Research Article

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Bioinorganic Nanohybrid Catalyst for Multistep Synthesis of

Acetaminophen, an Analgesic
Boi Hoa San,†,‡,§ Subramaniyam Ravichandran,‡,§ Kwang-su Park,‡ Vinod Kumar Subramani,‡
and Kyeong Kyu Kim*,†,‡
†
Sungkyunkwan Advanced Institute of Nanotechnology, Sungkyunkwan University, Suwon 440-746, Korea
‡
Department of Molecular Cell Biology, Sungkyunkwan University School of Medicine, Suwon 440-746, Korea
*
S Supporting Information
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ABSTRACT: A bioinorganic nanohybrid catalyst was synthesized by combining

esterase with a platinum nanoparticle (PtNP). The combination of two catalysts
Downloaded via UNIV OF SUNDERLAND on October 11, 2018 at 08:32:32 (UTC).

resulted in enhanced catalytic activities, esterase hydrolysis, and hydrogenation in

PtNPs, as compared to each catalyst alone. This hybrid catalyst can be successfully
used in the multistep synthesis of acetaminophen (paracetamol), an analgesic and
antipyretic drug, in a one-pot reaction with high yield and efficacy within a short
time, demonstrating that the nanobiohybrid catalyst offers advantages in the
synthesis of fine chemicals in industrial applications.

KEYWORDS: bioinorganic nanohybrid, platinum nanoparticle, catalysis, esterase, hydrolysis, hydrogenation, acetaminophen

1. INTRODUCTION tions1−4,13,15 in synthetic chemistry, pharmaceuticals, environ-

The synergistic integration of nanotechnology with biotechnol-
ogy enables improved functionality and extended applications
to be achieved in nanobiohybrid materials.1−4 Among various
platforms for constructing nanobiohybrids, the combination of
an enzyme and a highly active metallic nanoparticle is
particularly interesting because of their enhanced biocatalytic
functionality and capability for performing multiple reactions.
Enzymes are extremely efficient at catalyzing a wide range of
reactions with high yields and possess accurate substrate or
product specificity under mild reaction conditions.5 Accord-
ingly, there is a significant demand for their utility in food,
energy, and various industrial processes as well as in the
synthesis of fine chemicals for pharmaceutical, agricultural, and
electronic purposes.1,5 However, metallic nanoparticles (NPs)
made from highly active noble metals such as platinum and
palladium have been extensively investigated for their
applications in various fields such as chemical synthesis,6
hydrogen production,7,8 and biomedicine.9,10 The combination
of an enzyme and a metallic NP with complementary activities
can significantly enhance the biocatalytic activity, stability,
capability, and engineering performances of both components
in bioprocessing.11,12 In addition, it can contribute to creating
new functionality. The versatility and reaction specificity of
nanobiohybrid catalysts have been demonstrated with the
generation of multifunctional hybrid materials with novel
properties and enhanced functionalities,1,2,11,13,14 which make
them promising biocatalysts for numerous fascinating applica-
mental treatment, and food technology.
Oligomeric enzymes with a hollow interior have been used as
a template to construct nanobiohybrid catalysts using NPs
because of their advantage in synthesizing homogeneous NPs
with high stability, biocompatibility, and functional diver-
sities.10,16−24 In the case of nanoparticles encapsulated within
enzymes, it was observed that the enzyme activity was reduced
due to the presence of nanoparticles close to the active site of
enzymes.11 Therefore, it is required to overcome this limitation
by developing a nanobiohybrid with different structural
architecture.
In this study, we synthesized a novel nanobiohybrid catalyst
by combining oligomeric esterases (EST) with PtNPs that are
grown outside of proteins. By this approach, catalytic sites of
both platinum and enzymes are intact and fully accessible to the
substrates. We demonstrated its capability of catalyzing
multiple-step reactions in a novel one-pot reaction for the
production of acetaminophen (paracetamol), a widely used
antipyretic (Scheme 1), with enhanced catalytic activities. One-
pot reactions are the most efficient strategy for organic
synthesis and are highly desirable in the manufacture of
pharmaceuticals. The combination of a one-pot synthesis
reaction with green chemistry can result in significant benefits,
Received: October 10, 2016
Revised: October 19, 2016
Accepted: October 20, 2016
Published: October 31, 2016

© 2016 American Chemical Society 30058 DOI: 10.1021/acsami.6b12875

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Scheme 1. Schematic of Multistep Reaction Synthesis of Acetaminophen Catalyzed by EST−PtNP Complexesa
a
(1) Hydrolysis of 4-nitrophenyl acetate to produce 4-nitrophenol by enzymatic activity of EST−PtNPs. (2) Hydrogenation of 4-nitrophenol to 4-
aminophenol by hydrogenated activity of EST−PtNPs. (3) The formation of N-(4-hydroxyphenyl) acetamide (acetaminophen) in the presence of
acetic anhydride. (4) Direct synthesis of acetaminophen from 4-nitrophenol acetate in the presence of EST−PtNPs and acetic anhydride.
and this study comprehensively demonstrates the advantage of
using nanobiohybrid catalysts for industrial applications.
2. EXPERIMENTAL SECTION
2.1. Materials. All chemicals were purchased from Sigma-Aldrich
(St. Louis, MO) and used as received without further purification.
2.2. Sample Preparation. The preparation of EST and the
synthesis of the platinum nanoparticles (PtNPs) using EST were
described previously.11,25 In brief, the EST enzyme with hydrolytic
activity was prepared by cloning the gene encoding EST from
Mycobacterium smegmatis (GeneID: 4532379) into a pET-based vector
with a tobacco etch virus (TEV) protease-cleavable N-terminal hexa-
histidine tag. The recombinant plasmid, named pVFT1S-EST, was
transformed into Escherichia coli BL21 (DE3) cells (Novagen, Billerica,
MA). Cells were cultured in Luria−Bertani medium at 37 °C to an
OD600 of 0.5 before being induced with 0.5 mM isopropyl-b-D-
thiogalactopyranoside. The protein was first purified by metal affinity
chromatography on a HiTrap chelating column (GE Healthcare,
Pittsburgh, PA). The N-terminal His-tag was removed using TEV
protease in buffer A (20 mM Tris−HCl, pH 7.5, and 10 mM NaCl).
The cleaved protein was further purified by anion exchange
chromatography on a HiTrap Q column (GE Healthcare). The
purified protein was dialyzed against buffer B (20 mM Tris−HCl, pH
7.5) and concentrated using YM-3 ultrafiltration and Centricon
(Millipore, Billerica, MA). The purity of EST was determined using
15% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-
PAGE). In situ synthesis of PtNPs was performed in solution by
reducing PtII in the presence of EST at 22 °C.
To synthesize EST−PtNPs, purified recombinant EST protein was
mixed with K2PtCl4 in 10 mL of buffer (50 mM HEPES, pH 8.0)
followed by 1 h incubation with constant stirring. In the reaction
mixture, the final concentration of EST was 1 μM, and the final
concentration of K2PtCl4 was 0.5, 1.0, and 3.0 mM for the synthesis of
1.8, 2.6, and 3.8 nm EST−PtNPs, respectively. After 1 h of incubation
of protein and platinum precursor, 0.5 mL of 100 mM ice-cold NaBH4
solution was added into the reaction mixture, followed by 2 h of
incubation with constant stirring. The reaction mixture containing the
EST−PtNPs was concentrated using Centricon centrifuge (30 kDa
cutoff, Sartorius, Gottingen, Germany) at 16 100 rcf for 30 min at 4 °C
to remove aggregates from the supernatant that is used for further
analyses as the EST−PtNPs. The concentration of EST was
determined by the Bradford assay26 using bovine serum albumin to
generate the standard curve.
Citrate-capped PtNPs were synthesized according to reported
procedures with some modifications.27 Briefly, PtNPs were obtained
by reducing 1 mM H2PtCl6 with 10 mM NaBH4 in the presence of 3
mM sodium citrate in 50 mL of reaction mixture. The amounts of each
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component listed above were their final concentrations in the reaction
mixture. The NaBH4 solution was added drop-wise to the reaction
mixture with constant stirring for 1 h. The final product was kept at 4
°C until further use. The concentration of citrate-capped PtNPs was
determined by inductively coupled plasma optical emission spectros-
copy (ICP-OES).
2.3. Sample Characterization. The UV−visible absorption
spectra at 200−500 nm were measured using a UV-550 spectropho-
tometer (Jasco, Tokyo, Japan) with samples in a 10 mm light path
quartz cuvette. Transmission electron microscopy (TEM) images were
taken using JEOL JEM-3010 microscopes (JEOL, Tokyo, Japan)
operated at 300 kV. TEM samples were prepared by applying the
EST−PtNP solution to a copper grid covered with a thin carbon film
(JEOL, Tokyo, Japan) and dehydrating it overnight at room
temperature. To prepare negatively stained PS−PtNP samples, the
protein samples were stained on a copper grid with 2% uranyl acetate
for 30 s. The size of the PtNPs was estimated by averaging 200
individual particles using the Gatan Digital Micrograph software
(Gatan, Pleasanton, CA). Energy dispersive X-ray spectroscopy data
were obtained from the samples prepared for TEM.
Circular dichroism (CD) spectra of EST only and EST−PtNPs
were measured with a Jasco J-810 CD spectrometer at 25 °C in a 1
mm quartz cell. High-performance liquid chromatography (HPLC)
analysis was performed using a C18 reverse-phase column (Agilent
Technologies, Santa Clara, CA) in an Agilent 1100 HPLC system
(Agilent Technologies).
X-ray photoelectron spectroscopy (XPS) was performed on a
Thermo Scientific ESCALAB 250 Xi system. 2.6 nm EST−PtNPs were
drop-coated on a 1 cm2 silicon wafer that was previously cleaned by
ultrasonication in acetone. The samples were dried overnight before
XPS analysis. The binding energy was calibrated using the C 1s peak
intensity at 284.8 eV. The data was analyzed using CasaXPS and peak
fittings carried out using Gaussian−Lorentzian functions.
Liquid chromatography−mass spectrometry (LC−MS) was carried
out on an Agilent Model 1100 HPLC system fitted with an ion trap
mass analyzer. LC was carried out on a C18 reverse-phase column and
the mass analyzed using the ESI system in both positive and negative
ion modes. The multistep synthesis reaction mixture was filtered using
a 0.22 μm filter before analysis. A 1 g/mL solution of Acetaminophen
(Sigma) in DMSO was used as standard.
2.4. Hydrolytic Activity of EST−PtNPs. The hydrolytic activities
of the EST−PtNPs were determined using 4-nitrophenyl acetate as a
substrate. In a typical experiment, 200 μL of reaction mixture
containing 1 mM substrate and 1 μM soluble EST−PtNPs in 10 mM
Tris−HCl buffer at pH 7.0 was incubated at 37 °C in a 96 well plate.
The release of 4-nitrophenol was then monitored using a SpectraMax
Plus384 (Molecular Devices, Sunnyvale, CA) at a wavelength of 405
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Figure 1. Crystal structure (PDB ID: 2Q0Q) of EST. (A) Ribbon diagram of an EST octamer. The EST subunits are shown in different colors. (B)
Charge distribution on the exterior surface of EST; negative charges are red, and positive charges are blue. (C) Charge distribution on the internal
surface of EST. The dimensions of the inside and outside of EST are indicated.

Figure 2. TEM images of EST−PtNPs. (A) EST−PtNP complexes were confirmed using electron microscopy with 2% uranyl acetate staining. The
insets are representative images of EST−PtNP complexes at a 2:1 (left) and 3:1 (right) ratio between EST and PtNP. (B) EDX analysis of EST−
PtNP complex. (C−E) Ratio-controlled synthesis of EST−PtNPs. Platinum precursors were incubated with EST at varying molar ratios of 500:1,
1000:1, and 3000:1 for 2 h, resulting in complexes with average sizes of 1.8, 2.6, and 3.8 nm, respectively. The inset in (D) shows the magnified
image of an unstained 2.6 nm EST−PtNP. The periodicity of the lattice fringes was found to be 0.226 nm in the [111] direction. The bottom panels
in (C−E) are the corresponding size distribution histograms, with the numbers indicating the average size of the NPs.

nm for 30 min, and all experiments were repeated three times to
obtain a mean value.
2.5. Hydrogenation Activity of EST−PtNPs. The catalytic
activities of the EST−PtNPs were measured using 4-nitrophenol as a
substrate in the liquid phase.14 Briefly, a 9 mL reaction mixture
containing EST−PtNPs (20 ppm PtNP) and 0.15 M NaBH4 was
stirred for 15 min at RT. A total of 1 mL of 4-nitrophenol was added
to the mixture to yield a final concentration of 0.5 mM, and this
mixture was stirred until the reaction was completed. The reaction
progress was spectrophotometrically monitored at 405 nm using a
UV−vis spectrometer (UV550, JASCO, Tokyo, Japan) for 6 min. Each
50 μL aliquot of the sample was diluted in 450 μL of distilled water.
The hydrogenation activity was determined as a first-order reaction.
The hydrogenation activity of the sodium citrate-capped PtNPs at 20
ppm was assessed in a similar way.
2.6. Multistep Catalytic Activities. The synthesis of acetamino-
phen was carried out in a one pot reaction by mixing 1 mM of 4-
nitrophenyl acetate substrate with EST−PtNP complexes for a
concentration of 20 ppm Pt in a final reaction volume of 5 mL. To
measure the release of 4-nitrophenol from 4-nitrophenyl acetate, UV−
vis spectra with a wavelength ranging from 250 to 500 nm were
scanned for 5 min at 1 min intervals (UV550, JASCO, Tokyo, Japan).
Subsequently, 28.5 mg of NaBH4 was added to initiate the
hydrogenation reaction. The hydrogenation of 4-nitrophenol to 4-
aminophenol was also monitored at 405 nm via UV−vis scanning for 5
min at 1 min intervals. Finally, 0.12 g of acetic anhydride was added to
the reaction mixture to yield the final product of acetaminophen. The
formation of the final product was monitored at 5 min intervals for 30
min. All reactions were performed at 22 °C with vigorous stirring. For
all UV measurements, 50 μL aliquots of the sample were diluted in 450
μL of distilled water.
2.7. Calculation of the Yield of Final Product (Acetamino-
phen). Commercially available acetaminophen in powder form was
dissolved in dimethyl sulfoxide (DMSO) as a standard and injected
into a C18 reverse-phase column (Agilent Technologies, Santa Clara,
CA) in an Agilent 1100 HPLC system (Agilent Technologies). The
elution profile of acetaminophen was monitored at 250 nm. A standard
curve of acetaminophen was plotted using the peak area of the elution
peak on the y-axis and the number of moles on x-axis. The amount of
acetaminophen produced was calculated by extrapolating the values
through a calibration curve (R2 = 0.9999) (Figure S7). The yield of
acetaminophen production was obtained using the following equation:
yield of acetaminophen (%)
number of molecules of acetaminophen
= × 100
number of moles of 4‐nitrophenylacetate

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2.8. Calculation of the Turnover Frequency of the Multistep
Reaction. The turnover frequency (TOF) value of the EST−PtNP
complexes in multistep reaction was determined using the following
formula:13
number of moles of acetaminophen 1
TOF = ×
number of moles of Pt atom on the PtNP surface time
3. RESULTS AND DISCUSSION
3.1. Synthesis and Characterization of EST−PtNPs.
Esterase (EST) from M. smegmatis, which self-assembles into a
well-defined cube-like octameric complex28 with overall exterior
dimensions of 8.0 nm × 8.0 nm × 6.0 nm, has been introduced
as a PS for the synthesis of PtNPs. The cube-like architecture of
EST reveals a cavity with a diameter of approximately 2.5 nm
located in the center (Figure 1). EST, an SGNH hydrolase
enzyme, efficiently catalyzes acyl group transfer to alcohols
(alcoholysis) in aqueous conditions. It also preferably catalyzes
perhydrolysis rather than the hydrolysis reaction when peroxide
acts as the acceptor.28 A total of six channels, two large and four
small with diameters of 2 and 1 nm, respectively, presented on
each face of the cube are likely to serve as entries for the
substrate and exits for the product during catalysis. The active
sites of EST face inward in the cube-like cavity.
EST−PtNPs were synthesized using EST protein as a
template by slowly reducing K2PtCl4 with NaBH4 as a reducing
agent in an aqueous solution at 25 °C. The formation of PtNPs
was visibly observed by a color change from light yellow to dark
brown, which was also confirmed by UV−vis spectra
measurements (Figure S1). Consistent with previous re-
ports,9−11 the EST−PtNP solution showed a broad UV−vis
absorption spectrum with a maximum peak at 280 nm, a
representative fingerprint of the EST protein (Figure S1). On
the contrary, the two major peaks from the K2PtCl4 solution at
320 and 375 nm completely disappeared (Figure S1), indicating
the formation of PtNPs. EST−PtNPs were purified using high-
speed centrifugation (16 100 rcf) and were characterized using
TEM (Figure 2). As a control, PtNPs with a size of 2.4 nm were
chemically synthesized and stabilized by sodium citrate (Figure
S2).
TEM images of EST−PtNPs, stained with 2% uranyl acetate,
revealed PtNPs stabilized by several EST proteins (Figure 2A).
In some instances, one PtNP can be stabilized by either two or
three EST proteins (Figure 2A, inset). However, overall, the
distribution of EST around the PtNP remained heterogeneous.
We observed that the final EST−PtNPs were quite well-
dispersed; very little clumping was seen, which was the result of
stabilization of nanoparticles by the proteins. The molar ratio
between the platinum precursors and EST in the initial reaction
mixture largely contributes to the size of the PtNPs. When 1
μM of octameric esterase was incubated with platinum
precursor at the concentration of 500 μM, 1 mM, and 3 mM,
the size of resulting PtNPs in the EST−PtNP nanohybrids were
1.8 ± 0.3, 2.6 ± 0.4, and 3.8 ± 0.9 nm, respectively (Figures
2C−E). The high-resolution image revealed that the lattice
spacing was 0.226 nm, which corresponds to the (111) plane of
platinum exhibiting fcc crystal structure (Figure 2D, inset).
Energy-dispersive X-ray (EDX) analysis confirmed the presence
of platinum ion in the protein−NP complexes (Figure 2B).
X-ray Photoelectron Spectroscopy (XPS) was used to
analyze the surface characteristics of the EST−PtNP complex
(Figures 3 and S3). Region-specific XPS spectra were acquired
and fitted for the analysis of Pt, C, O, and N (Figure 3) using
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Figure 3. XPS region spectrum of EST−PtNP complex. (A) O 1s, (B)
Pt 4f7/2 and Pt 4f5/2, (C) C 1s, and (D) N 1s. The red and dotted lines
represent the fitted and experimental curves, respectively. Black, green,
blue, and brown lines represent the binding energy peaks.
Gaussian−Lorentzian curves (Table S1). The XPS peaks at
69.45 and 72.88 eV can be attributed to the Pt 4f7/2 and Pt 4f5/2
core levels, respectively. The separation between the 4f7/2 and
4f5/2 peaks was 3.4 eV, which is consistent with the previous
report.29 However, an additional peak in each core level was
observed, possibly due to the energy shift caused by protein
binding.
The C 1s peak was deconvoluted into three peaks at 284.70,
285.58, and 287.15 eV, which can be assigned to C−C and C−
H, CN, and CO bonds, respectively.30 The O 1s peaks
were fitted to three peaks at 530.56 and 531.54 eV and a weak
peak at 535.51 eV; these positions are consistent with earlier
literature.31 The binding energy at 531.54 eV is likely
corresponding to the oxygen atom in the COO− group
bound to the Pt surface. The region spectrum for N 1s was
fitted to peaks at 398.51 and 400.54 eV that can be assigned to
the chemical states of nitrogen NH2 and NH3+ connected to
carbon atoms, respectively.30,31 The NH3+ binding energy was
shifted relative to NH2, possibly due to the binding of NH3+
group to the PtNP surface.30
The circular dichroism (CD) spectra of EST−PtNPs and
EST revealed that the compositions of their secondary
structures are almost identical, indicating that the overall
conformation is well maintained during PtNP synthesis.
However, local conformational changes are expected from the
minor deviations in the two spectra (Figure S4).
Together with TEM analysis, these results suggest that,
unlike other known NPs deposited inside PSs,10,18,32 PtNPs are
preferably synthesized outside of EST, possibly because of the
difference between the charge distribution of EST and that of
other known PSs such as PepA, ferritin, and ClpP, which have
negatively charged residues inside.10,18,32 However, EST shows
a highly negatively charged surface, whereas its interior is
positively charged (Figure 1B,C). Considering that negatively
charged residues in PSs play a role in metal deposition in the
initial stage of NP synthesis, NPs are likely to grow from the
exterior of EST.
Considering all of the physicochemical properties of EST−
PtNPs, we propose the following mechanism for PtNP
synthesis in EST: (1) binding of platinum ions, incubation of
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Pt2+ ions with EST molecules causes Pt2+ ions to bind to the
EST surface via electrostatic interactions with the negatively
charged surface of EST, which serves as nucleation sites; (2)
nucleation of platinum crystal, addition of an excess amount of
strong reducing agent (NaBH4) subsequently reduces Pt2+ to
Pt0, after which Pt0 nuclei are formed on the surface of EST;
(3) growth of PtNPs, in which from the Pt0 nuclei, PtNPs
continue to grow until Pt2+ are depleted in the solution.
3.2. Evaluation of the Catalytic Ability of EST−PtNPs.
We first investigated hydrolytic and hydrogenation activities,
separately, in the EST−PtNP hybrid catalyst. 4-nitrophenyl
acetate was used as a substrate for the measurement of the
hydrolytic activity of EST−PtNPs. The release of 4-nitrophenol
from 4-nitrophenyl acetate was observed for 30 min (Figure 4).
Figure 4. Enzymatic activity comparison of 1.8, 2.6, and 3.8 nm EST−
PtNPs with EST only and a mixture of EST with citrate-stabilized 2.6
nm PtNPs. The hydrolysis of 4-nitrophenyl acetate to 4-nitrophenol
was monitored by taking the absorbance at 405 nm. The absorbance of
EST only was set as 100%, and the relative activity percent of other
conditions was calculated relative to EST only. All experiments were
done in duplicate; error bars represent standard deviation of three
individual trials (n = 3).
We compared the hydrolytic activities of EST−PtNPs of
different sizes to ascertain whether the size had any effect on
activity, which was one of the limitations of earlier nano-
biohybrid catalysts.11 The results showed that the hydrolytic
activity was independent of the size of the nanoparticle in the
EST−PtNP complex. Because the nanoparticles are stabilized
externally, the interior active site of EST cannot be affected by
the binding of PtNPs on the exterior surface. Surprisingly, the
hydrolytic activity of EST−PtNPs was significantly higher than
that of EST alone, suggesting that the substrate binding site of
EST in the hybrid complex is more exposed to the substrate
than that of EST alone possibly by conformation alteration
accompanied by PtNP binding, which was evidenced by CD
spectra (Figure S4). Consistently, it has been also reported that
gold nanoparticles can positively affect the catalytic activity of
lysozymes when they form an enzyme−NP conjugate by
inducing conformation changes in the lysozymes.33 Addition-
ally, the conjugation of protein to metal nanoparticles can also
increase enzyme activity by increasing collisions of the substrate
with the enzyme aided by the Brownian movements of
nanoparticles.12
In addition, to check whether the enhanced catalytic activity
was a result of true synergy between the nanoparticle and EST
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protein, we compared the activity of EST−PtNP complex with
a mixture containing EST and citrate-stabilized PtNPs (Figure
4). The average sizes of PtNPs in hybrid and citrate-stabilized
were 2.6 and 2.4 nm, respectively. The concentrations of
protein and Pt in the EST−PtNP complex were analyzed using
the Bradford assay and ICP-OES, respectively. When the
mixture of EST and citrate-stabilized PtNP were used for the
activity assay, their concentrations were adjusted to those in the
EST−PtNPs. The hydrolysis activity of the EST−PtNP
complex was significantly higher than that of the mixture
containing EST and citrate-stabilized PtNPs, indicating that
PtNPs in the EST−PtNP complex contributed to the enhanced
catalytic activity of EST.
Similarly, the hydrogenation activity of EST−PtNPs was
compared to that of the PtNPs in the mixture of EST by
monitoring the reaction rate of hydrogenation from p-
nitrophenol to p-aminophenol for 6 min (Figure 5A,C). The
Figure 5. Hydrogenation activities of (A) mixture of EST and 2.4 nm
citrate-stabilized PtNPs and different-sized EST−PtNPs (B−D). Inset
shows the rate constant.
hydrogenation activity of EST−PtNPs was higher than that of
citrate-stabilized PtNPs physically mixed with EST, indicating
that the PtNP stabilized by EST proteins is more active. This is
consistent with previous reports in which the catalytic activity
of metallic nanoparticles was enhanced when they were bound
to proteins.10,11 Taking these results together, it can be
concluded that EST−PtNPs possess higher hydrolytic and
hydrogenation activities as compared to the catalytic activities
of EST and PtNP in a simple mixture.
In addition, we also examined whether the size of PtNPs in
the nanobiohybrid catalyst affects the hydrogenation activity
(Figure 5). We observed that the rate constant of hydro-
genation was inversely proportional to the size of the
nanoparticles in the EST−PtNP complex (Figures 5B−D).
This result is consistent with the results of the size-dependent
activity change of Pt nanoparticles reported in other
literature.34 We also confirmed that the nanobiohybrid catalyst
was very stable over multiple cycles of reactions by verifying
that the hydrogenation activity did not decrease (Figure S5).
This result indicates that there had not been any catalyst
deactivation.35,36 It is well-known that leaching and catalyst
deactivation have a cause−effect relationship.37 Therefore, we
believe that there was no leaching. To verify this analysis, we
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filtered the reaction mixture through a membrane with 10 kDa
molecular weight cut off and analyzed the filtrate using ICP-
OES. However, we could not detect Pt in the filtrate, which
suggested that the amount of leached Pt was lower than the
detection limit of our machine. Therefore, we can conclude that
there was no leaching in current setup of experiment.
It has been well documented that the size of PtNPs
(especially <5 nm) strongly influences its catalytic activity.6,38,39
The question is then why did the EST−PtNPs show higher
catalytic activity than that of citrate−PtNPs although their sizes
are similar? We believe that the functional difference originated
from the number of available active sites. The PtNPs in EST−
PtNP complexes are stabilized and protected by EST proteins
because they form a thermodynamically stable protein−
nanoparticle entity. Therefore, the hybridization of EST is
likely to enhance the stability and dispersion of PtNPs in
aqueous environments (Figure 2). As a result, catalytic sites
present on the surface of PtNPs are fully exposed in an active
state. Moreover, the accessibility of substrates to the catalytic
sites on the PtNPs cannot be affected by EST because
substrates can freely access to the nanoparticle through solvent
channels present between EST subunits (Figure 1). However,
in the case of citrate-stabilized PtNPs, particles are not well
dispersed (Figure S2). Therefore, it is expected that the PtNP
surface will not be fully accessible to the substrates. Consistent
to this interpretation, it had been reported that the hydro-
genation activity of PtNPs is enhanced when they are
immobilized on a support due to increased dispersion when
compared to unsupported capped PtNPs.40 It is also reported
that the protein-stabilized PtNPs have shown up to 40%
enhanced ROS-scavenging activities than PtNPs stabilized by
organic stabilizer.10,11
3.3. Synthesis of Acetaminophen Using Combined
Catalytic Activity of EST−PtNPs. We then tested the
combined catalytic activities of EST−PtNPs using 4-nitro-
phenyl acetate as starting material. EST-mediated hydrolysis
yielded 4-nitrophenol, with the development of a yellow color
(Scheme 1-1 and Figure 6). Subsequent hydrogenation of 4-
nitrophenol was monitored by the disappearance of the yellow
color under reducing conditions (Scheme 1-2), showing that
this two-step reaction was completed by one bioinorganic
Figure 6. Multistep reaction catalyzed by the EST−PtNP complexes.
Hydrolysis of 4-nitrophenyl acetate to produce 4-nitrophenol was
monitored by a change in spectrum from 275 nm (orange) to 405 nm
(dark blue). Subsequent hydrogenation of 4-nitrophenol resulted in
the disappearance of an absorption peak at 405 nm and the emergence
of a new peak at 300 nm (pink). The N-(4-hydroxyphenyl) acetamide
(acetaminophen) was spontaneously formed in the presence of acetic
anhydride (light blue and gray). Black, red, green, and orange lines
indicate the absorption spectra of the acetaminophen, EST−PtNP
complexes, acetic anhydride, and 4-nitrophenyl acetate, respectively.
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nanohybrid catalyst. The hydrogenated product, 4-amino-
phenol, subsequently reacted with acetic anhydride to form
the final product, N-(4-hydroxyphenyl) acetamide (acetamino-
phen) (Scheme 1-3). When EST−PtNPs were incubated with
4-nitrophenyl acetate, NaBH4, and acetic anhydride, the final
product, acetaminophen, was spontaneously formed (Scheme
1-4). All reactions were performed in a single vessel at room
temperature without any specialized equipment. The reaction
started in an aqueous phase, but subsequent hydrolysis by EST
led to the formation of acetic acid. Therefore, the aqueous
phase was converted to an acidic aqueous phase. However,
because it is known that acetylation can be carried out in the
presence of weakly acidic environments, the entire reaction can
be carried out without the use of any organic solvent.41 For the
same reasons, lyophilization was not necessary before
acetylation. The whole reaction process seemed to be
completed within 10 min because no spectral change was
observed after 10 min of reaction (Figure 6). The reaction
mixture was syringe-filtered and analyzed using liquid
chromatography−mass spectrometry (LC−MS) to confirm
the formation of acetaminophen (Figure 7). In positive mode
Figure 7. Mass spectrum of the filtered multistep catalysis reaction
mixture in ESI (+) mode. The peak at 151.9 m/z represents the peak
for acetaminophen.
of LC−MS a peak was obtained at 151.9, which corresponds to
the calculated value of acetaminophen (C8H9NO2 [M + H]+
152.1). By the application of high-performance liquid
chromatography (HPLC) to quantify the final product, the
conversion yield was confirmed to be 96% (Figures S6 and S7)
and the turnover frequency (TOF) was 4.44 × 10−4 s−1 (see the
Data Analysis section in the Supporting Information). We also
performed the acetaminophen synthesis reaction in the
presence of a mixture of EST and citrate-stabilized PtNPs
(Figure S8). However, when the yield of reaction performed by
the mixture of EST and the citrate-stabilized PtNPs was
calculated, it turned out to be 75%. This result suggests that the
EST−PtNP nanobiohybrid catalyst is highly efficient and
advantageous in terms of the reaction yield in comparison
with the individual catalysts. Taken together, these results
demonstrate that the EST−PtNP bioinorganic nanohybrid
catalyst can be used to perform multistep reactions for the
synthesis of acetaminophen in a one-pot reaction with high
yield and efficacy within a short time.
Gopinathan et al. have previously shown a chemical route to
synthesize acetaminophen with 86% yield from the reaction of
hydroquinone with acetamide at elevated temperatures (280−
300 °C) in the presence of solid acid catalysts such as zeolite β
or a heteropolyacid.42 More recently, Joncour et al. have
demonstrated a green chemical route for direct synthesis of
acetaminophen with high yield (95%) from hydroquinone
using ammonium acetate as an amidating agent, but the
DOI: 10.1021/acsami.6b12875
ACS Appl. Mater. Interfaces 2016, 8, 30058−30065
ACS Applied Materials & Interfaces
reaction utilized high temperatures (220 °C) and long reaction
times (15 h).43 In this reaction, high temperature was applied
for the dehydration of ammonium acetate. Moreover, a high-
temperature reaction would be advantageous because reaction
rate is generally enhanced at high temperature. In our approach,
we designed the multistep reactions at room temperature and
atmospheric pressure in consideration of that the reaction was
conducted by the proteins that can easily lose their folding and
activity at the elevated temperature. However, although the
reaction was conducted at room temperature, we would be able
to obtain products with a high yield within short time. Briefly,
compared to previous methods that had been used for the
synthesis of acetaminophen, the current approach has
advantages in terms of cost, time, and environment because
all the reactions were performed in an aqueous phase under
room temperature and atmospheric pressure within a short
time (10 min) without using expensive or specialized
equipment. These advantages can and will be translated into
significant cost cuts for the industry. Accordingly, this method
can possibly facilitate the production of pharmaceutically active
compounds in a place where investment for building a new
production line or access to equipment is restricted.
4. CONCLUSIONS
In summary, we developed a straightforward method to
synthesize EST−PtNP, a nanobiohybrid catalyst, by growing
PtNPs in the presence of an oligomeric enzyme. EST−PtNPs
showed enhanced hydrolytic and hydrogenation abilities while
overcoming the size and stability limitations faced by earlier
biohybrid catalysts. We also demonstrated that the current
EST−PtNP nanobiohybrid catalyst could be used for a one-pot
synthesis of acetaminophen in a cost-effective and environ-
mentally friendly manner. This offers significant advantages
compared to the currently available synthetic processes, and
thus this study could potentially offer a new way of exploiting
the advantages of the combination of organometallic chemistry
and biocatalysis for various applications.
■
ASSOCIATED CONTENT
*
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acsami.6b12875.
Additional data analysis of TOF, UV−vis, CD, HPLC,
and XPS results of EST−PtNP; TEM analysis of citrate−
PtNP; the cyclic hydrogenation reaction; and the
acetaminophen standard curve. Table of fitting results
for region XPS spectra of Pt, N, O, and C. (PDF)
■
AUTHOR INFORMATION
Corresponding Author
*E-mail: kyeongkyu@skku.edu. Tel: +82-31-299-6136. Fax: 82-
31-299-6159.
Author Contributions
§ SiNW-FETs by Integrating Protein-Shelled CdSe Quantum Dots.
B.H.S. and S.R. contributed equally to this work.
Notes
The authors declare no competing financial interest.
■
ACKNOWLEDGMENTS
This work was supported by the Next Generation BioGreen 21
Program (SSAC PJ0110705).
30064
Research Article
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30065 DOI: 10.1021/acsami.6b12875

ACS Appl. Mater. Interfaces 2016, 8, 30058−30065