X. Liao, C. Zhu, D. Huang et al.
Spectrochimica Acta Part A: Molecular and Biomolecular Spectroscopy 256 (2021) 119733
Scheme 1. Synthetic routes for the target compound XB-1.
Japan). The reactions were monitored by thin-layer chromatogra-
phy and carried out on commercial Merck Kieselgel 60 F254 plates.
Column chromatography was performed on silica gel. H NMR
2.5. Molecular docking
1
Molecular docking was performed using a Sybyl-X 2.0 software.
The crystal structure of HSA (PDB code: 2BXD) was downloaded
from the Protein Data Bank. The crystal structure of HSA was opti-
mized with H added and charge added by AMBER7 FF99 method.
Site I ligand warfarin and site II ligand ibuprofen were selected
in PDB files (PDB codes: 2BXD and 2BXG) to generate the protomol
by using the software protocols, with the threshold kept at 0.5 and
the bloat was fixed as 0. Then structures of small molecules were
draw by Sybyl-X 2.0 package, and were subjected to the polar H
adding and being energy optimized with a tripos force field and
charged optimized with Gasteiger-Huckel method. Finally, mole-
cules were docked to the two main drug binding site I (Subdomain
IIA) and site II (Subdomain IIIA) of HSA, respectively. The top 20
preferred conformation of the structure with low energy of the
binding system were obtained, and the results with the highest
scoring function (Total binding scores) were selected for analysis.
spectra were obtained on an Agilent 400 MR spectrometer, while
C NMR spectra were obtained with proton decoupling on an Agi-
1
3
lent 400 MR DD2 (100 MHz). The chemical shifts were reported in
parts per million (ppm), the coupling constants (J) were expressed
in hertz (Hz) and signals were described as singlet (s), doublet (d),
triplet (t), as well as multiplet (m). The NMR data was analyzed by
MestReNova software. High-resolution mass spectra were
obtained on a Bruker SolariX 7.0 T spectrometer. Melting points
were recorded on a WRS-2A digital melting point apparatus.
2.2. Synthesis and characterization of XB-1
First, 1-(4-fluorophenyl)ethan-1-one (1) was nitrified in the
presence of concentrated nitric acid and sulfuric acid to afford 1-
4-fluoro-3-nitrophenyl)ethan-1-one (2), which was chlorinated
(
in acetonitrile with 2 equivalents of DCDMH at 35 °C for 18 h.
Finally, the fluorine group in 3 was then substituted by 4-
isopropylaniline to furnish the novel dichloroacetophenone XB-1,
3. Results and discussion
whose structure was confimed by NMR and HRMS (Figs. S1–S3).
3.1. Profiling the binding of XB-1 to HSA with absorption spectra
1
M.p. 133–134 °C; H NMR (400 MHz, CDCl
3
): d (ppm) 9.95 (s,
1
2
H), 9.02 (s, 1H), 8.07–8.04 (m, 1H), 7.35–7.33 (m, 2H), 7.22 (m,
H), 7.17 (d, J = 9.2 Hz, 1H), 6.58 (s, 1H), 3.01–2.94 (m, 1H), 1.29
In general, the absorption spectra analysis is considered as the
simple and straightforward method for profiling the interaction
information of a small molecule with biomacromolecule (e.g., serum
albumin, and DNA), which could effectively present the alterations
of biomacromolecule’s structure and surroundings [25]. On this
basis, thus, the absorption spectra of HSA-XB-1 system in PBS buffer
1
3
(
d, J = 7.2 Hz, 6H); C NMR (101 MHz, CDCl
3
): d (ppm) 183.29,
1
1
C
48.61, 147.45, 136.03, 134.50, 131.73, 130.37, 128.19, 125.61,
19.72, 116.27, 67.83, 33.94, 24.08. HRMS (ESI): calcd. for
ꢃ
17
H15Cl
2
N
2
O
3
[MꢃH] : 365.0465, found: 365.0456.
(
pH = 7.4, 10.0 mM) were firstly examined. As shown in Fig. 2, HSA
displayed two distinct absorption peaks at ~217 nm and ~280 nm,
respectively. In contrast, XB-1 showed four absorption peaks at
2.3. Fluorescence measurements
~228 nm, ~270 nm, ~350 nm, and ~445 nm, respectively. After incu-
In a 1.5 mL PE tube, 10.0
XB-1 in the range from 0 to 17.0
l
M HSA and various concentrations of
M were incubated at the indi-
bation of XB-1 with HSA in PBS buffer (pH = 7.4, 10.0 mM), the strong
absorption peak of HSA at ~217 nm was red-shifted to ~231 nm,
along with an obvious peak intensity decrease. According to previ-
l
cated temperature (273 K, 300 K, and 310 K) for 10.0 min. After
that, all test sample were applied to acquire the fluorescence
spectra upon an excitation wavelength of 280 nm and a scanning
wavelength range of 300–500 nm. The excitation and emission slit
widths are both 10 nm.
ous reports, H O, as a polar solvent, possessed a strong capacity to
2
impair the p–p* transition of C@O in the HSA’s polypeptide back-
bone structure, resulting in the peak intensity decrease of HSA in
therange from 200 nm to 220 nm [26,27]. Given such uniquefeature,
when XB-1 was incubated with HSA in aqueous solution, HSA mole-
cule occurred denaturation and the internal main chain was
uncurled, improving the likelihood of encounter between water
molecule and the internal amide moieties. As a result, a remarkable
decrease of peak intensity as well as an obvious red-shift at this
range were observed, demonstrating that XB-1 could effectively
cause the destruction of HSA’s secondary or tertiary structure. Addi-
tionally, the peak intensity of HSA at ~280 nm was increased and
blue-shifted from ~280 nm to ~275 nm, attributable to the remark-
able microenvironmental alterations of Trp and Tyr residues in HSA
[28]. By subtracting the absorption of XB-1 from HSA-XB-1, there
was a hypochromic shift in the absorption peak at ~280 nm by com-
2
.4. Absorption and CD measurements
The ultraviolet–visible absorption measurements were per-
formed on the TU-1901 UV–Vis spectrometer with a scan rate of
0 nm/s in the range of 200 nm – 600 nm at 300 K. Absorption
spectra were obtained with fixed concentrations of HSA
10.0 M) and XB-1 (40.0 M) in PBS buffer (pH = 7.4, 10.0 mM).
For CD measurements, 20.0 M HSA incubation with 20.0
XB-1 were dissolved with PBS buffer (pH = 7.4, 10.0 mM) in a
.5 mL PE tube at 300 K for 10.0 min. After that, the two samples
were carried out CD measurements in the range of 200–260 nm.
1
(
l
l
l
lM
1
paring with that of equimolar HSA, probably attributable to the p-p
3