Synthesis of heterogeneous enzyme-metal nanoparticle biohybrids in aqueous media and their applications in C-C bond formation and tandem catalysis

Article abstract of DOI:10.1039/c3cc42475h

The straightforward synthesis of novel enzyme-metalNP nanobiohybrids in aqueous medium was developed. These new nanobiohybrids were excellent multivalent catalysts combining both activities in various sets of synthetic reactions even at ultra-low concentrations (ppb amount).

Full text of DOI:10.1039/c3cc42475h

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COMMUNICATION
Synthesis of heterogeneous enzyme–metal nanoparticle
biohybrids in aqueous media and their applications in
C–C bond formation and tandem catalysis†
Cite this: Chem. Commun., 2013,
4
9, 6876
Received 5th April 2013,
Accepted 10th June 2013
a
b
b
a
Marco Filice,* Marzia Marciello, Maria del Puerto Morales and Jose M. Palomo*
DOI: 10.1039/c3cc42475h
www.rsc.org/chemcomm
The straightforward synthesis of novel enzyme–metalNP nanobiohybrids nanoparticle formation, (ii) a stabilizing and supporting agent (avoiding
in aqueous medium was developed. These new nanobiohybrids were nanoparticle aggregation) and (iii) a biocatalyst, all at the same time.
excellent multivalent catalysts combining both activities in various sets of These new nanobiohybrids were successfully applied as heterogeneous
synthetic reactions even at ultra-low concentrations (ppb amount).
catalysts in a set of different interesting reactions exploiting selectively
the catalytic activity of the metal, the enzyme or both at the same time
Nanocatalysts have undergone explosive growth during the past (domino and tandem reactions). Furthermore, these new catalysts were
1,2
decade, both in homogeneous and heterogeneous forms. The large recycled several times keeping their catalytic properties intact. Initially,
surface-to-volume ratio of nanomaterials compared to bulk materials the synthesis of the nanobiohybrids in aqueous medium was
3
makes them attractive candidates for their application as catalysts.
attempted by adding the commercial liquid Candida antarctica B lipase
For example, palladium nanoparticles (PdNPs) have played a pivotal (CAL-B) to an aqueous solution of fully water soluble Na PdCl salt at
2
4
4
role in a wide variety of C–C coupling reactions. Generally, the most room temperature and under gentle stirring. After 24 h, the initial clear
useful approaches for synthesis of nanoparticles usually employ solution turned into a slight cloudy suspension where only 10% of
harsh conditions. Therefore, biological methods could represent a the enzyme disappeared (by the Bradford method ) forming a hetero-
green alternative. For example, RNA sequences or polypeptides were geneous composite (Table 1 and Table S1, ESI†).
discovered to aid the formation of nanoparticles by using a reducing
5
10
6–7
agent (typically ascorbic acid or sodium borohydride). Amongst all
the biomacromolecules, enzymes are natural proteins with nanodi-
mensions (e.g., lipase from Candida antarctica B; 3 nm  4 nm Â
5
nm) that can catalyse a wide set of reactions with exquisite control of
regio- and stereochemistry. They have also been involved as reactants
8
9
in the synthesis of metal nanoparticles and hybrid nanostructures.
Therefore, considering the continuous demand for the development
of new catalysts with high-efficiency and a broad reaction scope, the
creation of heterogeneous hybrid nanocatalysts with an overall catalytic
Scheme 1 Preparation strategy for enzyme–metalNP biohybrids.
Using Pd(OAc) in the presence of different co-solvents, a
2
activity resulting from the combination of two different but comple- quantitative precipitation of the protein was observed after 24 h
mentary catalytic activities (noble metal NPs together with enzymes) (Table 1). An ICP-AES analysis of the supernatants revealed that
2+
could represent a straightforward breakthrough in this field.
6.45–7.57 mmol of Pd were entrapped into the protein. CALB–
Here we describe the synthesis of novel enzyme–metalNP nanobio- PdNP-(2–7) biohybrids were recovered by centrifugation, washed
hybrids, where NPs were generated in situ from an aqueous noble metal with distilled water and lyophilized.
salt solution (Scheme 1). The enzyme acted as a (i) reducing agent for
TGA of the CALB–PdNPs-2 lyophilized powder (Fig. S1, ESI†)
showed that 26% (w/w) of the amount of Pd comprised the solid
material, confirming the value previously obtained using ICP-AES.
As an initial representative example, the CALB–PdNPs-2
nanobiohybrid was fully characterized using SEM, EDX, XRD,
XPS and TEM (Fig. 1 and Fig. S2, ESI†). The EDX experiment (Fig. 1) as
well as SEM analysis revealed that the formed precipitate was
composed of an aggregate with a mesoporous amorphous suprastruc-
ture composed of palladium atoms dispersed into an organic matrix
(CAL-B) (Fig. S2a, ESI†). TEM analysis together with XRD and XPS
a
Departamento de Biocat a´ lisis, Instituto de Cat a´ lisis (ICP-CSIC), Marie Curie 2,
Cantoblanco, Campus UAM, 28049 Madrid, Spain. E-mail: marcof@icp.csic.es,
josempalomo@icp.csic.es
b
Departamento de Biomateriales y Materiales Bioinspirados, Instituto de Ciencia de
Materiales, (ICMM-CSIC), Sor Juana In ´e s de la Cruz 3, Campus UAM,
2
8049 Madrid, Spain
†
Electronic supplementary information (ESI) available: Experimental section,
characterization data of the different nanobiohybrids, recycling studies and
additional tables and figures. See DOI: 10.1039/c3cc42475h
6
876 Chem. Commun., 2013, 49, 6876--6878
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Table 1 Synthesis of enzyme–Pd nanobiohybrids
absence of any exogenous reducing agents, finally generating the
11–12
c
NPs.
FTIR experiments together with the pH-dependent zeta
Metal salt
Protein b
Metal amount
a
À1
potential measurement of native CAL-B and the CALB–PdNPs-2,
supported this idea (Fig. S5, ESI†) (For a more detailed discussion on
the nanobiohybrid formation mechanism, see ESI†).
Catalyst
Co-solvent (1 mg mL ) amount (%) (mmol)
CALB–PdNPs-1 —
Na
2
PdCl
4
10
99
99
99
99
nd
6.45
7
6.68
7.12
CALB–PdNPs-2 DMF
CALB–PdNPs-4 ACN
CALB–PdNPs-5 MeOH
CALB–PdNPs-6 THF
Pd(OAc)
Pd(OAc)
Pd(OAc)
Pd(OAc)
2
2
2
2
The synthesized metal NPs were very stable in aqueous solution
without any changes in particle size and morphology for three
months (data not shown) providing further evidence that the
enzyme network acts not only as a physical support and reducing
agent during the NP synthesis but also serves as a stabilizing agent.
The catalytic properties of CALB–metalNP biohybrids were
initially tested in the hydrolysis of 4-nitrophenyl butyrate 1 to
obtain the chromogenic 4-nitrophenol 2 (by enzyme catalysis),
in the reduction of 2 to 4-aminophenol 3 (by metal catalysis)
and in the domino one-pot transformation of 1 to 3 (by combo-
catalysis) as model reactions (Fig. 2 and Table S3, ESI†).
a
b
2
0% (v/v). Calculated by the Bradford assay of the supernatant after
c
2
4 h. Calculated using ICP-AES analysis of the supernatant after 24 h.
Fig. 1 Physicochemical characterization of nanobiohybrids (I) CALB–PdNPs-2,
II) CALB–AgNPs-1, (III) CALB–AuNPs-1 and (a) TEM images. (b) EDX pattern.
(
Fig. 2 Domino reaction for the synthesis of aminoarene 3.
experiments (Fig. S2, ESI†) confirmed the generation of PdNPs
embedded in the protein net (Fig. 1). The morphology and the
Among all the Pd nanohybrids, CALB–PdNPs-5 and CALB–
distribution of PdNPs were investigated using TEM and HRTEM. A PdNPs-6 recovered the highest enzymatic activity (around 47%) in the
dual particle size distribution was observed (Fig. 1 and Fig. S2, ESI†). hydrolysis of 1 compared to the starting soluble CALB. Almost no
The main fraction was composed of rather spherical particles with an enzymatic activity (o5%) was found in the CALB–AgNP biohybrids,
average diameter of 1.3 nm densely deposited throughout the hybrid whereas 25% of the initial enzymatic activity was recovered in the
composite. The minor fraction was composed of larger NPs with an CALB–AuNP biohybrids (Table S3, ESI†). For the catalytic reduction of 2,
average diameter of 4.5 nm randomly decorating the enzymatic net- the reaction rate constant (k) and the turnover frequency number (TOF)
work (Fig. S2, ESI†). The HRTEM image of the larger NPs clearly for the CALB–metalNP biohybrids were calculated, showing better
showed their atomic lattice, confirming the high crystallinity of the NPs enzymatic activity (Table S3, Fig. S6–S8, ESI†). The CALB–PdNPs-5
À1
(
Fig. S2, ESI†). The CALB–PdNP (3–6) biohybrids were also characterized biohybrid exhibited the highest k and TOF values (0.6 min and
À1
using EDX and TEM (Fig. S3, ESI†). In all the cases, the bimodal almost 150 min , respectively) with only slight differences compared
distribution in NPs was maintained although with slight differences in to CALB–PdNPs-6 (Table S3, ESI†). As far as we know, the TOF value is
13
distribution size (1.5–3.5 and 5.5–6.8 nm average size for main smaller the highest described in the literature for this reaction. k and TOF
À1
NPs and minor larger NPs, respectively (Fig. S3, ESI†)).
To expand the method, AgNO and HAuCl
precursors with the same experimental procedure previously devel- these values being comparable to the best ones reported in the
values for CALB–AgNPs-1 and CALB–AuNPs-1 were 0.28 min and
À1
À1
À1
3
4
were tested as about 10 min , and 0.31 min and about 28 min , respectively,
14
oped for CALB–PdNPs hybrids. Under the best conditions, quanti- literature for these metals. Consequently, CALB–PdNPs-5 was selected
tative (CALB–AgNPs-2) or almost quantitative (CALB–AuNPs-1) yields for the direct domino transformation of 1 to 3 (Fig. 2 and Fig. S6, ESI†)
of both biohybrids were achieved (Table S2, ESI†). ICP analysis confirming the good results previously achieved for separate.
+
3+
revealed that 37–41 mmol of Ag and about 19 mmol of Au were
We continued our study evaluating the catalytic performance of the
entrapped inside the protein net. EDX and TEM analyses confirmed CALB–PdNPs in C–C coupling reactions. The Suzuki–Miyaura cross
the presence of silver or gold spherical nanoparticles (Fig. 1 and coupling reaction in aqueous media was attempted using the CALB–
Fig. S4, ESI†) with a unique particle size distribution of about 8 nm PdNPs-2 biohybrid as the catalyst (considering the high surface area of
average diameter for CALB–AgNPs-1 and CALB–AuNPs-1, and 10 nm NPs owing to their small average diameter and highest catalytic activity
for CALB–AgNPs-2 and CALB–AuNPs-2 (Fig. S4, ESI†).
Therefore, considering all these results, a two-step mechanism transfer catalysts (PTC) were evaluated (Table 2 and Table S4, ESI†).
for the enzyme–noble metal NPs nanobiohybrid formation can be Upon using chlorobenzene, the reaction yield was negligible
in the reduction of 2) (Table 2). Different aryl-halides, bases and phase
15
proposed (Scheme S1, ESI†): (i) a first rapid adsorption of soluble in all the cases (Table 2, entry 1), whereas a 50–55% yield of
n+
Me ions on the enzyme, reducing its solubility and acting as a biphenyl 6 was obtained when aryl-bromide or iodide was used
cross-linker between the enzyme molecules (initial fast precipitation) (Table 2, entries 2 and 3). The addition of PTCs showed a
and afterwards, (ii) an ‘‘in situ’’ reduction of metal ions in the positive effect only when the reaction was performed in the
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a
Table 2 Suzuki-Miyaura coupling catalyzed by CALB–PdNPs-2
25% (v/v) water (Table 3). The biohybrids CALB–PdNPs-2, 5 and 6
exhibited similar results (data not shown).
Finally, the nanocatalysts were tested in a tandem catalysis
process (both enzymatic and Pd catalytic activities at the same
time) for the dynamic kinetic resolution (DKR) of rac-pheny-
lethylamine 10 (Table 4 and Table S7, ESI†).
Firstly, both reactions (hydrolysis and racemization) were
studied separately (Fig. S9, ESI†). Free lyophilized CALB was used
to study the enzymatic transesterification of 10, showing a very
high enantioselectivity toward the R enantiomer (ee > 99%). The
b
c
d
Entry
X
PTC
Base
Time (h)
Yield (%)
1
2
3
4
5
Cl
Br
I
Br
I
—
—
—
NaOH
NaOH
NaOH
NaOH
NaOH
48
24
24
2.5
38
2
50
55
99
52
TBACl
TBACl
a
Reaction conditions: 4 (0.5 mmol), 5 (0.55 mmol), H
30 ppb of Pd catalyst, 50 1C. Phase transfer catalyst; 0.165 mmol.
.5 eq. Calculated by HPLC analysis as described in the ESI.
2
O (1 mL), CALB–PdNPs-4 and CALB–PdNPs-5 were used in the Pd-racemiza-
b
1
c
d
tion process of the enantiopure S-10 achieving the rac-10 in both
cases (Table S8, ESI†). Therefore, the tandem enzyme–Pd catalysis
was performed using CALB–PdNPs-5 which was the best catalyst
in terms of enzymatic hydrolytic activity. Different parameters
such as acyl donors, use of molecular sieves or different bases
were evaluated (Table 4, Table S7, ESI†). In the best case, using
ethylacetate as the acylating agent, rac-10 was transformed by
CALB–PdNPs-5 into R-13 in almost quantitative yield and excellent
enantiopurity (ee > 99%) in 4 h. The CALB–PdNPs-5 was reused
for three cycles maintaining its activity and selectivity intact
1
a
Table 3 Heck coupling catalyzed by CALB–PdNPs-4
b
Co-solvent (%v/v, H
2
O)
T (1C)
Time (h)
Yield (%)
—
70
70
70
24
18
24
0
99
20
2
5
5
0
(Table S9, ESI†). Under the same conditions, the CALB–PdNPs-6
biohybrid showed the same result (Table 4).
a
Reaction conditions: 7 (0.274 mmol), 8 (0.55 mmol), DMF (1 mL), 1
mg of CALB–PdNPs-4 catalyst, 70 1C, triethylamine (TEA) (0.412 mmol).
Calculated by HPLC analysis as described in the ESI.
In summary, the straightforward in situ synthesis of metal
NPs, induced by enzymes, generates new hybrid catalysts that
could open a new way to rationally exploit the advantages
offered by the combination of organometallic chemistry and
b
presence of aryl-bromide achieving almost quantitative yields biocatalysis.
of 6 in the presence of TBACl (Table 2, entry 4), using 0.025% This research was supported by The Spanish National
mol/mol) of Pd (about 130 ppb of Pd), with a TON and TOF of Research Council (CSIC). Authors thank European Community
(
3
À1
16
876 and 1550 h , respectively. The nanobiohybrid was used (FP7-MULTIFUN) for the contract of M.M.
for 5 reaction cycles retrieving similar results (in terms of yield
and rate (TOF)) in each cycle and without significant loss of Notes and references
activity (Table S5, ESI†).
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2
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organic synthesis, the Heck reaction was studied (Table 3 and
Table S6, ESI†) selecting CALB–PdNPs-4 as the representative catalyst.
Different reaction parameters, such as temperature or presence
of water, were evaluated in order to soften the common harsh
reaction conditions (Table S6, ESI†). Under the best conditions,
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4
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2
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Propiso
Et
Et
—
—
TEA
5
4
4
4
88
98
91
98
90
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28
3
e
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4
Et
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Reaction conditions: 10 (0.01 mmol), 11 or 12 (0.06 mmol), toluene 16 These values are comparable with the best ones reported in litera-
a
b
c
(
1 mL) and 70 1C, 5 mg CALB–PdNPs-5. 0.07 mmol. Calculated by
RP-HPLC analysis. Determined by chiral HPLC. CALB–PdNPs-6 was
used as the catalyst.
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d
e
6
878 Chem. Commun., 2013, 49, 6876--6878
This journal is c The Royal Society of Chemistry 2013