fluoresc
DOI 10.1007/s10895-017-2164-5
ORIGINAL ARTICLE
Fluorescent Labeling of Protein Using Blue-Emitting
8-Amino-BODIPY Derivatives
Dokyoung Kim1,2,3 · Donghee Ma4 · Muwoong Kim3 · Yuna Jung3 · Na Hee Kim3 ·
Chiho Lee5 · Seo Won Cho4 · Sungnam Park5 · Youngbuhm Huh1,3 · Junyang Jung1,3
Kyo Han Ahn4
·
Received: 22 June 2017 / Accepted: 10 August 2017
© Springer Science+Business Media, LLC 2017
Abstract 8-Amino-BODIPY (boron-dipyrromethane) dyes
show bright blue fluorescence. Disclosed here are synthe-
sis and characterization of the photophysical properties of
a series of functionalized 8-Amino-BODIPY (BP1–4) for
protein labeling. The compact structure and solvent-insensi-
tive absorption property of the dye are desirable features for
protein labeling. For the model protein, bovine serum albu-
min (BSA), the labeling proceeds under mild condition via
amide bond formation or thiol-ene conjugation with main-
taining the bright blue fluorescence. The chromatography
and mass spectroscopy analysis clearly support the labeling
of the BODIPY dye on the BSA. The protein labeling with
blue-emitting BODIPY would be applicable for studying
protein dynamics and fluorescence resonance energy trans-
fer (FRET) with intrinsic biomolecules.
Keywords Protein labeling · Blue-emitting BODIPY ·
Fluorescent labeling · Bioconjugation
Introduction
Proteins play crucial roles in a variety of cellular events, and
the study of protein dynamics is critical to understanding of
sophisticated living systems [1]. Particularly, visualization
of protein movements has a great potential to reveal protein
functions and dynamics. So far, a number of visualization
methods have been developed to monitor protein behaviors
[2, 3]. Among them, a protein labeling method based on
fluorescence has come into spotlight due to its simplicity,
high sensitivity and reliability [4, 5].
The conjugation of protein with fluorescent signaling
unit have been carried out using labeling chemistries such
as click-chemistry, condensation reaction, ester/amide bond
formation, and thiol-ene reaction [6]. Also, various fluores-
cent signaling units (called dye) have been reported that in
a wide range of spectrum from visible (blue, green, red) to
near-infrared (NIR) [1, 7]. Fluorescein and rhodamine deriv-
atives are representative dyes in a green and red fluorescence
range, respectively. However, the development of blue-
emitting dye for protein labeling have been rarely explored
while it is important for studying the fluorescence resonance
energy transfer (FRET) from or to intrinsic biological fluo-
rophores such as tryptophan (Trp, λexi/emi =295/353 nm) and
flavin adenine dinucleotide (FAD, λexi/emi =450/540 nm) [8,
9]. A few blue-emitting dyes have been reported based on
coumarin backbone; Alexa Fluoro 350, AMCA, and Pacific
Blue (Fig. 1) [10–13]. Coumarin is a naturally occurring
benzopyran compound extracted from plants, and it have
shown interesting bio-activity and photophysical properties
[14]. These blue-emitting coumarin dyes are conjugated
Electronic supplementary material The online version of this
article (doi:10.1007/s10895-017-2164-5) contains supplementary
material, which is available to authorized users.
* Dokyoung Kim
dkim@khu.ac.kr
1
Department of Anatomy and Neurobiology, College
of Medicine, Kyung Hee University, Seoul 02447,
South Korea
2
Center for Converging Humanities, Kyung Hee University,
Seoul 02447, South Korea
3
Department of Biomedical Science, Graduate School, Kyung
Hee University, Seoul 02447, South Korea
4
Department of Chemistry, Pohang University of Science
and Technology (POSTECH), Pohang, Gyungbuk 37673,
South Korea
5
Department of Chemistry, Korea University, Seoul 02841,
South Korea
Vol.:(0123456789)
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To address this issue, a new series of blue-emitting dye
which shows environment-insensitive property making it a
promising protein labeling is desirable. Herein, we wish to
report the synthesis and characterization of novel deriva-
tives of blue-emitting 8-Amino-BODIPY dyes for protein
labeling (Fig. 2). BODIPY (boron-dipyrromethene) dyes are
a class of fluorescent dyes, which have valuable properties
such as environment-insensitive photophysical properties,
intense absorption and emission peaks [16]. Generally, the
photophysical properties of BODIPY dyes are dramatically
changed depend on the electron density at 2, 3, 8-posi-
tions [16]. Among them, the substitution products with
amino group at 8-position show strong blue fluorescence
at 409–467 nm wavelength (absorption at 370–400 nm). In
this study, we synthesized and analyzed the properties of
8-Amino-BODIPY derivative, and demonstrated the protein
labeling.
Fig. 1 Known blue-emitting dyes based on coumarin backbone
with protein via amide-bond formation, and resulting prod-
uct shows strong fluorescence in biological media. How-
ever, coumarin has a drawback that comes from structure;
typically electron donor–acceptor type dyes are sensitive to
environment changes such as viscosity, hydrophobicity, ion
concentration, pH, and polarity [15]. The changes can cause
unexpected fluorescence shift/quenching or affect the protein
function.
Experimental
Materials
All reagents and solvents were purchased from commercial
source (Sigma Aldrich; USA, TCI; Japan, Merck; USA),
and used without further purification. Anhydrous solvents
for organic synthesis were prepared by passing through a
Fig. 2 a Structures of 8-SMe-BODIPY (inset number: numbering of BODIPY dye), and 8-Amino-BODIPY. Inset photo was taken under UV
light (365 nm). b Structures of 8-Amino-BODIPY derivatives in this work
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solvent purification tower. Silica was used silica gel 60,
230–400 mesh, Merck (USA). Thin-layer chromatography
(TLC) was performed on pre-coated silica gel 60F-254 glass
plates, Merck (USA).
Synthesis/Method
8-thiomethyl-BODIPY (8-SMe-BODIPY) was prepared by
following the literature procedure [19], and the synthetic
routes to 8-Amino-BODIPY derivatives are outlined in
Scheme 1 (see Scheme S1–S4 in Supplementary Material for
the synthesis of linkers and protein conjugation procedure).
Methods and Instruments
1H and 13C NMR spectra were measured with a Bruker DPX-
300 and DPX-500 (Germany). Coupling constants (J value)
are reported in Hertz. Mass spectral analysis was recorded
with Jeol JMS 700 (Japan) and reported in units of mass
to charge (m/z). Chemical shifts are reported as δ in parts
per million (ppm). UV/Vis absorption spectra were obtained
using a HP 8453 UV/Vis spectrophotometer (USA). Fluo-
rescence spectra were recorded on a Photon Technology
International Fluorescence System (USA) with a 1-cm
standard quartz cell. High-resolution mass spectra of small
molecules were recorded on a JEOL JMS-700 spectrom-
eter (Japan) at the Korea Basic Science Center, Kyungpook
National University and the values are reported in units of
mass to charge (m/z). Mass data of protein and dye-labeled
protein were measured using MALDI-TOF mass spectrom-
etry, 4700 model proteomics analyzer with TOF/TOFTM ion
Optics, ABSCIEX corp (Canada). The data were acquired
with a Nd:AG laser with 200 Hz repetition rate, and up to
2000 shots were accumulated for each spectrum. LC analy-
sis was performed using a DIONEX P580A connected to
DIONEX UVD170S Detector (USA). Column: Phenomenex
(USA) C18 250×4.60 mm. Wavelength: 394 nm. Eluent: DI
H2O:acetonitrile=1:1 (v/v, 0.1% trifluoroacetic acid). Flow
N1-(4,4-Difluoro-4-bora-3a, 4a-diaza-sindacene)-ethane-1,2-di-
amine (BP-1) To a solution of 8-SMe-BODIPY (150 mg,
0.63 mmol) in anhydrous dichloromethane (12 mL) under
argon atmosphere was added ethylenediamine (0.42 mL,
6.3 mmol). The reaction mixture was stirred for 30 min at
room temperature (25 °C), and then treated with deionized
water (20 mL). The two layers were separated, and the aque-
ous layer was extracted with dichloromethane (3×20 mL).
The combined organic extracts were washed with brine,
dried over anhydrous Na2SO4 and concentrated. The residue
was purified by silica gel column chromatography (eluent:
10% methanol in dichloromethane) to afford BP-1 as a bright
1
yellow solid (150 mg, 95%). H NMR (CDCl3, 300 MHz,
293 K): δ 6.40 (2H, m), 7.05 (2H, m), 7.20 (2H, m), 9.77
(2H, s). 13C NMR (CDCl3, 75 MHz, 293 K): δ 150.7, 135.4,
132.4, 126.8, 124.8, 123.8, 117.6, 115.5, 114.3, 50.1, 40.4.
HRMS (FAB) m/z: C14H17BF2N6 found 250.12 (M+).
3-[(4,4-Difluoro-4-bora-3a, 4a-diaza-sindacene)-amino]-pro-
pionic Acid (BP-2) To a solution of 8-SMe-BODIPY
(183 mg, 0.769 mmol) in anhydrous tetrahydrofuran
(13.5 mL) under argon atmosphere was added β-alanine
(171 mg, 1.92 mmol) and triethylamine (0.27 mL,
1.92 mmol). The reaction mixture was stirred for 5 min at
room temperature (25 °C). After 5 min, deionized water
(1.5 mL) was added and the mixture was stirred for 1 h at
room temperature. The solvents were removed in vacuo and
the residue was extracted with ethyl acetate (3×10 mL), 1
N HCl, and deionized water. The combined organic extracts
were washed with brine, dried over anhydrous Na2SO4
and concentrated. The residue was purified by silica gel
column chromatography (eluent: 10% methanol in dichlo-
romethane) to afford BP-2 as a bright yellow solid (182 mg,
Rate: 1.0 mL min−1
.
Results and Discussion
Design
In 2010, Peña-Cabrera and co-workers reported conven-
tional synthetic routes and unique photophysical proper-
ties of 8-Amino-BODIPY derivatives [17, 18]. Originally,
8-thiomethyl-BODIPY (8-SMe-BODIPY) has green fluo-
rescence centered at 525 nm (the absorption maximum at
485 nm), and the amine-thiomethyl exchange reaction gener-
ate 8-Amino-BODIPY, which show bright blue fluorescence
at 409–467 nm wavelength (absorption at 370–400 nm)
with high quantum yield (Fig. 2a). We designed a series of
8-Amino-BODIPY derivatives that have protein conjugata-
ble moiety; amine (BP-1, via amide conjugation), carboxylic
acid (BP-2, via amide/ester conjugation), maleimide (BP-
3, via thiol-ene conjugation), and azide (BP-4, via copper-
catalyzed click conjugation) (Fig. 2b).
1
85%). H NMR (CDCl3, 300 MHz, 293 K): δ 6.40 (2H,
m), 7.05 (2H, m), 7.20 (2H, m), 9.77 (2H, s). 13C NMR
(CDCl3, 75 MHz, 293 K): δ 177.0, 141, 140.8, 140.5, 118.3,
118.0,122.0, 121.8, 112.0, 111.0, 41.8, 38.1. HRMS (FAB)
m/z: C12H12BF2N3O2 found 279.16 (M+).
4-(2,5-Dioxo-2,5-dihydro-pyrrol-1-yl)-benzoic Acid
(BP-3) To a solution of BP-1 (47 mg, 0.218 mmol) in
tetrahydrofuran (8 mL) were added N´-ethylcarbodiimide
hydrochloride (50 mg, 0.262 mmol), hydroxybenzotria-
zole (35 mg, 0.262 mmol), N-methylmorpholine (72 µL,
0.654 mmol) and compound 3 (60 mg, 0.24 mmol, see the
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Scheme 1 Reagent and condi-
tions. a thiophosgene, diethyl
ether, 0 °C, 10 min, 76%. b
methyl iodide, dichlorometh-
ane, 25 °C, 15 h. c BF3·Et2O,
triethylamine, dichlorometh-
ane, 25 C, 12 h, 71% (from
two steps). d dichlorometh-
ane, 25 °C, 30 min, 95%. e
triethylamine, tetrahydrofuran,
25 °C, 1 h, 85%. f dichlo-
romethane, 25 °C, 1 h, 91%. g
N′-ethylcarbodiimide hydro-
chloride, hydroxybenzotriazole,
N-methylmorpholine, tetrahy-
drofuran, 25 °C, 5 h, 61%
details in Supplementary Material). The mixture solution
was stirred for 5 h under argon at room temperature (25 °C).
Solvent was evaporated under vacuum, and the residue was
extracted with dichloromethane (3×10 mL) and deionized
water. The combined organic extracts were washed with
brine, dried over anhydrous Na2SO4 and concentrated. The
residue was purified by silica gel column chromatogra-
phy (eluent: 25% ethyl acetate in n-hexane) to afford BP-3
1H NMR (CDCl3, 300 MHz, 293 K):δ 1.47–1.79 (6H, m),
3.31–3.35 (2H, m), 3.43–3.50 (2H, m), 6.27 (1H, S). 13C
NMR (CDCl3, 75 MHz, 293 K): 148.16, 135.78, 132.52,
124.84, 123.78, 122.27, 114.65, 113.82, 51.06, 47.07, 28.34,
24.16. HRMS (FAB) m/z: C14H17BF2N6 found 318.16 (M+).
BSA Labeling with BP3 BP3 (10 µM) was incubated
with BSA (100 µM) in PBS buffer (pH 7.4, 1% CH3CN)
for 1 h incubation at 37 °C. The labeling of BP3 to BSA
was confirmed by the fluorescence measurement and HPLC
analysis.
1
as a bright yellow solid (98 mg, 61%). H NMR (CDCl3,
300 MHz, 293 K): δ 6.40 (2H, m), 7.05 (2H, m), 7.20 (2H,
m), 9.77 (2H, s). 13C NMR (CD3CN, 75 MHz, 293 K):
169.70, 169.59, 148.73, 135.12, 134.60, 134.02, 132.49,
130.74, 128.05, 126.20, 123.60, 117.29, 115.82, 114.41,
113.35, 50.16, 38.72. HRMS (FAB) m/z: C22H18BF2N5O3
found 449.15 (M+).
Cellular Fluorescence Imaging of BP2-lysozyme Prod-
uct 5 × 105 of TM3 mouse Leydig cell line was seeded
on 6-cm cell culture dish (SPL, Pocheon, Rep. of Korea)
and cultured in complete Dulbecco’s modified Eagle’s
medium (DMEM, Hyclone, Waltham, Massachusetts, USA)
containing 10% heat-inactivated fetal bovine serum (FBS,
Hyclone, Waltham, Massachusetts, USA) and 1 × Penicil-
lin/Streptomycin solution (Corning, Corning, New York,
USA) at 37 °C in a humidified atmosphere containing 5%
CO2 for 10 h. The cell was washed twice 1 × Phosphate
buffered saline (PBS, Welgene, Gyeongsan, Rep. of Korea),
followed by being applied with DMEM containing BP2-
lysozyme product (see the preparation method in Supple-
mentary Material, Scheme S4) for 2 h (37 °C, 5% CO2 and
Humidified condition). The dish was washed with 1×PBS
twice, and then filled with DMEM prior to the imaging. The
fluorescence signal was monitored with Axioimager upright
(5-Azido-pentyl)-(4,4-difluoro-4-bora-3a,4a-diaza-sind
acene)-amine (BP-4) To a solution of 8-SMe-BODIPY
(50 mg, 0.21 mmol) in anhydrous dichloromethane (4 mL)
under argon atmosphere was added compound 5 (41 mg,
0.32 mmol, see the details in Supplementary Material). The
reaction mixture was stirred for 1 h at room temperature
(25 °C), and then treated with deionized water (10 mL).
The two layers were separated, and the aqueous layer was
extracted with dichloromethane (3×10 mL). The combined
organic extracts were washed with brine, dried over anhy-
drous Na2SO4 and concentrated. The residue was purified
by silica gel column chromatography (eluent: 30% ethyl
acetate in n-hexane) to BP-4 as a yellow solid (61 mg, 91%).
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Table 1 Photophysical properties of BP dyes (10 µM) in different
microscope equipped at 37 °C in 5% CO2 controlled system.
(Zeiss, Jena, Germany).
solvents
Compound
BP1
Solvent
λabs (nm)
λem (nm)
Stokes
Φ
shift (nm)
Photophysical Properties
H2O
402
396
396
401
400
404
391
389
390
397
397
403
395
399
394
400
400
403
390
396
396
398
400
404
460
437
445
454
453
462
452
435
443
442
451
465
470
442
446
448
446
449
461
443
447
445
452
458
58
41
49
53
53
58
61
46
53
45
54
62
75
43
52
48
46
46
71
47
51
47
52
54
0.29
0.09
0.16
0.36
0.34
0.46
0.08
0.12
0.15
0.12
0.46
0.84
0.04
0.11
0.19
0.17
0.59
0.58
0.02
0.06
0.06
0.06
0.36
0.60
The experimental UV/Vis absorption and fluorescence emis-
sion spectra of BP1–4 (10 µM) are measured in various sol-
vents; protic (deionized water, methanol) and aprotic (dime-
thyl sulfoxide, acetonitrile, ethyl acetate, dichloromethane)
(Fig. 3, Supplementary Figs. S1–S10, Table 1).
DMSO
CH3CN
MeOH
EtOAc
CH2Cl2
H2O
DMSO
CH3CN
MeOH
EtOAc
CH2Cl2
H2O
DMSO
CH3CN
MeOH
EtOAc
CH2Cl2
H2O
DMSO
CH3CN
MeOH
EtOAc
CH2Cl2
BP1–4 showed strong broad absorption bands in the
range of 370–400 nm with no significant solvent depend-
ent hypso-/bathochromic shift. Moreover, the results rep-
resent no decrement of absorbance in protic solvents while
coumarin-based dyes are not. The environment non-sensitive
absorption properties of BP dyes can be considered as an
ideal FRET acceptor from the intrinsic biomolecules.
The strong blue fluorescence emission bands of BP1–4
were observed in the range of 409–467 nm under excitation
at the maximum absorption wavelength in the various sol-
vents. The center of emission spectra shows a little hypsoch-
romic shift by increasing solvent polarity (Supplementary
Figs. S1–S10, Table 1). Generally, primary or secondary
amine moiety reduce the fluorescence intensity of fluoro-
phore as a quencher via photo-induced electron transfer
(PeT) [21], but BP1 has relatively strong fluorescence with
high quantum yield even in the deionized water (Φ=0.29).
Slightly lower fluorescent quantum yields were observed for
BP2–4 in the high polarity or protic solvents (Table 1), but
the 8-Amino-BODIPY have shown sufficient fluorescence
bio-imaging capability in the reported works [22].
BP2
BP3
BP4
Φ: fluorescence quantum-yield, DMSO: dimethyl sulfoxide, CH3CN:
acetonitrile, MeOH: methanol, EtOAc: ethyl acetate, CH2Cl2: dichlo-
romethane. Fluorescence quantum yield (Φ) were determined using
9,10-diphenylanthracene (DPA, 10 µM) as a standard [20]. Quantum
yields were derived from the equation: Φs =Φr ×(ns2/nr2)×(Is/Ir)×(Ar
/As), where s is the sample (BP dyes), r is the standard (DPA), n is
refractive index, I is the integrated fluorescence emission, and A is the
absorbance at the excitation wavelength
The time-resolved fluorescence (TRF) study of amino-
BODIPY derivatives have been reported,[18] and nano-
second lifetime was characterized. We measured the
time-resolved fluorescence emission of BP1 and BP2 in
the deionized H2O, and they showed 1.31 and 0.24 ns life-
time, respectively, which are similar to the typical decay of
BODIPY (Fig. S11).
Protein Labeling with BP Dyes
As a model protein, a bovine serum albumin (BSA, Sigma,
Cat. No. A2058, mol wt~66 kDa) was used, which was incu-
bated with BP2 and BP3 for the test of labeling reactivity.
BP2 (10 µM) was pre-treated with N-hydroxysuccinim-
ide to form active-ester form (see details in Supplementary
Material), and then BSA solution (100 µM in pH 7.4 PBS;
Fig. 3 Normalized absorption and fluorescence emission spectra
of BP dyes (10 µM) in deionized water (DI H2O). The emission
spectra were obtained under excitation at the maximum absorption
wavelength
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J Fluoresc
phosphate-buffered saline buffer containing 1% acetoni-
trile). The lysine amine residues in the BSA (59 lysine in
the total 607 amino acid) undergo the amide bond formation
with active-ester of BP2, and the resulting product shows
bright blue fluorescent at 450 nm wavelength (Fig. 4). The
fluorescence enhancement (2 times) with a little hypsochro-
mic shift of emission maximum wavelength was observed
that presumably coming from (i) environment change of
BP2 on the BSA, and (ii) reduced quenching effect of car-
boxylic acid to amino-BODIPY dye [23]. We conducted
MALDI-TOF (matrix-assisted laser desorption ionization
time-of-flight) analysis to confirm the BSA labeling with
Fig. 6 High performance liquid chromatography (HPLC) spectra of
BP3 only (10 µM), and mixture of BP3 with BSA for 1 h incuba-
tion at 37 °C in PBS buffer (pH 7.4, 1% acetonitrile). BSA concentra-
tion: 0.5 eq (5 µM)–2.5 eq (25 µM). The intensity was measured at
394 nm. Eluent was deionized H2O:acetonitrile=1:1 (v/v, 0.1% trif-
luoroacetic acid), and flow rate was 1.0 mL min−1
Fig. 4 Fluorescence emission spectra of BP2 (10 µM) and BSA
labeled with BP2 [100 µM BSA, 24 h incubation at 37 °C in PBS
buffer (pH 7.4, 1% acetonitrile)]. See the details in Supplementary
Material (Scheme S4). The excitation wavelength was 394 nm
Fig. 5 a Fluorescence emission spectra of BSA only, BP3 (10 µM),
and BSA labeled with BP3 [100 µM BSA, 1 h incubation at 37 °C
in PBS buffer (pH 7.4, 1% acetonitrile)]. b Fluorescence emission
spectra of BP3 (10 µM) with BSA, amino acids, mercaptoethanol,
and metal ions (100 µM, respectively), for 1 h incubation at 37 °C in
PBS buffer (pH 7.4, 1% acetonitrile). The excitation wavelength was
394 nm
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Fig. 7 Fluorescence images of TM3 mouse Leydig cells treated with the BP2-labeled lysozyme product. Control: without treatment. Scale bar:
250 µm. Excitation and detection wavelength were 380 nm and 420 15 nm, respectively
the BP2 (Fig. S12). The mass change of BSA with the BP2
(m/z = 279.05) added was observed:m/z = 68014.34 (no
BP2) ~ m/z = 68562.52 (+ 2 BP2), and the result suggests
that on average 1–2 molecules of the BP2 were labelled per
each BSA.
BSA (Fig. 6). In the absence of BSA, the peak of BP3 was
appeared around 9 min. Upon the addition of BSA and incu-
bation for 1 h at 37 °C in PBS buffer (pH 7.4, 1% CH3CN),
a new peak was appeared around 2 min that corresponded
with BSA-BP3 conjugated product and 2.5 equivalent of
BSA gave complete conversion.
Next, we demonstrated the BSA labeling using BP3
which have a maleimide moiety for the thiol-ene conjuga-
tion. The primary structure of BSA has 35 cysteine residues,
and the 34 cysteines are existed as disulfide form. The only
one free cysteine residue, Cys34, which located in the crev-
ice on the protein surface domain-I can react with maleimide
moiety in the BP3. BP3 (10 µM) was incubated with BSA
solution (100 µM in pH 7.4 PBS buffer containing 1% ace-
tonitrile) for 1 h at 37 °C. The fluorescence measurement
of resulting product showed similar property changes likes
BP2, emission peak shifting (15 nm) with enhancement (2.2
times) (Fig. 5a). To analyze the fluorescence change of BP3
at various conditions, it was incubated with BSA, mercap-
toethanol (HS-CH2CH2-OH), amino acids (Phe, Lys, Ser,
Ala, Met, His, Hcy, Cys, GSH), and metal ions (Ca2+, Fe3+,
Mg2+, Zn2+, Cu2+), respectively (Fig. 5b). As we expected,
BSA-BP3 product showed fluorescence enhancement with
a peak shifting but the other thiol moiety containing sub-
stitutes such as mercaptoethanol, Hcy, Cys, GSH showed
no significant changes that indicating fluorescence enhance-
ment comes from the labeling-induced environment change
of BP dye in BSA.
Further experiment was conducted to confirm the fluo-
rescence imaging capability of the amino-BODIPY after
the labeling of protein. The lysozyme (Sigma, Cat. No.
10,837,059,001, mol wt ~ 14 kDa) was labelled with BP2
via amide-bond formation (6 lysine in the total 129 amino
acid). The labeling was verified by measurement of fluores-
cence and MALDI-TOF (1–2 molecules of the BP2 labelled
per each lysozyme on average). The resulting product was
lyophilized, and then incubated with TM3 mouse Leydig
cells for 2 h at 37 °C under 5% CO2. Then, the cells were
rinsed with PBS (phosphate buffered saline) buffer prior
to fluorescence imaging. The bright blue fluorescence was
observed inside the cells, which indicates that BP2-labeled
lysozyme penetrates the cells and maintain the fluorescence
in biological media (Fig. 7).
Conclusion
Disclosed here are the synthesis and photophysical proper-
ties of a series of 8-Amino-BODIPY (BP1–4) dyes as pro-
tein labeling reagents. The primary amine moiety reacts with
a thiomethyl group in 8-SMe-BODIPY proceeds fast (within
HPLC (high-performance liquid chromatography) was
conducted to verify the bond formation of the BP3 to the
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hours) under mild condition (25 °C) with high yield, and
the resulting 8-Amino-BODIPY derivatives shows bright
blue fluorescence at 409–467 nm wavelength (absorption
at 370–400 nm). The synthesized derivatives were applied
to the labeling of bovine serum albumin (BSA), a model
protein, via amide bond formation (BP2) or thiol-ene con-
jugation (BP3). The conjugation was successfully proceeds
in mild condition, and the resulting BSA-BP conjugated
product maintained the strong blue fluorescence. The HPLC
and MALDI-TOF mass analysis support the conjugation of
BP dyes to the amino acids of BSA. The solvent-insensitive
absorption and blue-emitting property of the BP dye are
desirable feature for protein labeling, therefore it would be
applicable for studying the protein dynamics in living cells
and fluorescence resonance energy transfer (FRET) with
intrinsic biomolecules.
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Acknowledgements This work was supported by a grant from Kyung
Hee University in 2017 (KHU-20170857). K.H.A thanks the finan-
cial support from the Ministry of Health & Welfare (HI13C1378) and
Global Research Laboratory Program (2014K1A1A2064569) through
the National Research Foundation (NRF) funded by the Ministry of
Science, ICT & Future Planning, Korea. D.K acknowledges finan-
cial support from the Basic Science Research Program of the Korea
National Research Foundation (NRF) funded by the Ministry of Edu-
cation (2016R1A6A3A03006343). Y.H thanks the Korea NRF (Grant
No. 2011-0030072).
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