Selective Two-Step Labeling of Proteins
FULL PAPER
Instruments: NMR spectra were recorded on a JEOL JNM-LA300 in-
strument at 300 MHz for 1H NMR and at 75 MHz for 13C NMR or a
JEOL JNM-LA400 instrument at 400 MHz for H NMR and at 100 MHz
After being cooled to room temperature, the reaction mixture was
poured into ice-cold H2O (900 mL). The precipitate was filtered off and
dried under vacuum at 908C, overnight, to afford a brown solid (21.0 g).
The solid was stirred in acetic anhydride (75 mL) and pyridine (4.5 mL),
and heated to reflux at 1008C for 1 h. The reaction mixture was allowed
to cool to room temperature, stirred for 4 h, and then filtered. The ob-
tained solid was dried under vacuum to afford a colorless solid (12.4 g).
This solid was dissolved in ethyl acetate (200 mL) and the solution was
washed twice with HCl (2m) aqueous solution and once with saturated
NaCl solution, then dried over MgSO4 and filtered. The filtrate was
evaporated to dryness to give 10.5 g of 2’,7’-dichloro-6-carboxyfluorescein
diacetate. 2’,7’-Dichloro-6-carboxyfluorescein diacetate (10.5 g) was dis-
solved in NaOH (2m) aqueous solution (400 mL), and the mixture was
stirred for 3 h at room temperature. After the reaction, the solution was
acidified to pH 2 with HCl (12m) aqueous solution. A yellow precipitate
formed and was collected by filtration and washed with water to give 5
1
for 13C NMR. Mass spectra (ESI+ or ESIꢀ) were measured with a JEOL
JMS-T100 LC AccuTOF for ESI. PCR reactions were performed with
Veriti 96-well Thermal Cycler (Applied Biosystems). Photoimages of
gels, EBFP and probe fluorescence were captured with a digital camera
(HS EX-FC1000, Casio). HPLC analyses were performed on an Inertsil
ODS-3 (4.6ꢂ250 mm) column (GL Sciences, Inc.) by using a HPLC
system composed of a pump (PU-980, Jasco) and a detector (MD-2015 or
FP-2025, Jasco) or a HPLC system composed of a pump (PU-2080,
Jasco) and a detector (MD-2018 or FP-2025, Jasco). Preparative HPLC
was performed on an Inertsil ODS-3 (10ꢂ250 mm) column (GL Sciences,
Inc.) by using a HPLC system composed of a pump (PU-2080, Jasco) and
a detector (MD-2015 or FP-2025, Jasco) or a HPLC system composed of
a pump (PU-2086, Jasco) and a detector (MD-2018, Jasco). DNA se-
quence analyses were performed on an ABI PRISM 3100 genetic ana-
lyzer (Applied Biosystems).
as
a
yellow solid (8.82 g, 19.8 mmol, 40% over 4 steps). 1H NMR
(300 MHz, DMSO): d=6.77 (d, J=8.8, 1.5 Hz, 2H), 6.62 (d, J=8.8 Hz,
2H), 6.73 (s, 2H), 7.07 (s, 2H), 7.78 (s, 1H), 8.13 (d, J=8.1 Hz, 1H),
8.25 ppm (d, J=8.1 Hz, 1H); 13C NMR (75 MHz, DMSO): d=103.9,
109.8, 116.4, 124.5, 125.7, 128.3, 129.4, 131.3, 137.5, 150.1, 151.7, 155.5,
166.0, 167.6 ppm; HRMS (ESI+): m/z: calcd for C21H11Cl2O7: 444.9882
[M+H+]; found: 444.9901.
UV–visible absorption and fluorescence emission spectral measurement:
UV–visible spectra were obtained on a spectrometer (UV-1650, Shimad-
zu, Japan). Fluorescence spectroscopic studies were performed on a fluo-
rescence spectrometer (F-4500, Hitachi, Japan). The slit width was 2.5 nm
for both excitation and emission. The photomultiplier voltage was 700 V.
All experiments were carried out at 298 K. Dyes were dissolved in
DMSO as stock solutions. NiSO4 was dissolved in distilled water as stock
solution. Peptides were dissolved in distilled water as stock solutions, ali-
quots were transferred into microtubes and kept at ꢀ208C for short-term
storage (less than 1 week). Glutathione (GSH) was dissolved in distilled
water and was freshly prepared every day before use. EBFP was dis-
solved in PBS as a stock solution and aliquots were stored at ꢀ708C.
Photochemical properties of dyes were examined in HEPES buffer
(100 mm, pH 7.4) containing less than 0.1% (v/v) DMSO as a co-solvent.
The final concentrations of dyes, Ni2+, peptides, GSH and EBFP are
given in the Figure legends.
Synthesis of 2: Compound 5 (445 mg, 1.00 mmol) and DIEA (348 mL,
2.00 mmol) were dissolved in DMF (10 mL). 2,4-Dinitrofluorobenzene
(372 mg, 2.00 mmol) was added dropwise to this solution, and the mix-
ture was stirred for 6 h at room temperature. The reaction mixture was
evaporated to dryness and the residue was partially purified by silica gel
chromatography with CH2Cl2/MeOH/AcOH (97.8:2.0:0.2). The crude
compound was purified by preparative HPLC under the following condi-
tions: A/B=40/60 (0 min) to 0/100 (20 min); (solvent A: H2O, 0.1%
TFA; solvent B: acetonitrile/H2O, 80:20, 0.1% TFA) to afford 2 as a
yellow solid (159.0 mg, 0.260 mmol, 26%). 1H NMR (300 MHz, DMSO):
d=6.86 (s, 1H), 6.93 (s, 1H), 7.29 (s, 1H), 7.43 (d, J=9.5 Hz, 1H), 7.61
(s, 1H), 7.88 (s, 1H), 8.16 (d, J=8.8 Hz, 1H), 8.28 (d, J=8.8 Hz, 1H),
8.50 (d, J=9.5 Hz, 1H), 8.97 (s, 1H), 11.25 ppm (s, 1H); 13C NMR
(75 MHz, DMSO): d=80.8, 103.6, 109.6, 110.9, 116.8, 117.5, 119.9, 120.0,
122.1, 124.6, 126.0, 128.5, 129.2, 129.9, 130.1, 131.5, 137.6, 139.3, 142.5,
149.6, 150.4, 150.8, 151.6, 153.0, 155.5, 166.1, 167.5 ppm; HRMS (ESI+):
m/z: calcd for C27H13Cl2N2O11: 610.9896 [M+H+]; found: 610.9868.
Quantum yield measurements: For determination of the relative fluores-
cence quantum yields (Ffl), fluorescein in 0.1m NaOH aq. (Ffl =0.85)
was used as a standard. Calculations were carried out according to Equa-
tion (1), where F is the quantum yield (subscript “st” stands for the refer-
ence and “x” for the sample), A is the absorbance at the excitation wave-
length, n is the refractive index, and D is the area (on an energy scale) of
the fluorescence spectra. The sample and the reference were excited at
the same wavelength (490 nm).
Synthesis of 3: Compound 5 (223 mg, 0.50 mmol) and DIEA (435 mL,
2.50 mmol) were dissolved in DMF (5 mL). 2-Fluoronitrobenzene
(263 mL, 2.50 mmol) was added dropwise to the solution, and the mixture
was stirred and heated to reflux at 1008C for 36 h. The reaction mixture
was evaporated to dryness and the residue was partially purified by silica
gel chromatography with CH2Cl2/MeOH/AcOH (95.8:4.0:0.2). The crude
compound was purified by preparative HPLC under the following condi-
tions: A/B=40/60 (0 min) to 0/100 (20 min); (solvent A: H2O, 0.1%
TFA; solvent B: acetonitrile/H2O, 80:20, 0.1% TFA) to give 3 as a
yellow solid (13.2 mg, 0.023 mmol, 5%). 1H NMR (400 MHz, DMSO):
d=6.80 (s, 1H), 6.88 (s, 1H), 7.10 (s, 1H), 7.15 (s, 1H), 7.34 (d, J=
7.8 Hz, 1H), 7.49 (t, J=7.8 Hz, 1H), 7.77 (t, J=7.8 Hz, 1H), 7.83 (s, 1H),
8.13 (d, J=7.8 Hz, 1H), 8.16 (d, J=7.8 Hz, 1H), 8.25 (d, J=7.8 Hz, 1H),
11.22 ppm (s, 1H); 13C NMR (100 MHz, DMSO): d=81.1, 103.6, 107.8,
109.6, 115.4, 116.7, 118.9, 121.6, 124.6, 125.8, 126.0, 126.2, 128.5, 129.3,
129.7, 131.4, 135.7, 137.6, 140.8, 147.6, 149.7, 150.2, 151.6, 153.1, 155.4,
166.1, 167.5 ppm; HRMS (ESI+): m/z: calcd for C27H14Cl2NO9: 566.0046
[M+H+]; found: 566.0051.
2
Fx=Fst ¼ ½Ast=Axꢁ ½nx2=nst ꢁ ½Dx=Dstꢁ
ð1Þ
Determination of peptide concentrations: Peptide concentrations were
determined by measuring absorbance at 280 nm of peptide solutions. Ab-
sorption coefficient, e, at 280 nm, was calculated by using Equation (2):
e280 nm ¼ ð#TrpÞð5500Þ þ ð#TyrÞð1490Þ þ ð#CysÞðl25Þ
ð2Þ
Determination of EBFP concentrations: The concentrations of His6-
EBFP and His6Cys-EBFP were determined by measuring absorbance at
380 nm of EBFP solution. Absorption coefficient e280 =31000 cmꢀ1 mꢀ1
was used for calculation.
Fluorescence imaging of microbeads with 1–Ni2+: Microbeads were incu-
bated with 1–Ni2+ (5 mm) complex in HEPES buffer (100 mm, pH 7.4)
containing DMSO (0.5%) as a co-solvent. Fluorescence images were cap-
tured 30 min after addition of 1–Ni2+. The imaging system was comprised
of an inverted fluorescence microscope (IX-71, Olympus) and EM-CCD
digital camera (C9100; Hamamatsu Photonics K.K.). Bright-field and
fluorescence images were captured by using MetaMorph software (Mo-
lecular Devices) with a 10ꢂ objective lens (UPlanApo 10ꢂ/0.40, Olym-
pus).
Synthesis of 4: Compound 5 (223 mg, 0.50 mmol) and DIEA (435 mL,
2.50 mmol) were dissolved in DMF (5 mL). 4-Fluoronitrobenzene
(265 mL, 2.50 mmol) was added dropwise to the solution, and the mixture
was stirred and heated to reflux at 1008C for 36 h. The reaction mixture
was evaporated to dryness and the residue was partially purified by silica
gel chromatography with CH2Cl2/MeOH/AcOH (95.8:4.0:0.2). The crude
compound was purified by preparative HPLC under the following condi-
tions: A/B=40/60 (0 min) to 0/100 (20 min): (solvent A: H2O, 0.1%
TFA; solvent B: acetonitrile/H2O, 80:20, 0.1% TFA) to afford 4 as a
Synthesis of 5:[19] Compound 5 was synthesized according to ref. [19]. 4-
Carboxyphthalic anhydride (9.6 g, 50.0 mmol) was added to a solution of
4-chlororesorcinol (14.4 g, 100 mmol) in methanesulfonic acid (1m,
100 mL). The mixture was heated at 908C in an open vessel, overnight.
1
yellow solid (6.7 mg, 0.012 mmol, 2%). H NMR (300 MHz, DMSO): d=
Chem. Eur. J. 2011, 17, 14763 – 14771
ꢁ 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
14769