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Surface-functionalized mesoporous nanoparticles as heterogeneous supports to transferbifunctional catalysts into organic solvents for tandem catalysis

Zhang, Ningning; Hübner, René; Wang, Yangxin; Zhang, En; Zhou, Yujian; Dong, Shengyi ;Wu, Changzhu

Published in:ACS Applied Nano Materials

DOI:10.1021/acsanm.8b01572

Publication date:2018

Document version:Accepted manuscript

Citation for pulished version (APA):Zhang, N., Hübner, R., Wang, Y., Zhang, E., Zhou, Y., Dong, S., & Wu, C. (2018). Surface-functionalizedmesoporous nanoparticles as heterogeneous supports to transfer bifunctional catalysts into organic solvents fortandem catalysis. ACS Applied Nano Materials, 1(11), 6378–6386. https://doi.org/10.1021/acsanm.8b01572

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Article

Surface-Functionalized Mesoporous Nanoparticlesas Heterogeneous Supports to Transfer Bifunctional

Catalysts into Organic Solvents for Tandem CatalysisNingning Zhang, René Hübner, Yangxin Wang, En Zhang, Yujian Zhou, Shengyi Dong, and Changzhu Wu

ACS Appl. Nano Mater., Just Accepted Manuscript • DOI: 10.1021/acsanm.8b01572 • Publication Date (Web): 30 Oct 2018

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1

Surface-Functionalized Mesoporous Nanoparticles

as Heterogeneous Supports to Transfer Bifunctional

Catalysts into Organic Solvents for Tandem

Catalysis

Ningning Zhang,a René Hübner,b Yangxin Wang,a En Zhang,c Yujian Zhou,d Shengyi Dong,e and

Changzhu Wu*f

a Institute of Microbiology, Technische Universität Dresden, Zellescher Weg 20b, Dresden 01217,

Germany.

b Institute of Ion Beam Physics and Materials Research, Helmholtz-Zentrum Dresden-Rossendorf,

Bautzner Landstraße 400, Dresden 01328, Germany.

c Department of Chemistry, Technische Universität Dresden, Bergstraße 66, Dresden 01069,

Germany.

d Department of Chemistry and Food Chemistry, Technische Universität Dresden,

Mommsenstraße 4, Dresden 01062, Germany.

e College of Chemistry and Chemical Engineering, Hunan University, Changsha 410082, Hunan,

P. R. China.

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f Danish Institute for Advanced Study (DIAS) and Department of Physics, Chemistry and

Pharmacy, University of Southern Denmark, Odense 5230, Denmark.

KEYWORDS: multifunctional biocatalyst, mesoporous silica nanoparticles (MSN), palladium

nanoparticles, lipase CalB, cascade reaction

ABSTRACT: The combination of chemo- and biocatalysts offers a powerful platform to address

synthetic challenges in chemistry, particularly in synthetic cascades. However, transferring both

of them into organic solvents remains technically difficult due to the enzyme inactivation and

catalyst precipitation. Herein, we designed a facile approach using functionalized mesoporous

silica nanoparticles (MSN) to transfer chemo- and biocatalysts into a variety of organic solvents.

As a proof-of-concept, two distinct catalysts, palladium nanoparticles (Pd NPs) and Candida

antarctica lipase B (CalB), were stepwise loaded into separate locations of the mesoporous

structure, which not only provided catalysts with heterogeneous supports for the recycling but also

avoided their mutual inactivation. Moreover, mesoporous particles were hydrophobized by surface

alkylation, resulting in a tailor-made particle hydrophobicity, which allowed bifunctional catalysts

to be dispersed in eight organic solvents. Eventually, these attractive material properties provided

the MSN-based bifunctional catalysts with remarkable catalytic performance for cascade reaction

synthesizing benzyl hexanoate in toluene. On a broader perspective, the success of this study opens

new avenues in the field of multifunctional catalysts where a plethora of other chemo- and

biocatalysts can be incorporated into surface-functionalized materials ranging from soft matters to

porous networks for synthetic purpose in organic solvents.

INTRODUCTION

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The fabrication of heterogeneous biocatalysts, through entrapping or immobilizing enzymes/whole

cells onto organic and inorganic supports, offers the potential to revolutionize biocatalyst

performance at the industrial process scale.1 In particular, the use of heterogeneous supports

simplifies biocatalyst recycling and promotes continuous process development.2-3 Today, any type

of industrial enzymes can be immobilized onto a “solid” carrier whose properties can be further

tuned by chemical or physical manipulation. In many examples, hydrogels4-5 and synthetic resins6-8

are used as enzyme hosts for improving catalyst stability and usability at ambient/elevated

pressures and temperatures. However, catalytic efficiency using these carriers is often

compromised by their macroscopic sizes and nonporous structures that give rise to a small

interfacial contact during reactions.9 In contrast to traditional materials, mesoporous particles

become ideal supports owing to their self-assembled porous structures that allow for high enzyme

loadings with large interface contact.10-11 Moreover, the porous networks can provide enzymes a

protective environment against harsh reaction conditions, such as extreme pH, elevated

temperature, and organic solvents.12-13

Recently, the mesoporous silica nanoparticles (MSN) with hierarchical pores or dendritic pores

were reported to be good supports for enzyme immobilization.14-20 In addition, mesoporous

materials were particularly explored to accommodate two catalytic species into their hierarchical

structures for cascade catalysis.21 Since there is no need to purify and isolate intermediates, such

bifunctional system can reduce operation time, production cost and waste, and meanwhile, enhance

overall yield.22-23 Many groups have adopted this concept by combining metal particles (e.g., Ru,

Au, Pt, and Pd) with other active species on the porous surface,24-25 achieving exceptional cascade

productivity towards, for instance, knoevenagel condensation26 and oxidation of cinnamyl

alcohol.27 Despite these successes, enzymes are sparingly combined with metal particles or

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organocatalysts for the same principle, because the incorporation of these distinct species (e.g.,

different stability and size) into a size-restricted porous material remains a challenge to date.

Starting in 2013, the Bäckvall group reported the first example of bifunctional biocatalysts,

where they pioneered a crosslinking process to co-immobilize Candida antarctica lipase B (CalB)

and palladium nanoparticles (Pd NPs) in siliceous mesocellular foams.28 Such co-immobilization

offered the two catalysts to cooperatively catalyze the dynamic kinetic resolution (DKR) of a

primary amine with improved yield and enantioselectivity. Very recently, Zhang et al. further

improved the fabrication approach by positional loading of Pd NPs and CalB into the inner core

and outer shell of mesoporous silica, respectively.29 The spatial separation of two active sites

thereby resulted in an even better yield. These two pioneering examples successfully illustrate

mesoporous materials to be suitable to support bifunctional metal-enzyme composition for

cooperative reactions. However, from a practical point of view, the current preparation protocols

are based on as-synthesized porous scaffolds that are neither tailor-made to specific reaction

conditions nor provide an optimal enzyme microenvironment, thus leaving much room for further

improvement.

Herein, we report a stepwise approach that allows loading Pd NPs and biocatalysts into

mesoporous silica nanoparticles (MSN) whose surface hydrophobicity is finely tuned via post-

modification with alkyl chains. The tailored material surface not only provides an interfacially

active hydrophobic microenvironment to Candida antarctica lipase B (CalB) but also enables to

transfer the bifunctional MSN from water into a wide range of organic solvents, making the system

suitable in a reaction medium of interest. Moreover, their hydrophobized surface allows high

protein loading because of the hydrophobic interactions and possible hydrogen bonding,30 and

facilitates nonpolar substance diffusion across the hierarchical mesoporous structure. As a result

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of these properties, the combination of lipase and Pd NPs in our system allows to catalyze a cascade

reaction with high efficiency.

RESULTS AND DISCUSSION

The synthesis route of the bifunctional biocatalyst is illustrated in Scheme 1. It started with

preparing mesoporous silica nanoparticles (MSN) according to the previously reported method.31

Pd(0) NPs were then loaded into these as-synthesized MSN particles by in-situ reduction of

Pd(AcO)2 in the presence of NaBH4 solution, obtaining a hybrid Pd@MSN. The original MSN

display a white color, while after the Pd NPs encapsulation, the MSN-based nanocomposites show

a sharp contrast color of black, which is a straightforward proof of successful Pd loading in MSN

(Figure S1). In order to change the particles from hydrophilic to hydrophobic, long-chain alkanes

were introduced to the surface of Pd@MSN by reacting with trimethoxy(octadecyl)silane

(TMODS), obtaining [email protected]

Scheme 1. Schematic illustration of the construction of CalB@Pd@mMSN.

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In addition to Pd NPs, CalB (Figure S2) was subsequently immobilized to the hydrophobized

Pd@mMSN via hydrophobic interactions, forming a bifunctional biocatalyst, denoted as

CalB@Pd@mMSN. In order to optimize the protein loading, Pd@mMSN particles were mixed

with different concentrations of CalB (5, 20, and 40 mg mL-1), achieving CalB-5@Pd@mMSN,

CalB-20@Pd@mMSN, and CalB-40@Pd@mMSN, respectively. The exact protein loading on

particles was determined by Bradford assay, showing that CalB-5@Pd@mMSN, CalB-

20@Pd@mMSN, and CalB-40@Pd@mMSN contain 3.65, 7.41, and 10.76 µg mg-1 CalB,

respectively (Figure S3). This finding illustrates the enzyme loading depending on the initial

amount of added enzymes and the adsorption capacity could reach 15 µg mg-1. Such dependence

could be also proved by thermogravimetric analysis (TGA) (Figure S4). In contrast, the

unfunctionalized Pd@MSN can only immobilize a little CalB (2.23 µg mg-1) even mixed with a

higher initial concentration of CalB, which further indicates the importance of surface-

modification. Besides, the Pd loading was determined by inductively coupled plasma optical

emission spectrometry (ICP-OES), showing 5.3% and 4.6% Pd content on Pd@MSN and

Pd@mMSN, respectively. Interestingly, after CalB adsorption on the CalB-5@Pd@mMSN, CalB-

20@Pd@mMSN, and CalB-40@Pd@mMSN, Pd content slightly decreased to about 4.2% which

is assumed to be due to the loss of unencapsulated Pd NPs from the MSN surface and the weight

increase of the particle by loading CalB. This assumption is proved by the fact that Pd loading

remains constant in the successive reaction process.

The mesoporous nanoparticles were initially characterized by powder X-ray diffraction (XRD).

The broad peak from 20 o < 2 < 23 o in the wide-angle XRD pattern can be ascribed to amorphous

silica (Figure 1a).34 The peaks at around 1.5 o and 2.4 o in the small-angle XRD pattern could be

assigned to the typical plane of MSN, thus confirming the successful preparation of mesoporous

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MSN (Figure S5).35 For the Pd-containing samples, the peaks centered at 2 = 39.4 o, 46.1 o, 68.3

o, and 81.1 o in the wide-angle XRD pattern belong to the 111, 200, 220, and 311 diffraction

maxima of metallic Pd characterized by its face-centered cubic structure, respectively.36-37 The

disappearance of the characteristic diffraction peaks at 1.5 o and 2.4 o in small-angle XRD is related

to the destruction of regular mesoporous structure of MSN caused by the incorporation of Pd,

which indicates the filling of the MSN pore channels by Pd NPs.31, 38 The specific surface areas

and porosities of the resultant materials were measured by the N2 adsorption/desorption

experiments. The curve of MSN represents a type-IV isotherm pattern, indicating mesoporosity.

39 In comparison with the parent MSN, the remarkable decrement in N2 adsorption capacity and

significant pore size change of Pd@MSN indicate that pores of the MSN are occupied by the

encapsulated Pd nanoparticles (Figure S6).34,38 The similar trends go to the Brunauer-Emmett-

Teller (BET) surface and pore volumes, which further confirms the loading of Pd inside pores

blocking the N2 adsorption (Table 1). The resultant composites after immobilizing CalB exhibit

the less reduced BET surface areas, which is due to the attachment of CalB on the surface of MSN

blocking some pore openings since the pores of MSN are too small to be accessible for CalB.40-41

To investigate whether the surface modification worked out, Pd@mMSN was analyzed by Fourier-

transform infrared spectroscopy (FTIR) (Figure 1b). The characteristic bands (2780 - 2900 cm-1)

for Pd@mMSN correspond to the C-H stretching mode, thus clearly confirming the presence of

alkane groups on MSN particles.42 Not only these surface-modified particles but also

CalB@Pd@mMSN were subjected to FTIR analysis. The intensified bands at 1640 and 1560 cm-1

(Figure S7), which belong to the stretching absorption of amide I group and amide II, are found

on CalB@Pd@mMSN, suggest the presence of this protein on MSN particles after enzyme

immobilization.43

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Figure 1. (a) Wide-angle XRD of MSN, Pd@MSN, Pd@mMSN, and CalB-20@Pd@mMSN. (b)

FTIR spectra of CalB, Pd@MSN, Pd@mMSN, and CalB-20@Pd@mMSN. (c) Water contact

angle images of Pd@MSN, Pd@mMSN, CalB-5@Pd@mMSN, CalB-20@Pd@mMSN, and

CalB-40@Pd@mMSN; photos of Pd@MSN and Pd@mMSN dispersed in toluene (100 mg mL-

1).

After characterizing the materials composition, we next investigated their hydrophobicity by

contact angle measurement. As shown in Figure 1c, the high contact angle (131.2 ± 1.3 o) for

Pd@mMSN is observed after surface alkylation, which starkly contrasts to the low contact angle

of 22.6 ± 1.0 o for unmodified Pd@MSN. This remarkable change of contact angle discloses the

successful alkylation on MSN. Such hydrophobicity change could affect the particle dispersibility

in solvents. For example, modified Pd@mMSN could be well dispersed in toluene while

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unmodified samples quickly aggregated. Interestingly, CalB-loaded Pd@mMSN resulted in a

decrease of particle hydrophobicity. For instance, CalB-5@Pd@mMSN has a contact angle of

115.9 ± 1.4 o, which is lower than that for original Pd@mMSN. The contact angle further decreases

with higher CalB loading on Pd@mMSN. This decreased contact angle indicates the hydrophilic

nature of enzymes that can be further used to tune the particle hydrophobicity. Nevertheless,

CalB@Pd@mMSN remains hydrophobic after enzyme immobilization, thus qualified for their

following use in organic solvents.

Table 1. Structural Properties of the Tested Catalysts

Sample name SBET a

(m2 g-1)

VTotalb

(cm3 g-1)

MSN 649 0.80

Pd@MSN 63 0.27

Pd@mMSN 32 0.43

CalB-5@Pd@mMSN 28.6 0.40

CalB-20@Pd@mMSN 27 0.22

CalB-40@Pd@mMSN 25.8 0.32

a SBET = BET Surface area, based on the adsorption data in the partial pressure of 0.05 < P/P0 < 0.20. b VTotal = Total pore volume, estimated from the adsorbed nitrogen volume at a relative pressure of about 0.95.

For organic synthesis, the choice of solvent media plays a crucial role in reaction efficiency. As

such, CalB-20@Pd@mMSN was dispersed into eight different organic solvents with a wide range

of polarity index. Figure 2 shows that the hybrid catalysts could be well-dispersed in all listed

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solvents. This finding suggests that surface-modified MSN can be adapted in many reaction media

of interest.44-45

Figure 2. Photos of CalB-20@Pd@mMSN well-dispersed in different organic solvents (100 mg

mL-1; numbers on vials mean polarity index; DCM – dichloromethane, EA – ethyl acetate, DMF

– N, N-dimethylformamide, DMSO – dimethyl sulfoxide).

Next, the morphology and structure of catalysts were investigated using scanning electron

microscopy (SEM) and transmission electron microscopy (TEM). The SEM images of MSN and

Pd@mMSN show almost identical morphology and particle diameters (Figure S8), which implies

that the Pd encapsulation process has no effect to MSN surface. Interestingly, after CalB

adsorption, the MSN surface becomes rather rough, presumably due to random protein distribution

on MSN (Figure 3a). Inspired by this morphological observation, further TEM analysis was done

to analyze the localization and composition of the hybrid catalysts. The bright-field TEM images

(Figure S9) prove the highly ordered mesostructure of the parent material and the destructed

mesostructures of CalB@Pd@mMSN, which further testifies the pore filling by Pd NPs. From the

bright-field TEM micrograph (Figure 3b), the Pd NPs with narrow size distribution (ca.

1.80 ± 0.60 nm in diameter) are clearly observed on the mesoporous silica particles of CalB-

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20@Pd@mMSN. A similar Pd NPs density and size distribution for Pd@MSN and Pd@mMSN

(Figure S10) suggest that the morphological variations caused by enzyme immobilization are

negligible. The high-angle annular dark-field scanning transmission electron microscopy

(HAADF-STEM) and spectrum imaging based on energy-dispersive X-ray spectroscopy (EDXS)

further confirm the homogeneous distribution of Pd NPs in Pd@mMSN (Figure S11) and CalB-

20@Pd@mMSN (Figure 3c). Since CalB is a multiple element-composed macromolecule, it is

difficult to directly map its existence on MSN if the density is quite low. However, with OsO4

staining,46 EDXS analysis could indirectly prove the presence of OsO4-bound CalB on CalB-

20@Pd@mMSN (Figure S12).

Figure 3. (a) SEM image of CalB-20@Pd@mMSN; (b) bright-field TEM image of CalB-

20@Pd@mMSN (inset: Pd NPs size distribution); (c) HAADF-STEM image and corresponding

Si, O and Pd element maps for CalB-20@Pd@mMSN.

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To directly observe proteins on MSN, CalB was labelled with fluorescein isothiocyanate (FITC)

to prepare CalB-FITC@Pd@mMSN. Fluorescence microscopy images show the green particles

of CalB-FITC@Pd@mMSN are dispersed in acetone, which, however, was not observed by light

microscopy (Figure 4b), thus proving the presence of CalB-FITC on MSN. In addition to CalB,

green fluorescent protein (GFP) and FITC-labelled glucose oxidase (FITC-GOD) were also

adsorbed onto Pd@mMSN and observed with optical and fluorescence microscopy. Figure 4c and

4d show that both GFP and GOD-FITC could readily reside on silica particles. The success of

immobilization of three distinct proteins illustrates that surface-modified mesoporous silica can be

used as a versatile platform for preparing bifunctional catalysts from diverse biocatalyst sources.

Figure 4. (a) Scheme illustrates that diverse proteins/enzymes can be immobilized to Pd@mMSN.

Optical (top) and fluorescence (bottom) microscopy images of Pd@mMSN after adsorbing (b)

FITC-CalB, (c) GFP, and (d) FITC-GOD.

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After stepwise catalysts loading, we first evaluated the catalytic performance of CalB in CalB-

20@Pd@mMSN by a typical esterification reaction where 1-octanol and octanoic acid were

converted to octyl octanoate in toluene (Figure 5a). The yield of the product octyl octanoate was

measured by gas chromatography (GC) using pure octyl octanoate as the standard (Figure S13).

The catalytic data in Figure 5b and 5c reveal that the lipase-catalyzed conversion by CalB-

20@Pd@mMSN is very fast, achieving almost 100% yield in only 20 min. In contrast, with the

same duration and reaction conditions, there was only 25% yield using the identical amount of free

CalB. In addition, CalB-20@Pd@mMSN shows a superior catalytic performance to the equal

amount of CalB immobilized on Novozym 435 (175 mg g-1 CalB loading),47 a commercial CalB

immobilisate. Besides, the control experiment with Pd@mMSN as catalyst produces no octyl

octanoate without CalB loading. The superior catalytic performance by CalB-20@Pd@mMSN is

attributed to the large surface contact of mesoporous carriers as well as their good dispersity in

toluene. Apart from the catalytic efficiency, protein thermal stability was also evaluated in the

reaction medium (Figure 5d). At 80 °C, immobilized CalB (both CalB-20@Pd@mMSN and

Novozym 435) displays no activity decrease in 24 h; however, without the protective carriers, free

CalB loses 70% activity after 24 h. The improved thermal stability of enzymes in CalB-

20@Pd@mMSN, therefore, suggests their potential as robust biocatalysts for industry-relevant

application in organic solvents.

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Figure 5. (a) Reaction scheme of esterification of 1-octanol and octanoic acid catalyzed by CalB.

(b) Time-dependent yield of octyl octanoate catalyzed by (1) free CalB, (2) Novozym 435, and (3)

CalB-20@Pd@mMSN at 25 °C in toluene. (c) Yield (20 min) of octyl octanoate using different

catalysts. (d) Thermal stability of (1) free CalB, (2) Novozym 435, and (3) CalB-20@Pd@mMSN

at 80 °C in toluene.

The positive performance of CalB in CalB-20@Pd@mMSN encouraged us to further assess the

catalytic efficiency of the immobilized Pd NPs. For this purpose, a Suzuki coupling reaction was

performed in a mixed solution of ethanol and water using iodobenzene and 4-acetylphenylboronic

acid as substrates and CalB-20@Pd@mMSN as catalyst (Scheme S1). By mass spectroscopy

analysis (Figure S14), successful product formation with 100% conversion within 3 h is

confirmed, indicating that Pd NPs remain catalytically active after the immobilization of two

catalysts. The slight 0.16% Pd leaching during the Suzuki reaction was determined by ICP-OES

analysis, which indicates the stability of the catalyst with negligible leaching.

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After activity proof of the two separate catalysts (CalB and Pd NPs) in CalB-20@Pd@mMSN,

their combined use was demonstrated by a one-pot cascade reaction, as illustrated in Figure 6a.

In the reaction, Pd NPs first reduce the benzaldehyde to benzyl alcohol under a hydrogen

atmosphere. The immobilized CalB then converts the intermediate into benzyl hexanoate. The

benefit of such design can avoid the isolation of active intermediates, thus providing a facile

approach synthesizing esters based on benzaldehyde in a mild way for industrial use. For

comparison, five control experiments were established to catalyze the cascade reaction under the

identical procedure: CalB-40@mMSN (with 17.78 µg mg-1 CalB loading, Figure S3) performed

with Pd@mMSN, free CalB performed with Pd@mMSN, CalB-40@Pd@MSN, CalB-

40@mMSN, and Pd@mMSN. The final product could be evaluated and identified by GC, 1H-

NMR, and 13C-NMR (Figure S15), respectively. As illustrated in Figure 6b and 6c, CalB-

20@Pd@mMSN is the most efficient catalyst for the cascade reaction achieving 76% yield within

2 h, which is higher than the CalB-5@Pd@mMSN with 54% yield, CalB-40@Pd@mMSN with

62% yield, and three controls. Interestingly, after 2 h, CalB-20@Pd@mMSN and CalB-

40@Pd@mMSN exhibit higher yields than CalB-5@Pd@mMSN. In control experiments, the

CalB-40@mMSN performed with Pd@mMSN gives rise to 51% yield (2h), which is lower

comparing with CalB@Pd@mMSN, indicating the cooperative effect of Pd and CalB together

immobilized on CalB@Pd@mMSN. Besides, there is only 14% (2 h) yield observed for free CalB

with Pd@mMSN cascade reaction which contains the same amount of protein as CalB-

20@Pd@mMSN, which is caused by the poor protein solubility in organic solvents. Besides, the

unfunctionalized CalB-40@Pd@MSN gives rise to the lowest conversion (< 1%) after 2 h, which

is mainly due to the poor dispersibility and lower CalB loading. Moreover, neither CalB-

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40@mMSN nor Pd@mMSN (Figure S16) can lead to the final product benzyl hexanoate, which

illustrates the necessity of cooperating Pd NPs and enzyme for cascade reaction.

In regard to practical applicability, reusability is a crucial parameter for the heterogeneous

catalyst. The recycling experiments were performed using CalB-20@Pd@mMSN as a catalyst in

the same cascade reaction. After four-time reuse, reaction yield (4 h) remains higher than 80%

compared to the first run (Figure 6d). This multiple reusability demonstrates that CalB-

20@Pd@mMSN is a robust and recycling heterogeneous catalyst that may be further applied in

other solvent conditions for the synthetic purpose.

Figure 6. (a) One-pot cascade reaction of benzaldehyde with ethyl hexanoate. (b) Time-dependent

yield of benzyl hexanoate catalyzed by different catalysts (1) CalB-5@Pd@mMSN; (2) CalB-

20@Pd@mMSN; (3) CalB-40@Pd@mMSN; (4) CalB-40@mMSN performed with Pd@mMSN;

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(5) free CalB performed with Pd@mMSN; (6) CalB-40@Pd@MSN. (c) Yield (2 h) of benzyl

hexanoate using different catalysts. (d) Reusability of CalB-20@Pd@mMSN.

In order to get insight into understanding its stability, CalB-20@Pd@mMSN was re-subjected

to FTIR analyses and TEM characterization after four-time reuse. It is found that the reaction

caused no change of FTIR spectra, indicating the remaining presence of the modified surface and

proteins on the hybrid catalysts (Figure S17). TEM characterization confirms that there are no

significant morphological changes or Pd aggregation occurring between the fresh and recovered

CalB-20@Pd@mMSN (Figure S18). These results imply that our bifunctional materials are robust

under the critical reaction conditions like organic solvents. Furthermore, catalyst leaching

experiments were carried out by analyzing the residual CalB and Pd NPs released from the cascade

reaction (6 h). Bradford assay and ICP-OES analysis show that there is only 0.11% Pd leaching

and no detectable protein leaching, which illustrates the high stability of the bifunctional materials.

CONCLUSIONS

In conclusion, we have designed bifunctional biocatalysts based on mesoporous silica

nanoparticles (MSN) where particle hydrophobicity was finely-tuned by surface alkylation, and

two catalysts (Pd nanoparticles and CalB enzyme) were separately loaded into compartmentalized

locations. This system, on the one hand, provides a large-surface-area platform to transfer catalysts

into diverse organic solvents, thus can be developed as a versatile tool to accommodate other

chemo- and biocatalysts in the reaction medium of interest. On the other hand, the combined use

of metal and biocatalysts is demonstrated by the one-pot tandem reaction in the synthesis of benzyl

hexanoate. This proof-of-principle example suggests the high potential using MSN-based

bifunctional catalysts for advanced synthesis in the future.

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EXPERIMENTAL SECTION

Materials. Unless otherwise stated, all chemicals were obtained from commercial suppliers and

used without further purification. Candida antarctica lipase B (CalB) was purchased from c-LEcta

GmbH (Leipzig, Germany), and the protein content was about 10 wt. % based on the Bradford

test.

Analytical techniques. Inductively coupled plasma-optical emission spectroscopy (ICP-OES)

was carried out on a Perkin Elmer Optima 7000DV optical emission spectrometer. Bradford assay

was carried out with TECAN infinite M200. Drop shape analysis system DSA 10 from Krüss was

used to characterize the particle hydrophobicity by pressing particle samples into a smooth tablet

for water contact angle measurements and each analysis was repeated for 5 times.

Thermogravimetric analysis (TGA) was carried out with Mettler-Toledo TGA instrument using a

10 °C min-1 ramp up to 800 °C in air atmosphere. FTIR spectra were recorded on a FT-IR

spectrometer Tensor II (Bruker) with an ATR unit. The fluorescence images were taken using the

Olympus Provis AX70. The small-angle X-ray diffraction experiments were performed on a

Bruker Nanostar diffractometer with Cu Kα1 radiation (λ = 154.06 pm) and a position sensitive

Histar 2D detector. The wide-angle powder X-ray diffraction (XRD) analysis was performed on a

PANalytical X’Pert Pro powder diffractometer with Debye-Scherrer geometry equipped with a

Ge(111)-monochromator, a rotating sample stage, and a PIXcel detector, using Cu Kα1 radiation

(λ = 154.06 pm). The data was collected in reflection mode using a divergence slit that kept the

illuminated sample area constant. Nitrogen adsorptions are measured volumetrically at 77 K on a

QuadraSorb with a sample mass of ca. 100 mg. The Brunauer-Emmett-Teller (BET) method was

used to calculate the specific surface area based on the adsorption data in the partial pressure of

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0.05<P/P0<0.20 and the total pore volume was estimated from the adsorbed nitrogen volume at a

relative pressure of about 0.95. The pore size distributions (PSDs) were evaluated by the

equilibrium branches of the isotherms based on Quenched Solid Density Functional Theory

(QSFDT, nitrogen on carbon slit/cylindrical equilibrium branch kernel). The GC analysis was

carried out with the Shimadzu GC 2010 Plus instrument equipped with a flame ionization detector

(FID) and an AOC-20i auto-injector). GC chiral column (Hydrodex γ DIMOM as stationary phase,

30 m × 0.25 mm) was applied to determine the final products. Gas chromatography with mass

spectrometry coupling (GC/MS) was performed on an Agilent Technologies 6890N GC system

equipped with a 5973 mass selective detector (electron impact, 70 eV). SEM images were acquired

by a Gemini 500 (Carl Zeiss, Germany) system. Bright-field TEM images were recorded on a

Titan 80-300 (FEI) microscope operated at an accelerating voltage of 300 kV. High-angle annular

dark-field scanning transmission electron microscopy (HAADF-STEM) imaging and spectrum

imaging based on energy-dispersive X-ray spectroscopy (EDXS) were performed at 200 kV with

a Talos F200X microscope equipped with an X-FEG electron source and a Super-X EDXS detector

system (FEI). Prior to TEM analysis, the specimen mounted in a high-visibility low-background

holder was placed for 2 s into a Model 1020 Plasma Cleaner (Fischione) to remove contamination.

TEM specimens were prepared by dropping the solution under investigation with several

microliters of sample well-dispersed in absolute ethanol onto a carbon-coated copper grid (400

mesh, S160-4, Plano GmbH) and drying it under ambient conditions. 1H and 13C NMR spectra

were recorded at 25 °C by using a Bruker AVANCE III NMR spectrometer at 400 and 100 MHz,

respectively.

Preparation of MSN. The MSN were prepared as the previous method.31 Typically, N-

cetyltrimethylammonium bromide (CTAB, 1 g, 2.74 mmol) was dissolved in 480 mL Millipore

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water, followed by adding sodium hydroxide solution (2.00 M, 3.50 mL) and heating to 80 °C.

Tetraethoxysilane (TEOS, 5 mL, 22.4 mmol) was then introduced dropwise to the above solution

under vigorous stirring. The mixture was allowed to react for 2 h, producing a white precipitate

which was thoroughly washed with deionized water and methanol and dried in air to get MSN. To

remove the surfactant template (CTAB), 1.5 g of the obtained MSN was refluxed in 160 mL

methanol containing 9 mL of HCl (37.4%) for 24 h. The resulting material was filtered and washed

again with deionized water and methanol to obtain the surfactant-free MSN materials. They were

then dried at 50 °C overnight to remove the remaining solvent and get white powder, denoted as

MSN.

Preparation of Pd@MSN. The as-prepared MSN (1 g) was dispersed in 10 mL acetonitrile

containing palladium (II) acetate (210.9 mg, 0.940 mmol) and stirred for 2 h at 500 rpm at room

temperature. The unbound Pd2+ was washed away with acetonitrile (3 × 50 mL). The Pd2+@MSN

was collected via centrifugation at 8000 rpm for 5 min and dried overnight at 50 oC. The obtained

yellowish powder was re-suspended in 50 mL deionized water. The sodium borohydride aqueous

solution (135 mg, 3.568 mmol) was dissolved in 2 mL deionized water and added to this

suspension to reduce the Pd2+ to Pd (0). The sediments were collected by centrifugation (8000 rpm,

5 min), washed with deionized water (3 × 50 mL), and dried at 50 oC overnight, defined as

Pd@MSN.

Preparation of Pd@mMSN. Pd@MSN (0.5 g) was dispersed in 21.5 mL toluene containing

trimethoxy(octadecyl)silane (TMODS, 3.54 mL, 8.36 mmol) via sonication, and the mixture was

refluxed for 15 h. After cooling to room temperature, the resultant solid was centrifuged at 8000

rpm for 5 min. The product was further washed with toluene (3 × 40 mL) and ethanol (3 × 40 mL),

reapectively, and dried at 50 °C overnight, obtaining Pd@mMSN.

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Preparation of CalB@Pd@mMSN. Pd@mMSN (300 g) was dispersed in 3 mL lipase CalB

(commercial enzyme, 10% protein content determined by Bradford assay) solution with different

concentration (5 mg mL-1, 20 mg mL-1, and 40 mg mL-1) and stirred at 500 rpm at room

temperature for 1 h. After that, the particles were collected by centrifugation for 5 min at 6000 rpm

and 4 °C. The final product was washed with deionized water (3 × 5 mL), followed by

lyophilisation, and stored at -20 °C before use. The supernatant was collected for Bradford assay

to determine the adsorbed CalB on solid. CalB-40@Pd@MSN and CalB-40@mMSN were

prepared with the same method using different solid supports except the CalB concentration is 40

mg mL-1.

Preparation of CalB-40@mMSN. As-synthesized MSN (0.5 g) was dispersed in 21.5 mL

toluene containing trimethoxy(octadecyl)silane (TMODS, 3.54 mL, 8.36 mmol) via sonication,

and the mixture was refluxed for 15 h. After cooling to room temperature, the resultant solid was

centrifuged at 8000 rpm for 5 min, and washed with toluene (3 × 40 mL) and ethanol (3 × 40 mL),

and dried at 50 °C overnight.

Preparation of OsO4-treated materials. CalB@Pd@mMSN particles were incubated in PBS

(10 mM, pH 7.2) buffer containing 2% formaldehyde and 0.1% glutaraldehyde at room

temperature for 30 min. After that, particles were washed with PBS (10 mM, pH 7.2) buffer (4 ×

1 mL) and Millipore water (4 × 1 mL), and then incubated with 0.2% OsO4 aqueous solution

overnight at room temperature. The Os-treated CalB@Pd@mMSN was washed with Millipore

water (3 × 1 mL), followed by lyophilisation. The final solid particles were dispersed in absolute

ethanol via sonication for TEM analysis.

Catalytic performance of immobilized CalB for esterification. The stock solution of 1-

octanol (100 mM) and octanoic acid (100 mM) in toluene (10 mL) was prepared. CalB-

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20@Pd@mMSN (10 mg) and 500 µL of the above stock solution were mixed in a closed vial and

shaken at 40 rpm and 25 °C. At intervals, aliquots (10 µL) were withdrawn, diluted into 100 µL

with toluene and then subjected to GC measurement. Control experiments using free CalB,

Novozym 435, and Pd@mMSN were performed following the same procedure.

Thermal stability of the immobilized CalB. CalB-20@Pd@mMSN, free CalB, and Novozym

435 were dispersed in 200 µL toluene in vials, respectively. The sealed vials were shaken at 500

rpm under 80 °C. At intervals (0 h, 1 h, 2 h, 4 h, 8 h, and 24 h), vials were removed from heating.

After cooling to room temperature, 300 µL stock solution containing 1-octanol (100 mM) and

octanoic acid (100 mM) was added into vials to perform the esterification at 40 rpm and 25 °C for

30 min. The reaction solution (10 µL) was withdrawn and diluted into 100 µL with toluene for GC

analysis.

Catalytic performance of Pd NPs for Suzuki coupling reaction. Iodobenzene (11.2 µL, 0.1

mmol), 4-acetylphenylboronic acid (30.79 mg, 0.2 mmol), and potassium carbonate (K2CO3, 41.46

mg, 0.3 mmol) were dissolved in the mixture (300 µL) of ethanol/H2O (v/v = 2/1). CalB-

20@Pd@mMSN (5 mg, 5 mol %) was then added to the mixture. Then vial was closed and, the

reaction was performed at 800 rpm and 60 °C for 3 h. The final product in the supernatant was

analyzed by GC/MS. After the reaction, the reactants were centrifuged at 8000 rpm for 5 min,

followed by washing with ethanol (3 × 1.0 mL). The residual solution from the reactions and the

washing was collected and then evaporated under 50 °C in vacuum. Subsequently, the resultant

residue was re-dissolved in deionized water (2 mL), filtered through 0.22 µm membrane filter, and

subjected to ICP-OES for the assessment of Pd leaching.

Typical procedure for cascade reaction. A toluene solution (200 µL) with benzaldehyde (250

mM) and ethyl hexanoate (500 mM) was added into to a Schlenk tube containing 20 mg catalyst.

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The tube was purged with H2 and the reaction mixture was stirred at room temperature with a

balloon of H2. At interval time, 5 μL reaction mixture was withdrawn and diluted to 100 μL with

toluene where the solid catalyst was removed by centrifugation at 12000 rpm for 5 min. The upper

liquid sample was analyzed by GC to determine yield. The identity of the product was confirmed

by comparison with standard compounds.

Preparation of benzyl hexanoate as a standard product for cascade reaction. A toluene

solution (4 mL) containing vinyl hexanoate (2.0 mmol) and benzyl alcohol (2.0 mmol) was added

into the container with Novozym 435 (20 mg). The mixture was stirred under room temperature

for 2 h. The solid Novozym 435 was removed by filtration after the reaction was finished. Then

the product was purified with a flash column with hexane and ethyl acetate (v/v = 25/1) working

as eluent, and the final product was obtained as a colourless liquid (0.369 g, yield 89%).

Reusability of CalB-20@Pd@mMSN. The reactions were performed following the same

procedure as the above-mentioned cascade reaction except that the reaction time was 4 h. After

the first run, the catalyst was recovered by centrifugation at 8000 rpm for 2 min, followed by

washing with toluene (3 × 1.0 mL) and drying at room temperature. The residual solution from the

reactions and the washing were collected and then evaporated under 50 °C in vacuum.

Subsequently, the resultant residue was re-dissolved in deionized water (1 mL) and subjected to

Bradford assay and ICP-OES for the assessment of leaching of CalB and Pd NPs, respectively.

The solid recovered from the reactions was used for the next run under the same reaction

conditions.

ASSOCIATED CONTENT

Supporting Information.

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Supporting Information Available: [Photographs of catalysts, Crystal structure of CalB, Bradford

assay, TGA curves, small-angle XRD pattern, N2 adsorption/desorption isotherms, magnified

FTIR spectra, HAADF-STEM images and element maps, EDX spectra, SEM images, calibration

curves for GC, mass spectra, 1H and 13C NMR spectrum.] This material is available free of charge

via the internet at http://pubs.acs.org.

AUTHOR INFORMATION

Corresponding Author

*Email for C. W.: [email protected]

ORCID

Changzhu Wu: 0000-0001-9405-5616

Notes

The authors declare no competing financial interest.

ACKNOWLEDGMENT

This work is financially supported by the DFG (WU 814/1-1). N. Z. thanks China Scholarship

Council (No. 201606210135) for the financial support. The funding of TEM Talos by the German

Federal Ministry of Education of Research (BMBF), Grant No. 03SF0451 in the framework of

HEMCP is acknowledged. We would like to thank Prof. Marion. B. Ansorge-Schumacher for

valuable discussions and support. N. Z. and C. W. thank Prof. Rainer Jordan for providing lab

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facility for relevant synthesis. Furthermore, the use of HZDR Ion Beam Center TEM facilities is

acknowledged.

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GRAPHIC ABSTRACT

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surface-functionalized mesoporous nanoparticles as ......1 surface-functionalized mesoporous...

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