Artificial Nanometalloenzymes for Cooperative Tandem Catalysis

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Energy, Environmental, and Catalysis Applications

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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ACS Paragon Plus Environment

ACS Applied Materials & Interfaces 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

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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

28

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

29

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

30

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

32

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

41 42 43 44 45


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46

1. INTRODUCTION

47

Natural metalloenzymes with high substrate specificity and high catalytic capacity

48

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

49

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

50

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

51

metalloenzymes which combine the advantages of natural enzymes and metal

52

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

53

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

54

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

55

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

56

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

57

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

58

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

59

immobilized on nanoporous materials exhibited high catalytic activity and

60

enantioselectivity.15 Bäckvall and coworkers16 prepared an artificial metalloenzyme

61

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

62

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

63

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

64

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

65

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

66

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

67

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



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68

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

69

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

70

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

71

design of artificial nanometalloenzyme.

72

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

73

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

74

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

75

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

76

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

77

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

78

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

79

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

80

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

81

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

82

enantiomerically

83

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

84

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

85

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

86

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

87

apparatus, obtaining a yield of 65%.

pure

product.29-30

Im

et

al.25

performed

the

DKR

of

88

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

89

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


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90

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

91

conditions of racemic reaction and increase the encapsulated load of hydrophobic

92

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

93

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

94

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

95

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

96

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

97

of primary amines and secondary alcohols.

98

2. RESULTS AND DISCUSSION

99

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

100

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

101

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

102

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

103

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

104

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

105

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

106

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

107

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

108

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

109

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

110

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

111

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



112

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

113

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

114

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

115

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

116

facilitates the formation of the artificial nanometalloenzyme. This artificial

117

nanometalloenzyme allowed the highly efficient DKR processes of primary amines

118

and secondary alcohols in cooperative tandem catalysis.

119

We reasonably designed a novel artificial nanometalloenzyme, as shown in

120

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

121

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

122

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

123

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

124

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

125

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

126

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

127

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

128

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

129

time.

130

2.2. Experimental characterization. Scanning electron microscopy (SEM)

131

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

132

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

133

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

134

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


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135

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

136

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

137

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

138

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

139

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

140

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

141

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

142

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

143

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

144

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

145

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

146

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

147

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

148

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

149

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

150

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

151

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

152

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

153

the nanocomposite. Elemental analyses by inductively coupled plasma mass

154

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

155

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

156

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



Page 8 of 35

157

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

158

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

159

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

160

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

161

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

162

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

163

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

164

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

165

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

166

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

167

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

168

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

169

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

170

structure.

171

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

172

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

173

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

174

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

175

of

176

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

177

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

178

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

porous

materials.

The

Horvaih–Kawazoe


(HK)

method

and

the

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179

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

180

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

181

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

182

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

183

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

184

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

185

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

186

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

187

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

188

transportation of substrates and products.

189

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

190

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

191

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

192

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

193

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

194

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

195

be

196

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

197

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

198

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

199

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

200

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

deconvolved

into

three

subpeaks.

The

Co


2p3/2

XPS

spectra

of


Page 10 of 35

201

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

202

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

203

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

204

high-resolution

205

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

206

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

207

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

208

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

209

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

210

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

211

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

212

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

213

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

214

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

215

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

216

nanocomposite.

217

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

218

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

219

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

220

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

221

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

222

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

Ru

3p

From

and

the

Ru

XPS

3d

of

spectrum

MiMBN,


of

Shvo's

catalyst

CALB@MiMBN,

and

and

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223

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

224

surface of nanocomposite.

225

2.3. Cooperative tandem catalysis. The artificial nanometalloenzyme

226

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

227

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

228

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

229

artificial

230

efficiency and ee value at 70 ℃

231

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

232

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

233

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

234

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

235

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

236

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

237

Racemization of racemic primary amines requires a higher temperature and

238

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

239

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

240

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

241

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

242

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

243

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



Page 12 of 35

245

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

246

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

247

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

251

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

252

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

255

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


catalyzed

by


the

and

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267


activation energy and TOF determine differential catalytic capacity.

268

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

271

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

280

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



289

Page 14 of 35

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


Page 15 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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.



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.


Page 17 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

362


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



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


406 407

AUTHOR INFORMATION

408

Corresponding Authors

409

*E-mail:

[email protected]


(a)

or

Page 19 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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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420

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Kinetic Resolution of 1-Phenylethanol over Ru Complexes Immobilized on

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PTA-Modified Gamma-Alumina and Novozym 435®. Chem. Eng. J. 2013, 234,

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in Chemoenzymatic Dynamic Kinetic Resolution of Amines - Inner or Outer Sphere

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of Chiral Secondary Alcohols: Process Optimization for Production of (R)-1-Indanol

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and (R)-1-Phenylethanol. Org. Process Res. Dev. 2008, 12, 192-195.

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Bn5CpRu(CO)(2)Cl and Its Application as Racemization Catalyst in Preparative-Scale

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Metalloenzymatic Dynamic Kinetic Resolution of 1-Phenylethanol. Pure Appl. Chem.

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Kinetic

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Enantioselectivity. J. Org. Chem. 2013, 78, 2571-2578.

and

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of


Secondary

Alcohols

with

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

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Enantioselective Catalyst for the Dynamic Kinetic Resolution of Secondary Alcohols.

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

571 572 573 574 575 576 577 578 579 580 581 582 583



Page 26 of 35

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


and

an

enzyme

CALb@MiMBN

kinetic

resolution

into

and

of

Page 27 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


Figures

609 610

611 612

Scheme 1.

613 614 615 616 617 618 619 620 621 622



a

b

c

d

e

f

623

624

625 626

Figure 1.

627 628 629 630 631


Page 28 of 35

Page 29 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


a

b b

632

c

d

633 634

Figure 2.

635 636 637 638 639 640 641 642



a

b

643

c

d

e

f

g

h

644

645

646 647

Figure 3.


Page 30 of 35

Page 31 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


648 649

Figure 4.

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



662 663

Figure 5.

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


Page 32 of 35

Page 33 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60


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



Page 34 of 35

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


Page 35 of 35 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

703


TOC

704

705


Artificial Nanometalloenzymes for Cooperative Tandem Catalysis

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