Flexible Method for Conjugation of Phenolic Lignin Model Compounds

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Flexible Method for Conjugation of Phenol Lignin Model Compounds to Carrier Proteins Ruili Gao, Fachuang Lu, Yimin Zhu, Michael G. Hahn, and John Ralph J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b04273 • Publication Date (Web): 03 Oct 2016 Downloaded from http://pubs.acs.org on October 4, 2016

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Journal of Agricultural and Food Chemistry

Flexible Method for Conjugation of Phenolic Lignin Model Compounds to Carrier Proteins

Ruili Gao,*,a,b Fachuang Lu,a,b Yimin Zhu,c Michael G. Hahn,d and John Ralph*,a,b a

Department of Biochemistry, University of Wisconsin-Madison, Madison, WI, 53706, USA. email: [email protected]

b

DOE Great Lakes Bioenergy Research Center, Wisconsin Energy Institute, University of Wisconsin-Madison, Madison, WI 53726, USA. email: [email protected]

c

Department of Chemistry, The Pennsylvania State University, Altoona College 3000 Ivyside Park, Altoona, PA 16601, USA

d

Complex Carbohydrate Research Center, The University of Georgia, Athens, Georgia 30602, US

ACS Paragon Plus Environment


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ABSTRACT

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Linking lignin model compounds to carrier proteins is required in order either to raise

3

antibodies to them or to structurally screen antibodies raised against lignins or models. We

4

describe a flexible method to link phenolic compounds of interest to cationic bovine serum

5

albumin (cBSA) without interfering with their important structural features. With the

6

guaiacylglycerol-β-guaiacyl ether dimer, for example, the linking was accomplished in

7

89% yield with the number of dimers per carrier protein being as high as 50; NMR

8

experiments on a 15N- and 13C-labeled conjugation product indicated that 13 dimers were

9

added to the native lysine residues and the remainder (~37) to the amine moieties on the

10

ethylenediamine linkers added to BSA; ~32% of the available primary amine groups on

11

cBSA were therefore conjugated to the hapten. This loading is suitable for attempting to

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raise new antibodies to plant lignins and for screening.

13 14

KEYWORDS: lignin model dimer, lignification, antibody, cBSA-conjugation, NMR

2 ACS Paragon Plus Environment

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INTRODUCTION

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Lignification, although crucial to plant growth and development, is a prime factor limiting

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the utilization of cell walls in agriculturally important plants in processes such as ruminant

18

digestibility and biomass feedstock conversion to liquid biofuels. Improving our

19

understanding of the complex lignin polymer structure becomes important both in

20

designing pretreatments to provide access to wall polysaccharides and in engineering

21

plants to minimize the recalcitrance caused by lignins.1-17 Over the past decade,

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considerable research has focused on the compatibility of non-traditional lignin monomer

23

(monolignol) components available during lignin polymerization,1,18-23 and on the

24

structural attributes of the resulting polymer that incorporate novel monomers throughout

25

cell wall development. Antibodies against lignin substructures have become effective tools

26

for delineating the in planta distributions of various components, for clarifying the

27

mechanisms of lignification in the plant cell wall, and for potentially giving new

28

information that cannot be obtained by chemical degradative and other analytical

29

methods.24-39 In immunological studies, a particular compound can be surveyed and

30

characterized according to the selectivity of an antigen-antibody reaction. The application

31

of polyclonal antibodies raised against pure guaiacyl and mixed guaiacyl-syringyl

32

synthetic lignins (DHPs or dehydrogenation polymers) has helped illustrate the lignin

33

guaiacyl and mixed guaiacyl-syringyl profile and how it is influenced by various genetic

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perturbations.31-35 A polyclonal antibody raised from a synthetic DHP polymer directed

35

specifically against syringyl units has also been reported.36

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To date, few immunological probes for lignin have been developed. Antibodies can be

37

raised directly to synthetic lignins or carefully selected isolated lignins. This is the basis of



38

the fairly successful polyclonal antibodies raised to synthetic guaiacyl (G) and syringyl (S)

39

lignins. Antibodies can also be raised to synthesized model compounds for either the

40

prominent or the more minor recognized interunit bonding structures in native lignins.

41

Successful polyclonal antibodies have been raised to a dibenzodioxocin model, for

42

example.37 Recently, monoclonal antibodies toward some lignin-related phenolics such as

43

p-coumarates were generated.38 Monoclonal antibodies generated against

44

dehydrodiconiferyl alcohol were used to localize β–5- or β–β-linked lignin structures in

45

plant cell walls.39 The availability of such antibodies has been a boon to the lignin

46

research community, finding uses in wide-ranging studies, but the range of such

47

antibodies for recognizing diverse and well-characterized structural epitopes in lignins is

48

limited.

49

In the case of low-molecular-mass dimers or oligomers, they must be non-disruptively

50

linked to a carrier protein, in order to raise and screen the antibodies, via means that will

51

not obliterate the structural features that need to be recognized. It therefore becomes

52

particularly important to find a method to load the low-molecular-mass lignin model

53

compounds onto a carrier protein. There are two methods reported. The primary method

54

reacts the amine group from lysine units in the protein with p-AHA (p-aminohippuric

55

acid), converts the product aromatic amines to diazonium ions via treatment with NaNO2,

56

and reacts these with phenolic models, with the addition (electrophilic aromatic

57

substitution) occurring ortho to the phenol.40 This alters the substitution on the aromatic

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ring and also has the obvious limitation that it can not be used for syringyl units that are

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already substituted at both positions ortho to the phenol. The requirement for two steps

60

involving the protein provides a challenge to characterizing the products. More


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importantly, there are only 60 lysines in standard BSA,41 some 30-35 of which are

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accessible for coupling reactions. The amount of hapten loading to the carrier protein is

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therefore typically ~20 per protein. A second method was used to raise polyclonal

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antibodies to the relatively recently discovered dibenzodioxocin units in lignins. A

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dibenzodioxocin model compound in which (non-native) carboxylic acids had been

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introduced into the side-chains was added to the carrier protein by the coupling reaction of

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the carboxylic acid with the lysine amine. However, the conjugation of the

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dibenzodioxocin model to BSA resulted in precipitation and only 6% was conjugated in

69

30% protein yield. Although carboxylic acids could, in principle, be introduced into side-

70

chains of most lignin model compounds, the non-standard nature of these models and the

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potential disruption of the recognition of native lignin structures limits the general

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usefulness of this approach. However, the methodology used37 provides the basis

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procedure for our method.

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The goal of our research here was to develop a flexible method that can be widely

75

used for the conjugation of lignin model compounds to a carrier protein, with high loading

76

of hapten. For our purposes, it was essential to establish the efficacy of the reactions

77

involved, and thus to find a way to characterize the protein conjugates.

78 79

MATERIALS AND METHODS

80

General

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All organic substrates and solvents were purchased from commercial sources and used

82

without further purification. Commercial cBSA was obtained from Fisher Scientific.

83

Flash-chromatography was performed with Biotage snap silica cartridges on an Isolera



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84

One (Biotage, Charlottesville, VA). NMR spectra were acquired on a Bruker Biospin

85

(Billerica, MA) AVANCE 500 MHz and 700 MHz spectrometers equipped with a 5 mm

86

cryogenic TCI 1H/13C/15N (500) or QCI 1H/31P/13C/15N (700) gradient probe with inverse

87

geometry (proton coil closest to the sample). Spectral processing was performed using

88

Bruker’s Topspin 3.1 (Mac) software. The central solvent peaks were used as internal

89

reference

90

implementations of 1D and 2D [gradient-selected correlation spectroscopy (COSY),

91

heteronuclear single-quantum coherence (HSQC), and heteronuclear multiple-bond

92

correlation (HMBC)] NMR experiments were used for routine structural assignments of

93

newly synthesized compounds. Dialysis was performed by using slide-A-Lyzer™ dialysis

94

cassette (10,000 MWCO, 3-12 mL capacity). MALDI-TOF MS was measured by Applied

95

Biosystems/MDS SCIEX 4800 MALDI TOF/TOF with OPtiBeamTM on-axis laser as

96

source.

(δH/δC:

acetone-d6, 2.04/29.84;

D2O,

4.79).

The

standard

Bruker

97 98

Preparation of cBSA

99

BSA (6 mg) was dissolved in 1.0 mL MES buffer (0.1 M, pH 4.7) and this solution was

100

slowly added to 1.0 mL ethylenediamine dihydrochloride (1 M in 0.1 M MES, pH 4.7).

101

Then 4 mg 1-ethyl-3-(dimethylaminopropyl)-carbodiimide (EDC) was added and the

102

mixture was stirred at room temperature for 6 h during which time another 8 mg EDC in

103

two 4 mg aliquots at 2 h intervals was added. The reaction solution was then dialyzed

104

against PBS buffer (0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4) and lyophilized.

105 106

Study of cBSA yield dependency on the amount of EDC


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A set of experiments was performed. (a) BSA (6 mg) was dissolved in 1.0 mL MES buffer

108

(0.1 M, pH 4.7) and this solution was slowly added to 1.0 mL ethylenediamine

109

dihydrochloride (1 M in 0.1 M MES, pH 4.7). Then 4 mg EDC was added and the mixture

110

was stirred at room temperature for 2 h. The reaction solution was then dialyzed against

111

PBS buffer (0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4) and lyophilized. (b) BSA (6

112

mg) was dissolved in 1.0 mL MES buffer (0.1 M, pH 4.7) and this solution was slowly

113

added to 1.0 mL ethylenediamine dihydrochloride (1 M in 0.1 M MES, pH 4.7). Then 4

114

mg EDC was added and the mixture was stirred at room temperature for 4 h during which

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another 4 mg aliquot EDC was added at 2 h intervals was added. The reaction solution

116

was then dialyzed against PBS buffer (0.02 M sodium phosphate, 0.15 M NaCl, pH 7.4)

117

and lyophilized. (c) BSA (6 mg) was dissolved in 1.0 mL MES buffer (0.1 M, pH 4.7) and

118

this solution was slowly added to 1.0 mL ethylenediamine dihydrochloride (1 M in 0.1 M

119

MES, pH 4.7). Then 4 mg EDC was added and the mixture was stirred at room

120

temperature for 6 h during which another 8 mg two 4 mg aliquot EDC was added at 2 h

121

intervals. The reaction solution was then dialyzed against PBS buffer (0.02 M sodium

122

phosphate, 0.15 M NaCl, pH 7.4) and lyophilized.

123 124

Model compounds

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The model compounds in Figure 1 were all synthesized by standard methods. The β-ether

126

dimeric models 1a and 2a, were prepared as previously.42 Dehydrodiconiferyl alcohol (3a,

127

β–5) was synthesized via 8–5-coupled dehydrodiferulate,43 followed by reduction of the

128

esters with DiBAL-H,44 as described for this exact compound in the Supplementary



129

Information in a previous report.45 Syringaresinol (4a, β–β) was produced via the radical

130

coupling of sinapyl alcohol.46

131 132

Phenol etherification and acid introduction to produce the hapten required for

133

conjugation

134

The conjugation reaction is illustrated with the β-ether model 1a to produce compound 1b

135

(Figure 2). The β–O–4-dimer (1a, 96 mg, 0.3 mmol) and 55 mg (0.33 mmol) ethyl

136

bromoacetate were dissolved in 10 mL acetone and then 207 mg (1.5 mmol) K2CO3 was

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added to the solution. The mixture was stirred overnight, after which solids were filtered

138

off and the solvent was evaporated. The crude product was dissolved in 10 mL 95%

139

ethanol and 0.5 mL 1 M NaOH was added. After stirring for 2 h, the solution was

140

acidified to pH ~1, checked using a 0-2 range pH test kit, with 1 M HCl and extracted with

141

ethyl acetate. The organic layer was washed with satd. NaCl solution and dried over

142

MgSO4. The solvent was evaporated and the crude product was purified over 50 g silica-

143

gel column by flash chromatography (CH2Cl2/MeOH/AcOH = 9:1:0.1%, v/v/v) to give

144

100 mg product 1b (yield 89%). Under the same conditions, the other lignin model

145

compounds (Figure 1) were successfully produced to the haptens for the conjugation with

146

83-87% yields. The haptens were characterized by 1D and 2D NMR.

147 148

Compound 1b. 1H NMR (acetone-d6) δ (ppm): 3.48 and 3.68 (2H, m, Hγ), 3.81 (3H, s, A-

149

OCH3), 3.85 (3H, s, B-OCH3), 4.20 (1H, m, Hβ), 4.67 (2H, S, CH2), 4.91 (1H, m, Hα),

150

6.85 (1H, m, B-H6), 6.90 (1H, d, J = 8.43 Hz, A-H5), 6.93-6.98 (2H, m, B-H1 and B-H5),

151

6.99 (1H, m, B-H2), 7.15 (1H, dd, J = 8.43 and 1.60 Hz, A-H6), 7.16 (1H, d, J = 1.60 Hz,


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A-H2). 13C NMR (acetone-d6) δ (ppm): 56.12 (B-OCH3), 56.26 (A-OCH3), 61.83 (Cγ),

153

66.73 (CH2CO), 73.58 (Cα), 88.28 (Cβ), 112.03 (A-C2), 113.34 (B-C2), 115.06 (A-C5),

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119.78 (A-C6 and B-C5), 121.93 (B-C6), 123.46 (B-C1), 136.56 (A-C1), 147.95 (A-C4),

155

149.63 (B-C4), 150.57 (A-C3), 151.76 (B-C3) and 170.41 (CO).

156 157

Compound 2b. 1H NMR (acetone-d6) δ (ppm): 2.27 (3H, S, CH3), 3.34 and 3.41 (2H, m,

158

Hγ), 3.84 (12H, s, OCH3), 4.20 (1H, m, Hβ), 4.50 (2H, S, CH2), 5.00 (1H, m, Hα), 6.53

159

(2H, B-H3), 6.81 (2H, A-H2). 13C NMR (acetone-d6) δ (ppm): 21.74 (CH3), 56.45

160

(OCH3), 61.53 (Cγ), 70.62 (CH2CO), 73.84 (Cα), 88.89 (Cβ), 104.98 (B-C3/C5), 107.01

161

(A-C2/C6), 134.62 (B-C4), 134.78 (A-C1), 136.19 (B-C1), 139.10 (A-C4), 152.89 (B-C2),

162

153.53 (A-C3) and 170.53 (CO).

163 164

Compound 3b. 1H NMR (acetone-d6) δ (ppm): 3.52 (2H, CH2), 3.80 (3H, s, B-OCH3),

165

3.85 (3H, s, A-OCH3), 3.88 (1H, m, Hβ), 3.91 (2H, CH2OH), 4.68 (2H, S, CH2), 5.60 (1H,

166

d, J = 6.77 Hz Hα), 6.24 (1H, ddd, J = 17.53, 7.3, 7.3, CH), 6.54 (1H, d, J = 17.53, CH),

167

6.90-6.96 (4H, A-H2 A-H6, A-H5, and B-H2) ), 7.07 (2H, B-H6). 13C NMR (acetone-d6)

168

δ (ppm): 54.76 (Cβ), 56.23 (OCH3), 63.37 (CH2CH), 64.59 (Cγ), 66.43 (CH2CO), 88.17

169

(Cα), 111.21 (A-C2), 115.14 (B-C2), 116.30 (A-C5), 118.80 (B-C6), 128.27 (A-C6),

170

130.55 (B-C5), 131.97 (B-C1), 136.88 (A-C1), 145.14 (B-C3), 148.81(A-C3), 150.65 (A-

171

C4) and 170.41 (CO).

172 173

Compound 4b. 1H NMR (acetone-d6) δ (ppm): 3.18 (2H, m, Hβ), 3.81 (12H, s, OCH3),

174

4.23 (4H, m, Hγ), 4.49 (2H, S, CH2), 4.71 (2H, m, Hα), 6.67 (4H, d, J = 1.60 Hz, A-



175

H2/H6 and B-H2/H6). 13C NMR (acetone-d6) δ (ppm): 55.76 (Cβ), 56.61 (OCH3), 72.41

176

(Cγ), 69.65 (CH2CO), 86.2 (Cα), 104.2 (A-C2 and B-C2), 133.21 (C1), 136.32 (B-C4),

177

148.21 (A-C4), 153.81 (A-C3/B-C3) and 170.91 (CO).

178 179

Reaction of model compound 1b with N-α-carbobenzoxy-L-lysine methyl ester

180

hydrochloride and characterization of the product 1c

181

L-Lys-OMe·HCl

182

and 125 mg model compound 1b (0.33 mmol) was dissolved in 2 mL THF. The latter

183

solution was slowly added to the lysine solution under stirring. Then 57 mg EDC (0.3

184

mmol) was added to the mixture. After stirring at room temperature for 6 h, the mixture

185

was extracted with ethyl acetate and dried over MgSO4. The crude product was purified by

186

flash chromatography (n-hexanes/EtOAc) to give 160 mg product 1c (yield 82%).

(100 mg, 0.3 mmol) was dissolved in 6 mL THF/0.1M MES buffer (1:2),

187 188

Compound 1c. 1H NMR (acetone-d6) δ (ppm): 1.42 (2H, m, H11), 1.53 (2H, m, H10), 1.76

189

(2H, m, H12), 3.27 (2H, m, H9), 3.48 and 3.68 (2H, m, Hγ), 3.66 (3H, s, C15H3), 3.81

190

(3H, s, A-OCH3), 3.85 (3H, s, B-OCH3), 4.17 (1H, m, NCH), 4.20 (1H, m, Hβ), 4.42 (2H,

191

s, C7H2), 4.94 (1H, m, Hα), 5.06 (2H, s, CH2Ph), 6.67 (1H, d, J = 7.34 Hz, HNC13), 6.85

192

(1H, m, B-H6), 6.90 (1H, d, J = 8.43 Hz, A-H5), 6.93-6.98 (2H, m, B-H1 and B-H5), 6.99

193

(1H, m, B-H2), 7.15 (1H, dd, J = 8.43 and 1.60 Hz, A-H6), 7.16 (1H, d, J = 1.60 Hz, A-

194

H2), 7.43 (1H, t, J = 5.43 Hz, HNC8). 13C NMR (acetone-d6) δ (ppm): 23.69 (C11), 29.83

195

(C10), 31.94 (C12), 38.84 (C9), 52.23 (C15), 54.98 (C13), 56.12 (B-OCH3), 56.26 (A-

196

OCH3), 61.83 (Cγ), 66.73 (CH2Ph), 70.54 (C7), 73.58 (Cα), 88.04 (Cβ), 112.03 (A-C2),

197

113.34 (B-C2), 116.29 (A-C5), 119.78 (A-C6 and B-C5), 121.93 (B-C6), 123.46 (B-C1),


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128.65 (C-C2), 129.20 (C-C4), 129.27 (C-C3), 137.34 (A-C1), 138.14 (C-C1), 147.81 (A-

199

C4), 149.55 (B-C4), 150.57 (A-C3), 151.76 (B-C3), 157.10 (NCO), 168.90 (C8) and

200

173.66 (C14). HR-MS (ESI) calcd for C34H42N2O11 [(M + H)+]: 655.2862; found:

201

655.2858.

202 203

Conjugation to cBSA or BSA

204

The procedure is illustrated for compound 1b. Hapten 1b (10 mg) was dissolved in 1.0 mL

205

THF and then added 0.5 mL MES buffer (0.1 M, pH 4.7). This solution was added

206

dropwise to 1.0 mL cBSA or BSA solution (10 mg cBSA or BSA in 1.0 mL 0.1 M MES

207

buffer, pH 4.7), and then 5 mg EDC was added to this mixture. The conjugate solution

208

was allowed to react for 2 h at room temperature. The reaction mixture was dialyzed

209

against 2 L PBS buffer (pH 7.4) for 6 h in 2 h intervals and then 2 L water for 4 h in 2 h

210

intervals, and finally lyophilized. The other conjugations of haptens to cBSA or BSA were

211

performed under the same conditions.

212 213

Preparation of 15N/13C-labeled conjugate sample for 3D HNCO NMR

214

The coupling product of the β–O–4-dimer 1a with ethyl bromoacetate (13C-labeled on CO)

215

was hydrolyzed by 1 M NaOH in 95% ethanol solution. After purification the 13C-labeled

216

hapten 1b was collected in 89% yield. 15N-labeled cBSA was synthesized from the

217

conjugation of 15N-labeled ethylenediamine to BSA by the same protocol as for making

218

cBSA. The conjugation of 13C-labeled hapten 1b to 15N-labeled cBSA was performed

219

following the typical protocol.

220

The NMR sample contained PBS buffer (20 mM sodium phosphate, 150 mM NaCl,



221

pH 7.4), 8% D2O (v/v), 50 µM 4,4-dimethyl-4-silapentane-1-sulfonic acid (DSS). DSS

222

was used as an internal reference. NMR data were collected at the National Magnetic

223

Resonance Facility at Madison (NMRFAM). NMR spectra were acquired at 25 °C using a

224

600 MHz Varian Unity-Inova spectrometer equipped with a z-gradient cryogenic probe.

225

3D acquisition used 64 increments in the 15N dimension and 64 increments in the 13C

226

dimension with 16 scans for each increment. TimeTN, the transfer delay to detect the

227

weak three-bond 3JNC' correlation across H-bonds between the amide N and the carbonyl C

228

was set to 66.5 ms.47,48 Sparky was used for data analysis.49

229 230

RESULTS AND DISCUSSION

231 232

Preparation and characterization of cBSA

233

Bovine serum albumin (BSA) has 60 Lys residues, along with 40 Asp and 59 Glu residues

234

that bear carboxylate sidechain groups that can be converted to further new primary

235

amines with ethylenediamine to increase the number of conjugatable primary amine

236

groups on the protein, as well as the net charge or pI, producing cationic bovine serum

237

albumin (cBSA). The highly positive charge on cBSA increases its immunogenicity, and

238

cBSA used as a carrier protein induces a similar increase in the production of antibodies

239

against any attached haptens.50-52 Here we modify the typical protocol by addition of EDC

240

at 2 h intervals (see Experimental Section) to generate a more uniform cBSA with a high

241

conversion of native carboxylic acid groups to amines, a product that is more suitable for

242

our purposes.53

243

The amount of ethylenediamine covalently added to BSA was evaluated by MALDI-


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TOF MS using sinapic acid as a matrix. Figure 3A shows the MALDI-TOF MS spectra of

245

BSA (top), cBSA from our protocol (middle), and commercial cBSA (bottom). The

246

molecular weights, estimated from the spectra, were approximately 66,658, 70,921, and

247

69,841. Obviously, the difference in molecular weight between a cBSA preparation and

248

the BSA parent corresponds to the mass of covalently bound ethylenediamine, i.e., the

249

ethylenediamine on BSA loading. From the MALDI-TOF MS spectra, our protocol can

250

generate cBSA with a loading of ~100 ethylenediamines; from the number of Asp and Glu

251

units in BSA (99 units), this is therefore an ~100% yield on the protein with 98% purity.

252

By comparison, the purchased commercial cBSA contained only 76 ethylenediamines

253

with ~60% purity. cBSA therefore contains the same level of lysine (60 units) as standard

254

BSA but contains a further 100 primary amines as a result of adding ethylenediamine to

255

the carboxylic acid groups. As a consequence, there are ~160 primary amines on cBSA

256

that can react with a carboxylic acid group on a hapten.

257

To investigate the effect of EDC additions on the yield of cBSA, a set of experiments

258

was performed by adjusting the amount of EDC added to the reaction mixture. Figure 3B

259

demonstrates the molecular weights of cBSA resulting from adding a different number of

260

aliquots (and different amounts) of EDC. The molecular weight of cBSA prepared

261

following the typical protocol by adding 4 mg EDC was 68,842 (top) which, when

262

compared to the molecular weight of BSA at 66,658, indicates that this cBSA contains 52

263

ethylenediamines. The middle spectrum in Figure 3B shows that a cBSA with a molecular

264

weight of 70,104 was generated by adding 8 mg EDC in two 4 mg aliquots at 2 h intervals,

265

corresponding to 82 added ethylenediamines. The bottom spectrum in Figure 3B shows

266

that ~100 of the carboxylic acid groups in BSA reacted with ethylenediamine to form



267

cBSA with a molecular weight of 70,921 after adding 12 mg EDC in three 4 mg aliquots

268

at 2 h intervals. It is therefore clear that the cationization degree of BSA, and the

269

homogeneity of the product, can be conveniently controlled by adjusting the EDC

270

addition.

271 272

Etherification of the model compound phenol and introduction of a carboxylic acid

273

Our aim was to provide a flexible derivatization method for the phenol in lignin model

274

compounds, etherifying the phenol and therefore mimicking most of the units in the lignin

275

polymer (that are also etherified), and at the same time introducing a new carboxylic acid

276

group. Such model compounds can then conjugate to the carrier proteins via the usual

277

amidation reactions between this carboxylic acid and primary amine groups on the

278

protein.54 With this goal, the hapten 1b was synthesized in 89% yield by adding ethyl

279

bromoacetate to the phenolic β–O–4-dimer 1a, followed by mild saponification. Structural

280

confirmation of the product’s authenticity was by the usual array of 1D and 2D NMR

281

methods. The other models were similarly derivatized, in yields of 83-87%.

282 283 284

Testing the efficacy of conjugation using (protected) lysine as a protein model Before the conjugation of the hapten to the carrier protein was attempted, we assured

285

ourselves that the coupling reaction of the derivatized model 1b with protected lysine was

286

clean and high yielding. The coupling reaction of the model compound 1b and protected

287

lysine (N-α-carbobenzoxy-L-lysine methyl ester hydrochloride) was performed in

288

THF/MES buffer solution to generate the product 1c (Figure 2) in 82% yield after

289

purification by flash chromatography. The product was characterized by 1D and 2D NMR.


Page 14 of 32

Page 15 of 32


290 291

Conjugation of model compounds to cBSA and BSA

292

The conjugation of model compound 1b to the cBSA was performed by the typical

293

protocol and the amount of model compound in its respective conjugates was evaluated by

294

MALDI-TOF MS. Figure 3C shows the MALDI-TOF MS spectra of cBSA (top), and the

295

cBSA conjugated with the β–O–4-dimer model compound 1b (middle). The molecular

296

weights estimated from the spectra were approximately 70,921 and 88,885, indicating that

297

~50 covalently bonded lignin model compound units were on each cBSA. By contrast,

298

only ~13 model dimers were conjugated to BSA (bottom) (representing conjugation to

299

~20% of the lysine units). Under the same conditions, the conjugation scope was

300

expanded by performing the conjugation of the three other lignin model compounds: the

301

syringyl β-aryl ether 2b (β–O–4 S–S), phenylcoumaran (β–5) model 3b and resinols (β–β)

302

model 4b with cBSA with a loading of ~44, ~113 and ~41 molecules of the model

303

compounds per molecule of carrier protein (Figure 3D). Recently, the conjugation of

304

dehydrodiconiferyl alcohol, pinoresinol, and the phenolic β-ether model 1a, to BSA by the

305

p-AHA (p-aminohippuric acid)-BSA method was reported, but the number of covalently

306

bonded lignin model compounds was only ~3-10.39 It has been conjectured, but not firmly

307

established or documented, that having a loading of ~50 model dimers per molecule of

308

carrier protein was ideal; we therefore conjectured that the improved method for

309

producing highly cationized cBSA, and the new method for derivatizing and conjugating

310

models to it, should provide more effective hapten-protein conjugates for use in the

311

process of raising and screening antibodies. This assumption can not be validated here,

312

and a recent paper determined that very high hapten loadings reduced immunogenicity,



313

albeit not in the BSA system.55 With reproducibility also reported to be an issue,56 the

314

method described here illustrates how to obtain very high loadings, but does not determine

315

the efficacy for monoclonal antibody production which is known to be particularly

316

difficult. Clearly, making lower loadings by simply using lower molar hapten:protein

317

ratios is possible by the same approach as described here.

318

Based on MALDI-TOF MS spectra, there are ~50 model dimers attached to cBSA, but

319

the question remains: where are the model compounds bound to cBSA? In cBSA, there are

320

60 amino groups from lysine and ~100 added primary amines from the ethylenediamine

321

linkers that can bond to the carboxylic acid on the model compound. These 50 model

322

compounds loaded onto cBSA may only bond to native lysines, to amine groups from the

323

added ethylene diamine, or may bond to both. To investigate where the model compounds

324

bond with cBSA, we first performed the conjugation of model compound 1b to BSA

325

under the same conditions as for the conjugation to the cBSA. Figure 3C (bottom) shows

326

that the molecular weight of this conjugation product is 71,421. Comparison with the

327

molecular weight of BSA (66,658) indicates that the number of bonded β–O–4-dimer

328

model compound units on BSA is 13, i.e., on 20% of the lysine units.

329

To confirm that the other dimers (~37 of them) were bonded to the small linker from

330

ethylenediamine, [U-15N]-labeled cBSA was synthesized by using [U-15N]-labeled

331

ethylenediamine, and the β–O–4-dimer model compound 1b was synthesized with a 13C-

332

label on the carbonyl carbon. The conjugation of the 13C-labeled dimer model compound

333

to the [U-15N]-labeled cBSA was performed following the typical protocol and the

334

molecular weight of this conjugation product was evaluated again by MALDI-TOF MS. In

335

addition, a 3D HNCO NMR experiment was performed using the reaction product of


Page 16 of 32

Page 17 of 32


336

unlabeled BSA, U-15N-labeled ethylenediamine, and 13C-labeled dimer model compound

337

(Figure S1). This triple resonance experiment detects through-backbone connectivities in

338

proteins between 15N and 1HN atoms of one residue together with the carbonyl carbon

339

(13CO) resonance of the preceding residue through 1J(N-H) and 1J(N-CO) coupling

340

constants.57 Only the model compound that is covalently bonded to the linker has 15N-13CO

341

coupling and gives an NMR correlation signal under this experiment. Conversely, any

342

model compound bound directly to BSA (14N-13CO) is not detectable in this experiment.

343

As shown in Figure S1, correlation signals were observed on both a C–H and an N–H

344

plane, indicating that the model compound had indeed conjugated to the labeled linker of

345

the derivatized cBSA. Thus, on the N–H plane (Figure S1b), the signal (8.00/114 ppm)

346

belongs to the 1H–15N connection. Figure S1a shows the signal on the C–H plane, the x-

347

axis is the 1H chemical shift in ppm and the y-axis is 13C chemical shift in ppm. On the C–

348

H plane, there is one signal (1H at 8.00 ppm belonging to 1H–15N and 13C at 174 ppm

349

belonging to 13CO). This signal started at 1H–15N, goes through the 15N–13C bond, and

350

then back through 13C–15N and finally back to 1H. This HNCO NMR data therefore

351

confirms that model compound 1b has indeed linked to cBSA by bonding to the small

352

linker’s primary amine.

353

In summary, we have described herein flexible new methodology to produce protein

354

conjugates of phenolic compounds, at high levels if desired, relevant to lignins in plant

355

cell walls. Such conjugates are useful for both exploring the production of antibodies from

356

protein-hapten conjugates, and for producing the assortment of protein-bound model

357

compounds that are required to screen antibodies for selected activities and to attempt to

358

identify specific recognition epitopes (or at least structural features that are recognized by



359

such antibodies). These activities are intended to support the production of monoclonal

360

antibodies that will be particularly useful in many areas of plant cell wall research.


Page 18 of 32

Page 19 of 32


361

ABBREVIATIONS

362

BSA

Bovine serum albumin

363

cBSA

Cationic bovine serum albumin

364

DHP

Dehydrogenation polymer (of monolignols, i.e., a synthetic lignin)

365

MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass

366

spectrometry

367

MES

2-(N-morpholino)ethanesulfonic acid

368

PBS

Phosphate buffered saline

369

EDC

1-Ethyl-3-(dimethylaminopropyl)-carbodiimide

370

DSS

4,4-Dimethyl-4-silapentane-1-sulfonic acid

371 372 373

ACKNOWLEDGEMENT

374

We are grateful to funding provided through the US Department of Energy, the Office of

375

Science (BER DOE-DE-SC0006930).

376 377

SUPPORTING INFORMATION AVAILABLE.

378

This material is available free of charge via the Internet at http://pubs.acs.org.

379 380



381 382

REFERENCES (1) Vanholme, R.; Morreel, K.; Darrah, C.; Oyarce, P.; Grabber, J. H.; Ralph, J.;

383

Boerjan, W., Metabolic engineering of novel lignin in biomass crops. New Phytol. 2012,

384

196, 978-1000.

385 386 387

(2) Vanholme, R.; Morreel, K.; Ralph, J.; Boerjan, W., Lignin engineering. Curr. Opin. Plant Biol. 2008, 11, 278-285. (3) Ralph, J.; Lundquist, K.; Brunow, G.; Lu, F.; Kim, H.; Schatz, P. F.; Marita, J. M.;

388

Hatfield, R. D.; Ralph, S. A.; Christensen, J. H.; Boerjan, W., Lignins: natural polymers

389

from oxidative coupling of 4-hydroxyphenylpropanoids. Phytochem. Rev. 2004, 3, 29-60.

390

(4) Boerjan, W.; Ralph, J.; Baucher, M., Lignin biosynthesis. Annual Reviews in Plant

391 392 393 394

Biology 2003, 54, 519-546. (5) Boudet, A. M., Lignins and lignification: Selected issues. Plant Physiol. Biochem. 2000, 38, 81-96. (6) Fu, C.; Mielenz, J. R.; Xiao, X.; Ge, Y.; Hamilton, C. Y.; Rodriguez, M., Jr.; Chen,

395

F.; Foston, M.; Ragauskas, A.; Bouton, J.; Dixon, R. A.; Wang, Z.-Y., Genetic

396

manipulation of lignin reduces recalcitrance and improves ethanol production from

397

switchgrass. Proc. Natl. Acad. Sci. 2011, 108, 3803-3808.

398

(7) Jackson, L. A.; Shadle, G. L.; Zhou, R.; Nakashima, J.; Chen, F.; Dixon, R. A.,

399

Improving saccharification efficiency of Alfalfa stems through modification of the

400

terminal stages of monolignol biosynthesis. Bioenergy Research 2008, 1, 180-192.

401 402

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(9) Li, X.; Chapple, C., Understanding lignification: challenges beyond monolignol biosynthesis. Plant Physiol. 2010, 154, 449-452. (10) Bonawitz, N. D.; Chapple, C., The genetics of lignin biosynthesis: connecting genotype to phenotype. Annu. Rev. Genet. 2010, 44, 337-363. (11) Li, X.; Weng, J. K.; Chapple, C., Improvement of biomass through lignin modification. Plant J. 2008, 54, 569-581. (12) Chapple, C.; Ladisch, M.; Meilan, R., Loosening lignin’s grip on biofuel production. Nat. Biotechnol. 2007, 25, 746-748. (13) Chapple, C.; Carpita, N., Plant cell walls as targets for biotechnology. Cur. Opin. in Plant Biol. 1998, 1, 179-185.

413

(14) Chundawat, S. P. S.; Donohoe, B. S.; da Costa Sousa, L.; Elder, T.; Agarwal, U.

414

P.; Lu, F.; Ralph, J.; Himmel, M. E.; Balan, V.; Dale, B. E., Multi-scale visualization and

415

characterization of lignocellulosic plant cell wall deconstruction during thermochemical

416

pretreatment. Energy and Environmental Science 2011, 4, 973-984.

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(15) Simmons, B. A.; Loqué, D.; Ralph, J., Advances in modifying lignin for enhanced biofuel production. Curr. Opin. Plant Biol. 2010, 13, 313-320. (16) Ralph, J., Perturbing Lignification. In The Compromised Wood Workshop 2007,

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Entwistle, K.; Harris, P. J.; Walker, J., Eds. Wood Technology Research Centre,

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University of Canterbury, New Zealand: Canterbury, 2007; pp 85-112.

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(17) Shen, H.; Poovaiah, C. R.; Ziebell, A.; Tschaplinski, T. J.; Pattathil, S.; Gjersing,

423

E.; Engle, N. L.; Katahira, R.; Pu, Y.; Sykes, R.; Chen, F.; Ragauskas, A. J.; Mielenz, J.

424

R.; Hahn, M. G.; Davis, M.; Stewart, C. N.; Dixon, R. A., Enhanced characteristics of



425

genetically modified switchgrass (Panicum virgatum L.) for high biofuel production.

426

Biotechnology for Biofuels 2013, 6, 1-15.

427

(18) Ralph, J., What makes a good monolignol substitute? In The Science and Lore of

428

the Plant Cell Wall Biosynthesis, Structure and Function, Hayashi, T., Ed. Universal

429

Publishers (BrownWalker Press): Boca Raton, FL, 2006; pp 285-293.

430

(19) Tobimatsu, Y.; Elumalai, S.; Grabber, J. H.; Davidson, C. L.; Pan, X.; Ralph, J.,

431

Hydroxycinnamate conjugates as potential monolignol replacements: in vitro lignification

432

and cell wall studies with rosmarinic acid. ChemSusChem 2012, 5, 676-686.

433

(20) Grabber, J. H.; Ress, D.; Ralph, J., Identifying new lignin bioengineering targets:

434

Impact of epicatechin, quercetin glycoside, and gallate derivatives on the lignification and

435

fermentation of maize cell walls. J. Agric. Food Chem. 2012, 60, 5152-5160.

436

(21) Elumalai, S.; Tobimatsu, Y.; Grabber, J. H.; Pan, X.; Ralph, J., Epigallocatechin

437

gallate incorporation into lignin enhances the alkaline delignification and enzymatic

438

saccharification of cell walls. Biotechnology for Biofuels 2012, 5, 1-13.

439

(22) Grabber, J. H.; Schatz, P. F.; Kim, H.; Lu, F.; Ralph, J., Identifying new lignin

440

bioengineering targets: 1. Monolignol substitute impacts on lignin formation and cell wall

441

fermentability. BMC Plant Biol. 2010, 10, 1-13.

442

(23) Grabber, J. H.; Hatfield, R. D.; Lu, F.; Ralph, J., Coniferyl ferulate incorporation

443

into lignin enhances the alkaline delignification and enzymatic degradation of maize cell

444

walls. Biomacromolecules 2008, 9, 2510-2516.

445 446

(24) Chabannes, M.; Ruel, K.; Yoshinaga, A.; Chabbert, B.; Jauneau, A.; Josseleau, J.P.; Boudet, A.-M., In situ analysis of specifically engineered tobacco lignins reveals a


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447

differential impact of individual transformations on the spatial patterns of lignin

448

deposition at the cellular and sub-cellular levels. Plant J. 2001, 28, 271-282.

449

(25) Pincon, G., Chabannes, M., Lapierre, C., Pollet, B., Ruel, K., Joseleau, J. P.,

450

Boudet, A. M., Legrand, M., Simultaneous down-regulation of caffeic/5-hydroxy ferulic

451

acid-O-methyltransferase I and cinnamoyl-coenzyme A reductase in the progeny from a

452

cross between tobacco lines homozygous for each transgene. Consequences for plant

453

development and lignin synthesis. Plant Physiol. 2001, 126, 145-155.

454

(26) Rohde, A.; Morreel, K.; Ralph, J.; Goeminne, G.; Hostyn, V.; De Rycke, R.;

455

Kushnir, S.; Van Doorsselaere, J.; Joseleau, J. P.; Vuylsteke, M.; Van Driessche, G.; Van

456

Beeumen, J.; Messens, E.; Boerjan, W., Molecular phenotyping of the pal1 and pal2

457

mutants of Arabidopsis thaliana reveals far-reaching consequences on phenylpropanoid,

458

amino acid, and carbohydrate metabolism. Plant Cell 2004, 16, 2749-2771.

459 460 461

(27) Ruel, K.; Burlat, V.; Joseleau, J. P., Relationship between ultrastructural topochemistry of lignin and wood properties. Iawa Journal 1999, 20, 203-211. (28) Goujon, T.; Ferret, V.; Mila, I.; Pollet, B.; Ruel, K.; Burlat, V.; Joseleau, J. P.;

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Barrière, Y.; Lapierre, C.; Jouanin, L., Down-regulation of the AtCCR1 gene in

463

Arabidopsis thaliana: effects on phenotype, lignins and cell wall degradability. Planta

464

2003, 217, 218-228.

465

(29) Ruel, K.; Montiel, M. D.; Goujon, T.; Jouanin, L.; Burlat, V.; Joseleau, J. P.,

466

Interrelation between lignin deposition and polysaccharide matrices during the assembly

467

of plant cell walls. Plant Biol. 2002, 4, 2-8.

468 469

(30) Donaldson, L. A., Lignification and lignin topochemistry – an ultrastructural view. Phytochemistry 2001, 57, 859-873.



470

(31) Joseleau, J. P.; Imai, T.; Kuroda, K.; Ruel, K., Detection in situ and

471

characterization of lignin in the G-layer of tension wood fibres of Populus deltoides.

472

Planta 2004, 219, 338-345.

473

(32) Joseleau, J. P.; Ruel, K., Study of lignification by noninvasive techniques in

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growing maize internodes – An investigation by Fourier transform infrared cross-

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polarization magic angle spinning 13C-nuclear magnetic resonance spectroscopy and

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immunocytochemical transmission electron microscopy. Plant Physiol. 1997, 114, 1123-

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1133.

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(33) Ruel, K., Chabannes, M., Boudet, A. M., Legrand, M. Joseleau, J. P.,

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Reassessment of qualitative changes in lignification of transgenic tobacco plants and their

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impact on cell wall assembly. Phytochemistry 2001, 57, 875-882.

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(34) Leplé, J.-C.; Dauwe, R.; Morreel, K.; Storme, V.; Lapierre, C.; Pollet, B.;

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Naumann, A.; Kang, K.-Y.; Kim, H.; Ruel, K.; Lefèbvre, A.; Joseleau, J.-P.; Grima-

483

Pettenati, J.; De Rycke, R.; Andersson-Gunneras, S.; Erban, A.; Fehrle, I.; Petit-Conil, M.;

484

Kopka, J.; Polle, A.; Messens, E.; Sundberg, B.; Mansfield, S. D.; Ralph, J.; Pilate, G.;

485

Boerjan, W., Downregulation of cinnamoyl-coenzyme A reductase in poplar: Multiple-

486

level phenotyping reveals effects on cell wall polymer metabolism and structure. Plant

487

Cell 2007, 19, 3669-3691.

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(35) Ruel, K., Faix, O., Joseleau, J. P., New immunogold probes for studying the

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distribution of the different lignin types during plant-cell wall biogenesis. J. Trace

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Microprobe Tech. 1994, 12, 247-265.

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(36) Joseleau, J. P., Faix, O., Kuroda, K. I., Ruel, K., A polyclonal antibody directed against syringylpropane epitopes of native lignins. C. R. Biol. 2004, 327, 809-815.


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(37) Kukkola, E. M.; Koutaniemi, S.; Gustafsson, M.; Karhunen, P.; Ruel, K.; Lundell,

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T. K.; Saranpaa, P.; Brunow, G.; Teeri, T. H.; Fagerstedt, K. V., Localization of

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dibenzodioxocin substructures in lignifying Norway spruce xylem by transmission

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electron microscopy-immunogold labeling. Planta 2003, 217, 229-237.

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(38) Tranquet, O., Saulnier, Luc, Utille, J. P., Ralph, J., Guillon, F., Monoclonal antibodies to p-coumarate. Phytochemistry 2009, 70, 1366-1373. (39) Kiyoto, S., Yoshinaga, A., Tanaka, N., Wada, M., Kamitakahara, H., Takabe, K.,

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Immunolocalization of 8-5' and 8-8' linked structures of lignin in cell walls of

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Chamaecyparis obtusa using monoclonal antibodies. Planta 2013, 237, 705-715.

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(40) Nagasaki, T., Yasuda, S., Imai, T., Preparation of antibody against agatharesinol, a norlignan, using a hapten-carrier conjugate. Phytochemistry 2002, 58, 833-840. (41) Huang, B. X.; Kim, H. Y.; Dass, C., Probing three-dimensional structure of bovine

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serum albumin by chemical cross-linking and mass spectrometry. J. Am. Soc. Mass

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Spectrom. 2004, 15, 1237-1247.

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(42) Nakatsubo, F.; Sato, K.; Higuchi, T., Synthesis of guaiacylglycerol-b-guaiacyl ether. Holzforschung 1975, 29, 165-8. (43) Ralph, J.; Garcia-Conesa, M. T.; Williamson, G., Simple preparation of 8-5coupled diferulate. J. Agric. Food Chem. 1998, 46, 2531-2532. (44) Quideau, S.; Ralph, J., Facile large-scale synthesis of coniferyl, sinapyl, and pcoumaryl alcohol. J. Agric. Food Chem. 1992, 40, 1108-1110. (45) Morreel, K.; Kim, H.; Lu, F.; Dima, O.; Akiyama, T.; Vanholme, R.; Niculaes, C.; Goeminne, G.; Inzé, D.; Messens, E.; Ralph, J.; Boerjan, W., Mass-spectrometry-based



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fragmentation as an identification tool in lignomics. Analytical Chemistry 2010, 82, 8095-

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8105.

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(46) Freudenberg, K.; Neish, A. C., Constitution and biosynthesis of lignin. SpringerVerlag: Berlin-Heidelberg-New York, 1968. (47) Cordier, F., Grzesiek, S., Direct observation of hydrogen bonds in proteins by

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interresidue (3h)J(NC') scalar couplings. J. Am. Chem. Soc. 1999, 121, 1601-1602.

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(48) Cordier, F., Nisius, L., Dingley, A. J., Grzesiek, S., Direct detection of N-

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H[...]O=C hydrogen bonds in biomolecules by NMR spectroscopy. Nat. Protoc. 2008, 3,

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235-41.

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(49) Goddard, T. D., Kneller, D. G., SPARKY 3, Sparky is a software package

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developed at the University of San Francisco for visualizing and analyzing

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multidimensional (2D, 3D or 4D) NMR spectra.

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(50) Muckerheide, A., Apple, R. J., Pesce, A. J., Michael, J. G., Cationization of protein antigens. I. Alteration of immunogenic properties. J. Immunol 1987, 138, 833-837. (51) Apple, R. J., Domen, P. L., Muckerheide, A., Michael, J. G., Cationization of

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protein antigens. IV. Increased antigen uptake by antigen-presenting cells. J. Immunol

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1988, 140, 3290-3295.

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(52) Domen, P. L., Muckerheide, A., Michael, J. G., Cationization of protein antigens III. Abrogation of oral tolerance. J. Immunol 1987, 139, 3195-3198. (53) Hermanson, G. T., Bioconjugate Techniques Second Edition ed.; Academic Press: San Diego, Calif, USA, 2008. (54) Brinkley, M., A brief survey of methods for preparing protein conjugates with dyes, haptens, and cross-linking reagents. Bioconj. Chem. 1992, 3, 2-13.


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(55) Pryde, D. C.; Jones, L. H.; Gervais, D. P.; Stead, D. R.; Blakemore, D. C.; Selby,

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M. D.; Brown, A. D.; Coe, J. W.; Badland, M.; Beal, D. M.; Glen, R.; Wharton, Y.;

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Miller, G. J.; White, P.; Zhang, N.; Benoit, M.; Robertson, K.; Merson, J. R.; Davis, H. L.;

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McCluskie, M. J., Selection of a novel anti-nicotine vaccine: influence of antigen design

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(56) Singh, K. V.; Kaur, J.; Varshney, G. C.; Raje, M.; Suri, C. R., Synthesis and

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545

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546

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547

dimensional NMR experiments with improved sensitivity. Journal of Magnetic

548

Resonance, Series B 1994, 103, 203-216.

549



550

Scheme and Figures

551 552

Figure 1. Lignin model compounds used for the conjugation with carrier proteins:

553

guaiacyl β-Aryl ether (1a, 1b, β–O–4-G); syringyl β-Aryl ether (2a, 2b, β–O–4-S);

554

phenylcoumaran dehydrodiconiferyl alcohol (3a, 3b, β–5); and resinol syringaresinol (4a,

555

4b, β–β).

556 557

Figure 2. Reaction of model compound with (protected) lysine. i) Ethyl bromoacetate,

558

K2CO3, acetone; ii) NaOH/95% ethanol, 89%; iii) EDC/THF/ 0.1 M MES, pH 4.7, 82%.

559 560

Figure 3. MALDI-TOF MS spectra. (A) BSA and cBSA comparisons. Top: BSA; middle:

561

cBSA from the modified protocol described; bottom: cBSA from Fisher Scientific. (B)

562

cBSA loading. cBSA was prepared by adding, top: 4 mg EDC; middle: 8 mg EDC;

563

bottom: 12 mg EDC. (C) Proteins and model 1b conjugates. Top: cBSA; middle: cBSA

564

with model compound conjugated; bottom: BSA with model compound conjugated. (D)

565

Proteins with models 2b – 4b conjugated. cBSA conjugated with; top: syringyl β-aryl

566

ether 2b (β–O–4 S–S); middle: dehydrodiconiferyl alcohol 3b (β–5); bottom:

567

syringaresinol 4b (β–β).

568


Page 28 of 32

Page 29 of 32


Figure 1.



Figure 2.


Page 30 of 32

Page 31 of 32


100

100

66,658

A)

60 40 20

103,169 0 100

70,921

60 40 20

60 40 20

47,485

0 100

0 100

69,841

70,921

80 % Intensity

80 % Intensity

70,104

80 % Intensity

% Intensity

80

56,638

60 40

65,316 46,134

0 40,000

50,000

74,245

60,000

70,000

80,000 90,000 Mass (m/z)

100,000

100

47,485

50,000

60,000

70,000

D)

80,000 90,000 Mass (m/z)

100,000

110,000

90,045

% Intensity

80

60 40 20

40

0 40,000

110,000

70,921

C)

60

20

83,469

80 % Intensity

40 20

0 100

100

60

99,669

88,748

20

68,842

B)

80 % Intensity

% Intensity

80

60 40 20

47,485

0 100

0 100

88885

116,031 80

80 % Intensity

% Intensity

58,382 60 40 20

0 100

71,421

89,437 80 % Intensity

80 % Intensity

40 20

0 100

60 40 20

60 40 20

95,641 0 40,000

60

50,000

60,000

70,000

80,000 90,000 Mass (m/z)

107,344

100,000

110,000

0 50K

60K

70K

80K

Figure 3.


90K

100K 110K 120K 130K 140K 150K Mass (m/z)


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Flexible Method for Conjugation of Phenolic Lignin Model Compounds

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