48
G. Wu et al. / Journal of Molecular Structure 1045 (2013) 47–54
While the structure of BSA varies upon the addition of surfac-
Mix diethylene glycol diester (0.015 mol) and bromotetradec-
ane (0.045 mol) with acetonitrile, and reflux the solution for
40 h. Recrystallize the product in dichloromethane/acetone (1:20)
at least three times, collect white powder (the gemini surfactant)
and dry it in vacuum at 30 °C for 12 h. The structure of EQ14-2-
14 was confirmed by MS spectrometry and characterized by melt-
ing point, IR and NMR spectrometry. (Figs. S1–S4).
tants, there surely are changes happening inside the surfactants.
Zhou et al. [19] studied the complexes of C.I. Acid Orange 7 with
HSA by 1H NMR. All the chemical shifts of 1H signals moved
up-field and the spin–lattice relaxation times (T1) of C.I. Acid Or-
ange 7 decreased by the addition of HSA. The data confirmed that
C.I. Acid Orange 7 indeed interacted with HSA, and hydrophobic
portion of C.I. Acid Orange 7 should be embedded into the hydro-
phobic pocket of HSA. Research by Wu et al. [20] indicated that all
the 1H signals of naphthol shifted up-field when adding BSA, which
EQ14-2-14: white powder; Yield: 62%; MP (°C): 77–78. 1H NMR
(400 MHz, D2O, TMS): d 9.614(s, 1H), 9.360 (d, 1H, J = 4.4 Hz), 9.107
(d, 1H, J = 8 Hz), 8.357–8.326 (dd, 1H, J1 = 6.4 Hz, J2 = 6 Hz), 4.883
(s, 2H), 4.589 (s, 2H), 4.008 (s, 2H), 2.004 (s, 2H), 1.257–1.105
indicated p–p stacking occurring between naphthol and aromatic
residues of BSA. And the protons far from the hydroxyl group
up-field shifted significantly, which suggested that these protons
were close to the aromatic ring in the binding site. However, little
study is reported referring to BSA and gemini surfactants owing to
the expensive instrument, dear reagents, complicated method and
so on.
(m, 22H), 0.627 (s, 3H). 13C NMR (100 MHz, CDCl3,TMS):
d
161.57, 148.38, 145.78, 145.59, 130.34, 129.06, 68.71, 66.04,
62.55, 31.89, 31.85, 29.68, 29.66, 29.63, 29.58, 29.42, 29.34,
29.17, 26.09, 22.66, 14.09; FT-IR (KBr, cmꢁ1): 3425, 2924, 2854,
1736, 1638, 1462, 1378, 1301, 1178,1122, 1023,872, 749, 680,
539. MS (m/z): [M-Br]+ 355.2798.
Therefore, the purpose of the present work is to synthesize a
new gemini surfactant (EQ14-2-14) and probe the bilateral action
between BSA and the gemini surfactant by DPI, FTIR, 1H NMR and
molecular docking, so as to get a more comprehensive understand
about the interaction between proteins and gemini surfactants.
2.3. FTIR spectroscopy
All FTIR spectra were taken on a Nicolet 6700 FTIR spectrometer
(ThermoFisher Scientific, Massachusetts, USA) via the attenuated
total reflection (ATR) with resolution of 4 cmꢁ1 and 64 scans. The
spectrum of D2O was collected first, and then it was subtracted
from the spectrum of EQ14-2-14/D2O solution to get the FTIR spec-
trum of EQ14-2-14. The spectrum of EQ14-2-14/D2O was sub-
tracted from that of BSA/EQ14-2-14/D2O to get the FTIR
spectrum of BSA binding with EQ14-2-14. A quantitative analysis
of the spectra of BSA before and after the addition of EQ14-2-14
was processed by Fourier self-deconvolution, second-derivation
and Gaussian curve-fitting.
2. Materials and methods
2.1. Materials
Nicotinic acid (CR), thionyl chloride (AR), diglycol (AR) and bro-
motetradecane (AR) were purchased from Chengdu Kelong Chem-
ical Reagent Company, BSA from Shanghai Fankel Biological
Technology CO., Ltd., tris–HCl buffer (1.0 M, pH 7.4) from Beijing
Solarbio Science & Technology CO., Ltd., and deuterium oxide with
purity of 99.994% from Qingdao Tenglong Weibo Technology Co.,
Ltd. All other chemicals were of analytical grade and used as sup-
plied without further purification.
2.4. Nuclear magnetic resonance measurements
All solutions of EQ14-2-14 and BSA were prepared at pH 7.4 in
0.05 M sodium phosphate buffer in D2O. The concentration of
EQ14-2-14 stock solution was 1.0 ꢂ 10ꢁ2 M. The concentration of
BSA was 5 ꢂ 10ꢁ3 M. 1H NMR measurements at different molar ra-
tio ([BSA]:[EQ14-2-14]) were recorded on a Bruker Advance 400
spectrometer (Rheinstetten, Germany) by using a probe tuned at
400 MHz and 5 mm tubes at 298 K. Chemical shift of EQ14-2-14
proton resonances were measured in relation to the TMS signal
at 0 ppm. Presaturation method was used for water suppression.
T1 of protons were determined by conventional inversion-recovery
method. Maximum error of chemical shift and relaxation time was
0.0001 ppm and 0.001 s, respectively.
2.2. Preparation and characterization of EQ14-2-14
Scheme 1 presents the way for preparing the gemini surfactant.
The procedure is as follow.
Add thionyl chloride (0.2 mol) to nicotinic acid (0.1 mol) under
stirring, and reflux the mixture for 4 h. Remove excess thionyl
chloride by a rotary evaporator, add dropwise the mixture of digly-
col (0.045 mol) and chloroform into the flask under agitating at
65 °C, reflux the mixture for 10 h, then evaporate the solvent.
Add saturated Na2CO3 solution to the residue to adjust the solution
to neutrality or alkalescence. Extract the mixture with ethyl ace-
tate, then, purify the extractive by column chromatography (petro-
leum ether: ethyl acetate = 3:1) and collect pale yellow liquid
(Diethylene glycol diester).
2.5. DPI measurements
The details of Dual polarization interferometry (Ana Light
Nano2000, Farfield Group Ltd., Manchester, U.K.) and theory has
Scheme 1. Preparation of the EQ14-2-14.