Ultra-small Pd(0) nanoparticles into a designed semisynthetic lipase: An efficient and recyclable heterogeneous biohybrid catalyst for the heck reaction under mild conditions

Article abstract of DOI:10.3390/molecules23092358

A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus thermocatenulatus lipase (GTL) variant genetically and then chemically

Full text of DOI:10.3390/molecules23092358

molecules
Article
Ultra-Small Pd(0) Nanoparticles into a Designed
Semisynthetic Lipase: An Efficient and Recyclable
Heterogeneous Biohybrid Catalyst for the Heck
Reaction under Mild Conditions
David Lopez-Tejedor 1 , Blanca de las Rivas 2 and Jose M. Palomo 1,*
1
Department of Biocatalysis, Institute of Catalysis (CSIC), Marie Curie 2, Cantoblanco Campus UAM,
2
8049 Madrid, Spain; david.lopez@csic.es
Institute of Food Science, Technology and Nutrition (ICTAN-CSIC), José Antonio Novais 10,
8040 Madrid, Spain; blanca.r@csic.es
Correspondence: josempalomo@icp.csic.es; Tel.: +34-91-585-4768
2
2
*
ꢀ
ꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀ
ꢁꢂꢃꢄꢅꢆ
Received: 27 July 2018; Accepted: 13 September 2018; Published: 14 September 2018
Abstract: A novel heterogeneous enzyme-palladium (Pd) (0) nanoparticles (PdNPs) bionanohybrid
has been synthesized by an efficient, green, and straightforward methodology. A designed Geobacillus
thermocatenulatus lipase (GTL) variant genetically and then chemically modified by the introduction of
a tailor-made cysteine-containing complementary peptide- was used as the stabilizing and reducing
agent for the in situ formation of ultra-small PdNPs nanoparticles embedded on the protein structure.
This bionanohybrid was an excellent catalyst in the synthesis of trans-ethyl cinnamate by Heck
◦
reaction at 65 C. It showed the best catalytic performance in dimethylformamide (DMF) containing
1
0–25% of water as a solvent but was also able to catalyze the reaction in pure DMF or with a higher
amount of water as co-solvent. The recyclability and stability were excellent, maintaining more than
0% of catalytic activity after five cycles of use.
9
Keywords: heterogeneous catalyst; palladium nanoparticles; semisynthetic enzyme; lipase; Heck reaction
1
. Introduction
The Heck reaction is one of the most studied coupling reactions for its usefulness, allowing the
catalytic construction of C–C bonds under relatively mild conditions [1,2]. It has been widely used
for the synthesis of a huge spectrum of molecules, natural products, polymers, and pharmaceuticals
on both the laboratory and industrial scale. Some of the most notable products obtained using this
method are Naproxen, Montelukast (Singulair), octyl-4-methoxycinnamate (present on UVB sunscreen),
Electriptan or Leuconolam (Figure 1) [3,4].
Molecules 2018, 23, 2358; doi:10.3390/molecules23092358
www.mdpi.com/journal/molecules

Molecules 2018, 23, 2358
2 of 11
Figure 1. Pharmaceuticals synthesized by using Heck cross-coupling reaction.
Several metals and a huge range of ligands have been studied as catalysts for the reaction, with
palladium (Pd) the preferred metal due to its tolerance to a wide variety of functional groups and ability
to form C–C bonds between functionalized substrates, proceeding with stereo and regioselectivity and,
usually, with good to excellent yields [2]. Investigation towards palladium-catalyzed Heck reaction
has been focused on greener approaches to the reaction, performing it in aqueous media, and being
able to recover and reuse the catalyst, which is quite important for industrial purposes [5–8].
In particular, the application of Pd nanoparticles as catalysts presents important advantages
compared to other Pd molecules due to the high surface-to-volume ratio and very active surface
atoms [9,10].
As described for other types of Pd complexes, optimization in the synthetic conditions to avoid
hard or extreme conditions in the synthesis of Pd nanoparticles is greatly desirable. The application
of biomolecules to induce the synthesis of these nanoparticles has been recently described [11].
The application of carbohydrates, peptides, polymers or microorganisms has been successfully
applied [6–8,11–14]. However, in some cases, the generation of large size nanoparticles, not well
dispersed or even a mixture in the nanoparticle morphology by using these biomolecules, have
made the application of enzymes as a biological entity the best alternative to the ones mentioned
before [11,15].
However, for possible industrial implementation of this type of catalyst, enzyme production is a
key point. Although this can be obtained from commercial suppliers, it is important to have a good
and consistent production source to obtain enough enzyme to be translated into high volumes of the
final nanobiomaterials. In addition, enzymes with good stability might be interesting for application
on reactions in harsh conditions, high temperatures or in the presence of organic solvents.
In this work, we have synthesized a novel palladium (Pd) (0) nanoparticle (PdNPs)-enzyme
nanobiohybrid using a tailor-made designed enzyme to induce the formation of ultra-small Pd NPs
into the protein network.
Thermoalkalophilic lipase from Geobacillus thermocatenulatus (GTL) was genetically modified by
introducing an unique cysteine (Cys) in a particular position and was overexpressed in Eschericia coli.
The recombinant enzyme was then modified site-specifically by using a cysteine-containing peptide via
disulfide exchange chemistry. This novel bionanohybrid was characterized by transmission electron
microscopy (TEM) and X-ray diffraction (XRD) to confirm the existence of zero valent Pd and the final
size and morphology of the nanoparticles.

Molecules 2018, 23, 2358
3 of 11
The potential synthetic application of this Pd-enzyme bionanohybrid as nanocatalyst was
evaluated in Heck Cross-Coupling reaction under different conditions. Stability and reusability
of the heterogeneous catalyst were also evaluated.
2
. Results and Discussion
2
.1. Synthesis and Characterization of the Bionanohybrid
First, a tailor-made enzyme was created following the protocol previously described [16,17]. Lipase
from Geobacillus thermocatenulatus (GTL) was genetically modified by site-directed mutagenesis, changing
Ala193 to Cys (Figure 2), without alteration in the properties of the enzyme. This GTLσ-A193C variant
was overexpressed in Escherichia coli and purified, which permitted to obtain a high amount of protein.
Then, this new enzyme solution was combined with palladium acetate to create a new kind of
bionanohybrid; nanocatalysts formed by in situ synthesized palladium nanoparticles in the protein
network [18]. The formation of the bionanohybrid can be easily recognized by the increase in turbidity
of the solution, which is precipitated after centrifugation.
Figure 2. Preparation of the Geobacillus thermocatenulatus (GTL)σ-A193Cp- palladium (0) nanoparticles
(PdNPs) bionanohybrid.
In this case, no aggregate was obtained when the GTLσ-A193C and palladium salt mixture was
incubated for 24 h at room temperature, with the solution remaining completely transparent. This
result indicated that no bionanohybrid could be formed when using this genetically modified protein.
Therefore, this GTLσ-A193C was site-specific chemically modified at the cysteine by a
Cys-containing peptide (Ac-Cys(PheGly) PheCONH ) (Figure 2). First, the cysteine on the protein
2
2
(
C193) was protected with 2-dipyridiyldisulfide before the reaction. Then the peptide was conjugated
to the Cys193 by a disulfide bond exchange. This modification generated a new semisynthetic GTL
lipase (GTL -A193Cp) with improved properties and specifically increased the hydrophobicity of the
σ
final protein structure [16].
Thus, the protocol for the bionanohybrid synthesis was applied using this new enzyme
GTLσ-A193Cp. In this case, the solution turned cloudy in the first hour of incubation and finally
aggregate was obtained after 24 h (Figure 2). The solid was recovered by centrifugation, washed and
lyophilized. X-ray diffraction (XRD) analysis showed that zero-valent Pd was the only species present
in the bionanohybrid (Figure 3).

Molecules 2018, 23, 2358
4 of 11
Figure 3. X-ray diffraction (XRD) pattern of GTLσ-A193Cp-PdNPs bionanohybrid.
The content of palladium in the solid was 40 wt.% measured by inductively coupled plasma
atomic emission spectrometry (ICP-OES). More than 99% of the enzyme offered was remaining in the
final bionanohybrid confirmed by Bradford assay [19]. TEM analysis showed that ultra-small spherical
palladium nanoparticles (PdNPs) were synthesized into the protein network of GTL
size distribution of 2 to 4 nm, but mainly with a size around 3 nm (Figure 4).
σ-A193Cp with a
Figure 4. Characterization of GTLσ-A193Cp-PdNPs bionanohybrid. (a–c) transmission electron
microscopy (TEM) and high resolution TEM (HRTEM) micrographs. (d) Pd NPs size distribution.

Molecules 2018, 23, 2358
5 of 11
The proposed mechanism for the formation of this enzyme-metal nanobiohybrid consists in two
2+
different steps, (i) The ionic binding of the Pd ions with different aminoacid residues of the protein,
and (ii) the nucleation, reduction of Pd2 to Pd(0) with the final growth of the nanoparticle. One of the
important points in this mechanism is the role of aminoacids and small peptide sequences into the
protein involved during the nanobiohybrid formation. The phenomenon that peptides sequences can
be reduced metal ions has been well studied using tailor-made synthetic small peptides. Therefore,
the protein in question must contain particular peptides sequences, the so-called ideal peptides, which
has to contain amino acids with moderate binding affinity for both metal ions and formed metal
particles (i.e., amino acids presenting hydrophobic or charged side chains (with the charge sign
opposite to that showed by the metal ions)) together with neighboring amino acids showing a strong
reducing ability (i.e., especially amino acids presenting hydrophobic side chains, or particularly alcohol
residues such as serines, threonines or tyrosines) [18].
+
Thus, adding the metal salt to the enzyme solutions, due to the binding ability of the
identified sequences, rapid adsorption of soluble Pd ions on the enzyme surface was observed.
Furthermore, the adsorbed metal ions act as a cross-linker between the enzyme’s molecules, reducing
their solubility and finally inducing the initial fast precipitation. When the metal ions are adsorbed,
the amino acids in protein surrounding them caused the nucleation.
The structural analysis of the chemically modified variant demonstrated that the site-specific
location of the hydrophobic peptide (Figure 5) was critical, considering the necessity of hydrophilic
groups together to hydrophobic ones for the reducing step. Serine, threonine, tyrosine or even
methionine were surrounded by the peptide (Figure 5). These groups were able to reduce the adsorbed
2+
Pd to Pd(0), which is mandatory for the nucleation process [18].
In contrast, the native variant without the peptide also presented a hydrophobic area on the
lid side (Figure S1, see Supplementary Materials), and although it could be possible from the initial
2+
adsorption of Pd , no adequate hydrophilic groups were surrounding that position which could
demonstrate why the native variant was unable to form the bionanohybrid.
Figure 5. Crystal structure of the active form of the artificial enzyme GTLσ-A193Cp. Cartoon of the
peptide sequence and the selected alcoholic protein residues. A193C-catalytic Ser114 is marked in
yellow, lid in blue, peptide in red and the different Ser, Thr, Tyr or Met residues in green. All pictures
were created by using Pymol.

Molecules 2018, 23, 2358
6 of 11
2
.2. Heck Reaction
Thus, this new GTL
σ
-A193Cp-PdNPs bionanohybrid was tested as a heterogeneous catalyst in
Heck reaction using iodobenzene (1) and ethyl acrylate (2) as substrates (Scheme 1). The reaction was
performed at moderate conditions, in an aqueous solution, containing 75% (v/v) dimethylformamide
◦
(
DMF) and 65 C.
Scheme 1. C–C Heck reaction catalyzed by bionanohybrid.
The C–C reaction was evaluated using different bases in the reaction (Table 1). The application
of dimethylaminopyridine (DMAP) as the base resulted in no conversion (Table 1, entry 1).
The nanocatalyst exhibited around 20% conversion after 24 h when the combination of DMAP and
triethylamine was used (Table 1, entry 2). The best result was obtained using triethylamine, where 78%
−
1
of ethyl cinnamate (3) was obtained with a TOF value of 2.96 h , more than three times compared
with the use of DMAP and with a complete reaction (>99%) at 48 h (Table 1, entry 3). In addition,
diisopropylethylamine (DIPEA) was evaluated, although the achieved conversion was slightly lower
−
1
after 24 h compared to the one with triethylamine (Table 1) with a TOF value of 2.32 h . In all cases
where a reaction was obtained, a high selectivity was achieved, producing the trans-isomer exclusively.
Table 1. Heck coupling of iodobenzene with ethyl acrylate catalyzed by GTL
σ
-A193Cp-PdNPs
bionanohybrid [a]
.
Conversion of 3
TOF
Selectivity
trans/cis (%)
Entry
Base
TON
−
1
[
%]
(h
)
1
DMAP
DMAP/NEt3
0
-
18.27
71.2(91.33)
55.7
-
-
2
3
4
5
6
7
20
78(>99) [
61
0.76
2.96 (1.90)
2.32
>99
>99
>99
>99
>99
>99
b]
NEt3
DIPEA
NEt3
NEt3
NEt3
[
c]
44
40.2
6.4
15.52
1.67
0.27
0.64
[
d]
7
[
e]
17
[
a]
Conditions:
1
(0.274 mmol),
2
(0.55 mmol), 1 mL DMF/H
2
O (75/25), 1.5 equiv base, 1 mg of nanocatalyst, 24 h,
◦
[b]
[c]
[d]
[e]
6
5 C. after 48 h. 5 equiv base.
10 equiv base. 10 equiv 2.
The increase in the amount of triethylamine in the reaction catalyzed by GTL
bionanohybrid was studied. This addition caused a slight negative effect in the final conversion value,
with 5 eq obtaining 41% of , but especially after 10 eq, where only 7% of was achieved after 24 h
Table 1, entries 5 and 6), a TOF value of 0.27 h , 15 times lower compared when 1.5 equivalent was
σ-A193Cp-PdNPs
3
3
−
1
(
used. The increase of 2 also showed a negative effect on the final conversion value (Table 1, entry 7).
For a possible industrial application, the recyclability of the heterogeneous nanocatalyst was
studied. The GTL
σ
-A193Cp-PdNPs bionanohybrid was used in at least five cycles of Heck reaction at
◦
2
5% water in DMF at 65 C. The nanocatalysts kept 90–95% of the initial activity while also maintaining
a complete trans selectivity (Figure 6).

Molecules 2018, 23, 2358
7 of 11
Figure 6. Recyclability of GTL
σ-A193Cp-PdNPs bionanohybrid in the Heck reaction. Conditions:
◦
1
(0.274 mmol), 2 (0.55 mmol), 1 mL DMF/H O (75/25), 1.5 equiv NEt , 1 mg of nanocatalyst, 65 C.
2
3
To evaluate the capacity of this nanocatalyst, the Heck reaction was performed changing the
volume of water in the reaction medium. The reduction in the volume of water in the medium was
first attempted. Surprisingly, the bionanohybrid catalyzed the reaction faster when only 10% water
−
1
was used, obtaining 86% conversion of 3 after 24 h, with a TOF value of 3.27 h (Table 2, entry 2).
Table 2. Heck coupling of iodobenzene with ethyl acrylate catalyzed by GTLσ-A193Cp-PdNPs
bionanohybrid as catalyst at different aqueous content. [a]
Co-Solvent
T
[ C]
Conversion
of 3 [%]
TOF
Selectivity
trans/cis (%)
Entry
TON
Reference
◦
−1
[
% v/v, H O]
(h
)
2
1
2
3
4
5
7
0
65
65
65
65
70
70
56
86
78
50
0
51.15
78.54
71.23
45.65
-
2.13
>99
>99
>99
>99
-
This work
This work
This work
This work
[18]
10
25
40
0
3.27
2.96
1.90
-
50
20
18.27
0.76
>99
[18]
[a]
◦
Conditions: 1 (0.274 mmol), 2 (0.55 mmol), 1 mL DMF, 1.5 equiv NEt3, 1 mg of nanocatalysts, 24 h, 65 C.
A very interesting result was also obtained when the reaction was performed in pure DMF as the
solvent. The GTLσ-A193Cp-PdNPs bionanohybrid catalyzed the Heck reaction with 56% conversion
−
1
after 24 h, TOF value of 2.13 h , with exclusively trans selectivity (Table 2, entry 1). This differed
from the results previously obtained using bionanohybrid synthesized using other lipase where no
conversion was achieved even at higher temperatures (Table 2, entry 5). This phenomenon could
demonstrate that the application of a highly stable protein variant of GTL allows broader applicability
of the final Pd NPs biohybrid in synthetic chemistry.
This argument was also corroborated when higher volumes of water were added to DMF as the
solvent. This bionanohybrid also catalyzed the Heck reaction in the presence of 40% of water in DMF
◦
−1
at 65 C, with a TOF value of 1.90 h (Table 2, entry 4), better than the previous comparison (Table 2,
entry 7). In addition, in all cases, the trans-selectivity was conserved.
To demonstrate the broad scope of applicability of this new bionanohybrid, Suzuki cross-coupling
reaction between bromobenzene and phenylboronic acid in aqueous media at mild conditions reaction
in pure water was attempted (Table S1, see Supplementary Materials). The GTL
σ
-A193Cp-PdNPs
◦
showed good performance producing 70% conversion of biphenyl in 24 h at 50 C in aqueous media,
with a TOF value of 0.43 (Table S1). Furthermore, Sonogashira reaction between propargyl alcohol and
iodoanisole was performed (Table S2).

Molecules 2018, 23, 2358
8 of 11
3
. Materials and Methods
3
.1. General
Butyl-Sepharose® 4 Fast Flow from GE Healthcare (Uppsala, Sweden), 2-dipyridyldisulfide
(
(
(
2-PDS), dithiothreitol (DTT), sodium borohydride, dimethylsulfoxide (DMSO), dimethylformamide
DMF), dimethylaminopyridine (DMAP), diisopropylethylamine (DIPEA), p-nitrophenylpropionate
pNPP), Pd(OAc) , ethyl-acrylate, iodobenzene, cis and trans ethyl cinnamate, and triethylamine were
2
from Sigma (St. Louis, MO, USA). Sucrose monolaurate was from Mitsubishi-Kagaku (Tokyo, Japan).
AcN-Cys(SH)-Phe-Gly-Phe-Gly-Phe-CONH was purchased from Isogen (De Meern, Netherlands).
2
Inductively coupled plasma atomic emission spectrometry (ICP-OES) of the acidic digestion of the
solid powder of bionanohybrid was performed on a Perkin Elmer OPTIMA 2100 DV equipment.
The transmission electron microscopy (TEM), high resolution TEM microscopy (HRTEM) were
performed on a JEOL 2100F microscope equipped with an EDX detector INCA x-sight (Oxford
Instruments, Tokyo, Japan). The X-ray diffraction (XRD) pattern was obtained using a Texture Analysis
Diffractometer D8 Advance (Bruker) with Cu Kα radiation.
3
.2. Site-Directed Mutagenesis, Cloning, and Expression of Geobacillus Thermocatenulatus Lipase (GTL)
All site-directed mutagenesis experiments were carried out by polymerase chain reaction (PCR)
using mutagenic primers. To introduce the amino acid change, the corresponding pair of primers
was used as a homologous primer pair in a PCR reaction using a specific plasmid as template and
Prime Start HS Takara DNA polymerase. The product of the PCR was digested with endonuclease
DpnI that exclusively restricts methylated DNA [20]. E. coli DH10B cells were transformed directly
with the digested product. The plasmid with mutated GTL was identified by sequencing and then
transformed into E. coli BL21 (DE3) cells to express the corresponding proteins. First, C65S was created,
and the resulting plasmid was used as a template to create the double mutant C65S/C296S-GTL
(GTLσ). This plasmid (pT1BGTLmutCys) was used as a template to construct the additional mutation
(
1
A193C), creating the mutant GTL
93-5,5 -GAAAGCGtgcGCTGTCGCCAG; Ala/cys 193-3 5 -CTGGCGACAGCg caCGCTTTC). The gene
σ
-A193C using different mutagenic primers: A193C (Ala/cys
0
0
corresponding to the mature lipase from G. thermocatenulatus (GTL) was cloned into a pT1 expression
vector as previously described in Reference [20]. Cells carrying the recombinant plasmid pT1GTL were
◦
◦
grown at 30 C and over expression was induced by raising the temperature to 42 C for 20 h.
3
.3. Purification of GTLσ-A193C
The crude extract from E. coli containing the GTL variant was diluted with 10 mM sodium
phosphate at pH 7.0 to a concentration of 0.5 mg/mL. Then, butyl-Sepharose was added in a
◦
1
/20 (v/v) proportion and gently stirred overnight at 25 C. Periodically, the activity of suspensions
and supernatants were measured by the pNPP assay described above. After that, the adsorbed lipase
preparation was abundantly washed with distilled water. Purification overall yield was 80% confirmed
by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis and a decrease in
the supernatant activity.
3
.4. Irreversible Site-Specific Chemical Modification of GTLσ-A193C by Thiol-Containing Peptides
GTL -A193C adsorbed on butyl-Sepharose (0.2 g) was incubated in 2 mL of DTT solution (50 mM
σ
in 25 mM sodium phosphate buffer) at pH 8 for 30 min, to avoid oxidation and permit the posterior
disulfide exchange. After, the reduced biocatalyst was washed with distilled water until the DTT
smell disappears. Then 0.2 g of the reduced biocatalyst was added to 3 mL of 2-PDS solution (1.5 mM
substrate in a mixture of DMSO (5% v/v) in 25mM phosphate buffer) at pH 8.0 for 1 h, and then
the suspension was abundantly washed with distilled water. The full cysteine PDS activation was
confirmed by spectrophotometric assay. Then, 1 mL of AcN-Cys(SH)-Phe-Gly-Phe-Gly-Phe-CONH2
(p) solution (0.2 mg/mL) was added to the 2 mL of PDS-GTL variant for 1 h. The peptide (p) was

Molecules 2018, 23, 2358
9 of 11
previously treated with a solution of sodium borohydride (1 mg/mL) in 250 mM phosphate at pH
0.0 for 30 min (to conserve the thiol group in a reductive form), and then pH was adjusted to 8.0 with
1
diluted HCl solution to destroy the remaining sodium borohydride.
After that, the chemically modified GTL variant was incubated in 2 mL of 1.25% (v/v) sucrose
monolaureate in 250 mM phosphate at pH 8.0 for 1 h, and the full desorption of the modified
GTL
σ-A193Cp variant was confirmed by activity assay and SDS electrophoresis. This solution had an
approximate protein concentration of 0.9 mg/mL.
3
.5. Enzymatic Activity of the Artificial GTL Variants on Hydrolysis of p-Nitrophenyl Propionate (pNPP)
The activity of the GTL variants was analyzed spectrophotometrically measuring the increment
−
1
−1
in absorbance at 348 nm produced by the release of p-nitrophenol (pNP) (
є
= 5150 M cm ) in the
◦
hydrolysis of 0.4 mM pNPP in 25 mM sodium phosphate at pH 7 and 25 C. To initialize the reaction,
0
.05–0.2 mL of lipase solution or suspension was added to 2.5 mL of substrate solution.
3
.6. Synthesis of GTLσ-A193Cp-PdNPs Bionanohybrid
Pd(OAc) (5mg) was dissolved in 1 mL of DMF and then added to 4 mL of GTLσ-A193Cp solution
2
(
the previously desorbed enzyme solution was diluted 1:1 with distilled water, obtaining 0.45 mg/mL
◦
solution) under vigorous magnetic stirring at 25 C. The amount of solvent (20% v/v) was needed to
ensure the full dissolution of the palladium salt in aqueous media. The solution was kept on gentle
magnetic stirring for 24 h at room temperature. The resulting final suspension was separated by
◦
centrifugation (8000 rpm; 4 C; 35 min), and the recovered pellet was washed two times with a 20%
v/v DMF/distilled water solution for the removal of the remainders of palladium salt, and two more
times with 5 mL of distilled water. After the last washing, the suspension was lyophilized to obtain
the solid for characterization and use in the reaction.
3
.7. General Procedure for Heck Reaction
The reaction mixture was prepared in a screw-sealed glass vessel, containing 1 mL of solvent
solution (either 1 mL of pure DMF or DMF/water), 0.274 mmol (30
µ
L) of iodobenzene and 0.55 mmol
◦
(
59
µL) of ethylacrylate. This solution was preheated at 65 C for 5 min under magnetic stirring for the
complete solubilization of the substrates; then, 1 mg of palladium catalyst was added to the mixture
(
containing 0.4 mg of Pd) as well as 0.412 mmol of a base (either triethylamine, DMAP or DIPEA) and
◦
was kept under vigorous magnetic stirring at 65 C for the indicated times. Samples (80
µL) were taken
at different times, and the reaction was followed by HPLC. Samples were first centrifuged at 8000 rpm
for 5 min, and then 10 L was diluted 1000 times in a solution of acetonitrile:water 1:1. The reaction
outgoing was monitored by HPLC analysis of the reaction samples at different times. The analysis
conditions were performed with a Kromasil-C8 (150 4.6 mm and 5 m Ø), at a flow of 1.0 mL/min;
: 254 nm; and a mobile phase: 50% (v/v) ACN in MilliQ water. In these conditions, the retention
µ
×
µ
λ
times were: 4.18 min for ethyl acrylate (2), 11.1 min for (E)-trans ethyl cinnamate (3) and 13.25 min
for iodobenzene (
1). The E configuration was confirmed by HPLC using the (E) and (Z)-3 standards.
Retention time for the (Z)-3 was 10.35 min (see Supplementary Materials).
3
.8. Recyclability Test
The protocol for Heck reaction mentioned previously was followed, using triethylamine as a
base and 75% (v/v) DMF in water as a solvent. After each cycle (stopping the reaction around 50%
conversion), the reaction solution was centrifuged, and the nanobiohybrid was washed two times with
DMF and left to dry for 15 min in a fume hood. This recyclability test was performed for five cycles.

Molecules 2018, 23, 2358
10 of 11
4
. Conclusions
Herein we have demonstrated the applicability of a new kind of Pd bionanohybrid in the
Heck reaction.
A recombinant lipase from Geobacillus thermocatenulatus was selected, which was genetically
modified by introducing a cysteine at position 193 and then this cysteine modified with a tailor-made
peptide. This designed semisynthetic lipase was applied successfully to produce a heterogeneous
bionanohybrid formed by ultra-small Pd nanoparticles embedded on the protein network.
The applicability of a recombinant protein to produce the hybrid would permit to the accessibility of
reproducible and multi-milligram scaled bionanohybrid. The use of a thermoalkalophilic enzyme in
the formation of the nanobiohybrid is an advantage, which was demonstrated by this new hybrid
◦
catalyzing the Heck reaction in pure DMF at 65 C, in comparison with other bionanohybrid using
another lipase where no conversion was observed at these conditions [18]. The bionanohybrid was
excellent using 10% water (v/v) as co-solvent in DMF and was quite stable being recyclable without
losing the activity or trans-selectivity.
Therefore, these results demonstrate that this could be an excellent candidate for application in
organic synthesis, even in harsh conditions.
Supplementary Materials: The following are available online: Figure S1: Crystal structure of GTL native and
modified together with the lid area marked key amino acids, Table S1: Suzuki-Miyaura cross coupling of
bromobenzene (BB) with phenylboronic acid (PBA) catalyzed by bionanohybrid, Table S2: Suzuki-Miyaura cross
coupling of bromobenzene (BB) with phenylboronic acid (PBA) catalyzed by bionanohybrid.
Author Contributions: D.L.-T. and B.d.l.R. performed the experiments; J.M.P. designed and supervised the study
and experiments, and J.M.P. and D.L.-T. wrote the manuscript.
Funding: This work has been sponsored by the Spanish Government (Project AGL2017-84614-C2-2-R) and CSIC
Project CSIC-PIE 201880E011).
Acknowledgments: The authors thank the support by the Spanish National Research Council (CSIC).
Conflicts of Interest: The authors declare no conflicts of interest.
References
1
.
Beletskaya, I.P.; Cheprakov, A.V. Heck reaction as a sharpening stone of palladium catalysis. Chem. Rev.
000, 100, 3009–3066. [CrossRef] [PubMed]
2
2
3
.
.
Jagtap, S. Heck Reaction—State of the Art. Catalysts 2017, 7, 267. [CrossRef]
Magano, J.; Dunetz, J.R. Large-scale applications of transition metal-catalyzed couplings for the synthesis of
pharmaceuticals. Chem. Rev. 2011, 111, 2177–2250. [CrossRef] [PubMed]
4
5
.
.
De Vries, J.G. The Heck reaction in the production of fine chemicals. Can. J. Chem. 2001, 79, 1086–1092.
[CrossRef]
Evangelisti, C.; Panziera, N.; Pertici, P.; Vitulli, G.; Salvadori, P.; Battocchio, C.; Polzonetti, G. Palladium
nanoparticles supported on polyvinylpyridine: Catalytic activity in Heck-type reactions and XPS structural
studies. J. Catal. 2009, 262, 287–293. [CrossRef]
6
7
.
.
Zhu, J.; Zhou, J.; Zhao, T.; Zhou, X.; Chen, D.; Yuan, W. Carbon nanofiber-supported palladium nanoparticles
as potential recyclable catalysts for the Heck reaction. Appl. Catal. A-Gen. 2009, 352, 243–250. [CrossRef]
Firouzabadi, H.; Iranpoor, N.; Gholinejad, M.; Akbari, S.; Jeddi, N. Palladium nanoparticles supported on
agarose-functionalized magnetic nanoparticles of Fe O as a recyclable catalyst for C–C bond formation
3
4
via Suzuki–Miyaura, Heck–Mizoroki and Sonogashira–Hagihara coupling reactions. RSC Adv. 2014, 4,
7060–17070. [CrossRef]
1
8
.
.
Firouzabadi, H.; Iranpoor, N.; Ghaderi, A. Solvent-free Mizoroki-Heck reaction catalyzed by palladium
nano-particles deposited on gelatin as the reductant, ligand and the non-toxic and degradable natural
product support. J. Mol. Catal. A Chem. 2011, 347, 38–45. [CrossRef]
Lamblin, M.; Nassar-Hardy, L.; Hierso, J.C.; Fouquet, E.; Felpin, F.X. Recyclable heterogeneous palladium
catalysts in pure water: Sustainable developments in Suzuki, Heck, Sonogashira and Tsuji-Trost reactions.
Adv. Synth. Catal. 2010, 352, 33–79. [CrossRef]
9

Molecules 2018, 23, 2358
11 of 11
1
0. Khalafi-Nezhad, A.; Panahi, F. Immobilized palladium nanoparticles on a silica-starch substrate (PNP–SSS):
As an efficient heterogeneous catalyst for Heck and copper-free Sonogashira reactions in water. Green Chem.
011, 13, 2408. [CrossRef]
2
1
1. Palomo, J.M.; Filice, M. Biosynthesis of Metal Nanoparticles: Novel Efficient Heterogeneous Nanocatalysts.
Nanomaterials 2016, 6, 84. [CrossRef] [PubMed]
1
2. Ghorbani-Choghamarani, A.; Taherinia, Z. Synthesis of peptide nanofibers decorated with palladium
nanoparticles and its application as an efficient catalyst for the synthesis of sulfides via reaction of aryl
halides with thiourea or 2-mercaptobenzothiazole. RSC Adv. 2016, 6, 59410–59421. [CrossRef]
3. Ghorbani-Choghamarani, A.; Taherinia, Z. Synthesis of biaryls using palladium nanoparticles immobilized
on peptide nanofibers as catalyst and hydroxybenzotriazole as novel phenylating reagent. Chin. J. Catal.
1
2
017, 38, 469–474. [CrossRef]
1
4. Jomma, E.Y.; Ding, S.N. One-Pot hydrothermal synthesis of magnetite prussian blue Nano-Composites and
their application to fabricate glucose biosensor. Sensors 2016, 16, 243. [CrossRef] [PubMed]
5. Prastaro, A.; Ceci, P.; Chiancone, E.; Boffi, A.; Cirilli, R.; Colone, M.; Fabrizi, G.; Stringaro, A.; Cacchi, S.
Suzuki-Miyaura cross-coupling catalyzed by protein-stabilized palladium nanoparticles under aerobic
conditions in water: Application to a one-pot chemoenzymatic enantioselective synthesis of chiral biaryl
alcohols. Green Chem. 2009, 11, 1929. [CrossRef]
1
1
1
1
6. Romero, O.; De las Rivas, B.; Lopez-Tejedor, D.; Palomo, J.M. Effect of Site-Specific Peptide-Tag Labeling on
the Biocatalytic Properties of Thermoalkalophilic Lipase from Geobacillus thermocatenulatus. ChemBioChem
2
018, 19, 369–378. [CrossRef] [PubMed]
7. Romero, O.; Filice, M.; De las Rivas, B.; Carrasco-Lopez, C.; Klett, J.; Morreale, A.; Hermoso, J.A.; Guisan, J.M.;
Abian, O.; Palomo, J.M. Semisynthetic peptide-lipase conjugates for improved biotransformations.
Chem. Commun. 2012, 48, 9053–9055. [CrossRef] [PubMed]
8. Filice, M.; Marciello, M.; del Puerto Morales, M.; Palomo, J.M. Synthesis of heterogeneous enzyme–metal
nanoparticle biohybrids in aqueous media and their applications in C–C bond formation and tandem
catalysis. Chem. Commun. 2013, 49, 6876. [CrossRef] [PubMed]
1
2
9. Bradford, M.M. A rapid and sensitive method for the quantitation of microgram quantities of protein
utilizing the principle of protein-dye binding. Anal. Biochem. 1976, 72, 248–254. [CrossRef]
0. Schmidt-Dannert, C.; Rúa, M.L.; Atomi, H.; Schmid, R.D. Thermoalkalophilic lipase of Bacillus thermocatenulatus.
I. Molecular cloning, nucleotide sequence, purification and some properties. Biochim. Biophys. Acta Lipids Lipid
Met. 1996, 1301, 105–114. [CrossRef]
Sample Availability: Samples of the compounds are not available from the authors.
©
2018 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).