Synthesis and Assembly of Laccase-polymer Giant Amphiphiles by

Subscriber access provided by - Access paid by the | UCSB Libraries

Article

Synthesis and Assembly of Laccase-polymer Giant Amphiphiles by Self-catalyzed CuAAC Click Chemistry Chunyang Bao, Yueheng Yin, and Qiang Zhang Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.8b00087 • Publication Date (Web): 21 Mar 2018 Downloaded from http://pubs.acs.org on March 23, 2018

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

Biomacromolecules is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.

Page 1 of 56 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

Biomacromolecules

1

Synthesis and Assembly of Laccase-polymer Giant Amphiphiles

2

by Self-catalyzed CuAAC Click Chemistry

3

Chunyang Bao,† ‡ Yueheng Yin,† ‡ Qiang Zhang†‡*

4

†

5

Environmental and Biological Engineering, Nanjing University of Science and Technology,

6

Nanjing 210094, P. R. China.

7

‡

8

Nanjing University of Science and Technology, Nanjing 210094, P. R. China.

9

KEYWORDS: Giant amphiphiles; bionanoreactor; Cu(0)-LRP; laccase; click chemistry

Jiangsu Key Laboratory of Chemical Pollution Control and Resources Reuse, School of

Institute of Polymer Ecomaterials, School of Environmental and Biological Engineering,

10 11

Manuscript prepared for Biomacromolecules

12 13

Corresponding author: Prof. Qiang Zhang, Tel. (Fax): 0086-18652966493, Email:

14

[email protected]

15 16

January 18, 2018

ACS Paragon Plus Environment

1

Biomacromolecules 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

Page 2 of 56

17

ABSTRACT

18

Covalent coupling of hydrophobic polymers to the exterior of hydrophilic proteins would

19

mediate unique macroscopic assembly of bioconjugates to generate amphiphilic superstructures

20

as novel nanoreactors or biocompatible drug delivery system. The main objective of this study

21

was to develop novel strategy for the synthesis of protein-polymer giant amphiphiles by the

22

combination of copper mediated living radical polymerization and azide-alkyne cycloaddition

23

reaction (CuAAC). Azide-functionalized succinimidyl ester was first synthesized for the facile

24

introduction of azide group to proteins such as albumin from bovine serum (BSA) and laccase

25

from Trametes versicolor. Alkyne-terminal polymers with varied hydrophobicity were

26

synthesized by using commercial copper wire as the activators from a trimethylsilyl protected

27

alkyne-functionalized initiator in DMSO under ambient temperature. The conjugation of alkyne-

28

functionalized polymers to the azide-functionalized laccase could be conducted even without

29

additional copper catalyst, which indicated a successful self-catalyzed CuAAC reaction. The

30

synthesized amphiphiles were found to aggregate into spherical nanoparticles in water and

31

showed strong relevance to the hydrophobicity of coupled polymers. The giant amphiphiles

32

showed decreased enzyme activity yet better stability during storage after chemical modification

33

and self-assembly. These findings will deepen our understanding on protein folding,

34

macroscopic self-assembly and support potential applications in bionanoreactor, enzyme

35

immobilization and water purification.

36


2

Page 3 of 56 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

Biomacromolecules

37

Introduction

38

Protein-polymer conjugates have emerged as novel hybrid materials in pharmacy,

39

nanotechnology and biotechnology since the past few decades due to their unique combined

40

properties derived from proteins and polymers.1-4 PEGylation as one of the most representative

41

conjugation strategy could significantly improve some in vivo pharmacokinetic properties of

42

therapeutic proteins including extended circulation half-life, increased water solubility and

43

thermal stability as well as the reduced immunogenicity etc.5-8 Although poly(ethylene glycol)

44

(PEG) is the only existing synthetic polymer approved by Food and Drug Administration (FDA)

45

for conjugation with protein drugs, much more exciting properties could be attained when

46

replacing PEG with other functional polymers.7,

47

shown extraordinary ability in stabilizing proteins under lyophilization, heat stresses, vacuum

48

conditions and even high-energy radiation.10, 11 Coupling of heparin-mimicking sulfated polymer

49

to basic fibroblast growth factor could enhance the stability of conjugates against heat or acidic

50

process, storage and proteolytic degradation.12, 13 Such hydrophilic polymers generally do not

51

interfere with the binding pocket in the interior of the helix bundle when attached to the exterior

52

surface of proteins / peptides, which would only slightly decrease helix folding or even promote

53

the folding thus coiled-coil associations were retained.14-16 Although most studies up to now have

54

focused on the hydrophilic protein-polymer conjugates, amphiphilic protein-polymer conjugates

55

with covalently linked hydrophobic polymers could mediate the construction of novel

56

supramolecular structures, which are of great importance to our understanding of multi-length

57

scale assembly in multi-component systems.15 Conjugation of hydrophobic polymers to peptides

58

may unfold peptide and induce a conformational transition of α-helix to β-sheet.

59

presence of hydrophobic polymers could also enhance the solubility and processability of

9

Trehalose-containing glycopolymers have

6, 17

The


3


Page 4 of 56

60

conjugates in organic solvents and control the self-organization of peptides to generate uniform

61

or janus type nanotubes.15, 18, 19

62

Giant amphiphiles are defined as amphiphilic macromolecular surfactant with protein as the

63

polar head group and synthetic polymer as the nonpolar tail.20-22 These amphiphiles were first

64

synthesized via a “grafting to” strategy, either by biotin-streptavidin association, cofactor

65

reconstitution or thiol-maleimide addition reaction.20-23 Such strategy benefits from the pre-

66

synthesized polymers with well-defined structure and molecular weight (MW) and allows the

67

precise control over the assemble structures or tuning of aggregate morphologies. Later

68

developed “grafting from” strategy, which mainly utilized controlled radical polymerization such

69

as atom transfer radical polymerization (ATRP) and reversible addition-fragmentation chain

70

transfer polymerization (RAFT) etc., could directly initiate the propagation of polymer chains

71

from the surface of modified proteins.24-30 This approach avoids the tedious purification required

72

for removal of excess unreacted polymers after “grafting to” modification and could be

73

performed in aqueous solution without organic solvents, which is good for the retention of

74

protein activity.24 These giant amphiphiles could self-assemble into hierarchical superstructures

75

including spheres, fibres or vesicles in a similar manner as low MW amphiphiles.21, 24 It needs to

76

be noted that some assemblies are permeable for the encapsulated proteins and possess the

77

potential as nanoreactors.24 Various albumin-polymer spherical nanoparticles or micelles

78

prepared by Michael addition reaction have shown efficient delivery for macromolecular

79

platinum drugs with enhanced cellular-uptake behaviour by the cancerous cells.31-33

80

Click chemistry is an ideal synthesis approach for protein-polymer conjugates as binding two

81

macromolecular building blocks in a facile, selective, mild and high-yield reaction without by-

82

products.

23, 34-36

As a pioneer of click chemistry, copper mediated azide-alkyne cycloaddition


4

Page 5 of 56 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

Biomacromolecules

83

reaction (CuAAC) has been introduced to synthesize the giant amphiphiles by post-

84

functionalization of hydrophilic protein–polymer conjugates.23, 25 The hydrophobic group was

85

introduced in mild aqueous solution without organic solvents, which refrain from proteins

86

denaturation. Besides, the hydrophobicity could also be tuned by the ratio of introduced

87

functional groups, which would impel the self-assembly of amphiphiles to generate various

88

morphologies.23, 25 To overcome the problems associated with copper residues during the click

89

reaction, strain-promoted azide–alkyne cycloaddition (SPAAC) reaction without additional

90

catalyst was developed for synthesizing biomaterials and enzymes immobilization under mild

91

conditions even in the living system.37,

92

regioselectivity and may lead to non-specific labelling.39 Many other copper-free click reactions

93

were also developed for protein conjugation with functional polymers, such as thiol-ene reaction,

94

thiol-halogen ligation, Diels-Alder reaction and multicomponent reactions (four-component Ugi

95

reaction etc.).40-43 Compared with the traditional chemical reactions, enzyme-catalyzed reactions

96

are well known due to the high efficiency, ultrafast reaction rate and high specificity. Many

97

enzymes, such as peroxidases and lipase, have already been used for the oxidative

98

polymerization of phenol derivatives or ring-opening polymerization of cyclic monomers as well

99

as RAFT polymerization for multiblock and ultrahigh-molecular-weight polymers with oxygen

100

tolerance.44-47 Laccase, a copper-containing protein, have shown great success in mediating

101

ATRP of N-vinylimidazole and poly(ethylene glycol) methyl ether methacrylate (PEGMA) in

102

water.48, 49 It seems reasonable to hypothesize that laccase could also catalyze CuAAC under

103

similar reaction conditions as ATRP without additional copper catalyst. However, to the best of

104

our knowledge, limited relevant research have been performed. We believe the challenge mainly

38

However, SPAAC was reported to have poor


5


Page 6 of 56

105

derives from the separation and recovery of high MW & water-soluble enzyme from this system

106

in order to avoid product contamination.

107

This study investigated the possibility of using giant amphiphiles as the enzymatic nanoreactors

108

to encapsulate proteins into the varied assemblies to realize immobilization. Novel strategies

109

were developed for the synthesis and assembly of laccase-polymer giant amphiphiles to form

110

hierarchical superstructures. Facile synthetic route to azide-functionalized succinimidyl ester was

111

first developed for the azide functionalization of proteins. Zero-valent copper mediated living

112

radical polymerization (Cu(0)-LRP) was then utilized for the synthesis of alkyne-terminal

113

polymers with tuned hydrophilicity. Subsequent conjugation reactions to the azide-functionalized

114

laccase were examined in the presence or absence of additional copper catalyst. Moreover, the

115

effect of modification and self-assembly on enzyme activity were also respectively evaluated.

116

Experimental section

117

Materials

118

Laccase from Trametes versicolor (≥ 0.5 U/mg) was supplied by Sigma-Aldrich (America) and

119

purified

120

(dimethylamino)ethyl)amine

121

methylpropanoate and 3-azido-propan-1-ol were synthesized according to literature procedure

122

and stored in the freezer under a nitrogen atmosphere.50-52 1-(2′-propargyl) D-mannose and 1-(2′-

123

propargyl) D-galactose were prepared according to procedures described in previous literature.53,

124

54

125

and rinsed thoroughly with water, dried under nitrogen before use. Membrane dialysis (1K and

126

50K MWCO) was obtained from Spectrum Laboratories. All other reagents and solvents, such as

by

dialysis

against

water

for

(Me6TREN),

24

h

following

lyophilization.

3-(trimethylsilyl)prop-2-yn-1-yl

Tris(2-

2-bromo-2-

Copper wire (diameter=0.25 mm) was pre-treated by washing in hydrochloric acid for 15 min


6

Page 7 of 56 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

Biomacromolecules

127

albumin from bovine serum (BSA, 96%), 2, 2'-azinobis(3-ethylbenzothiazoline-6-sulfonic acid

128

ammonium salt (ABTS, 98%), Di(ethylene glycol) ethyl ether acrylate (DEGEEA, ≥90%), Poly

129

(ethylene glycol) methyl ether acrylate (PEGA480, number-average molecular weight / Mn 480)

130

and Copper(II) bromide (CuBr2, 99%) etc. were obtained at the highest purity available from

131

Aladdin (China) and used without further purification unless otherwise stated.

132

Analytical techniques

133

The MW and the MW distribution (Mw/Mn) of linear polymers were determined by Waters 1515

134

size exclusion chromatography (SEC) in N, N-dimethylbenzamide (DMF) at 40 °C with a flow

135

rate of 1.00 mL min-1, which was equipped with 2414 refractive index (RI) and 2489 UV

136

detectors, a 20 µm guard column (4.6 mm ×30 mm, 100 - 10K) followed by three Waters

137

Styragel columns (HR1, HR3 & HR4) and autosampler. Narrow linear polystyrene standards in

138

range of 540 to 7.4×105 g·mol-1 were used to calibrate the system. Aqueous SEC (Waters 1515)

139

was used for the characterization of protein-polymer conjugates and it was equipped with 2414

140

RI & 2489 UV detector, an Ultrahydrogel guard column and three Waters Ultrahydrogel

141

columns (250, 500 & 1000 PKGD) and autosampler, using aqueous solution of sodium dodecyl

142

sulfate (SDS, 2%) as the eluent at 25 °C with a flow rate of 1.00 mL min-1. Hydrophilic

143

poly(ethylene glycol) (1.0×102 – 2.2×104 g·mol-1) and dextran (5.0×103 - 7.5×105 g·mol-1) were

144

used to calibrate the aqueous SEC system. All samples were passed through 0.45 µm PTFE filter

145

before analysis. UV/Vis spectrophotometer (SHIMADZU UV-2600) was utilized to measure the

146

lower critical solution temperature (LCST) of thermoresponsive polymers at the wavelength of

147

500 nm and the heating rate for the thermostatically controlled cuvette was 1 °C min-1. The

148

LCST was defined as the temperature corresponding to 50% decreases of transmittance. The


7


Page 8 of 56

149

enzyme activity of laccase was calculated according to the degradation of ABTS in defined

150

buffer solution and temperature using SHIMADZU UV-2600 UV/ Vis spectrophotometer to

151

measure the absorbance at 420 nm. 1H and

152

Bruker AV 500M spectrometer using deuterated solvents obtained from Aladdin. Fourier

153

transform infrared (FTIR) spectra were recorded on a Nicolet iS5 FTIR spectrometer using an

154

iD7 diamond attenuated total reflectance optical base. Transition electron microscopy (TEM)

155

images were acquired by FEI TECNAI G2 20 TEM microscope equipped with LaB6 filament.

156

The turbidity of the nanoparticles were detected by WGZ-2000 turbidity meter (Beijing Warwick

157

Industrial Science and technology, China). The size and size distribution of polymer and

158

conjugate particles were measured by dynamic light scattering (DLS) using a ZetaPALS variable

159

temperature analyzer (Brookhaven Instruments, UK). Elemental analysis was performed using

160

Elementar Vario MICRO.

161

Synthesis of 3-azidopropyl (2,5-dioxopyrrolidin-1-yl) carbonate

162

The functional succinimidyl ester was synthesized via similar procedure as previous report.28, 55

163

To a 100 mL round-bottom flask equipped with a magnetic stir bar and a rubber septum, 3-azido-

164

propan-1-ol (1.42 g, 0.014 mol) and triethylamine (2.4 mL, 0.017 mol) were dissolved in 10 mL

165

anhydrous acetonitrile and N, N’- disuccinimidyl carbonate (4.35 g, 0.017 mol) was then added.

166

The reaction mixture was stirred at ambient temperature under nitrogen protection for one day.

167

Subsequently the acetonitrile was removed via rotary evaporation and the residue product was

168

dissolved in 100 mL dichloromethane (DCM) following extraction with 3×100 mL water for

169

three times. The DCM solution was dried with anhydrous magnesium sulfate and following

170

filtration the solvent was removed via rotary evaporation. The oily product was then purified via

171

silica gel column chromatography using petroleum ether / DCM as the eluent (petroleum ether /

13

C NMR spectra were recorded at 25 °C with a


8

Page 9 of 56 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

Biomacromolecules

172

DCM = 1 / 1, Rf

173

obtained as colourless viscous oil under ambient temperature and white solid under -18 oC after

174

drying under vacuum (2.53 g, 75%).

175

1

176

2H), 2.85 (s, 4H), 2.05 – 2 (p, J = 6.28, 6.28 Hz, 2H) ppm.

177

13

178

FTIR ν: 2096 (N≡N), 1734 (C=O), 1430 (C-N), 1362 (C-H), 1196 (C-C) cm-1.

179

ESI-MS m/z calcd for C8H10N4O5Na+ (M + Na) 265.07, found 265.03.

180

Conjugation of 3-azidopropyl (2,5-dioxopyrrolidin-1-yl) carbonate with proteins

181

To a vial equipped a magnetic stir bar and a rubber septum, BSA (0.5 g, 0.008 mmol) or laccase

182

(0.5 g, 0.008 mmol) was dissolved in 10 mL phosphate buffer solution (200 mM, pH=8.0). 3-

183

azidopropyl (2,5-dioxopyrrolidin-1-yl) carbonate (61 mg, 0.25 mmol) was dissolved in 1 mL

184

DMSO and added dropwise to protein solution and the mixture was stirred at 4 oC for one day.

185

Subsequently the formed white suspension was transferred to a dialysis tube (MWCO 50 KDa)

186

and dialyzed against water for two days. The final product was recovered as white powder after

187

lyophilisation. The product was named as BSA-N3 or laccase-N3.

188

Synthesis of alkyne-terminal polymers by Cu(0)-LRP

189

To a 25 mL vial fitted with a magnetic stir bar and a rubber stopper, monomer (either DEGEEA,

190

(1918 mg, 10.2 mmol, 20 eq); or PEGA480 (4896 mg, 10.2 mmol, 20 eq); or (DEGEEA (1918

191

mg, 10.2 mmol, 20 eq) and PEGA480 (960 mg, 2 mmol, 4 eq) mixture), CuBr2 (11.2 mg, 0.05

product

= 0.3). The final 3-azidopropyl (2,5-dioxopyrrolidin-1-yl) carbonate was

H NMR (CDCl3, 500 MHz, 298 k): δ 4.44-4.42 (t, J = 6.22 Hz, 2H), 3.5-3.47 (t, J = 6.42 Hz,

C NMR (CDCl3, 125 MHz, 298 k): δ 168.86(2), 151.53, 68.23, 47.52, 27.99, 25.51(2) ppm.


9


Page 10 of 56

192

mmol, 0.1 eq), Me6TREN (20.7 mg, 0.09 mmol, 0.18 eq), 3-(trimethylsilyl)prop-2-yn-1-yl 2-

193

bromo-2-methylpropanoate (141 mg, 0.51 mmol, 1 eq) and DMSO (2 mL) were charged and the

194

mixture was bubbled with nitrogen for 15 min. After that, pre-activated copper wire (3 cm, 13

195

mg) was carefully added to the solution under nitrogen protection. The vial was sealed and the

196

light green solution was allowed to polymerize at 25 °C for 6 hours. Samples of the reaction

197

mixture were then taken for 1H NMR and SEC characterization. The viscous solution was

198

directly transferred to a dialysis tube (MWCO 1000 Da) for dialysis against water for two days to

199

remove the solvent and catalyst. The final product was recovered as viscous solid after

200

lyophilization.

201

The deprotection of trimethylsilyl group was performed according to previous report.56 In a

202

typical reaction, poly(DEGEEA) (800 mg, ~ 0.13 mmol according to Mn, NMR) and acetic acid (36

203

mg, 0.6 mmol) was solubilized in 20 mL THF under nitrogen protection and cooled to -20 oC.

204

0.6 mL tetrabutylammonium fluoride solution (TBAF, 1.0 M in THF) was then added dropwise

205

and the reaction was allowed to react under -20 oC for 0.5 hour and then at ambient temperature

206

for further 24 hours. After that, the solution was transferred to dialysis tube (MWCO 1000 Da)

207

for dialysis against water / MeOH (3/1) for one day and then pure water for another two days

208

before lyophilization.

209

Calculation of azide groups per protein by 1H NMR spectroscopy

210

In a small vial, laccase-N3 (20 mg), 1-(2′-propargyl) D-mannose (7 mg, 0.032 mmol), copper(II)

211

sulfate pentahydrate (CuSO4, 1.2 mg, 0.005 mmol) and DMSO (5 μL, as internal standard) were

212

solubilized in 1.5 mL D2O and degassed via nitrogen bubbling for 10 min. Then 0.4 mL of this

213

solution was taken for 1H NMR spectroscopy. After that, 0.1 mL degassed aqueous solution


10

Page 11 of 56 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

Biomacromolecules

214

containing (+)-sodium L-ascorbate (NaAsc, 2 mg, 0.01 mmol) was added and the suspension was

215

allowed to stir under ambient temperature for 24 h. After reaction, sample from this solution was

216

taken for 1H NMR spectroscopy. The number of azide groups per protein was determined by

217

calculating the consumption of 1-(2′-propargyl) D-mannose during click reaction. The final

218

products were recovered by dialyzed against water using a high MWCO (50 K Da) dialysis tube

219

following lyophilization. A control experiment was conducted without the addition of CuSO4

220

under identical conditions. The reaction with 1-(2′-propargyl) D-galactose was also performed

221

in the presence or absence of CuSO4 in similar way.

222

Synthesis of protein-polymer conjugates by CuAAC click reactions

223

The CuAAC reaction with laccase-N3 was performed in the presence or absence of CuSO4. In

224

one typical reaction, laccase-N3 (40 mg), alkyne-functional reagents (propargyl alcohol or

225

alkyne-terminal polymer, all as 40 mg) and CuSO4 (1.5 mg, 0.006 mmol) were added to 2.5 mL

226

water and the suspension was degassed via nitrogen bubbling for 10 min. After that, 0.5 mL

227

degassed aqueous solution of NaAsc (2.5 mg, 0.012 mmol) was added and the suspension was

228

allowed to stir under ambient temperature or ice / water cool (for reaction with poly(DEGEEA)

229

only) for 24 hours. The reaction mixture was then dialyzed against ice / water for two days using

230

a high MWCO (50 K Da) dialysis tube and the conjugates were then recovered via lyophilization.

231

The BSA conjugates were synthesized using CuSO4 / NaAsc as the catalyst. BSA-N3 (40 mg),

232

alkyne-functional reagents (propargyl alcohol, 1-(2′-propargyl) D-mannose or alkyne-terminal

233

poly(PEGA480), all as 40 mg) and CuSO4 (1.5 mg, 0.006 mmol) were added to 2.5 mL water and

234

the suspension was degassed via nitrogen bubbling for 10 min. After that, 0.5 mL degassed

235

aqueous solution of NaAsc (2.5 mg, 0.012 mmol) was added and the suspension was allowed to


11


Page 12 of 56

236

stir under ambient temperature for 24 hours. The reaction mixture was then dialyzed against

237

water for two days using a high MWCO (50 K Da) dialysis tube and the conjugates were then

238

recovered via lyophilization.

239

Activity assays of laccase and conjugates

240

The enzyme activity of laccase was determined using ABTS as the substrate according to

241

previously reported procedures by UV/Vis spectroscopy, typically measuring the absorbance at

242

420 nm for a defined sample after defined period and the laccase activity was deduced according

243

to the kinetic parameters.57 The equation used for the calculation of the activity of free laccase

244

and immobilized laccase is shown as follow: ∆ ∆∆ = ∆

245

∆ was the concentration of the sample per unit of molar concentration and ∆ ⁄ ∆ represented

246

the activity of absorbance change (∆) at a specific time interval (∆). The extinction coefficient

247

for the oxidation of ABTS at 420 nm is 36×10-3 M-1 cm-1 and the path length of the optical cell

248

used is 1 cm.58 1 Unit was defined as the formation of 1 mM of product per minute. The enzyme

249

activity was determined under different temperature (20, 50, 70 oC) and at varied time periods

250

(1-5 days) for both the free laccase and the conjugates.

251 252 253


12

Page 13 of 56 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

Biomacromolecules

254

255

Results and discussion

256

Synthesis and characterization of functional succinimidyl ester for the azidation of

257

proteins.

258 259

Scheme 1. Scheme representation for the synthesis of functional succinimidyl ester and azidation

260

of proteins.

261

Succinimidyl ester chemistry is one of the most frequently used bioconjugation strategy to

262

covalently couple amine-containing biomacromolecules (e. g. proteins or peptides) with

263

functional polymers via amide linkages. Acid derivatives with azide functional groups have been

264

preciously treated with N-hydroxysuccinimide to give the corresponding activated esters with an

265

amine-reactive component at one end and an azide group at the opposite end.59 Herein we

266

utilized a different approach (Scheme 1), by reacting an azide compound with a free hydroxyl

267

terminus with N, N′-disuccinimidyl carbonate to generate the targeted azide-functionalized

268

succinimidyl ester 1 (3-azidopropyl (2,5-dioxopyrrolidin-1-yl) carbonate), which was designed

269

to conjugate with the terminal amine and lysine residues in the protein. This one-step reaction

270

following purification gave product 1 in a conversion of 75% and high purity (Figure S1, S2).


13


Page 14 of 56

271 272

Figure 1. FTIR spectra (A) and SEC elution traces (B) of BSA, BSA-N3, BSA @ propargyl

273

alcohol and BSA@ poly(PEGA480) conjugates.

274

The azide linker (Compound 1) was first reacted with BSA, which is a typical water-soluble

275

protein with high purity, under slightly alkaline condition (PBS buffer, 0.2 M, pH = 8.0) in order

276

to promote the conjugation. Massive precipitates were formed in the solution soon after the

277

addition of azide linker, indicating that the successful chemistry modification thoroughly

278

changed the structure of BSA and further leaded to dramatic change in solubility. After

279

purification via dialysis against water, the FTIR spectrum (Figure 1 A) of lyophilized product

280

revealed typical peak of azide group at ~ 2100 cm-1. The BSA-N3 was further denatured and

281

solubilized in SDS solution for characterization by aqueous SEC. As shown in Figure 1 B, SEC

282

analysis demonstrated a slight yet clear shift of peak elution time from 12.25 min of pristine

283

BSA to 12.23 min of BSA-N3, which was due to the increase of MW and hydrodynamic volume

284

after reaction. The azide-functionalized BSA tends to have low solubility in water and it’s

285

hypothesized that the NHS-amine reaction modified the hydrophilic surface area of BSA with

286

hydrophobic short alkyl groups, which decreased the solubility of conjugates.

287


14

Page 15 of 56 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

Biomacromolecules

288 289

Figure 2. FTIR (A), aqueous SEC elution traces (B), turbidity (C), size distribution by DLS (D)

290

and TEM image (D, inset, laccase-N3) of laccase, laccase-N3 and laccase conjugates.

291

Subsequently, succinimidyl ester 1 was used for the azide-functionalization of laccase. As shown

292

in Figure 2 A, FTIR spectrum of laccase-N3 also showed the appearance of azide absorbance

293

band at ~ 2100 cm-1. It needs to be noted that the commercial laccase used in this research is a

294

natural extract from Trametes versicolor and two main peaks were observed on the SEC traces

295

(Figure 2 B). After reaction, SEC analysis revealed a slight shift of elution traces to higher MW

296

region (from 14.82 min to 14.70 min, Figure 2 B). Likewise, laccase-N3 also showed limited

297

solubility in water due to the modification with hydrophobic alkyl chain. Turbidity test of

298

Laccase-N3 in water demonstrated a fast decrease of turbidity to almost zero in ~ 60 minutes


15


Page 16 of 56

299

(Figure 2 C), indicating the functionalized protein cannot disperse stably in water. DLS revealed

300

the presence of nanoparticles with diameter of ~ 280 nm and a broad size distribution (PDI =

301

0.55, Figure 2 D) in the suspension, which suggested that the nanoparticles may not have

302

uniform size. Further TEM characterization (Figure 2 D, inset) found clusters of small

303

nanoparticles to form large, irregular aggregates with size at micrometre level, which further

304

proved the fast decrease of turbidity.

305

All these results proved the successful synthesis of functional succinimidyl ester and subsequent

306

azidation of proteins. The immobilization of hydrophobic alkyl chain to the amine groups of

307

different proteins would significantly decrease their water solubility and dispersion stability in

308

water.

309

Synthesis and characterization of hydrophobic, hydrophilic and thermoresponsive

310

polymers with terminal alkyne groups by Cu(0)-LRP.

311 312

Scheme 2. Synthesis of functional polymers with terminal alkyne groups by Cu(0)-LRP.

313

Although underlying debates still exist for the reaction mechanism of Cu(0)-LRP as single

314

electron transfer living radical polymerization (SET-LRP) or supplemental activator and

315

reducing agent atom transfer radical polymerization (SARA ATRP), Cu(0)-LRP proved to be a

316

facile route for fast and controlled radical polymerization of different monomers in DMSO. 60-63

317

Herein Cu(0)-LRP was utilized for the synthesis of functional polymers with terminal alkyne

318

groups. Initiator 2 (Scheme 2, 3-(trimethylsilyl)prop-2-yn-1-yl 2-bromo-2-methylpropanoate)


16

Page 17 of 56 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

Biomacromolecules

319

with a trimethylsilyl-protected alkyne group was used for the polymerization, which was mainly

320

used to prevent the alkyne-alkyne (i.e., Glaser) coupling side reactions during the post-

321

polymerization workup after the synthesis of alkyne-functional polymer by ATRP.52,

322

typical polymerization of DEGEEA in DMSO at ambient temperature, commercial copper wire

323

was used as the activator with initially added CuBr2 / Me6TREN as the deactivator under

324

following reaction conditions: [2]: [DEGEEA]: [CuBr2]: [Me6TREN] = 1: 20: 0.1: 0.18. The

325

polymerization almost reached full conversion (98%, Figure S 3) after 6 h. As shown in Figure 3

326

A, SEC analysis revealed the successful synthesis of well-defined polymer with narrow MW

327

distribution (Mn, SEC = 10100, Mw / Mn = 1.08) without significant radical coupling termination

328

observed. The small shoulder peak at relatively high MW region was due to the presence of trace

329

amounts of diacrylate in the monomer. Figure 3 B shows the 1H NMR spectra of poly(DEGEEA)

330

and the peak assignments are indicated in the figure, which also revealed that the Mn, NMR was

331

5917 by comparing the integra of polymer backbone protons with that of the trimethylsilyl group.

332

This experimental MW is higher than the theoretical value (Mn,

333

efficiency during the Cu(0)-LRP is ~ 68%，which suggests that some side reactions such as

334

disproportionation or coupling termination may happen especially at the beginning of

335

polymerization.

theo

64

In a

= 4037). The initiator


17


Page 18 of 56

336 337

Figure 3. DMF SEC elution traces (A), 1H NMR spectra (B), temperature dependence of the

338

optical transmittance at 500 nm (C, 1 mg mL−1) and DLS (D, alkyne terminal poly(DEGEEA)-r-

339

(PEGA480), 1 mg mL−1) of alkyne-terminal polymers.

340

The terminal trimethylsilyl protection group was then removed under acidic conditions in the

341

presence of TBAF and the alkyne polymer could be recovered after dialysis against water

342

following lyophilization. As shown in Figure 3B, 1H NMR spectra of alkyne poly(DEGEEA)

343

revealed the total disappearance of trimethylsilyl peak at ~ 0.1 ppm after deprotection without

344

change of other groups. A slight MW increase was observed on SEC with Mn increased from

345

10100 to 10400 while maintaining narrow MW distribution (Figure 3A). This unusual

346

phenomenon has also been observed in the previous reports when using DMF as the eluent of

347

SEC.65


18

Page 19 of 56 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

Biomacromolecules

348

Poly(DEGEEA) has a relatively low LCST (close to 0 oC) and is hydrophobic at ambient

349

temperature. Thus poly(PEGA480) and poly(DEGEEA)-r-(PEGA480), which are hydrophilic or

350

thermoresponsive, were synthesized by Cu(0)-LRP under similar reaction conditions in order to

351

supply polymers with different solubility for conjugation with proteins. All the polymerizations

352

were allowed to reach full or very close to full conversion, while SEC characterization all proved

353

the successful synthesis of well-defined polymers with controlled MW and narrow MW

354

distribution (Figure 3A). 1H NMR spectra proved the synthesis of targeted polymers as well as

355

the deprotection of trimethylsilyl groups according to the total disappearance of resonance at ~

356

0.1 ppm (Figure S4, S5). For the copolymer poly(DEGEEA)-r-(PEGA480) with terminal

357

trimethylsilyl group, a LCST at ~ 44 oC (1 mg mL-1) was observed and after deprotection the

358

LCST of alkyne polymer dramatically increased to ~ 49 oC (Figure 3C), which suggests that

359

even such a small protection group significantly contributes to the thermo phase transition

360

behaviour. Further DLS (Figure 3D) characterization of alkyne-terminal poly(DEGEEA)-r-

361

(PEGA480) under different temperature indicated the self-assembly of thermoresponsive polymer.

362

Nanoparticles with size at 258 nm and a narrow size distribution of 0.03 were formed when the

363

temperature increased to 70

364

demonstrates the importance of hydrophobicity to generate uniform assemblies.

365

In summary, three alkyne-terminal polymers have been successfully synthesized by Cu(0)-LRP

366

in DMSO from a trimethylsilyl protected initiator following facile deprotection. These polymers

367

have well-defined properties and different solubility in water for further conjugation with

368

proteins.

o

C. The decreased PDI with the increase of temperature

369


19


Page 20 of 56

370

Synthesis and characterization of protein-polymer conjugates via CuAAC click reaction

371

To evaluate the possibility of the reaction between azide-functionalized proteins and alkyne

372

polymers, a model reaction with BSA using propargyl alcohol was first conducted. The reaction

373

was performed in water using CuSO4 and NaAsc as the catalyst. After reaction, the FTIR

374

spectrum (Figure 1A) of BSA @ propargyl alcohol conjugate revealed the total disappearance of

375

absorption band of azide group. As shown in Figure 1B, SEC characterization further showed the

376

shift of peak elution time from 12.23 min to 12.20 min, confirming the occurrence of click

377

reaction and increase of MW. When conjugated with the alkyne-terminal poly(PEGA480), FTIR

378

spectrum (Figure 1A) of obtained product showed the appearance of new absorption band

379

associating with the polymer such as C=O peak at 1730 cm-1 and C-H stretching vibration at

380

2865 cm-1 combined with the total disappearance of azide group. However, the solubility of BSA

381

@ poly(PEGA480) conjugate in water or SDS solution became even worse and the conjugate

382

could be filtered during the sample treatment prior to the SEC analysis thus no MW change

383

could be compared.

384

Subsequently propargyl alcohol was reacted with laccase-N3 in the presence of CuSO4 / NaAsc.

385

The reaction was also successful as revealed by the total disappearance of azide absorption band

386

on the FTIR spectrum (Figure 2 A) and MW increase on the SEC (Figure 2 B). However, it is

387

worth noting that laccase is a multi-copper-containing protein and has been directly used as the

388

catalyst for ATRP.48, 49 Thus we are inspired to conduct the conjugation of propargyl alcohol to

389

laccase without the addition of CuSO4 but only NaAsc as the reducing agent to generate active

390

Cu(I) catalyst for the click reaction. As shown in Figure 2 A & B, both the FTIR and SEC

391

characterization revealed similar spectra as the product obtained under the catalysis of CuSO4 /


20

Page 21 of 56 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

Biomacromolecules

392

NaAsc, which demonstrated that the click reaction could still happen without the presence of

393

additional copper catalyst.

394 395

Scheme 3. Self-catalyzed click reaction for the glycosylation modification of laccase-N3.

396

To further verify the occurrence of enzymatic click reaction and self-catalyzed conjugation, the

397

reaction of laccase-N3 with copper-free alkyne-functionalized monosaccharides were performed

398

in the presence or absence of CuSO4 (Scheme 3). As shown in Figure 4A, 1H NMR spectroscopy

399

was used to follow the consumption of 1-(2′-propargyl) D-mannose using D2O as the deuterated

400

solvent and DMSO as the internal standard. The integra of alkyne hydrogen at 2.80 - 2.95 ppm,

401

which is an anomeric mixture, decreased from 12.38 to 6.53 compared with the DMSO standard.

402

FTIR spectra (Figure 4B) of the final product showed total disappearance of azide groups. This

403

indicated the occurrence of click reaction with total consumption of azide groups and residual of

404

excess 1-(2 ′ -propargyl) D-mannose. The number of azide groups per protein could be

405

calculated using the following equation: × !""# 12.38 − 6.53 = × 64000 + 127 × 12.38 218

406

Where n is the number of azide groups per protein, and !""# represent the

407

weight of laccase-N3 (20 mg) and 1-(2′-propargyl) D-mannose (7 mg) used in the reaction


21


Page 22 of 56

408

respectively. 64000 (Da) and 218 (Da) is the MW of laccase and 1-(2′-propargyl) D-mannose,

409

separately, while 127 (Da) represents the MW increase from amine to azide group after NHS

410

reaction. Under our test, n is calculated as ~ 54, which means that each laccase is functionalized

411

with 54 azide groups.

412

It was also proved that even without the addition of CuSO4, the click reaction could proceed to

413

full conversion as FTIR spectra (Figure 4 B) of the final product revealed the disappearance of

414

all azide groups. The n value was calculated as ~ 66 by 1H NMR spectroscopy (Figure S 6) when

415

the reaction was performed without additional copper catalyst, which is close to previous value

416

(~ 54). The relative error could be from the measurement of chemicals and integra of hydrogen

417

peak during experiment and the n should be seen as an average value of azide groups per protein.

418 419

Figure 4. 1H NMR spectra (A) of samples taken during the click reaction of laccase-N3 with 1-(2′

420

-propargyl) D-mannose using CuSO4 as the additional catalyst and the FTIR spectra (B) of

421

laccase @ saccharides conjugates.

422

Repeated experiments using a different alkyne-functionalized saccharide, 1-(2′-propargyl) D-

423

galactose were also conducted. The FTIR spectra (Figure 4 B) of final products proved the total


22

Page 23 of 56 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

Biomacromolecules

424

disappearance of azide groups for the reactions in the presence or absence of CuSO4. Although

425

hydrophilic saccharides were covalently linked to the laccase-N3, the final product after click

426

reaction tends to be only able to suspend in water and SDS solution thus no SEC or NMR

427

characterization was available for this kind of conjugates.

428

As control experiments, BSA-N3 was also used for the click reaction with propargyl alcohol and

429

1-(2-(propargyl) D-mannose in similar manner. Under the catalyst of CuSO4 / NaAsc the

430

CuAAC reaction could go to full conversion as revealed in FTIR spectra (Figure S 7) by the total

431

disappearance of azide resonance at ~ 2100 cm-1. However, the FTIR spectra (Figure S 7) of

432

final products indicated that the azide groups still exist when the reactions were performed

433

without the addition of CuSO4. SEC (Figure 1 B) revealed a clear shift of elution trace for the

434

conjugates synthesized under the catalysis of CuSO4, while elution trace of the product

435

synthesized without the addition of CuSO4 almost overlapped with that of the BSA-N3,

436

indicating the reaction did not occur. Based on all these results from control experiments, the

437

successful click reaction of laccase-N3 with different copper-free chemicals proved that laccase

438

itself could act as an efficient catalyst for CuAAC.

439 440

Scheme 4. Synthesis of protein-polymer conjugates by CuAAC click reactions.

441

Subsequently, different alkyne polymers including poly(DEGEEA), poly(PEGA480) and

442

poly(DEGEEA)-r-(PEGA480) were conjugated to the azide-functionalized laccase (Scheme 4).


23


Page 24 of 56

443

The reaction with poly(DEGEEA) was first performed in water by the catalysis of CuSO4 /

444

NaAsc under ice/water cool in order to solubilize the thermoresponsive polymer. The yields of

445

all click reactions were summarized in Table S 1.

446 447

Figure 5. TEM images (A: laccase @ poly(DEGEEA); B: laccase @ poly(PEGA480); C: laccase

448

@ poly(DEGEEA)-r-(PEGA480)), FTIR (D) & 1H NMR (E, product obtained under the catalysis

449

of CuSO4) spectra of laccase-polymer conjugates.

450

Although laccase-N3 could not stably disperse in water, the obtained laccase @ poly(DEGEEA)

451

conjugates could form emulsion-like suspension and the turbidity test revealed a much higher

452

turbidity value compared with laccase-N3 under the same concentration (1 mg mL-1) and no

453

decrease of turbidity was observed in 4 hours (Figure 2 C), indicating the conjugates may form

454

stable particles in the water. Further DLS analysis (Figure 2 D) demonstrated the presence of

455

nanoparticle with increased diameter at 562 nm and much narrower size distribution (PDI =0.08)


24

Page 25 of 56 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

Biomacromolecules

456

compared with laccase-N3. TEM characterization (Figure 5 A) confirmed the presence of sphere-

457

like nanoparticles with diameter ranging from ~ 300 to ~ 700 nm. Due to the relatively big size,

458

we believe these assemblies are aggregated nanoparticles rather than giant micelles. To prove the

459

success of click reaction, FTIR and 1H NMR spectroscopy was utilized to characterize the

460

conjugates. As shown in Figure 5 D, the azide absorbance totally vanished and typical

461

absorbance bands from the polymer groups such as the C=O (1730 cm-1) and C-H stretching

462

vibration at 2865 cm-1 appeared after click reaction. 1H NMR spectra of conjugates revealed the

463

appearance of polymer-related peaks as listed in the figure and resonance of proton from typical

464

triazole ring at 8.1 ppm. All these powerful evidences proved the successful synthesis of laccase-

465

poly(DEGEEA) conjugates by CuAAC click reaction, rather than a sole mixture of laccase-N3

466

and unreacted polymers.

467

Hydrophobic interaction has been considered as one of the most important factors during the

468

folding of globular proteins in aqueous system. To some extent, it makes the dominant

469

contribution to protein stability even than hydrogen-bonding does.66, 67 It is hypothesized that the

470

conjugation of hydrophobic polymer to the protein surface, which contains different charged,

471

hydrophobic and hydrophilic amino acids, will lead to the change of protein solubility and self-

472

assembly of conjugates. It is a challenge to define the position and distribution of proteins in the

473

nanospheres as the aggregation of hydrophobic poly(DEGEEA) may encapsulate laccase into the

474

core, mainly because the immobilization of polymer cannot be precisely controlled via the

475

random NHS-amine reaction. However, some laccase or at least a part area of the laccase, such

476

as the hydrophilic or the charged region should be exposed on the surface in order to make the

477

nanospheres disperse stably in water even without the addition of any surfactants.


25


Page 26 of 56

478 479

Figure 6. SEC elution traces (A) and DLS (B) of laccase-polymer conjugates.

480

To verify the self-assembly behaviour of conjugates immobilized with hydrophilic polymers,

481

click reactions with alkyne-terminal poly(PEGA480) were performed using NaAsc to generate

482

active catalyst in the presence and absence of additional CuSO4. As shown in Figure 5 D, FTIR

483

spectra of both products showed disappearance (in the presence of CuSO4) or diminish (in the

484

absence of CuSO4) of the absorbance band of azide group, indicating the occurrence of click

485

reaction. Although SEC revealed significant MW increasing compared with the laccase-N3 as

486

proved by the significant shift to higher MW region, the elution traces (Figure 6 A) of obtained

487

laccase @ poly(PEGA480) in the presence and absence of CuSO4 are different. The product

488

synthesized under the catalysis of CuSO4 / NaAsc tends to have higher MW (such as the

489

significant peak at ~ 10.8 min, Figure 6 A), while the one without additional CuSO4 even

490

revealed the presence of unreacted laccase-N3 (such as the peak at ~ 14.6 min). It is worth noting

491

that SEC here may not reveal the total information on the MW and MW distribution of

492

conjugates as slight insoluble solids were observed in the SDS solution, which would be filtered

493

off when passing through the filter before the sample was injected into the SEC system. The 1H

494

NMR spectra (Figure S 8) of laccase @ poly(PEGA480) showed appearance of typical peaks from


26

Page 27 of 56 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

Biomacromolecules

495

the polymer after click reaction, such as the resonances from protons of polymer backbones at

496

0.5-2.4 ppm and protons of repeated ethylene glycol unit at 3.0-4.2 ppm. However, typical peak

497

from the triazole ring proton were not significant at ~ 8 ppm, possibly due to the relatively low

498

concentration during NMR test as well as the low content compared with the repeated ethylene

499

glycol units.

500

It’s hypothesized that the alkyne polymers may not efficiently reach the copper atoms

501

surrounded by dense peptides due to the steric effect. Thus the click reaction did not go to full

502

conversion after same reaction time compared with the one in the presence of CuSO4.

503

Nevertheless, the obtained conjugates showed better dispersion stability in water and the

504

decrease of turbidity was much smaller compared with laccase-N3 as shown in Figure 2C. DLS

505

revealed a significant increase of size from 280 nm (laccase-N3) to 589 nm or 756 nm, however,

506

the size distribution also became broader (0.98 or 0.92, Figure S9). TEM characterization (Figure

507

5 B) showed the presence of sphere-like particles with varied size from tens of nanometre to

508

hundreds of nanometre, which further proved the broad size distribution in DLS. These results

509

suggest that the coupling of poly(PEGA480) increased the hydrophilicity of protein-polymer

510

conjugates as well as the dispersion stability in water. The lack of hydrophobicity may prevent

511

highly-ordered self-assembly of the conjugates into uniform structures.

512

Above-mentioned results demonstrate that hydrophobicity is not only important for the protein-

513

folding but also dominates the self-assembly of protein-polymer conjugates. To further prove

514

this hypothesis, conjugation of thermoresponsive poly(DEGEEA)-r-(PEGA480) with laccase was

515

conducted in order to evaluate the self-assembly behaviour of conjugates under temperatures

516

below or above the LCST. 1H NMR spectra (Figure S10) proved the successful click reaction by

517

the appearance of typical peaks from the polymer backbones at 1 - 4 ppm and the triazole ring


27


Page 28 of 56

518

proton at 8.1 ppm and FTIR spectra (Figure 5 D) showed the total disappearance of azide

519

absorbance band. After reaction, the LCST (Figure 3 C) of conjugates increased from ~ 49 oC to

520

~ 58 oC compared to the alkyne polymer, indicating that the functional groups from the protein

521

significantly interact with the polymer moieties, which affect the thermal phase transition

522

behaviours. The dispersion stability of conjugates was also increased as the turbidity could be

523

stable during test period under 25 oC with only slight decrease at the beginning (Figure 2 C). It is

524

important to note that DLS (Figure 6 B) revealed multiple distribution of particles with size from

525

~ 40 nm to ~ 200 nm and ~ 600 nm and a relatively high PDI value (0.68) under 25 oC,

526

indicating that the soluble polymers would stabilize the conjugates even at molecular level and

527

prevent them from forming uniform assemblies. TEM image (Figure 5 C) of laccase @

528

poly(DEGEEA)-r-(PEGA480) under 25 oC showed the presence of similar sphere-like particles as

529

laccase @ poly (PEGA480), which has size distribution from tens of nanometres to hundreds of

530

nanometres. When the temperature was increased to a value (55 oC) close to the LCST, much of

531

the polymers would change from a swollen hydrophilic state to a collapsed hydrophobic state

532

and this change will drive the assembly of polymer as well as the folding of proteins. DLS

533

showed a main distribution at ~ 245 nm and the PDI decreased to 0.34. When the temperature

534

was further increased to 70 oC, DLS (Figure 6 B) revealed only a single distribution at ~ 284 nm

535

and a lower PDI of 0.19. These results prove the successful synthesis of protein-polymer

536

conjugates via self-catalyzed CuAAC reaction and support our hypothesis that hydrophobicity is

537

important for the assembly of giant amphiphiles.

538

Enzyme activity of modified laccase and laccase-polymer conjugates

539

Three types of copper, including type I or blue type copper, type II or usually type copper and

540

type III or coupling double-core copper were included in the laccase from Trametes versicolor


28

Page 29 of 56 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

Biomacromolecules

541

and type I or II copper could be removed when additional chelating agents exist.68, 69 Laccase has

542

been used for the detection of ascorbic acid or as the catalyst for ATRP, during which the

543

reduced enzyme activity after repeated use and electrostatic complexation of the polymer were

544

observed.

545

presence of reducing agents etc. was first tested.

48, 49, 70

In this research, the enzyme activity of laccase after modification or in the

546 547

Figure 7. Enzyme activity of laccase (1 mg mL-1) in the presence of CuSO4 (0.05 mg mL-1),

548

NaAsc (0.1 mg mL-1), CuCl2 (0.05 mg mL-1) or Na2SO4 (0.1 mg mL-1) before and after dialysis

549

(A) and enzyme activity of laccase-related conjugates (B). All tests were conducted in PBS

550

buffer (0.2 M, pH = 3.0) at 20 oC. Error bars represent standard deviations calculated from

551

triplicates.

552

Laccase was mixed with excess CuSO4, NaAsc, or both in order to check the effect of those

553

chemicals on laccase. The activity was tested directly in the presence of these chemicals or after

554

purification via dialysis. As shown in Figure 7, a significant decrease of laccase activity from

555

1.5± 0.05 U mg-1 (Figure 7) to 0.55 ± 0.12, 0.44 ± 0.04, 0.73 ± 0.14 U mg-1 was found in the

556

presence of CuSO4, NaAsc, or both (Figure 7 A) before dialysis, respectively. A further decrease

557

of activity was observed after removing these chemicals via dialysis and lyophilization. It’s our


29


Page 30 of 56

558

hypothesis that CuSO4 may denature protein or the excess SO42- may coordinate with the copper

559

in the laccase thus affect the activity. NaAsc would reduce the Cu2+ of laccase to Cu+ and it is

560

interesting that laccase still showed relative high activity even in the presence of excess NaAsc

561

that theoretically could induce a complete reduction, indicating the high stability of copper

562

coordination structure inside of the laccase. We further checked the enzyme activity of laccase in

563

the presence of CuCl2 or Na2SO4 or both before and after dialysis. In the presence of these salts

564

the enzyme activity decreased to 0.11 ± 0.01, 0.12 ± 0.01, 0.09 ± 0.01 U mg-1 separately as

565

shown in Figure 7A. While after dialysis the enzyme activity significantly increased to 0.36 ±

566

0.01, 0.39 ± 0.02, 0.31 ± 0.01 U mg-1 separately. This indicates that the presence of CuCl2 or

567

Na2SO4 also inhibits the activity of laccase and after removal of such salts the activity could be

568

recovered. It is also noted that after dialysis the activity was similar as that of laccase after

569

treated with CuSO4 or NaAsc. This phenomenon means that anions that may coordinate with the

570

copper such as Cl-1 and SO42- should be responsible for the loss of laccase activity.

571

When laccase was modified into laccase-N3, the primary structure of laccase changed to be

572

aggregation in water and the enzyme activity decreased to 1.08 ± 0.05 U mg-1. After coupling

573

with alkyne polymers, the weight content laccase (X) in the conjugate was roughly calculated by

574

elemental analysis, which should be included in the activity calculation of laccase-polymer

575

conjugates. For example, the elemental content of laccase @ poly(DEGEEA) ([C]: [H]: [N] =

576

48.85%: 7.60%: 0.67%) showed a significant decrease of nitrogen content and increase of carbon

577

& hydrogen content compared with laccase-N3 ([C]: [H]: [N] = 39.94%: 6.38%: 1.49%) after

578

coupled with poly(DEGEEA) ([C]: [H]: [N] = 55.44%: 8.86%: 0%), which further proved the

579

success of click reaction. So the weight ratio (X) of laccase in the conjugate could be calculated

580

according to carbon content change: 39.94 X + (1-X) 55.44 = 48.85, or hydrogen content change:


30

Page 31 of 56 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

Biomacromolecules

581

6.38 X + (1-X) 8.86 = 7.60, or nitrogen content change: 1.48 X = 0.67, which will lead to a series

582

of close value of laccase content as 43%, 51% or 45% respectively. Due to the high carbon

583

content in the conjugates, we utilized a value calculated according to the change of carbon

584

content in order to reduce the relative calculated error. Nevertheless, the test showed that after

585

conjugation with the poly(DEGEEA), propargyl alcohol and poly(DEGEEA)-r-(PEGA480), the

586

activity sharply decreased to 0.33 ± 0.01; 0.16 ± 0.02 and 0.10 ± 0.02 U mg-1. Similar trend has

587

also been observed when attaching a hydrophobic poly(styrene) tail to lipase, which was ascribed

588

to the destabilization of the active conformation of the enzyme.21 We speculate that this is also

589

due to the self-assembly of conjugates, which encapsulate much enzyme into the core of the

590

nanoparticles and prevent the efficient access of substrate to the active sites of enzyme.

591

However, the conjugates forming more stable assemblies with narrower PDI, such as laccase @

592

poly(DEGEEA), have shown higher enzyme activity than the other conjugates. We believe that

593

the stable assemblies would exposed more laccase on the surface and thus have more

594

accessibility site to the small-molecular substrate. It is quite interesting that the laccase @

595

saccharide showed similar activity as laccase, which is much higher than the other conjugates

596

including propargyl alcohol etc. as shown in Figure 7 B. Laccase itself is a glycoprotein and we

597

hypothesize that the click reaction with alkyne-functionalized saccharides loaded more

598

saccharides onto the protein, which may help to maintain its activity. This phenomenon needs

599

further research to confirm the relationship between enzyme activity and glycosylation.


31


Page 32 of 56

600 601

Figure 8. Enzyme activity of laccase and conjugates under different temperatures (pH = 3.0) (A)

602

and the relative activity kinetic (B) of laccase and conjugates incubated in PBS buffer (pH=3.0,

603

0.2 M) at 20 oC during 5 days. Error bars represent standard deviations calculated from

604

triplicates.

605

We further checked the effect of temperature on the enzyme activity. For the native laccase and

606

azide-functionalized laccase, they both showed the highest activity at 50 oC (Figure 8 A). When

607

attached with thermoresponsive poly(DEGEEA)-r-(PEGA480), the activity of conjugate at 50 oC

608

(0.08 ± 0.01 U mg-1) was even lower than that at 25 oC (0.13 ± 0.01 U mg-1) most probably due

609

to the collapse of polymer chain above the LCST, which will cover more catalytic centre of the

610

enzyme. The activity of laccase (1.69 ± 0.33 U mg-1) or laccase-N3 (0.50 ± 0.08 U mg-1) at 70 oC

611

still maintains at relatively high level. However, the laccase @ poly(DEGEEA)-r-(PEGA480)

612

conjugate showed very low activity (0.004 ± 0.002 U mg-1 ) under 70 oC, which further indicated

613

that the collapsed and aggregated polymers will prevent the exposure of the catalytic centre to

614

the substrate. When we used the laccase @ saccharides for the activity test under different

615

temperature and pH, the enzyme activity showed the highest value at 50 ℃ and pH 3.0 (Figure 9).


32

Page 33 of 56 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

Biomacromolecules

616

Under all these tests, the activity of laccase @ saccharides was found to be similar as that of the

617

unmodified laccase and slightly higher than laccase-N3.

618 619

Figure 9. The activity of laccase @ saccharides under different temperature (A, pH 3.0) and pH

620

(B, under ambient temperature (~ 15 ℃)). All tests were conducted in PBS buffer (0.2 M). Error

621

bars represent standard deviations calculated from triplicates.

622

Although the conjugates showed relatively lower enzyme activity than the free laccase, higher

623

stability was found for the laccase @ poly(PEGA480) conjugates under ambient temperature. As

624

shown in Figure 8 B, during a five-day test laccase showed a significant decrease of ~ 1 /3

625

activity the second day while the conjugates kept ~ 90% of the original activity. After four days,

626

laccase only showed ~ 42% relative activity while the conjugate still has ~ 2/3 of the initial

627

activity. We hypothesized that the conjugated polymers could increase the stability of protein

628

against environmental stress during a long time use. It is believed that the conjugation of

629

polymer to the protein may form a polymer net around the protein’s surface, which will embrace

630

the protein inside and may prevent the protein from subunit dissociation or denaturation during

631

the acute change of environmentally relevant stressors. Previous report showed that the

632

responsive nature of conjugated polymer allowed thermal regulation of the biological behaviour


33


Page 34 of 56

633

of the protein.71 By conjugation with trehalose glycopolymers or sulfonate-containing polymers,

634

the proteins showed superior stability to a series of environmentally relevant stressors including

635

heat, lyophilization or acid treatment etc.10,

636

agglomeration was found for some dextran-type polymer through the molecular crowding

637

effect.72, 73 It is possible that the aggregation of thermoresponsive polymers above the LCST

638

could lead to the compaction of laccase, which will be against the thermal unfolding during

639

heating and thus protein’s structure and activity could be maintained.

640

Conclusions

641

In conclusion, we have successfully synthesized protein-polymer giant amphiphiles via a

642

combination of Cu(0)-LRP and CuAAC click chemistry. The azide-functionalized succinimidyl

643

ester was synthesized in a one-step reaction with high conversion for the azidation of BSA and

644

laccase. Due to the short hydrophobic alkane linker, the azide-functionalized BSA and laccase

645

showed poor solubility in water and further proved the importance of linker type for the

646

solubility of conjugates. Well-defined polymers with controlled MW and narrow MW

647

distribution were synthesized by Cu(0)-LRP with almost full conversion within few hours under

648

mild reaction conditions. The trimethylsilyl protection groups proved to be important for the

649

LCST of thermoresponsive copolymer and could be removed via a facile TBAF process to

650

generate terminal alkyne groups quantitatively for subsequent click reaction. Reduction of

651

laccase by NaAsc could generate active copper catalyst for the CuAAC click reaction even

652

without the addition of external copper source such as CuSO4, although it may not reach

653

complete conversion due to the steric effect when conjugated with macromolecules. To the best

654

of our knowledge, this is the first report of enzymatic CuAAC click reaction and self-catalyzed

12

Increase of protein stability induced by


34

Page 35 of 56 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

Biomacromolecules

655

bioconjugation. The obtained giant amphiphiles could aggregate into spherical nanoparticles in

656

water and hydrophobicity was proved to be the most important factor during the self-assembly. It

657

revealed a decrease of activity after the chemical modification of enzyme or subsequent self-

658

assembly of conjugates, probably due to the destabilization of the active conformation of enzyme

659

or encapsulation of enzyme into the core of nanoparticles, which suggests that formation of pore

660

could be an available choice to increase the activity. We believe such giant amphiphiles will not

661

only deepen our understanding on protein folding and macroscopic self-assembly but also will

662

find potential applications in bionanoreactor, enzyme immobilization and water purification.

663

ASSOCIATED CONTENT

664

Supporting Information.

665

The Supporting Information is available free of charge on the ACS Publications website.

666

1

H NMR spectrum for the analysis of conversion during the Cu(0)-LRP of DEGEEA in DMSO,

667

1

H NMR spectra of the final poly(PEGA480) or poly(DEGEEA)-r-(PEGA480) products with

668

terminal alkyne groups, DLS of laccase-poly(PEGA480) conjugate and 1H NMR spectra of

669

laccase@ poly(DEGEEA)-r-(PEGA480) conjugate (PDF).

670

AUTHOR INFORMATION

671

Corresponding Author

672

* E-mail: [email protected] (Qiang Zhang).

673

ORCID

674

Qiang Zhang: 0000-0003-4596-9993


35


Page 36 of 56

675

Author Contributions

676

The manuscript was written through contributions of all authors. All authors have given approval

677

to the final version of the manuscript.

678

Funding Sources

679

Financial support from the Natural Science Foundation of China (21504044), Natural Science

680

Foundation of Jiangsu Province (BK20150769), Fundamental Research Funds for the Central

681

Universities (30916011203) and China Postdoctoral Science Foundation (157453) are greatly

682

acknowledged.

683

Notes

684

The authors declare no competing financial interest.

685

ACKNOWLEDGMENT

686

This project was under financial support from the Natural Science Foundation of China

687

(21504044), Natural Science Foundation of Jiangsu Province (BK20150769), Fundamental

688

Research Funds for the Central Universities (30916011203) and China Postdoctoral Science

689

Foundation (157453). Q. Z. appreciate the support from the Thousand Talents Plan and Jiangsu

690

Innovation and Entrepreneurship Program.

691

REFERENCES

692 693 694 695 696 697 698 699

1. Heredia, K. L.; Bontempo, D.; Ly, T.; Byers, J. T.; Halstenberg, S.; Maynard, H. D., In Situ Preparation of Protein−“Smart” Polymer Conjugates with Retention of Bioactivity. J. Am. Chem. Soc. 2005, 127, (48), 16955-16960. 2. Gauthier, M. A.; Klok, H.-A., Peptide/protein-polymer conjugates: synthetic strategies and design concepts. Chem. Commun. 2008, (23), 2591-2611. 3. Boyer, C.; Bulmus, V.; Liu, J.; Davis, T. P.; Stenzel, M. H.; Barner-Kowollik, C., WellDefined Protein−Polymer Conjugates via in Situ RAFT Polymerization. J. Am. Chem. Soc. 2007, 129, (22), 7145-7154.


36

Page 37 of 56 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

700 701 702 703 704 705 706 707 708 709 710 711 712 713 714 715 716 717 718 719 720 721 722 723 724 725 726 727 728 729 730 731 732 733 734 735 736 737 738 739 740 741 742 743 744 745

Biomacromolecules

4. Lele, B. S.; Murata, H.; Matyjaszewski, K.; Russell, A. J., Synthesis of Uniform Protein−Polymer Conjugates. Biomacromolecules 2005, 6, (6), 3380-3387. 5. Caliceti, P.; Veronese, F. M., Pharmacokinetic and biodistribution properties of poly(ethylene glycol)–protein conjugates. Adv. Drug Delivery Rev. 2003, 55, (10), 1261-1277. 6. Tao, L.; Mantovani, G.; Lecolley, F.; Haddleton, D. M., α-Aldehyde Terminally Functional Methacrylic Polymers from Living Radical Polymerization: Application in Protein Conjugation “Pegylation”. J. Am. Chem. Soc. 2004, 126, (41), 13220-13221. 7. Pelegri-O’Day, E. M.; Lin, E.-W.; Maynard, H. D., Therapeutic Protein–Polymer Conjugates: Advancing Beyond PEGylation. J. Am. Chem. Soc. 2014, 136, (41), 14323-14332. 8. Bailon, P.; Palleroni, A.; Schaffer, C. A.; Spence, C. L.; Fung, W.-J.; Porter, J. E.; Ehrlich, G. K.; Pan, W.; Xu, Z.-X.; Modi, M. W.; Farid, A.; Berthold, W.; Graves, M., Rational Design of a Potent, Long-Lasting Form of Interferon: A 40 kDa Branched Polyethylene GlycolConjugated Interferon α-2a for the Treatment of Hepatitis C. Bioconjug. Chem. 2001, 12, (2), 195-202. 9. Alconcel, S. N. S.; Baas, A. S.; Maynard, H. D., FDA-approved poly(ethylene glycol)protein conjugate drugs. Polym. Chem. 2011, 2, (7), 1442-1448. 10. Mancini, R. J.; Lee, J.; Maynard, H. D., Trehalose Glycopolymers for Stabilization of Protein Conjugates to Environmental Stressors. J. Am. Chem. Soc. 2012, 134, (20), 8474-8479. 11. Bat, E.; Lee, J.; Lau, U. Y.; Maynard, H. D., Trehalose glycopolymer resists allow direct writing of protein patterns by electron-beam lithography. Nat. Commun. 2015, 6, 6654-6661. 12. Nguyen, T. H.; Kim, S.-H.; Decker, C. G.; Wong, D. Y.; Loo, J. A.; Maynard, H. D., A heparin-mimicking polymer conjugate stabilizes basic fibroblast growth factor. Nat. Chem. 2013, 5, 221-227. 13. Paluck, S. J.; Nguyen, T. H.; Maynard, H. D., Heparin-Mimicking Polymers: Synthesis and Biological Applications. Biomacromolecules 2016, 17, (11), 3417-3440. 14. Shu, J. Y.; Tan, C.; DeGrado, W. F.; Xu, T., New Design of Helix Bundle Peptide−Polymer Conjugates. Biomacromolecules 2008, 9, (8), 2111-2117. 15. Xu, T.; Shu, J., Coiled-coil helix bundle, a peptide tertiary structural motif toward hybrid functional materials. Soft Matter 2010, 6, (2), 212-217. 16. Shu, J. Y.; Huang, Y.-J.; Tan, C.; Presley, A. D.; Chang, J.; Xu, T., Amphiphilic Peptide−Polymer Conjugates Based on the Coiled-Coil Helix Bundle. Biomacromolecules 2010, 11, (6), 1443-1452. 17. Dube, N.; Presley, A. D.; Shu, J. Y.; Xu, T., Amphiphilic Peptide–Polymer Conjugates with Side-Conjugation. Macromol. Rapid. Commun. 2011, 32, (4), 344-353. 18. Couet, J.; Samuel, J. D. J. S.; Kopyshev, A.; Santer, S.; Biesalski, M., Peptide–Polymer Hybrid Nanotubes. Angew. Chem. Int. Ed. 2005, 44, (21), 3297-3301. 19. Danial, M.; Tran, C. M. N.; Young, P. G.; Perrier, S.; Jolliffe, K. A., Janus cyclic peptide-polymer nanotubes. Nat. Commun. 2013, 4, 2780-2792. 20. Boerakker, M. J.; Hannink, J. M.; Bomans, P. H. H.; Frederik, P. M.; Nolte, R. J. M.; Meijer, E. M.; Sommerdijk, N. A. J. M., Giant Amphiphiles by Cofactor Reconstitution. Angew. Chem. Int. Ed. 2002, 41, (22), 4239-4241. 21. Velonia, K.; Rowan, A. E.; Nolte, R. J. M., Lipase Polystyrene Giant Amphiphiles. J. Am. Chem. Soc. 2002, 124, (16), 4224-4225. 22. Hannink, J. M.; Cornelissen, J. J. L. M.; Farrera, J. A.; Foubert, P.; De Schryver, F. C.; Sommerdijk, N. A. J. M.; Nolte, R. J. M., Protein–Polymer Hybrid Amphiphiles. Angew. Chem. Int. Ed. 2001, 40, (24), 4732-4734.


37


746 747 748 749 750 751 752 753 754 755 756 757 758 759 760 761 762 763 764 765 766 767 768 769 770 771 772 773 774 775 776 777 778 779 780 781 782 783 784 785 786 787 788 789

Page 38 of 56

23. Le Droumaguet, B.; Mantovani, G.; Haddleton, D. M.; Velonia, K., Formation of giant amphiphiles by post-functionalization of hydrophilic protein-polymer conjugates. J. Mater. Chem. 2007, 17, (19), 1916-1922. 24. Le Droumaguet, B.; Velonia, K., In Situ ATRP-Mediated Hierarchical Formation of Giant Amphiphile Bionanoreactors. Angew. Chem. Int. Ed. 2008, 47, (33), 6263-6266. 25. Daskalaki, E.; Le Droumaguet, B.; Gerard, D.; Velonia, K., Multifunctional Giant Amphiphiles via simultaneous copper(i)-catalyzed azide-alkyne cycloaddition and living radical polymerization. Chem. Commun. 2012, 48, (10), 1586-1588. 26. Liu, X.; Gao, W., In Situ Growth of Self-Assembled Protein–Polymer Nanovesicles for Enhanced Intracellular Protein Delivery. ACS Appl. Mater. Interfaces 2017, 9, (3), 2023-2028. 27. Vanparijs, N.; De Coen, R.; Laplace, D.; Louage, B.; Maji, S.; Lybaert, L.; Hoogenboom, R.; De Geest, B. G., Transiently responsive protein-polymer conjugates via a 'grafting-from' RAFT approach for intracellular co-delivery of proteins and immune-modulators. Chem. Commun. 2015, 51, (73), 13972-13975. 28. Zhang, Q.; Li, M.; Zhu, C.; Nurumbetov, G.; Li, Z.; Wilson, P.; Kempe, K.; Haddleton, D. M., Well-Defined Protein/Peptide–Polymer Conjugates by Aqueous Cu-LRP: Synthesis and Controlled Self-Assembly. J. Am. Chem. Soc. 2015, 137, (29), 9344-9353. 29. Li, H.; Li, M.; Yu, X.; Bapat, A. P.; Sumerlin, B. S., Block copolymer conjugates prepared by sequentially grafting from proteinsvia RAFT. Polym. Chem. 2011, 2, (7), 15311535. 30. Averick, S.; Simakova, A.; Park, S.; Konkolewicz, D.; Magenau, A. J. D.; Mehl, R. A.; Matyjaszewski, K., ATRP under Biologically Relevant Conditions: Grafting from a Protein. ACS Macro Lett. 2011, 1, (1), 6-10. 31. Jiang, Y.; Liang, M.; Svejkar, D.; Hart-Smith, G.; Lu, H.; Scarano, W.; Stenzel, M. H., Albumin-micelles via a one-pot technology platform for the delivery of drugs. Chem. Commun. 2014, 50, (48), 6394-6397. 32. Dag, A.; Jiang, Y.; Karim, K. J. A.; Hart-Smith, G.; Scarano, W.; Stenzel, M. H., Polymer–Albumin Conjugate for the Facilitated Delivery of Macromolecular Platinum Drugs. Macromol. Rapid. Commun. 2015, 36, (10), 890-897. 33. Jiang, Y.; Lu, H.; Dag, A.; Hart-Smith, G.; Stenzel, M. H., Albumin-polymer conjugate nanoparticles and their interactions with prostate cancer cells in 2D and 3D culture: comparison between PMMA and PCL. J. Mater. Chem. B 2016, 4, (11), 2017-2027. 34. Kolb, H. C.; Finn, M. G.; Sharpless, K. B., Click chemistry: Diverse chemical function from a few good reactions. Angew. Chem. Int. Ed. 2001, 40, (11), 2004-2021. 35. Moatsou, D.; Li, J.; Ranji, A.; Pitto-Barry, A.; Ntai, I.; Jewett, M. C.; O’Reilly, R. K., Self-Assembly of Temperature-Responsive Protein–Polymer Bioconjugates. Bioconjug. Chem. 2015, 26, (9), 1890-1899. 36. Tang, W.; Becker, M. L., "Click" reactions: a versatile toolbox for the synthesis of peptide-conjugates. Chem. Soc. Rev. 2014, 43, (20), 7013-7039. 37. Agard, N. J.; Prescher, J. A.; Bertozzi, C. R., A Strain-Promoted [3 + 2] Azide−Alkyne Cycloaddition for Covalent Modification of Biomolecules in Living Systems. J. Am. Chem. Soc. 2004, 126, (46), 15046-15047. 38. Früh, S. M.; Steuerwald, D.; Simon, U.; Vogel, V., Covalent Cargo Loading to Molecular Shuttles via Copper-free “Click Chemistry”. Biomacromolecules 2012, 13, (12), 3908-3911.


38

Page 39 of 56 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

790 791 792 793 794 795 796 797 798 799 800 801 802 803 804 805 806 807 808 809 810 811 812 813 814 815 816 817 818 819 820 821 822 823 824 825 826 827 828 829 830 831 832 833 834 835

Biomacromolecules

39. van Geel, R.; Pruijn, G. J. M.; van Delft, F. L.; Boelens, W. C., Preventing Thiol-Yne Addition Improves the Specificity of Strain-Promoted Azide–Alkyne Cycloaddition. Bioconjug. Chem. 2012, 23, (3), 392-398. 40. Dondoni, A.; Massi, A.; Nanni, P.; Roda, A., A New Ligation Strategy for Peptide and Protein Glycosylation: Photoinduced Thiol–Ene Coupling. Chem. Eur. J. 2009, 15, (43), 1144411449. 41. Wu, H.; Yang, B.; Zhao, Y.; Wei, Y.; Wang, Z.; Wang, X.; Tao, L., Fluorescent proteinreactive polymers via one-pot combination of the Ugi reaction and RAFT polymerization. Polym. Chem. 2016, 7, (30), 4867-4872. 42. Jones, M. W.; Strickland, R. A.; Schumacher, F. F.; Caddick, S.; Baker, J. R.; Gibson, M. I.; Haddleton, D. M., Polymeric Dibromomaleimides As Extremely Efficient Disulfide Bridging Bioconjugation and Pegylation Agents. J. Am. Chem. Soc. 2012, 134, (3), 1847-1852. 43. de Araújo, A. D.; Palomo, J. M.; Cramer, J.; Köhn, M.; Schröder, H.; Wacker, R.; Niemeyer, C.; Alexandrov, K.; Waldmann, H., Diels–Alder Ligation and Surface Immobilization of Proteins. Angew. Chem. Int. Ed. 2006, 45, (2), 296-301. 44. Kobayashi, S.; Uyama, H.; Kimura, S., Enzymatic Polymerization. Chem. Rev. 2001, 101, (12), 3793-3818. 45. Zhang, B.; Wang, X.; Zhu, A.; Ma, K.; Lv, Y.; Wang, X.; An, Z., Enzyme-Initiated Reversible Addition–Fragmentation Chain Transfer Polymerization. Macromolecules 2015, 48, (21), 7792-7802. 46. Liu, Z.; Lv, Y.; An, Z., Enzymatic Cascade Catalysis for the Synthesis of Multiblock and Ultrahigh-Molecular-Weight Polymers with Oxygen Tolerance. Angew. Chem. Int. Ed. 2017, 56, (44), 13852-13856. 47. Wang, X.-H.; Wu, M.-X.; Jiang, W.; Yuan, B.-L.; Tang, J.; Yang, Y.-W., NanoflowerShaped Biocatalyst with Peroxidase Activity Enhances the Reversible Addition–Fragmentation Chain Transfer Polymerization of Methacrylate Monomers. Macromolecules 2018, 51, (3), 716723. 48. Ng, Y.-H.; di Lena, F.; Chai, C. L. L., PolyPEGA with predetermined molecular weights from enzyme-mediated radical polymerization in water. Chem. Commun. 2011, 47, (22), 64646466. 49. Fodor, C.; Gajewska, B.; Rifaie-Graham, O.; Apebende, E. A.; Pollard, J.; Bruns, N., Laccase-catalyzed controlled radical polymerization of N-vinylimidazole. Polym. Chem. 2016, 7, (43), 6617-6625. 50. Mantovani, G.; Ladmiral, V.; Tao, L.; Haddleton, D. M., One-pot tandem living radical polymerisation-Huisgens cycloaddition process ("click") catalysed by N-alkyl-2pyridylmethanimine/Cu(i)Br complexes. Chem. Commun. 2005, (16), 2089-2091. 51. Ciampolini, M.; Nardi, N., Five-Coordinated High-Spin Complexes of Bivalent Cobalt, Nickel, andCopper with Tris(2-dimethylaminoethyl)amine. Inorg. Chem. 1966, 5, (1), 41-44. 52. Storms-Miller, W. K.; Pugh, C., Prop-2-yn-1-yl 2-Bromo-2-methylpropanoate: Identification and Suppression of Side Reactions of a Commonly Used Terminal AlkyneFunctional ATRP Initiator. Macromolecules 2015, 48, (12), 3803-3810. 53. Roy, B.; Mukhopadhyay, B., Sulfuric acid immobilized on silica: an excellent catalyst for Fischer type glycosylation. Tetrahedron Letters 2007, 48, (22), 3783-3787. 54. Zhang, Q.; Slavin, S.; Jones, M. W.; Haddleton, A. J.; Haddleton, D. M., Terminal functional glycopolymers via a combination of catalytic chain transfer polymerisation (CCTP) followed by three consecutive click reactions. Polym. Chem. 2012, 3, (4), 1016-1023.


39


836 837 838 839 840 841 842 843 844 845 846 847 848 849 850 851 852 853 854 855 856 857 858 859 860 861 862 863 864 865 866 867 868 869 870 871 872 873 874 875 876 877 878 879 880 881

Page 40 of 56

55. Magnusson, J. P.; Bersani, S.; Salmaso, S.; Alexander, C.; Caliceti, P., In Situ Growth of Side-Chain PEG Polymers from Functionalized Human Growth Hormone—A New Technique for Preparation of Enhanced Protein−Polymer Conjugates. Bioconjug. Chem. 2010, 21, (4), 671678. 56. Ladmiral, V.; Mantovani, G.; Clarkson, G. J.; Cauet, S.; Irwin, J. L.; Haddleton, D. M., Synthesis of Neoglycopolymers by a Combination of “Click Chemistry” and Living Radical Polymerization. J. Am. Chem. Soc. 2006, 128, (14), 4823-4830. 57. Pich, A.; Bhattacharya, S.; Adler, H.-J. P.; Wage, T.; Taubenberger, A.; Li, Z.; van Pee, K.-H.; Böhmer, U.; Bley, T., Composite Magnetic Particles as Carriers for Laccase from Trametes versicolor. Macromol. Biosci. 2006, 6, (4), 301-310. 58. Dai, Y.; Niu, J.; Liu, J.; Yin, L.; Xu, J., In situ encapsulation of laccase in microfibers by emulsion electrospinning: Preparation, characterization, and application. Bioresour. Technol. 2010, 101, (23), 8942-8947. 59. Grandjean, C.; Boutonnier, A.; Guerreiro, C.; Fournier, J.-M.; Mulard, L. A., On the Preparation of Carbohydrate−Protein Conjugates Using the Traceless Staudinger Ligation. J. Org. Chem. 2005, 70, (18), 7123-7132. 60. Konkolewicz, D.; Wang, Y.; Krys, P.; Zhong, M.; Isse, A. A.; Gennaro, A.; Matyjaszewski, K., SARA ATRP or SET-LRP. End of controversy? Polym. Chem. 2014, 5, (15), 4396-4417. 61. Lligadas, G.; Grama, S.; Percec, V., Recent Developments in the Synthesis of Biomacromolecules and their Conjugates by Single Electron Transfer–Living Radical Polymerization. Biomacromolecules 2017, 18, (4), 1039-1063. 62. Soeriyadi, A. H.; Boyer, C.; Nyström, F.; Zetterlund, P. B.; Whittaker, M. R., High-Order Multiblock Copolymers via Iterative Cu(0)-Mediated Radical Polymerizations (SET-LRP): Toward Biological Precision. J. Am. Chem. Soc. 2011, 133, (29), 11128-11131. 63. Zhang, Q.; Collins, J.; Anastasaki, A.; Wallis, R.; Mitchell, D. A.; Becer, C. R.; Haddleton, D. M., Sequence-Controlled Multi-Block Glycopolymers to Inhibit DC-SIGN-gp120 Binding. Angew. Chem. Int. Ed. 2013, 52, (16), 4435-4439. 64. Leophairatana, P.; Samanta, S.; De Silva, C. C.; Koberstein, J. T., Preventing Alkyne– Alkyne (i.e., Glaser) Coupling Associated with the ATRP Synthesis of Alkyne-Functional Polymers/Macromonomers and for Alkynes under Click (i.e., CuAAC) Reaction Conditions. J. Am. Chem. Soc. 2017, 139, (10), 3756-3766. 65. Zhang, Q.; Anastasaki, A.; Li, G.-Z.; Haddleton, A. J.; Wilson, P.; Haddleton, D. M., Multiblock sequence-controlled glycopolymers via Cu(0)-LRP following efficient thiol-halogen, thiol-epoxy and CuAAC reactions. Polym. Chem. 2014, 5, (12), 3876-3883. 66. Kellis Jr, J. T.; Nyberg, K.; S˘ail, D. a.; Fersht, A. R., Contribution of hydrophobic interactions to protein stability. Nature 1988, 333, 784-786. 67. Pace, C. N.; Fu, H.; Fryar, K. L.; Landua, J.; Trevino, S. R.; Shirley, B. A.; Hendricks, M. M.; Iimura, S.; Gajiwala, K.; Scholtz, J. M.; Grimsley, G. R., Contribution of Hydrophobic Interactions to Protein Stability. J. Mol. Biol. 2011, 408, (3), 514-528. 68. Mayer, A. M.; Staples, R. C., Laccase: new functions for an old enzyme. Phytochemistry 2002, 60, (6), 551-565. 69. Riva, S., Laccases: blue enzymes for green chemistry. Trends Biotechnol. 2006, 24, (5), 219-226. 70. Wang, Z.; Xu, Q.; Wang, J.-H.; Yang, Q.; Yu, J.-H.; Zhao, Y.-D., Ascorbic acid biosensor based on laccase immobilized on an electrode modified with a self-assembled


40

Page 41 of 56 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

882 883 884 885 886 887 888 889 890 891 892 893

Biomacromolecules

monolayer and coated with functionalized quantum dots. Microchim. Acta 2009, 165, (3), 387392. 71. De, P.; Li, M.; Gondi, S. R.; Sumerlin, B. S., Temperature-Regulated Activity of Responsive Polymer−Protein Conjugates Prepared by Grafting-from via RAFT Polymerization. J. Am. Chem. Soc. 2008, 130, (34), 11288-11289. 72. de Lencastre Novaes, L. C.; Mazzola, P. G.; Pessoa, A.; Vessoni Penna, T. C., Investigation of charged polymer influence on green fluorescent protein thermal stability. New Biotechnol. 2011, 28, (4), 391-395. 73. Engel, R.; Westphal, A. H.; Huberts, D. H. E. W.; Nabuurs, S. M.; Lindhoud, S.; Visser, A. J. W. G.; van Mierlo, C. P. M., Macromolecular Crowding Compacts Unfolded Apoflavodoxin and Causes Severe Aggregation of the Off-pathway Intermediate during Apoflavodoxin Folding. J. Biol. Chem. 2008, 283, (41), 27383-27394.

894 895 896 897 898 899 900 901 902 903 904 905


41


Page 42 of 56

906 907

Table of Contents Graphic

908 909

Synthesis and assembly of laccase-polymer giant amphiphiles by a combination of Cu(0)-LRP &

910

self-catalyzed CuAAC click chemistry.


42

Page 43 of 56 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

Biomacromolecules

Figure 1. FTIR spectra (A) and SEC elution traces (B) of BSA, BSA-N3, BSA @ propargyl alcohol and BSA@ poly(PEGA480) conjugates. 1411x496mm (72 x 72 DPI)



Figure 2. FTIR (A), aqueous SEC elution traces (B), turbidity (C), size distribution by DLS (D) and TEM image (D, inset, laccase-N3) of laccase, laccase-N3 and laccase conjugates. 1411x989mm (72 x 72 DPI)


Page 44 of 56

Page 45 of 56 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

Biomacromolecules

Figure 3. DMF SEC elution traces (A), 1H NMR spectra (B), temperature dependence of the optical transmittance at 500 nm (C, 1 mg mL−1) and DLS (D, alkyne terminal poly(DEGEEA)-r-(PEGA480), 1 mg mL−1) of alkyne-terminal polymers. 1411x983mm (72 x 72 DPI)



Figure 4. 1H NMR spectra (A) of samples taken during the click reaction of laccase-N3 with 1-(2′-propargyl) D-mannose using CuSO4 as the additional catalyst and the FTIR spectra (B) of laccase @ saccharides conjugates. 1411x498mm (72 x 72 DPI)


Page 46 of 56

Page 47 of 56 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

Biomacromolecules

Figure 5. TEM images (A: laccase @ poly(DEGEEA); B: laccase @ poly(PEGA480); C: laccase @ poly(DEGEEA)-r-(PEGA480)), FTIR (D) & 1H NMR (E, product obtained under the catalysis of CuSO4) spectra of laccase-polymer conjugates. 1411x804mm (72 x 72 DPI)



Figure 6. SEC elution traces (A) and DLS (B) of laccase-polymer conjugates. 1411x496mm (72 x 72 DPI)


Page 48 of 56

Page 49 of 56 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

Biomacromolecules

Figure 7. Enzyme activity of laccase (1 mg mL-1) in the presence of CuSO4 (0.05 mg mL-1), NaAsc (0.1 mg mL-1), CuCl2 (0.05 mg mL-1) or Na2SO4 (0.1 mg mL-1) before and after dialysis (A) and enzyme activity of laccase-related conjugates (B). All tests were conducted in PBS buffer (0.2 M, pH = 3.0) at 20 oC. Error bars represent standard deviations calculated from triplicates. 1411x492mm (72 x 72 DPI)



Figure 8. Enzyme activity of laccase and conjugates under different temperatures (pH = 3.0) (A) and the relative activity kinetic (B) of laccase and conjugates incubated in PBS buffer (pH=3.0, 0.2 M) at 20 oC during 5 days. Error bars represent standard deviations calculated from triplicates. 1411x492mm (72 x 72 DPI)


Page 50 of 56

Page 51 of 56 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

Biomacromolecules

Figure 9. The activity of laccase @ saccharides under different temperature (A, pH 3.0) and pH (B, under ambient temperature (~ 15 ℃)). All tests were conducted in PBS buffer (0.2 M). Error bars represent standard deviations calculated from triplicates. 1411x498mm (72 x 72 DPI)



Scheme 1. Scheme representation for the synthesis of functional succinimidyl ester and azidation of proteins. 249x34mm (300 x 300 DPI)


Page 52 of 56

Page 53 of 56 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

Biomacromolecules

Scheme 2. Synthesis of functional polymers with terminal alkyne groups by Cu(0)-LRP. 243x37mm (300 x 300 DPI)



Scheme 3. Self-catalyzed click reaction for the glycosylation modification of laccase-N3. 271x67mm (300 x 300 DPI)


Page 54 of 56

Page 55 of 56 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

Biomacromolecules

Scheme 4. Synthesis of protein-polymer conjugates by CuAAC click reactions. 236x42mm (300 x 300 DPI)



TOC 231x56mm (300 x 300 DPI)


Page 56 of 56

Synthesis and Assembly of Laccase-polymer Giant Amphiphiles by

Recommend Documents