L. Zhang et al. / Journal of Molecular Catalysis B: Enzymatic 87 (2013) 30–36
31
new polymer supported palladium catalyst. Finally, the catalytic
2.4. Titration of hydroxyl group in phenol polymer
performances of the catalyst for Heck reactions of various aryl
halides were studied.
The ratio of phenylene and oxyphenylene units (Ph/Ox) in as-
prepared phenol polymer was determined by titration. Briefly,
0.10 g of as-prepared phenol polymer was dissolved in 5.0 mL of
pyridine containing 2.5% acetic anhydride. Resultant solution was
kept at 95 ◦C for 1 h under gentle magnetic stirring. After cooling
to room temperature, 0.5 mL of water was added and the mixture
was heated again at 95 ◦C for 10 min. Resultant solution was titrated
with 0.20 M sodium hydroxide solution in ethanol in the presence
of phenolphthalein as an indicator [32].
2. Experimental
2.1. Materials
Horseradish peroxidase (RZ = 2.5, activity = 200 U/mg) was
purchased from Shanghai Guoyuan Biotechnology Co., Ltd.
and used without further purification. Iodobenzene and p-
chloronitrobenzene were chemical pure and were purchased
from Sinopharm Chemical Reagent Co., Ltd. p-Iodoanisole
(98%, Lancaster), p-iodotoluene (98%, J & K Scientific Ltd.), p-
iodonitrobenzene (98%, Alfa Aesar), p-bromonitrobenzene (98%, J
& K Scientific Ltd.) and p-bromoanisole (97%, Lancaster) were used
without further purification. All other chemicals employed in this
work were obtained from various commercial suppliers and were
used as received.
2.5. Preparation of aminated phenol polymer supported
palladium complex
1.0 g of phenol polymer was dissolved in 10 mL of ethanol, then
10 mL of epoxy chloropropane and 20 mL of NaOH (20% aqueous
solution) were added. The mixture was stirred at 50 ◦C for 5 h. The
black phenol polymer turned into yellow powder. The powder was
filtrated, washedwith plentyofwaterandethanol. Afterbeing dried
at 60 ◦C in vacuum for 8 h, 1.2 g of epoxidated phenol polymer was
obtained.
2.2. Measurements
1.0 g of epoxidated phenol polymer was added into 20 mL of
H2O. The pH value of the mixture was adjusted to 10 with NaOH
solution. After 5 mL of triethylene-tetramine was added, the mix-
ture was heated to 50 ◦C and stirred for 8 h. The powder was
collected and dried at 60 ◦C in vacuum for 8 h to give the aminated
phenol polymer (abbreviated as PP-N).
0.5 g of aminated phenol polymer was added to the solution of
PdCl2 (0.05 g) in ethanol (20 mL). The mixture was stirred at 60 ◦C
for 72 h in air. After being cooled to room temperature, the reaction
mixture was filtered and washed with ethanol (3× 20 mL), H2O (3×
20 mL) following. Then, the powder was dried at 60 ◦C in vacuum for
8 h to give 0.52 g of aminated phenol polymer supported palladium
complex (abbreviated as PP-N-Pd).
Infrared (IR) spectra were recorded on an Avatar 360 Fourier
transform infrared spectroscopy (Nicolet Company, USA). Ther-
mogravimetric (TG) analyses in nitrogen and air atmosphere
were performed on a TGA/SDTA851e instrument (Mettler-Toledo
Company, Switzerland) at a heating rate of 10 ◦C/min. The con-
centrations of phenol during the enzymatic polymerization were
analyzed using a high-performance liquid chromatograph (HPLC;
Agilent 1100, American) equipped with a C18-reverse phase col-
umn (2.1 mm × 150 mm, 5 m). The eluent was composed of
acetonitrile, distilled water and acetic acid (volume ratio 45:55:0.1,
flow rate is 0.4 mL/min). Phenol was detected at 280 nm using an
ultraviolet (UV) absorbance detector. Palladium content in PP-N-Pd
was measured on an inductively coupled plasma (ICP) spectrome-
ter (Optima 2100DV, Perkin-Elmer, America). X-ray photoelectron
spectra(XPS) were acquiredonanAXISULTRA spectrometer (Kratos
Analytical, England) using mono-AlK␣ radiation. The C1s photo-
electron line was used for energy calibration and the C1s binding
energy was taken as 284.8 eV.
2.6. Typical procedure for the Heck reaction of aryl iodides with
acrylic acid
In a 50 mL of round bottomed flask, PP-N-Pd (0.015 g), acrylic
acid (1.5 mmol), iodobenzene (1.0 mmol), (n-Bu)3N (3.0 mmol) and
solvent (0.2 mL) were added. The mixture was stirred and heated
to 90 ◦C in air for 3 h. After the reaction mixture was cooled to room
temperature, H2O (25 mL) and Na2CO3 (1.0 g) were added. The mix-
ture was stirred for 10 min, and then PP-N-Pd was separated by
filtration. The filtrate was treated with 3 M HCl (5 mL) and the pre-
cipitate appeared. Then, the precipitate was filtered, washed with
H2O and dried in air to give the product.
2.3. Enzymatic polymerization of phenol in aqueous micelle
system
A proper amount of SDS and 5 mmol (0.47 g) of phenol were dis-
solved in 45 mL of phosphate buffer (0.1 M, pH 7.0). 1 mg of HRP was
dissolved in 5 mL of phosphate buffer. Then, the enzyme solution
and phenol solution containing SDS were mixed. To the resultant
mixed solution, hydrogen peroxide (5%, 0.25 mL) was added every
5 min for a total of 14 times under mild magnetic stirring at room
temperature. Resultant black mixture was stirred for an additional
0.5 h, followed by vacuum-filtering and thoroughly washing with
water to remove HRP, SDBS, residual hydrogen peroxide and phe-
nol. As-obtained black material was dried in vacuum at 60 ◦C to give
the desired phenol polymer.
When the effect of dosage of HRP on the yield of phenol polymer
was investigated, diluted enzyme solution was used. First, 2 mg of
HRP was dissolved in 10 mL of phosphate buffer. 4, 3, 2 and 1 mL
of the enzyme solution was taken out respectively. Then, the four
samples of enzyme solution were all diluted to 5 mL with phosphate
buffer. Finally, these diluted enzyme solutions were respectively
mixed with 45 mL of phenol (5 mmol) solution containing SDS. As
a result, for 5 mmol of phenol, the dosage of HRP is 0.8, 0.6, 0.4 and
0.2 mg respectively.
2.7. Typical procedure for the Heck reaction of aryl halides with
styrene
PP-N-Pd (0.015 g), styrene (2.25 mmol), iodobenzene
(1.5 mmol), (n-Bu)3N (3.0 mmol) and solvent (0.2 mL) were
added into a 50 mL round bottomed three necked flask. The reac-
tion mixture was stirred at 90 ◦C in air for 3 h. Then, the mixture
was cooled to room temperature and ether (30 mL) was added.
The ether layer was separated, concentrated, and recrystallized to
give the final product.
3. Results and discussion
3.1. Preparation and characterization of phenol polymer
We first examined the effect of SDS on HRP-catalyzed polymer-
ization of phenol in phosphate buffer at room temperature in air.