Artificial Nanometalloenzyme for Cooperative Tandem Catalysis - ACS

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Artificial Nanometalloenzyme for Cooperative Tandem Catalysis Hui Li, Chenggang Qiu, Xun Cao, Yuanyuan Lu, Ganlu Li, Xun He, Qiuhao Lu, Kequan Chen, Pingkai Ouyang, and Weimin Tan ACS Appl. Mater. Interfaces, Just Accepted Manuscript • DOI: 10.1021/acsami.9b03616 • Publication Date (Web): 15 Apr 2019 Downloaded from http://pubs.acs.org on April 15, 2019

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ACS Applied Materials & Interfaces

Artificial Nanometalloenzyme for Cooperative Tandem Catalysis

1 2

Hui Li,†,‡ Chenggang Qiu,†,‡ Xun Cao,†,‡ Yuanyuan Lu,†,‡ Ganlu Li,†,‡ Xun He,†,‡

3

Qiuhao Lu,†,‡ Kequan Chen,*,†,‡ Pingkai Ouyang,†,‡ and Weimin Tan∥

4 5

† College

6

Nanjing, 211816, China

7



8

211816, China

9

∥National

10

of Biotechnology and Pharmaceutical Engineering, Nanjing Tech University,

State Key Laboratory of Materials-Oriented Chemical Engineering, Nanjing,

Engineering Research Center for Coatings, CNOOC Changzhou Paint and

Coatings Industry Research Institute Co., Ltd, Changzhou 213016, P.R.China

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Abstract: Artificial metalloenzymes which combine the advantages of natural

25

enzymes and metal catalysts have been getting more research attention. As a proof of

26

concept, an artificial nanometalloenzyme (CALB-Shvo@MiMBN) was prepared by

27

co-encapsulation of metallo-organic catalyst and enzyme in a soft nanocomposite

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consisting of 2-methylimidazole, metal ion, and biosurfactant in mild reaction

29

conditions using one-pot self-assembly method. The artificial nanometalloenzyme

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with lipase acted as the core and metallo-organic catalyst embedded in micropore

31

exhibited a spherical structure of 30-50 nm in diameter. The artificial

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nanometalloenzyme showed high catalytic efficiency in dynamic kinetic resolution of

33

racemic primary amines or secondary alcohols, compared to one-pot catalytic reaction

34

of

35

nanometalloenzyme holds great promise for integrated enzymatic and heterogeneous

36

catalysis.

immobilized

lipase

and

free

metallo-organic

catalyst.

This

artificial

37 38 39

Keywords: Artificial nanometalloenzyme; 2-Methylimidazole metal-biosurfactant

40

nanocomposite; Cooperative tandem catalysis; Dynamic kinetic resolution; Lipase

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1. INTRODUCTION

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Natural metalloenzymes with high substrate specificity and high catalytic capacity

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have attracted a great deal of research interests in medical, chemical, and biological

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fields. Unfortunately, one major drawback of natural metalloenzymes is that they

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often exhibit a narrow substrate range, which limits their use in synthesis.1-3 Artificial

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metalloenzymes which combine the advantages of natural enzymes and metal

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catalysts have achieved significant progress in the last decade.4-6 Artificial

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metalloenzymes have so far been successfully applied to many types of catalytic

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reactions. In most cases, the construction of artificial metalloenzymes involves

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procedures of anchoring a metal catalyst to a protein or polypeptide by either a

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covalent bond, dative, or supramolecular interaction.7-9

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Nanomaterials as carriers for enzyme immobilization10, 11 are also promising for

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developing artificial metalloenzymes.12-14 For example, the metallo-organic catalyst

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immobilized on nanoporous materials exhibited high catalytic activity and

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enantioselectivity.15 Bäckvall and coworkers16 prepared an artificial metalloenzyme

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using silica mesocellular foams (MCFs) for co-immobilization of lipase and

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Palladium (Pd) nanoparticles with applications in dynamic kinetic resolution (DKR)

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of primary amines. Metal-biosurfactant nanocomposites (MBNs), a class of hybrid

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nanomaterials consist of metal ions and biosurfactant ligands, have showed promise in

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enzyme immobilization, protein encapsulation, drug delivery, and biomedicine.17-19

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Compared to hard nanomaterials such as metal-organic frameworks (MOFs) and

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nanoflowers, MBNs, as a new kind of soft hydrophobic nanomaterials, is very

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suitable for inclusion of hydrophobic catalysts and catalytic reactions in non-aqueous

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systems.19-21 Owing to the amphipathic confined environment, morphological

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variability, and metal-ligand diversity, MBNs could be an ideal carrier for the rational

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design of artificial nanometalloenzyme.

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Enantiomerically pure amines and alcohols are widely used as building blocks in

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synthetic organic chemistry, and there is a great challenge of synthesizing

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enantiomerically pure compounds in an efficient and convenient manner.22-24 Shvo's

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catalyst, a cyclopentadienone-ligated diruthenium complex, is an efficient catalyst for

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the racemization of primary amines and secondary alcohols. Shvo's catalyst has

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therefore been used in one-pot reaction with lipase for synthesizing enantiomerically

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pure compounds in DKR processes.25-27 In DKR processes, lipase selectively

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transforms only one enantiomer of racemic mixture into the corresponding product,

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while the metallo-organic catalyst such as Shvo's catalyst catalyzed the racemization

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of the rest enantiomer, resulting in 100% maximum theoretical yield of the

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enantiomerically

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(±)-1-phenylethanol using the Shvo's catalyst immobilized on PTA-modified

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γ-alumina and Novozym435, obtaining a yield of 86.4% at 60℃ for 2 h. Mavrynsky et

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al.26 performed the DKR of (±)-1-phenylethylamine on a large scale using

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Novozyme435 and free Shvo's catalyst at 105℃ and 130 mbar for 24 h in a soxhlet

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apparatus, obtaining a yield of 65%.

pure

product.29-30

Im

et

al.25

performed

the

DKR

of

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In this paper, MBNs, as a carrier of artificial nanometalloenzyme, was used for

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synchronizing encapsulated lipase and Shvo's catalyst in mild reaction conditions

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using one-pot self-assembly method. In order to adapt to the alkaline catalytic

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conditions of racemic reaction and increase the encapsulated load of hydrophobic

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catalyst, alkaline 2-methylimidazole (Mi) was added to the self-assembly conditions

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as a metal coordination reagent. 2-Methylimidazole metal-biosurfactant nanocomposite

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(MiMBN) increased the encapsulated load of lipase and Shvo's catalyst. Compared to

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one-pot catalytic reaction of immobilized CALB and free Shvo's catalyst, the artificial

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nanometalloenzyme CALB-Shvo@MiMBN improved the catalytic efficiency in DKR

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of primary amines and secondary alcohols.

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2. RESULTS AND DISCUSSION

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2.1. Synthesis of CALB-Shvo@MiMBN. In this study, we reported an artificial

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nanometalloenzyme, which was constructed by the co-encapsulation of lipase and

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Shvo's catalyst in nanocomposites consisting of Mi, metal ion, and biosurfactant

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(Scheme 1). In the nanocomposites, Mi can coordinate with metal ions to generate a

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meta-organic framework,31-33 which thereby increased the loading amounts and

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activities of lipase and Shvo's catalyst (Figure. S1 and Table S1). Nitrogen

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adsorption–desorption isotherms of metal-biosurfactant nanocomposite (MBN) and

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2-methylimidazole metal-biosurfactant nanocomposite (MiMBN) revealed differences

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in nanostructures. The MBN and MiMBN displayed a type IV isotherm with H3

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hysteresis, a type of porous materials with slit aperture openings (Figure. S1). The

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introduction of Mi into MBN in one-pot self-assembly process decreased the pore

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diameter from 26.51 to 20.74 nm, increased the pore volume from 0.2804 to 0.5639

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cm3 g-1, and increased the specific surface area from 42.03 to 92.72 m2 g-1 (Table S1).

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Mi located in the pore diameter occupied the pore size of the slit aperture openings

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and reduced the pore diameter. Mi located inside the pore volume enlarged the pore

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volume at the same time. The increase in the pore volume and the specific surface

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area favors encapsulating larger amounts of lipase and Shvo's catalyst and thereby

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facilitates the formation of the artificial nanometalloenzyme. This artificial

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nanometalloenzyme allowed the highly efficient DKR processes of primary amines

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and secondary alcohols in cooperative tandem catalysis.

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We reasonably designed a novel artificial nanometalloenzyme, as shown in

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Scheme 1. Enzyme acted as the core which induced the self-assembly of NaDC, Mi

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and Co2+ around the protein surface.19,34,35 The metal ion was the linker to assemble

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the nanocomposite. Mi generated metal ligand bonds with metal ions, which

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promoted encapsulation of a larger amount of the lipase. The CH3– group of Mi

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increased the internal hydrophobicity of MBN, which promoted encapsulating a larger

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amount of Shvo's catalyst. Mi increased the pore volume and the hydrophobicity of

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the hydrophobic area of MBN, which also increased the amount of encapsulated

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Shvo's catalyst (Scheme 1), in one-pot self-assembly system. Due to the hydrophobic

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interaction with NaDC, the Shvo's catalyst was encapsulated in MiMBN at the same

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time.

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2.2. Experimental characterization. Scanning electron microscopy (SEM)

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images and transmission electron microscopy (TEM) images (Figure. 1) suggested

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that the MiMBN exhibited a typical coiled rod-shaped morphology with an average

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length of >1 μm and a diameter of ~50 nm (Figure. 1a-b). The CALB@MiMBN

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exhibited a typical spherical structure of 30-50 nm in diameter (Figure. 1cd). The

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CALB-Shvo@MiMBN showed a similar morphology as CALB@MiMBN in SEM

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image (Figure. 1e), but there were obvious differences in TEM images (Figure. 1f). It

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is possible that the Shvo's catalyst was encapsulated in the hydrophobic pore of the

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flexible framework structure. Energy-dispersive X-ray spectroscopy (EDS) mapping

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under TEM (Figure. S2) also displayed the density of Ru and S elements which are

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from Shvo's catalyst and CALB, proving the successful encapsulation.

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Fourier-transform infrared spectroscopy (FTIR) (Figure. 2a) confirmed the

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chemical composition of the CALB-Shvo@MiMBN, indicating the formation of

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complexes and the presence of CALB and Shvo's catalyst in nanocomposites. We

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observed absorption bands of free CALB at 1675 cm−1 corresponding to C=O

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stretching in the amide-I region and of free Shvo's catalyst at 1963, 2005, and 2041

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cm−1 corresponding to γ C=O stretching. Therefore, the characteristic bands of

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CALB-Shvo@MiMBN at 1675, 1963, 2005, and 2041 cm−1 demonstrated the

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presence of CALB and Shvo’s catalyst in the nanocomposite.36,37 Thermal gravity

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analysis (TGA) in a nitrogen atmosphere of MiMBN, CALB@MiMBN, and

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CALB-Shvo@MiMBN revealed the different decomposition processes (Figure. 2b).

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The CALB-Shvo@MiMBN exhibited 15% higher weight loss than the MiMBN in the

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temperature range of 350-450℃, proving the presence of CALB and Shvo's catalyst in

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the nanocomposite. Elemental analyses by inductively coupled plasma mass

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spectrometry (ICP-MS) and protein concentration analyses by the Bradford method

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showed that the loading amounts of CALB and Shvo's catalyst on the

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CALB-Shvo@MiMBN were 15.21 wt% and 32.42 wt%, respectively (Table S2).

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Molar ratio of CALB and Shvo's catalyst on the CALB-Shvo@MiMBN was 1:65.7,

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which indicated that the high ratio Shvo's catalyst could promote racemization. These

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results confirmed that the loading amounts of lipase in MiMBN was much higher than

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that in MBN19, suggesting that MiMBN was efficient for immobilization of CALB

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and Shvo's catalyst, as 90% of the feeding enzyme and 81% of the feeding Shvo's

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catalyst were successfully encapsulated. We estimated that the volume occupied by

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lipase was larger than 675 nm3. The size of CALB is 3 nm×4 nm×5 nm and lower

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than the volume occupied by lipase. So we believed that there are several enzymes in

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the artificial nanometalloenzyme. The content of Co2+ and Na+ in MiMBN and

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CALB-Shvo@MiMBN was determined with ICP-MS as 174.9 mg g-1 and 1.718 mg

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g-1, and 74.11 mg g-1 and 0.4721 mg g-1, respectively (Table S2). The results were

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consistent with the TGA analysis. X-ray diffraction (XRD) results indicated that the

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MiMBN had no obvious crystal peaks (Figure. S3), indicating an amorphous

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structure.

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To further prove that the Shvo's catalyst was embedded in the nanocomposite

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microporous structure, nitrogen adsorption–desorption isotherms of CALB@MiMBN

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and CALB-Shvo@MiMBN were performed (Figure. 2cd). The CALB@MiMBN

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and CALB-Shvo@MiMBN displayed a type IV isotherm with H3 hysteresis, typical

175

of

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Barrett–Joyner–Halenda (BJH) method were used to calculate the micropore and

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mesopore size distributions of CALB@MiMBN and CALB-Shvo@MiMBN,

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respectively (Figure. 2d).37,38 As shown in Table S3, the CALB-Shvo@MiMBN had a

porous

materials.

The

Horvaih–Kawazoe

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(HK)

method

and

the

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larger BET surface area of 37.04 m2 g-1, micropore volume of 0.0121 cm3 g-1, and

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mesopore volume of 0.1439 cm3 g-1 compared to the CALB@MiMBN. However, the

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micropore diameter of CALB-Shvo@MiMBN (0.07868 nm) was smaller than that of

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CALB@MiMBN (1.131 nm). The micropore volume ratio of total pore volume of

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CALB-Shvo@MiMBN was 7.756%, smaller than that of CALB@MiMBN (10.52%).

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We observed the decrease in micropore volume ratio of CALB-Shvo@MiMBN,

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which proved that the microporous structure was occupied by Shvo's catalyst. The

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introduction of Shvo's catalyst in CALB@MiMBN increased the mesopore diameter

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from 3.052 to 17.52 nm (Table S3 and Figure. S5), which is favorable for the

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transportation of substrates and products.

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X-ray photoelectron spectroscopy (XPS) was performed to investigate the

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chemical composition of MiMBN, CALB@MiMBN, and CALB-Shvo@MiMBN. In

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XPS analysis (Figure. 3a), the MiMBN, CALB@MiMBN and CALB-Shvo@MiMBN

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showed a predominant narrow C 1s peak at ca. 284.8 eV, an O 1s peak at ca. 532.2 eV,

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an N 1s peak at ca. 400.1 eV, and a Co 2p peak at ca. 781.2 eV. The Co 2p3/2 XPS

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spectra of MiMBN, CALB@MiMBN, and CALB-Shvo@MiMBN (Figure. 3bd) can

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be

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CALB-Shvo@MiMBN at 780.8 eV corresponded to a Co-O bond, deriving from

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coordination of Co2+ with NaDC; the peak at 782.3 eV was attributed to the Co-N

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bond, deriving from coordination of Co2+ with Mi; and the peak at 785.6 eV was a

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satellite peak. The Co 2p3/2 XPS spectra of the CALB@MiMBN at 780.7, 782.0, and

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785.3 eV corresponded to the Co-O bond, Co-N bond, and satellite peak, respectively.

deconvolved

into

three

subpeaks.

The

Co

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2p3/2

XPS

spectra

of

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The Co 2p3/2 XPS spectra of MiMBN at 781.0, 782.2, and 785.4 eV were attributed to

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the Co-O bond, Co-N bond, and satellite peak, respectively. The peak shifts of Co

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2p3/2 were caused by adsorption of Co2+ with lipase.39,40 Figure. 3eh showed the

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high-resolution

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CALB-Shvo@MiMBN. XPS wide scan spectra of Shvo's catalyst was shown in Fig.

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S5. The Ru 3p peak in the CALB-Shvo@MiMBN (Figure. 3f) showed weak signal

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strength compared to the Shvo's catalyst (Figure. 3e), and the Ru 3d peak in the

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CALB-Shvo@MiMBN (Figure. 3h) had different peak signals from the Shvo's

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catalyst (Figure. 3g), which proved that the Shvo's catalyst (Figure. 3e) was located in

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the pores of the nanocomposite.41,42 The C 1s, N 1s and O 1s peaks of MiMBN,

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CALB@MiMBN, and CALB-Shvo@MiMBN were given in Figure. S6. We can

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observe the C=O/COO-bond in the O 1s XPS spectra (Figure. S6) of

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CALB@MiMBN because adsorption and metal coordination of Co2+ by lipase may

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result in C=O/COO- bonds being exposed to the surface of nanocomposite. The

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addition of Shvo's catalyst covered C=O/COO- groups exposed to the surface of

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nanocomposite.

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CALB-Shvo@MiMBN, we found that adsorption and metal coordination of Co2+ by

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encapsulated lipase and Shvo's catalyst influenced the peak shifts.43,44 XPS of

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CALB@MiMBN and CALB-Shvo@MiMBN (Figure 3a) did not detect P and S

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characteristic element peaks, which indicated that CALB is not on the surface and is

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on the inside of nanocomposite. Previous studies have shown that enzymes, as core,

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are located within nanocomposite.19-21 XPS of CALB-Shvo@MiMBN (Figure 3a)

Ru

3p

From

and

the

Ru

XPS

3d

of

spectrum

MiMBN,

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of

Shvo's

catalyst

CALB@MiMBN,

and

and

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detected Ru characteristic element peaks, which proved that Shvo's catalyst is on the

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surface of nanocomposite.

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2.3. Cooperative tandem catalysis. The artificial nanometalloenzyme

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CALB-Shvo@MiMBN was subjected to DKR of racemic primary amines or

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secondary alcohols. In a typical experiment, we performed the DKR of

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(±)-1-phenylethanol under argon (1 bar) in dry toluene. The results showed that the

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artificial

230

efficiency and ee value at 70 ℃

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CALB-Shvo@MiMBN was 10% and 12% higher than that of one-pot catalytic

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reactions of CALB@MiMBN and free Shvo's catalyst as well as Novozym435 and

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free Shvo's catalyst, respectively. High catalytic efficiency was maintained even

234

though the additive amount of CALB-Shvo@MiMBN and reaction temperature were

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reduced (Table 1). The results demonstrated that the CALB-Shvo@MiMBN is a

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highly efficient catalyst for DKR of (±)-1-phenylethanol.

nanometalloenzyme

CALB-Shvo@MiMBN

had

excellent

catalytic

as the reaction time at 2 h, and yield of

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Racemization of racemic primary amines requires a higher temperature and

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longer reaction time than that of racemic secondary alcohols, and produces more

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by-products using the same catalyst.45,46 We carried out the DKR process of

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(±)-1-phenylethylamine under argon (130 mbar) in dry toluene using the

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CALB-Shvo@MiMBN (Table 2). The results showed that the CALB-Shvo@MiMBN

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effectively catalyzed the DKR of racemic primary amine at 90℃ as the reaction time

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at 6 h, the yield of CALB-Shvo@MiMBN was 4% and 6% higher than that of one-pot

244

catalytic reaction of immobilized CALB and free Shvo's catalyst. However, the

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decreased reaction temperature and the additive amount of the CALB-Shvo@MiMBN

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were not conducive to the catalytic reaction. Racemization catalyzed by Shvo's

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catalyst is a rate-limiting step; an increase in reaction time gave a marginally

248

improved conversion. In the initial reaction stage, increasing the amount of catalyst

249

can enhance the catalytic efficiency, but the total catalytic efficiency does not increase

250

significantly at the same catalytic long time. Figure 4 showed the reaction time curves

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of DKR of (±)-1-phenylethylamine using three different catalysts. It is observed that

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the CALB-Shvo@MiMBN was the best catalyst for DKR of (±)-1-phenylethylamine,

253

compared to the one-pot catalytic reactions of immobilized CALB and free Shvo's

254

catalyst. The catalytic efficiency of CALB-Shvo@MiMBN increased 17.4% and

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7.8% compared with the one-pot catalytic reactions of CALB@MiMBN/Shvo and

256

Novozym435/Shvo at 90℃ as the reaction time at 4 h, respectively. Lipase can

257

rapidly consume one chiral enantiomer, and slowly catalyze another chiral enantiomer.

258

At long reaction time, the DKR of (±)-1-phenylethylamine catalyzed by

259

CALB-Shvo@MiMBN would show slightly lower ee value than in one-pot catalytic

260

reactions of CALB@MiMBN/Shvo and Novozym435/Shvo. The value of apparent

261

activation energy was calculated according to the Arrhenius equation. In the DKR of

262

(±)-1-phenylethanol

263

CALB-Shvo@MiMBN, the value of apparent activation energy was 10.68 and 14.37

264

kJ

265

(±)-1-phenylethylamine, the value of TOF (defined as µmol product per µmol CALB

266

protein per hour) was 1708 and 1182 h-1, respectively. Differences of apparent

mol-1,

and

respectively.

(±)-1-phenylethylamine

In

the

DKR

of

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catalyzed

by

(±)-1-phenylethanol

the

and

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activation energy and TOF determine differential catalytic capacity.

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The artificial nanometalloenzyme increased the reaction rate and shortened the

269

reaction time compared to the reported one-pot reaction system using free Shvo's

270

catalyst and Novozym435 under the same conditions.23,26 According to the loading

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amounts of CALB and Shvo's catalyst on the CALB-Shvo@MiMBN, the diameter of

272

CALB-Shvo@MiMBN, and the density of 1.06 g cm-3 of CALB-Shvo@MiMBN, We

273

estimated that the distance between CALB and Shvo's catalyst was less than 8.5 nm.

274

The artificial nanometalloenzyme CALB-Shvo@MiMBN had the better catalytic

275

efficiency than one-pot catalytic reactions in cooperative catalysis because of the

276

shorter distances between two functions. The racemic primary amines or secondary

277

alcohols entered the artificial nanometalloenzyme and exposed to CALB, leading to

278

the selective acylation of (R)-enantiomers. The rest (S)-enantiomers of primary

279

amines or secondary alcohols were then racemized by Shvo's catalyst encapsulated in

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the artificial nanometalloenzyme, followed by the CALB-catalyzed selective

281

acylation. NaDC as a surfactant could enhance the activity of lipase in selective

282

acylation of (R)-enantiomer of the racemic secondary alcohols or primary amines, and

283

then the (S)-enantiomer of racemic secondary alcohol or primary amines rapidly

284

accumulated. Mi increased the alkaline characteristics of CALB-Shvo@MiMBN

285

microenvironment, which helps to reduce the formation of by-products of

286

racemization and increase the catalytic efficiency of racemization. A high local

287

concentration of Shvo's catalyst inside CALB-Shvo@MiMBN improved the

288

racemization efficiency. In addition the proximity of CALB and Shvo's catalyst

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increased the transportation of reaction intermediate in the tandem catalysis.47-49

290

After five consecutive reuses, the CALB-Shvo@MiMBN still maintained its

291

catalytic activity. Harsh reaction conditions which caused partial deactivation of

292

artificial nanometalloenzyme in the DKR of (±)-1-phenylethylamine, the recyclability

293

of CALB-Shvo@MiMBN in DKR of (±)-1-phenylethylamine was worse than that of

294

(±)-1-phenylethanol (Figure 5). Organic solvents and magnetic mechanical stirring

295

would cause the leaching of encapsulated lipase and Shvo's catalyst of

296

[email protected] Results of leaching showed that the Shvo's catalyst and

297

lipase have slight leaching in toluene at each reuse. However, leaching of lipase and

298

Shvo's catalyst in DKR of (±)-1-phenylethanol was lower than that of

299

(±)-1-phenylethylamine (Figure S7). Leaching led to a decrease in catalytic activity,

300

but the remaining encapsulated lipase and Shvo's catalyst in CALB-Shvo@MiMBN

301

still maintained a high catalytic activity. Catalytic conditions are the key factors

302

determining

303

nanometalloenzyme has potential for industrial application

304

3. CONCLUSIONS

the

lifetime

of

artificial

nanometalloenzyme.

The

artificial

305

We developed a novel highly efficient artificial nanometalloenzyme by

306

synchronous encapsulating a metallo-organic catalyst and an enzyme into MiMBN.

307

Mi increased the alkaline characteristics of CALB-Shvo@MiMBN microenvironment

308

and improved the encapsulated load of lipase and Shvo's catalyst, which helps to

309

reduce the formation of by-products of racemization and increase the catalytic

310

efficiency of racemization. The proximity effect of Shvo's catalyst and CALB resulted

311

in an enhanced efficiency in the DKR of racemic primary amines and secondary

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312

alcohols. The design of co-encapsulating enzymes and heterogeneous catalysts in

313

MiMBNs holds great promise for creating novel bio-chemo hybrid catalysts with

314

many potential applications in cooperative tandem catalysis.

315

4. EXPERIMENTAL SECTION

316

4.1.

Materials.

1-Hydroxytetraphenylcyclopentadienyl-

(tetraphenyl-2,4-

317

cyclopentadien-1-one)-μ-hydrotetracarbonyldiruthenium (II) (Shvo's catalyst) was

318

purchased from Sigma–Aldrich (St. Louis, MO, USA). Sodium deoxycholate,

319

(R)-2-methoxy-N-(1-phenylethyl)

320

acetamide,

321

(±)-1-phenylethanol and isopropenyl acetate were purchased from Aladdin (Shanghai,

322

China). 2-Methylimidazole (Mi) was purchased from TCI (Tokyo, Japan).

323

Lipozyme®CALB L and Novozym435 were obtained from Novozymes (Copenhagen,

324

Denmark). Cobalt chloride hexahydrate (CoCl2·6H2O), Na2CO3 and potassium

325

tert-butoxide

326

Engineering-technological Research and Development Center (Guangzhou, China).

327

All other reagents were of analytical reagent grade, and used as received. Ultrapure

328

water (18.2 MΩ; Millpore Co., USA) was used throughout the experiment.

toluene,

isopropyl

(tBuOK)

was

acetamide, acetate,

got

(S)-2-methoxy-N-(1-phenylethyl)

pentadecane,

from

(±)-1-phenylethylamine,

Guangdong

Chemical

Reagent

329

4.2. Synthesis of MiMBN, CALB@MiMBN and CALB-Shvo@MiMBN. In a

330

typical experiment, 10 mL of CoCl2·6H2O aqueous solution (20 mM) was added into

331

10 mL of aqueous NaDC (15 mM) solution with 15 mM Mi, 12 μM Shvo's catalyst

332

(dissolved in N,N-dimethylformamide) and 2 mL CALB-L soultion. The mixture was

333

stirred at 300 rpm for 30 min at 25℃, followed by centrifugation at 7000 rpm for 10

334

min and washing twice by water to obtain nanocomposite. MiMBN does not contain

335

lipase and Shvo's catalyst, CALB@MiMBN contains lipase that does not contain

336

Shvo's catalyst and CALB-Shvo@MiMBN contains lipase and Shvo's catalyst.

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Page 16 of 35

337

4.3. Characterization. Field-emission scanning electron microscopy (FE-SEM)

338

(SEM) was performed using a Nova NanoSEM 450 (FEI, USA) using an accelerating

339

voltage of 5 kV with fit magnification. Transmission electron microscopy (TEM) was

340

carried out using a JEM-200CX analytical transmission electron microscope (Japan).

341

A Micromeritics 3Flex surface analyzer (USA) was used to analyze BET surface area,

342

pore volume and pore diameter and adsorption-desorption isotherm. Element contents

343

were analyzed by using the inductively coupled plasma mass spectrometry (ICP-MS)

344

from Aglient 7500a (USA). The chemical functional groups were analyzed by a

345

Thermo Corporation Nexus FTIR spectrophotometer (USA). TGA was performed on

346

a TGA Q500 thermogravimetric analyzer (TA, USA). The sample was heated from 25

347

°C to 450 °C at a rate of 10 °C/min under N2 atmosphere. X-ray diffraction (XRD)

348

was performed on a Rigaku Ultima IV diffractometer with Cu Kα X-rays. X-ray

349

photoelectron spectroscopy (XPS) was performed using a Kratos AXIS Ultra DLD

350

XPS system equipped with a hemispherical energy analyzer and a monochromatic Al

351

Kα source. The source was operated at 15 keV and 150 W; pass energy was fixed at

352

40 eV for the high-resolution scans. All samples were prepared as pressed powders

353

supported on a metal bar for the XPS measurements.

354

4.4.

General

procedure

for

dynamic

kinetic

resolution

of

355

(±)-1-phenylethanol. A dried 10-mL Schlenk tube was charged with the

356

CALB-Shvo@MiMBN (50 mg or 25 mg), dry Na2CO3 (25 mg) and tBuOK (25 mg).

357

Dry toluene (1 mL), (±)-1-phenylethanol (0.30 mmol), isopropenyl acetate (2.0 equiv),

358

and pentadecane (15 μL) were added subsequently. The vessel was closed, evacuated,

359

and backfilled with argon gas three times. The reaction mixture was stirred at 50, 60

360

and 70 °C for 2 and 4 h at 300 rpm. The reaction was cooled to room temperature; the

361

solids were removed by centrifugation and samples were diluted by toluene.

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362

ACS Applied Materials & Interfaces

4.5.

General

procedure

for

dynamic

kinetic

resolution

of

363

(±)-1-phenylethylamine. A dried 10-mL Schlenk tube was charged with the

364

CALB-Shvo@MiMBN (50 mg or 25 mg) and dry Na2CO3 (50 mg). Dry toluene (1

365

mL), (±)-1-phenylethylamine (0.30 mmol), isopropyl acetate (4.0 equiv), and

366

pentadecane (15 μL) were added subsequently. The vessel was closed, evacuated, and

367

backfilled with argon three times. The reaction mixture was stirred at 80 and 90 °C

368

for 6, 8, and 12 h at 300 rpm. The reaction was cooled to room temperature; the solids

369

were removed by centrifugation and samples were diluted by toluene.

370

4.6. Recyclability and leaching test of lipase and Shvo's catalyst of

371

CALB-Shvo@MiMBN. According to the general procedure for DKR of

372

(±)-1-phenylethanol or (±)-1-phenylethylamine, recyclability and leaching of lipase

373

and Shvo's catalyst of CALB-Shvo@MiMBN were tested. For (±)-1-phenylethanol,

374

the reaction was performed at 60°C for 2 h at 300 rpm; for (±)-1-phenylethylamine,

375

the reaction was performed at 90°C for 4 h at 300 rpm. After each test, toluene was

376

used to wash the CALB-Shvo@MiMBN three times to carry out the next test.

377

ICP-MS and Bradford method was used to determine Shvo's catalyst and lipase.

378

4.7. Determination of chiral gas chromatography. The enantiomeric excess

379

was determined by analytical GC (Agilent, USA) employing a CP-Chirasil-DEX CB

380

column (25 m × 0.32 mm, Agilent, USA). Chiral GC-analysis: The carrier gas was

381

helium; the velocity was 1.6 mL/min, injector and detector 250°C, program: 100°C /5

382

min/ 155°C /3°C min-1, 5 min/ 200°C /20°C min-1, 5 min.

383 384

Supporting Information

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Page 18 of 35

385

Figure S1 (a) Nitrogen adsorption-desorption isotherms of MBN and MiMBN at 77

386

K; (b) Barrett−Joyner−Halenda (BJH) pore size distribution calculated from the

387

adsorption branch of the isotherms of MBN and MiMBN.

388

Figure S2. Energy-dispersive X-ray spectroscopy (EDS) diagram in TEM of

389

CALB-Shvo@MiMBN.

390

Figure S3. X-Ray Diffraction (XRD) diagrams of MiMBN, CALB@MiMBN, and

391

CALB-Shvo@MiMBN.

392

Figure S4. Density Functional Theory (DFT) method mesopore size distribution from

393

BET of CALB@MiMBN and CALB-Shvo@MiMBN.

394

Figure S5. XPS spectra of Shvo's catalyst.

395

Figure S6. C 1s, N 1s, and O 1s spectra of (a, d, g) MiMBN, (b, e, f)

396

CALB@MiMBN, and (c, f, i) CALB-Shvo@MiMBN from XPS.

397

Figure S7. The leaching of lipase and Shvo's catalyst of CALB-Shvo@MiMBN

398

during

399

(±)-1-phenylethylamine (b).

400

Table S1. Brunauer−Emmett−Teller (BET) surface area, pore volume, and pore

401

diameter of MBN and MiMBN.

402

Table S2. The content of CALB, Shvo's catalyst, Co2+, and Na+ in MiMBN and

403

CALB-Shvo@MiMBN.

404

Table S3. Brunauer−Emmett−Teller (BET) surface area, pore volume, and pore

405

diameter of CALB@MiMBN and CALB-Shvo@MiMBN.

dynamic

kinetic

resolution

of

(±)-1-phenylethanol

406 407

AUTHOR INFORMATION

408

Corresponding Authors

409

*E-mail:

[email protected]

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(a)

or

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410

Notes

411

The authors declare no competing financial interest.

412 413

ACKNOWLEDGMENTS

414

The authors acknowledge the financial supports of the National Natural Science

415

Foundation of China (21706126, 21606127, and 21576134), the National Key

416

Research and Development Program (2016YFA0204300), and the Jiangsu Synergetic

417

Innovation Center for Advanced Bio-Manufacture (XTE1853).

418 419

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Resolution of Alcohols and Amines. Eur. J. Org. Chem. 2010, 41, 999-1015.

557

(46) Hoben, C. E.; Kanupp, L.; Bäckvall, J. E. Practical Chemoenzymatic Dynamic

558

Kinetic Resolution of Primary Amines via Transfer of a Readily Removable

559

Benzyloxycarbonyl Group. Tetrahedron Lett. 2008, 49, 977-979.

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(47) Kim, H.; Choi, Y. K.; Lee, J.; Lee, E.; Park, J.; Kim, M. J.

561

Ionic-Surfactant-Coated Burkholderia cepacia Lipase as a Highly Active and

562

Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols.

563

Angew. Chem. Int. Ed. 2011, 50, 10944-10948.

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(48) Pollock, C. L.; Fox, K. J.; Lacroix, S. D.; McDonagh, J.; Marr, P. C.; Nethercott,

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A. M.; Pennycook, A.; Qian, S.; Robinson, L.; Saunders, G. C.; Marr, A. C.

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Minimizing Side Reactions in Chemoenzymatic Dynamic Kinetic Resolution:

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Organometallic and Material Strategies. Dalton Trans. 2012, 41, 13423-13428.

568

(49) Zisis, T.; Freddolino, P. L.; Turunen, P.; van Teeseling, M. C. F.; Rowan, A. E.;

569

Blank, K. G. Interfacial Activation of Candida antarctica Lipase B: Combined

570

Evidence from Experiment and Simulation. Biochemistry 2015, 54, 5969-5979.

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Figure legends

584 585

Scheme 1. Preparation and structure of the artificial nanometalloenzyme via

586

co-encapsulation

587

2-methylimidazole-metal-biosurfactant nanocomposite.

588

Figure 1. (a) Scanning electron microscopy (SEM) image and (b) transmission

589

electron microscopy (TEM) image of MiMBN. (c) SEM image and (d) TEM) image

590

of CALB@MiMBN. (e) SEM image and (f) TEM image of CALB-Shvo@MiMBN.

591

Figure 2. (a) Thermal gravity analysis (TGA) curves of MiMBN, CALB@MiMBN,

592

and CALB-Shvo@MiMBN in a nitrogen atmosphere. (b) Fourier-transform infrared

593

spectroscopy (FTIR) of free CALB, free Shvo's catalyst, MiMBN, CALB@MiMBN,

594

and CALB-Shvo@MiMBN. (c) Nitrogen adsorption–desorption isotherms from

595

microporous

596

CALB-Shvo@MiMBN at 77 K; (d) Horvaih−Kawazoe (HK) micropore and

597

Barrett−Joyner−Halenda (BJH) mesopore size distributions calculated from the

598

adsorption branch of the isotherms of CALB@MiMBN and CALB-Shvo@MiMBN.

599

Figure 3. (a) XPS analysis of MiMBN, CALB@MiMBN and CALB-Shvo@MiMBN.

600

(b-d) Co 2p spectra of MiMBN, CALB@MiMBN and CALB-Shvo@MiMBN. (e-f)

601

Ru 3p spectra of Shvo's catalyst and CALB-Shvo@MiMBN. (g-h) Ru 3d spectra of

602

Shvo's catalyst and CALB-Shvo@MiMBN.

603

Figure

604

(±)-1-phenylethylamine by CALB-Shvo@MiMBN, CALB@MiMBN/Shvo, and

605

Novozym435/Shvo.

606

Figure 5. Recyclability study for dynamic kinetic resolution of (±)-1-phenylethanol

607

or (±)-1-phenylethylamine by CALB-Shvo@MiMBN.

4.

of

a

metallo-organic

Brunner−Emmet−Teller

Comparative

study

catalyst

(BET)

for

of

dynamic

608

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and

an

enzyme

CALb@MiMBN

kinetic

resolution

into

and

of

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Figures

609 610

611 612

Scheme 1.

613 614 615 616 617 618 619 620 621 622

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a

b

c

d

e

f

623

624

625 626

Figure 1.

627 628 629 630 631

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a

b b

632

c

d

633 634

Figure 2.

635 636 637 638 639 640 641 642

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a

b

643

c

d

e

f

g

h

644

645

646 647

Figure 3.

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648 649

Figure 4.

650 651 652 653 654 655 656 657 658 659 660 661

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662 663

Figure 5.

664 665 666 667 668 669 670 671 672 673 674 675 676 677

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ACS Applied Materials & Interfaces

Table

678 679

Table 1. Summary of results from the DKR of (±)-1-phenylethanol catalyzed by the

680

CALB-Shvo@MiMBN, the one-pot reaction of CALB@MiMBN and free Shvo's

681

catalyst and the one-pot reaction of Novozym435 and free Shvo's catalyst.[a]

682

Entry

Catalyst

T [℃]

t [h]

Yield [%][b]

ee [%][b]

1

CALB-Shvo@MiMBN

70

2

92

99

2

CALB-Shvo@MiMBN

70

4

98

99

3

CALB-Shvo@MiMBN

60

2

89

99

4

CALB-shvo@MiMBN

60

4

95

99

5

CALB-Shvo@MiMBN

50

2

83

99

6

CALB-Shvo@MiMBN

50

4

92

99

7

CALB-Shvo@MiMBN[c]

70

4

95

99

8

CALB@MiMBN/Shvo

70

2

82

99

9

Novozym435/Shvo

70

2

80

99

683

[a] Reaction conditions: All of the reactions were carried out in dry toluene (1 mL)

684

under 1 bar argon gas environment, catalyst (50 mg), (±)-1-phenylethanol (0.30

685

mmol), isopropenyl acetate (2.0 equiv), dry Na2CO3 (25 mg), tBuOK (25 mg),

686

pentadecane (15 μL) as internal standard, and 300 rpm. [b] Determined by GC

687

analysis. [c] Catalyst was 25 mg.

688

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689

Table 2. Summary of results from the DKR of (±)-1-phenylethylamine catalyzed by

690

the CALB-Shvo@MiMBN, the one-pot reaction of CALB@MiMBN and free Shvo's

691

catalyst and the one-pot reaction of Novozym435 and free Shvo's catalyst.[a] Entry

Catalyst

T[℃]

t [h]

Yield [%][b]

ee[%][b]

1

CALB-Shvo@MiMBN

90

6

76

98

2

CALB-Shvo@MiMBN

90

8

78

96

3

CALB-Shvo@MiMBN

90

12

79

92

4

CALB-Shvo@MiMBN

80

12

67

96

5

CALB-Shvo@MiMBN[c]

90

8

69

95

6

CALB@MiMBN/Shvo

90

8

72

94

7

Novozym435/Shvo

90

8

68

98

692

[a] Reaction conditions: All of the reactions were carried out in dry toluene (1 mL)

693

under 130 mbar argon gas environment, catalyst (50 mg), ( ± )-1-phenylethylamine

694

(0.30 mmol), isopropyl acetate (4.0 equiv), dry Na2CO3 (50 mg), pentadecane (15 μL)

695

as internal standard, and 300 rpm. [b] Determined by GC analysis. [c] Catalyst was 25

696

mg.

697 698 699 700 701 702

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705

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