2
Y. Li et al. / Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 227 (2020) 117565
The soft retinal chromophore backbone without functional
Synthesis of V2: The retinol (2.1 g, 7.34 mmol) and Na2CO3
(11.66 g, 0.11 mol) was dissolved in 130 mL THF and cooled to 0 ꢁC in
ice bar. The MnO2 (9.57 g, 0.11 mol) was slowly added into above
cooling mixture. The reaction mixture was continually stirred for
1 h after removing the ice bar. The reaction residue was separated
by vacuum filtration, retaining filtrate [27]. The crud product was
concentrated and further purified by silica gel column chroma-
tography, obtaining light yellow waxy solid 0.6 g, yield 29%.1H NMR
substituents is not suitable for binding copper ions [26]. Previously,
we developed a fluorescent probe for Cu2þ based on o-phenyl-
enediamino [24] as chelator, which exhibited excellent selectivity.
In this work, a novel fluorescent probe was designed and synthe-
sized, the o-phenylenediamino as a Schiff base chelator, the retinal
as the reporter. The probe with Schiff base actually still has good
solubility, due to the soft chromophore retinal. As expected, the soft
probe becomes rigid in the process of sensing copper ions, pro-
moting significant changes in the fluorescence.
(400 MHz, CDCl3)
d
10.11 (d, J ¼ 8.2 Hz,1H), 7.14 (dd, J ¼ 15.1,11.5 Hz,
1H), 6.36 (dd, J ¼ 15.5, 11.5 Hz, 2H), 6.22e6.13 (m, 2H), 5.97 (d,
J ¼ 8.2 Hz, 1H), 2.33 (d, J ¼ 0.9 Hz, 3H), 2.07e1.98 (m, 5H), 1.72 (s,
3H), 1.65e1.60 (m, 3H), 1.49e1.45 (m, 2H), 1.04 (s, 6H).
2. Experimental
Synthesis of V1: The retinal (0.30 g, 1.06 mmol) and o-Phenyl-
enediamine (0.34 g, 3.18 mmol) was refluxed in 35 mL acetonitril
for 2 h. The solvent was evaporated by rotary evaporator, getting
crud product. The crud product was recrystallized in 20 mL petro-
leum ether and 2 mL ethanol [24], cooling and vacuum filtration,
obtaining orange solid 0.35 g, yield 87.5%. 1H NMR (400 MHz,
2.1. Reagents
Retinol acetate, MnO2, o-Phenylenediamine were purchased
from J&K Chemical Ltd. Tetrahydrofuran, ethyl acetate, acetonitrile,
metal ions purchased from Sinopharm Chemical Reagent Co., Ltd.
(Shanghai). Human serum was from Solarbio. Other chemicals were
provided from Sigma-Aldrich. All commercially purchased chemi-
cal reagent materials were used without further purification.
DMSO‑d6)
d
8.64 (d, J ¼ 9.7 Hz, 1H), 7.08 (dd, J ¼ 8.0, 1.2 Hz, 1H),
7.02e6.88 (m, 2H), 6.67 (dd, J ¼ 8.0, 1.3 Hz, 1H), 6.60e6.45 (m, 2H),
6.44e6.33 (m, 1H), 6.24 (dt, J ¼ 29.7, 11.7 Hz, 3H), 5.06 (s, 2H), 2.20
(s, 3H), 2.06e1.94 (m, 5H), 1.70 (s, 3H), 1.58 (dt, J ¼ 8.5, 4.7 Hz, 2H),
1.49e1.39 (m, 2H), 1.02 (s, 6H). 13CNMR (101 MHz, CDCl3)
d 155.25,
2.2. General method
154.42, 143.70, 137.63, 136.96, 135.45, 134.27, 131.43, 131.22, 129.85,
124.84, 114.79, 112.57, 79.28, 39.65, 34.33, 33.29, 29.02, 21.87, 19.17,
14.05, 13.18. HR-MS: calcd for C26H36Nþ2 [MþH]þ, 375.2800; found:
375.2803.
1H NMR (400MHZ) and 13C NMR (100MHZ) spectra data were
recorded on a Bruker UltrashiedTM 400MHZ Plus nuclear magnetic
resonance spectrometer using DMSO‑d6/CDCl3 as solvent and tet-
ramethylsilane as an internal standard. High-resolution mass
spectra were carried out on a Bruker Micro TOF II 10257 instrument.
Fourier transform infrared (FT-IR) spectra were performed by using
NICOLET NEXUS 470 spectrometer (KBr discs) in the 4000-
400 cmꢀ1 region. UVevisible spectra were recorded on a Shimadzu
UV-2600 spectrometer. Steady-state fluorescence spectra were
recorded on Hitachi F-4600 spectrophotometers. The pH of buffed
solution was determined using a PHS-3C digital pH-meter (Leici,
Shanghai, China).
2.4. Procedures of the metal ion sensing
The solutions of metal ions (10.0 mM) were prepared in doubly
distilled water by the dissolution of their corresponding nitrate
salts, including Al3þ, Ca2þ, Cr3þ, Mn2þ, Fe3þ, Be2þ, Ni2þ, Cu2þ, Zn2þ
,
Agþ, Cd2þ, Ba2þ, Hg2þ, Pb2þ, Kþ, Na2þ, Mg2þ. Stock solution of V1
(1.0 ꢂ 10ꢀ3 mol/L) was prepared in acetonitrile or DMSO by dis-
solving the corresponding amount of V1 powder and then diluted
to 1.0 ꢂ 10ꢀ5 mol/L with acetonitrile, DMSO/stroke-physiological
saline solution (1:1000, v/v; pH ¼ 6.5). Human serum was added
into the test solution according to the mass ratio. In titration ex-
periments, test solution of V1 (2 mL) was filled in a quartz optical
2.3. Synthesis
The synthesis route of V1 is shown in Scheme 1.
Synthesis of V3: The commercial retinol acetate V4 (10.0 g) was
dissolved in 75.0 mL NaOH solution (1 M) and 75.0 mL THF. Then
the mixture is stirred at the room temperature for 12 h under ni-
trogen atmosphere. After the reaction finished, the reaction
mixture was transferred to separating funnel, standing and sepa-
ration of organic and aqueous phases. The aqueous phase was
washed by THF and ethyl acetate three times. The organic layer was
combined and dried by Na2SO4. The organic solvent was evaporated
by rotary evaporator, obtaining 4.5 g light yellow oil, yield 60%. The
crud product was used in the following reaction without purifica-
tion [27].
cell of 1 cm optical path length, and then aliquots of 2 mL of
Cu(NO3)2 solution (1.0 ꢂ 10ꢀ2 mol/L) were gradually added by using
a micro-pipette. Spectral data were recorded for 5 min after addi-
tion of the metal ions at steady temperature (298 K). For fluores-
cence measurements, the excitation wavelength was at 400 nm
using Xe lamp as an excitation source, the fluorescence emission
channel was from 410 to 800 nm. In pH titration experiments, stock
solution of V1 was added to the sodium phosphate buffer aqueous
solution with different pH values. The V1eCu2þ complex repara-
tion, 1.12 mg (3 mmol) V1 and 0.6 mg (3.6 mmol) Cu(NO3)2 was
dissolved in 5 ml acetonitrile and stirred for 5 mints, and then the
Scheme 1. Synthesis of compound V1.