A novel reactive dyeing method for silk fibroin with aromatic primary amine-containing dyes based on the Mannich reaction

Article abstract of DOI:10.1016/j.dyepig.2019.04.061

Chemical modification of silk fibroin is a useful method for improving its performance. There are many existing methods of protein modification, some of which are limited for application to silk by the disadvantages of severe reaction conditions, poor reaction selectivity and problems with characterization of the product. In this paper, a series of dyes containing aromatic primary amine groups was designed and synthesized for modification of silk fibroin via the Mannich reaction. This three-component Mannich modification showed several advantages, such as mild reaction conditions, good selectivity, easy characterization and high fastness. In particular, the degree of modification could be established visually according to the color yield and the color fixation on silk fibroin. Therefore, the Mannich reaction of silk with dyes containing aromatic primary amine groups may become a novel reactive dyeing method. Studies of structure-performance relationships showed that suitable dyes for dyeing of silk should meet the following criteria: (1)High nucleophilicity of the aromatic primary amine group. (2)No high electron cloud density center around the amino group.

Full text of DOI:10.1016/j.dyepig.2019.04.061

Accepted Manuscript
A novel reactive dyeing method for silk fibroin with aromatic primary amine-containing
dyes based on the Mannich reaction
Weiguo Chen, Pu Gao, Hua Jiang, Zhihua Cui
PII:
S0143-7208(18)31626-7
https://doi.org/10.1016/j.dyepig.2019.04.061
DYPI 7516
DOI:
Reference:
To appear in: Dyes and Pigments
Received Date: 24 July 2018
Revised Date: 2 April 2019
Accepted Date: 25 April 2019
Please cite this article as: Chen W, Gao P, Jiang H, Cui Z, A novel reactive dyeing method for silk
fibroin with aromatic primary amine-containing dyes based on the Mannich reaction, Dyes and Pigments
(2019), doi: https://doi.org/10.1016/j.dyepig.2019.04.061.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
1
2
3
4
5
6
7
8
9
A novel reactive dyeing method for silk fibroin with aromatic primary
amine-containing dyes based on the Mannich reaction
Weiguo Chena,b, Pu Gaoa, Hua Jianga,b, Zhihua Cuia,b,*
a
Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University,
Hangzhou 310018, China
b Key Laboratory of Advanced Textile Materials and Manufacturing Technology, Ministry of Education of China, Hangzhou 310018,
China
*Corresponding author
Email address: zhhcui@zstu.edu.cn
10
11
12
13
14
15
16
17
18
19
20
21
22
Abstract: Chemical modification of silk fibroin is a useful method for improving its performance.
There are many existing methods of protein modification, some of which are limited for application
to silk by the disadvantages of severe reaction conditions, poor reaction selectivity and problems
with characterization of the product. In this paper, a series of dyes containing aromatic primary
amine groups was designed and synthesized for modification of silk fibroin via the Mannich reaction.
This three-component Mannich modification showed several advantages, such as mild reaction
conditions, good selectivity, easy characterization and high fastness. In particular, the degree of
modification could be established visually according to the color yield and the color fixation on silk
fibroin. Therefore, the Mannich reaction of silk with dyes containing aromatic primary amine groups
may become a novel reactive dyeing method. Studies of structure-performance relationships showed
that suitable dyes for dyeing of silk should meet the following criteria: (1) High nucleophilicity of
the aromatic primary amine group. (2) No high electron cloud density center around the amino
group.
1

ACCEPTED MANUSCRIPT
Keywords: Coloration characterization; Silk fibroin; Mannich reaction; Aromatic primary amine dye;
reactive；Protein modification.
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
1. Introduction
The chemical modification of proteins through the formation of covalent bonds is an efficient
method for the introduction of various functional fragments, including (1) crosslinking reactions, (2)
chemical modifications of side chains, such as esterification, diazo-coupling reaction and acylation
reaction, (3) grafting modifications. However, the existing methods are always accompanied by
several negative impacts, such as low selectivity [1], poor performance, severe reaction conditions
and unwanted damage to the protein macromolecule [2]. In addition, quantitative analysis of the
degree of modification is difficult because of the complex procedure for the effective separation and
characterization of the modified products [3].
Tyrosine residues in proteins provide reactive sites in the ortho-position of the phenolic side
chains, where diazonium coupling [4,5] or the Mannich reaction can take place with high selectivity
and efficiency [6-9]. Diazonium coupling modification can transform colorless diazonium
compounds to azo pigments in-situ, thus making it possible to observe the reaction completion
visually. However, it is not easy to undertake quantitative analysis by measuring the red-shift in the
UV-vis spectrum, because of the extension of π-conjugation. In contrast, a non-conjugated single
bond was formed to link the protein and functional fragment in a three-component Mannich
modification. In terms of detection of the functional fragment, this method allows much more
accuracy. Furthermore, other advantages, such as high rates of conversion and mild reaction
conditions, make the Mannich reaction a promising strategy for the modification of proteins.
2

ACCEPTED MANUSCRIPT
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
64
65
66
Among numerous natural fibrous proteins, silk fibroin, with excellent biological compatibility and
easily chemical modification, has attracted much attention in the fields of regenerated silk fibroin
film [10], cell cultures [11], drug carriers [12,13], biological scaffolds [14,15], biosensors [16],
biomedicine [17], soft micro-optic and photonic applications [18], foods [19] and cosmetics [19].
Silk fibroin is rich in tyrosine residues (6 mol%) [20], providing favorable conditions for Mannich
modification. In order to establish a simple evaluation method for chemical modification and create a
novel reactive dyeing method with good performance simultaneously, dyestuffs containing aromatic
primary amine groups were applied as functional fragments instead of simple arylamine compounds.
For screening suitable dye structures, a series of aromatic primary amine dyes (APAD) were
designed, synthesized and evaluated with regard to their color yield and color fixation properties.
The structure-performance relationship of APAD was also studied, thus laying the foundation for
exploring further applications of the Mannich modification method on silk.
2. Experimental section
2.1 Materials and instruments
Silk fibroin fabrics (40 g/m2) used in experiments were procured from commercial sources. H2
(C.I. Acid Black 1) and H3 (C.I. Acid Green 20) were supplied by Hengsheng Chemical Company
Ltd, Hangzhou, China. The chemical reagents used for the synthesis of dyes were all A.R. grade and
bought from Aladdin Chemicals Company Ltd, Shanghai, China.
UV-vis spectra were obtained using a Cary 50 spectrometer in the range 200 – 700 nm. Fourier
transform infrared (FTIR-ATR) absorption spectra were recorded on a Nicolet 5700 FT-IR
1
spectrometer, using 32 scans at an effective resolution of 4 cm-1. H NMR spectra were determined
on an Advance Bruker DMX400 MHz NMR spectrometer (using tetramethylsilane as internal
3

ACCEPTED MANUSCRIPT
67
68
69
70
71
72
73
74
75
76
77
78
79
80
81
82
83
84
85
86
87
88
reference) at room temperature in DMSO. Electrospray ionization mass spectra (ESI-MS) were
recorded on a Thermo LCQ Fleet mass spectrometer in a scan range of 200-2000 amu.
2.2 Preparation of APAD
2.2.1 Synthesis of D1, D2, D3
In the synthesis of D1, D2 and D3, the same coupling component was used, and the
diazonium components of dyes D1, D2 and D3 were prepared using ortho, meta and
para-nitroaniline, respectively (see Fig. 1). For example, in the synthesis of D1, the o-nitroaniline
was blended with 40 mL water and 16 mL conc. hydrochloric acid. The resulting solution was cooled
to 0-5 ºC and was added to a solution of 0.345 g sodium nitrite in 20 mL water over a period of 30
min. The resulting mixture was stirred at 0-5 ºC for 30 min. Then urea was added to solution until no
blue color was apparent on starch / potassium iodide paper in the range of 2 seconds.
A solution of 0.07 mol of 4-(3-methyl-5-oxo-4,5-dihydropyrazol-1-yl) benzenesulfonic acid and 3
g NaOH in 200 mL water was filtered to remove the solid materials, and the diazo component was
added in deionized water at 0-5 ºC, pH 8.0. After the resulting mixture was stirred for 2 h, conc. HCl
was added to acid out the coupling product, and the crystalline product was washed and then dried.
Then, 0.01 mol coupling product was dissolved in 30 mL water in a 100 mL three necks flask, and
the solution was kept at 75 ºC. A solution of 4.8 g Na2S·9H2O, 1.68 g NaHCO3 in 25 mL water was
added over a period of 1 h and stirred for 4 h. The mixture was cooled to room temperature and the
dye D1 was obtained by acid precipitation and filtration.
1
D1: λmax 434 nm. FTIR (ATR, cm-1): 3385 (NH2), 1164, 1028 (-SO3H). H NMR (400 MHz,
DMSO-d6) δ 7.94 (d, J = 7.3 Hz, 2H), 7.66 (d, J = 7.0 Hz, 2H), 7.46 (d, J = 7.4 Hz, 1H), 7.02 (t, J =
7.9 Hz, 1H), 6.91 (d, J = 7.5 Hz, 1H), 6.78 (s, 1H), 5.35 (s, 2H), 2.32 (s, 3H). ESI MS (m/z, %):
4

ACCEPTED MANUSCRIPT
89
90
372.2 (M-H, 100).
D2: λmax 400 nm. FTIR (ATR, cm-1): 3385 (NH2), 1158, 1033(-SO3H). H NMR (400 MHz,
DMSO-d6) δ 8.04 (d, J = 8.6 Hz, 2H), 7.53 (d, J = 8.6 Hz, 2H), 6.98 (t, J = 7.8 Hz, 1H), 6.80 (d, J =
8.2Hz, 1H),6.74 (d, J = 7.7 Hz, 1H), 6.35 (d, J = 7.3 Hz, 1H), 5.41 (s, 2H), 4.97 (s, 1H), 2.29 (s, 3H).
ESI MS (m/z, %): 372.2 (M-H, 100).
1
91
92
93
94
D3: λmax 480 nm. FTIR (ATR, cm-1): 3388 (NH2), 2918 (CH3), 1641 (C=O), 1545, 1489 (Ar-H),
1161, 1032(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 13.64 (s, 1H), 7.89 (d, J = 8.7 Hz, 2H), 7.65
(d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.7 Hz, 2H), 6.66 (d, J = 8.7 Hz, 2H), 5.57 (s, 2H), 2.28 (s, 3H). ESI
MS (m/z, %): 372.2 (M-H, 100).
95
96
97
98
2.2.2 Synthesis of X1, X2 and X3
99
The synthetic routes of dyes X1, X2 and X3 were the same as that of D1. The coupling component
was sodium 2-naphthol-6-disulfonate, and the diazonium component was prepared by o (m,
p)-nitroaniline respectively (see Fig. 1).
100
101
102
103
104
105
106
107
108
109
110
X1：λmax 500 nm. FTIR (ATR, cm-1): 3474, 3353 (NH2), 3040, 2908, 1591, 1503(Ar-H), 1157,
1024 (-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 14.57 (s, 1H), 8.50 (d, J = 8.7 Hz, 1H), 8.15 (s, 1H),
8.06 (d, J = 9.0 Hz, 1H), 7.86 (d, J = 8.7 Hz, 1H), 7.79 (d, J = 8.1 Hz, 1H), 7.27 (d, J = 7.1 Hz, 1H),
7.22 (d, J = 9.1 Hz, 1H), 7.07 (d, J = 9.5 Hz, 1H), 6.88 (t, J = 7.4 Hz, 1H). ESI MS (m/z, %): 342.08
(M-H, 100).
X2: λmax 493 nm. FTIR (ATR, cm-1): 3457, 3359 (NH2), 2980, 2886, 1601, 1514 (Ar-H), 1124,
1
1020 (-SO3H). H NMR (400 MHz, DMSO-d6) δ 15.89 (s, 1H), 8.44 (d, J = 8.5 Hz, 1H), 8.05 (d,
1H), 7.94 (s, 1H), 7.81 (d, J = 8.5 Hz, 1H), 7.44 (s, 1H), 7.35 (t, J = 8.4 Hz, 1H), 6.93 (d, J = 9.4 Hz,
1H), 6.90 (s, 1H), 6.88 (d, J = 8.4 Hz, 1H). ESI MS (m/z, %): 342.16 (M-H, 100).
5

ACCEPTED MANUSCRIPT
111
112
113
114
115
116
117
118
119
120
121
122
123
124
125
126
127
128
129
130
131
132
X3: λmax 493 nm. FTIR (ATR, cm-1): 3442, 3348 (NH2), 2969, 2881, 1591, 1470 (Ar-H), 1293,
1091 (-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 11.09 (s, 1H), 8.11 (d, J = 8.6 Hz, 1H), 7.93 (s, 2H),
7.80 (d, J = 8.2 Hz, 2H), 7.32 (d, J = 8.9 Hz, 1H), 7.17 (d, J = 9.0 Hz, 1H), 6.79 (d, J = 8.5 Hz, 1H),
6.58 (d, J = 8.3 Hz ,1H). ESI MS (m/z, %): 342.22 (M-H, 100).
2.2.3 Synthesis of R1, R2, R3
The synthetic routes of dyes R1, R2 and R3 were the same as that of D1. The coupling component
(R1, R2, R3) was sodium 2-naphthol-3, 6-disulfonate, and the diazonium component was prepared
by o (m, p)-nitroaniline, respectively (see Fig. 1).
R1：λmax 519 nm. FTIR (ATR, cm-1): 3436 (NH2), 3238, 3073, 1624, 1487 (Ar-H), 1184, 1036
(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 8.45 (d, J = 7.8 Hz, 1H), 8.28 (s, 1H), 8.06 (s, 1H), 7.96
(d, J = 8.0 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.23 (t, J = 8.0 Hz, 1H), 7.01 (d, 7.91 Hz , 1H), 6.82 (s,
1H). ESI MS (m/z, %): 210.50 (M-2H), 100).
R2: λmax 507 nm. FTIR (ATR, cm-1): 3342 (NH2), 2980, 2902, 1607, 1480(Ar-H), 1184,
1053(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 8.36 (d, J = 8.5 Hz, 1H), 8.25 (s, 1H), 7.94 (s, 1H),
7.78 (d, J = 8.3 Hz 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.12 (s, 1H), 6.90 (d, J = 7.7 Hz, 1H), 6.58 (d, J =
8.5 Hz, 1H), 5.89 (s, 1H), 5.75 (s, 2H). ESI MS (m/z, %): 210.50 (M-2H, 100).
R3: λmax 508 nm. FTIR (ATR, cm-1): 3474, 3353 (NH2), 3501, 2980, 1601, 1503(Ar-H), 1157,
1024(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 8.57 (d, J = 8.6 Hz, 1H), 8.23 (s, 1H), 8.00 (s, 1H),
7.94 (d, J = 9.2 Hz, 2H), 7.80 (t, J = 9.8 Hz, 3H), 6.87 (s, 2H), 6.59 (d, J = 9.2 Hz, 2H). ESI MS
(m/z, %): 210.60 (M-2H, 100).
2.2.4 Synthesis of H1, γ1 and J1
H1 (γ1 and J1) was prepared by coupling reaction of aniline diazonium salt and
6

ACCEPTED MANUSCRIPT
133
134
135
136
137
138
139
140
141
142
143
144
145
146
147
148
149
1-naphthol-8-amino-3,6-disulfonic
acid
(or
2-amino-8-naphthol-6-sulfonic
acid,
3-amino-8-naphthol-6-sulfonic acid). The synthetic routes of dyes H1, γ1 and J1 were similar to that
of D1, but the reduction reaction of -NO2 to -NH2 by Na2S is not needed (see Fig. 1).
H1：λmax 540 nm. FTIR (ATR, cm-1): 3419 (NH2), 1635,1491(Ar-H), 1178, 1036(-SO3H).1H NMR
(400 MHz, DMSO-d6) δ 15.43 (s, 1H), 7.95 (s, 1H), 7.68 (d, J = 7.9 Hz, 2H), 7.41 (t, J = 7.6 Hz,
2H), 7.25 (s, 1H), 7.14 (t, J = 7.4 Hz, 1H), 7.04 (s, 1H), 6.84 (s, 1H), 5.42 (s, 2H). ESI MS (m/z,%):
(210.56 (M-2H)/2,100).
γ1: λmax 410 nm. FTIR(ATR, cm-1): 3342 (-NH2), 2975, 2898, 1624, 1497(Ar-H), 1151,
1036(-SO3H).1H NMR (400 MHz, DMSO-d6) δ 15.97 (s, 1H), 7.76 (d, J = 7.9 Hz, 2H), 7.45 (t, J =
7.9 Hz, 2H), 7.41 (d, J = 8.6 Hz, 1H), 7.37 (s, 1H), 7.36 (s, 1H), 7.23 (t, J = 7.2 Hz, 1H), 6.95 (d, J =
9.2 Hz, 1H), 5.71 (s, 2H). ESI MS (m/z, %): 342.15 (M-H, 100).
J1：λmax 480 nm. FTIR (ATR, cm-1): 3342 (-NH2), 2980, 2902, 1607, 1480 (Ar-H), 1184, 1053
(-SO3H). 1H NMR (400 MHz, DMSO-d6) δ 16.02 (s, 1H), 7.93 (d, J = 8.9 Hz, 1H), 7.65 (d, J = 7.6
Hz, 2H), 7.41 (t, J = 7.8 Hz, 3H), 7.22 (s, 1H), 7.15 (d, J = 7.1 Hz, 1H), 6.68 (t, J = 9.6 Hz, 1H), 6.63
(s, 1H), 6.38 (s, 1H), 5.42 (s, 2H). ESI MS (m/z, %): 342.16 (M-H, 100).
H2 (C.I. Acid Black 1) and H3 (C.I. Acid Green 20) were screened out as controls from
commercial dyes. Their structures were shown in Fig. 1.
150
7

ACCEPTED MANUSCRIPT
151
152
153
154
155
Fig. 1. The Synthetic routes and structures of APAD
156
2.3 Modification of silk fibroin with formaldehyde by the Mannich reaction
157
158
159
160
Modification of silk fabric was performed by adopting the following procedures. 2 mL of 1% w/v
dye solution and a stoichiometric amount of 0.1% w/v formaldehyde solution were mixed in a 1:3
molar ratio of dye to formaldehyde, and acetic acid-sodium acetate buffer solution of pH 4.5 was
added to make the volume up to 50 mL. 1.0 g of silk fabric was added and stirred for 10 h at 30°C.
8

ACCEPTED MANUSCRIPT
Finally, the dyed fabric was washed with water, and the UV-visible absorbance of the residual
solution was determined.
161
162
163
164
165
166
167
168
169
2.4 Modification of silk fibroin without formaldehyde
The procedure was similar to that described in Section 2.3, except that there was no formaldehyde
added.
2.5 Measurement of color yield (K/S values) on silk fabric
The color yield of the modified silk fabrics was evaluated with a Datacolor SF600 Plus
Spectrophotometer, in which D65 illuminant and 10 degrees visual field were employed.
2.6 Measurement of exhaustion rate
170
171
172
After the modification process, the absorbances of the original and residual dye solutions were
measured, and the exhaustion rate (E) was calculated by Eq. (1).
E /% = (A - A′) / A
(1)
173
174
175
176
177
178
179
180
181
182
183
Where E is the exhaustion rate, A and A′ are the absorbance at λmax of original and residual dye
solutions, respectively.
2.7 Measurement of color fixation though extraction with DMF
N,N-dimethylformamide (DMF) is an aprotic polar solvent with good dye solubility characteristics,
and most of the lower molecular weight organics could be dissolved in it. The non-covalent bond
interactions, including ionic bonds, van der Waals and hydrogen bonds between the dye molecules
and silk protein are easily dissociated in DMF. However, DMF has little effect on covalent bonds.
Therefore, DMF has been widely used to extract dyes bound by non-covalent bond on fabrics [22].
In this paper, the DMF extraction method was used to measure the combination mode and binding
efficiency between the dye molecules and silk fibroin.
Dried modified silk fabric was immersed in 300 mL DMF and extracted at 95 ºC for 30 min, then
9

ACCEPTED MANUSCRIPT
184
185
186
187
188
189
190
191
192
193
194
195
196
197
198
199
200
201
202
203
204
205
the silk fabric was separated from DMF solution and washed with water. Subsequently, the fabric
was dried and subjected to the same treatment for two more times. K/S values of the sample were
measured after each treatment. In order to evaluate and compare the modification efficiency of
different dyes, the color fixation based on color yield (F) was defined and calculated by Eq. (2).
F /% = (K/S)1 / (K/S)0
(2)
Where F was the color fixation calculated by K/S values, which was measured at maximum
absorption wavelength of dyes on silk fabric. (K/S)0 and (K/S)1 were the K/S values of dyed silk
fabrics before and after DMF extraction.
2.8 Measurement of color fixation through dissolution and dialysis
The dyed silk fabric was cut into 0.5×0.5 cm2 pieces. Accurately weighed 0.2500 g of dyed silk
fabric and 50 mL water were added in an Erlenmeyer flask, the flask was kept at 75 °C for 10 min,
then 8 g LiBr was added and the fabric dissolved completely after 20 min. The resulting solution was
enclosed in a dialysis bag with a magneton and kept stirring, and the dialysis bag was immersed in
water. The water was changed every 5 h until the absorbance of solution in dialysis bag change
no longer after about 130 h, and then the dialysis bag was taken out. The solutions before and after
dialysis were tested on UV-vis spectrophotometer. The color fixation based on dialysis method (Fd)
was defined and calculated by Eq. (3).
Fd /% = (A1-A2) / (A1′-A0′)
(3)
Where Fd is the color fixation calculated by method of dialysis. A1 and A0 are the absorbance of
dialysate from modified and unmodified samples, respectively. A1′ and A0′ is absorbance of
non-dialyzed modified and unmodified samples, respectively.
3 Results and discussion
10

ACCEPTED MANUSCRIPT
206
207
208
209
210
211
212
213
214
215
216
217
218
219
220
221
222
223
224
225
226
227
3.1 Design and synthesis of APAD
In order to explore the effect of APAD molecular structure on modification performance, 14 dyes
containing aromatic primary amine group were applied in modification of silk fibrous fabric. These
dyes can be classified as two types: aniline (D1-D3, R1-R3, X1-X3) and naphthylamine dyes (H1,
J1, γ1, H2, H3). For D1-D3, the amino groups were positioned at ortho, meta and para relative to
the azo group, respectively. The position of the amino group of R1-R3 and X1-X3 was similar to
D1-D3. The influence of dye structure was examined by the difference in coloration obtained for the
three-dye series. The naphthylamine dyes H1, J1, γ1, H2 and H3 were applied to study the
structure-performance relationship of amino groups on naphthalene.
The degree of reaction could be visually assessed by examination of the colored fabrics, and K/S
values were used to quantify the modification efficiency. Since K/S value of modified silk fabric is
related to the depth of shade of the dye, the higher the K/S value is, the higher the dye exhaustion rate
is. Vigorous extraction with DMF could remove dyes absorbed on silk fibre by non-covalent bonds
[21], and therefore the quantity of dye covalently bound to the silk fibre could be roughly calculated
by measuring K/S values of DMF-extracted fabric.
3.2 Structure-performance relationship
3.2.1 Effect of amino position on benzene ring on the modification results of silk fibrous protein
Silk fibre was modified by APAD with amino groups at different positions in the benzene ring.
The results are shown in Fig. 2 and Table 1. Here, three series of dyes D1-D3, X1-X3 and R1-R3
were included with the same molecular backbone in each series. The amino position of D1, X1 and
R1(hereinafter referred to as “ortho-position dyes”) is at ortho-position of diazo group on benzene
ring when that of D2, X2 and R2 (hereinafter referred to as “meta-position dyes” ) is at meta-position,
11

ACCEPTED MANUSCRIPT
228
and that of D3, X3 and R3(hereinafter referred to as “ para-position dyes” ) is at para-position.
6
18
1b
1a
D1
D2
D3
D1'
D2'
D3'
16
14
12
10
8
X1
X2
X3
X1'
X2'
X3'
4
2
0
6
4
2
0
350
400
450
500
550
600
650
700
350
400
450
500
550
600
650
700
wavelength/nm
Wavelength/nm
229
8
6
4
2
0
1c
R1
R2
R3
R1'
R2'
R3'
350
400
450
500
550
600
650
700
Wavelength/nm
230
231
The K/S values curves of modified samples from D1-D3, X1-X3 and R1-R3 is shown in 1a, 1b, 1c. Samples before
and after DMF extraction are displayed by D1-D3, X1-X3, R1-R3 and D1′-D3′ , X1′-X3′, R1′-R3′ , respectively.
Fig. 2. Effect of amino position in benzene ring on K/S values of the Mannich modifications
232
233
234
235
236
Table 1. Effect of amino position in benzene on color effect
Dyes
D1
λmax/nm
434
E/%
3.0
K0
K1
F/%
48.84
50.57
0.389
9.69
0.19
4.90
D2
400
61.2
12

ACCEPTED MANUSCRIPT
D3
X1
X2
X3
R1
R2
R3
480
500
493
493
519
507
508
87.5
47.0
45.0
52.0
2.0
17.34
4.80
4.60
5.57
0.74
5.70
8.00
13.56
0.97
1.20
2.30
0.17
3.50
5.11
78.20
20.21
26.09
41.29
22.97
61.40
63.88
48.8
50.3
237
238
239
240
241
242
243
244
245
246
247
248
249
250
251
The exhaustion rate reflects the equilibrium state of the exchange of dye between aqueous solution
and the silk protein. Hence, the exhaustion rate is positively correlated with the color yield, i.e. the
higher the exhaustion rate is, the higher the color yield (K/S values) is.
As can be seen from Fig. 2, the ortho-position dyes displayed no coloration of the silk, but the
meta- and para-position dyes have been strongly bound to the silk protein even after being extracted
with DMF, illustrating that the silk proteins have been successfully modified by dye molecules. The
color fixation of para-position dyes was higher than meta- (D3>D2, X3 >X2, R3> R2).
The data for ortho-position dyes were different from other dyes. Among the nine dyes, only these
three dyes hardly colored the silk protein fibers, and their K/S values were almost zero. Thus, a
further study was made by measuring the absorbance of dye solution with and without formaldehyde
for the ortho-position dyes. The results are shown in Fig. 3. The absorbance of dye solution without
formaldehyde has clear absorbance in the visible region. But after adding formaldehyde, the
absorbance has changed, and the curve became a nearly straight line in the visible region. It indicated
that the dye had a noticeable decolorization reaction with formaldehyde. Moreover, the absorption in
13

ACCEPTED MANUSCRIPT
the ultraviolet region (200-380 nm) also changed significantly, indicating that dye and formaldehyde
had undergone a reaction which destroyed the dye's chromophoric group and generated a new
structure.
252
253
254
2.5
1.1
2b
2a
1.0
0.9
0.8
0.7
0.6
0.5
0.4
0.3
0.2
0.1
0.0
2.0
1.5
1.0
0.5
0.0
X1 dye solution
X1 dye solution with formaldehyde
D1 dye solution
D1 dye solution with formaldehyde
200
300
400
500
600
700
200
300
400
500
600
700
Wavelength /nm
Wavelength /nm
255
5
4
3
2
1
0
2c
R1 dye solution
R1 dye solution with formaldehyde
200
300
400
500
/nm
600
700
Wavelength
256
372.08
296.01
100
90
80
70
60
50
40
30
20
10
0
100
90
80
70
60
50
40
30
20
10
0
2d
2e
299.00
318.10
325.93
351.23
358.02
387.96
414.01
430.09
292.18
300
460.32
450
339.32
413.92
499.56
500
370.28
394.00
400
436.03
476.15 492.15
500
350
450
300
350
400
m/z
m/z
257
258
259
14

ACCEPTED MANUSCRIPT
2a, 2b and 2c are the UV-visible absorbance curves of the D1, X1 and R1 influenced by formaldehyde, respectively,
2d and 2e are solutions of dye D1 and D1 with formaldehyde respectively, 2F is the possible reaction between D1
and formaldehyde.
260
261
262
263
264
265
266
267
268
269
270
271
272
273
274
275
276
277
278
279
280
281
Fig. 3. Reaction between ortho-position dyes and formaldehyde
The mass spectrum of D1 solution was obtained (see 2d and 2e ) to study the decolorization
phenomenon. After treatment with formaldehyde, the [M-H]- molecular ion peak m/z 372
disappeared, replaced by strong quasi molecular ion peaks of m/z 296, which was relatively reduced
m/z 76. This may be due to a reaction of the ortho- amino group and formaldehyde, a Schiff base
was produced and to attack the N atom of azo linkage, which can be destroyed by rearranging and
eliminating reaction, resulting in the decolorization phenomenon. The possible reaction is shown in
2f.
3.2.2 Influence of amino position on naphthalene ring on the modification results of silk fibrous
protein
Silk fibroin modification results of APAD with amino groups in different positions on the
naphthalene ring are shown in Fig. 4 ( 3d-3e ) and Table 2. It can be seen that color fixations of all
the five modified samples are high before extraction, and decreased to a certain degree after
extraction, especially for H1 and H2, whose K/S values significantly decreased to near zero.
Obviously, the adsorption between dyes (H1, H2) and silk fibroin depends only on physical
adsorption rather than covalent bonding. In contrast, J1 and γ1 have been bound to silk fibroin
protein by covalent bonds because the residue K/S values of J1 and γ1 on silk fabric are still high
after DMF extraction. The difference in modification performance originates from
15

ACCEPTED MANUSCRIPT
282
283
284
structural differences. It is possible that the intramolecular Mannich reaction occurs between amino
group and hydroxyl group on H-acid in the presence of formaldehyde (see Fig. 4) and leads to an
obvious color fixation decrease on silk fabric.
10
16
3a
3b
γ1
γ1'
14
12
10
8
H1
H1'
5
6
4
2
0
0
350
400
450
500
550
600
650
700
350
400
450
500
550
600
650
700
Wavelength/nm
Wavelength/nm
285
18
16
14
12
10
8
14
3c
3d
H2
H2'
12
10
8
J1
J1'
6
6
4
4
2
2
0
0
350
400
450
500
550
600
650
700
350
400
450
500
550
600
650
700
Wavelength/nm
Wavelength/nm
286
16
14
12
10
8
3e
H3
H3'
6
4
2
0
350
400
450
500
550
600
650
700
Wavelength/nm
287
288
289
3a, 3b, 3c, 3d, 3e are the K/S values curve of H1, γ1, J1, H2 and H3 respectively. Samples before and after DMF
290
extraction is displayed by H1, γ1, J1, H2, H3 and H1′, J1′, γ1′, H2′, H3′, respectively. 3f is the intramolecular
16

ACCEPTED MANUSCRIPT
reaction of H1 between Schiff base and hydroxyl group.
291
292
293
294
295
296
297
298
Fig. 4. Influence of amino position in naphthalene ring on K/S values of the Mannich modifications and
Although H1 and H2 containing H-acid hardly react with silk fibroin, H3 shows good color
fixation. Since the structure of H3 includes H-acid structure and also contains an aniline group, the
reactivity of H3 may be confined to the amino group on the benzene ring.
Table 2. Influence of amino position in naphthalene ring on color effect
Dyes λmax/nm E/%
K0
K1
F/%
H1
γ1
540
410
480
630
620
45.04 9.12 0.88 9.65
75.80 15.14 9.03 59.64
70.29 13.67 3.62 26.48
82.38 16.98 0.72 4.24
78.30 14.68 7.00 47.68
J1
H2
H3
299
300
301
302
303
304
305
306
3.3 The effect of the Mulliken charges distribution on the color fixation
3.3.1 The effect of the Mulliken charges of N atom of APAD amino group on color fixation
Based on the above experimental results, Gauss software was employed to calculate the Mulliken
charges of amino N atom (hereinafter referred to as Mulliken charges) by the BYLYP/3-21G method
and the results were shown in Table 3. According to the literature [18], D1, D2 and D3 usually exist
in a hydrazone structure (see Fig. 5, 4a). The nuclear magnetic data of X1, X2, X3, γ1 and J1 exist a
chemical shift ( δ ) at low field (X1: 14.57, X2: 15.89, X3: 11.09, γ1: 15.97, J1: 16.02 ), showing
17

ACCEPTED MANUSCRIPT
307
308
309
310
311
they also exist in the hydrazone form [19]. Therefore, D1, D2, D3, X1, X2, X3, γ1 and J1 were
calculated in the hydrazone form. R1, R2 and R3 were calculated in the azo form. As mentioned in
section 3.2.2 about H3, whose reaction site is located on the amino group of benzene ring, so it was
clarified into aniline. The results were shown in Table 3, and the calculation results of D1-D3 were
exhibited in Fig.5(4b, 4c, 4d) as a paradigmatic.
312
313
314
18

ACCEPTED MANUSCRIPT
315
316
4a is the Azo-hydrazone tautomerism of azo dyes. 4b,4c,4d is the Mulliken charges distribution of D1, D3, D3
317
318
319
respectively.
Fig. 5. The Mulliken charges distribution of D1, D3, D3
Table 3. Mulliken charges of APAD
Dyes
Form
Mulliken charges of Mulliken charges of
aniline
-0.766
-0.726
-0.831
-0.718
-0.726
-0.722
-0.839
-0.987
-0.824
—
naphthylamine
D1
D2
D3
X1
X2
X3
R1
R2
R3
H1
Hydrazone
Hydrazone
Hydrazone
Hydrazone
Hydrazone
Hydrazone
Azo
—
—
—
—
—
—
—
Azo
—
Azo
—
Azo
-0.864
19

ACCEPTED MANUSCRIPT
γ1
J1
Hydrazone
—
—
-0.832
-0.83
Hydrazone
Azo
H2
H3
—
-0.696
-0.885
Azo
-0.823
320
321
322
323
324
The relationship between the Mulliken charges and the color fixation of silk fabrics is shown in
Fig. 6. Due to the obvious decolorization of ortho-position dyes (D1, X1 and R1), the color fixation
cannot be obtained. And as discussed in section 3.2.2, H1 and H2 could not be regard as Mannich
modification on silk. Therefore, data for D1, X1, R1, H1 and H2 have not been shown in Fig.6.
100
Aniline dyes
Naphthylamine dyes
90
80
D3
70
R3
γ
R2
60
50
40
30
20
10
0
1
D2
X3
H3
J1
X2
70
75
80
85
90
95
100
Mulliken charges (*100)
325
326
Fig.6. Relationship between Mulliken charges distribution and color fixation (F)
327
328
329
330
331
It could be seen that for aniline dyes (D2, D3, X2, R2, R3, H3), with the exception of H3, the color
fixation increased with the increase of the Mulliken charges and then decreased. However the color
fixation of H3 appears different, because the molecule has two amino groups, and the amino group
of H-acid structure can expend one more equivalent weight formaldehyde than other APAD, so its
20

ACCEPTED MANUSCRIPT
332
333
334
335
336
337
338
339
340
341
342
343
344
345
346
347
348
349
color fixation is relatively lower. When the Mulliken charge number was at -0.831, the color fixation
reached a maximum. Therefore, it can be concluded that there exists a most suitable range for
Mulliken charges. The range is supposed to be above -0.831 (D3) but below -0.987 (R2). Within this
range, the aromatic primary amine compounds could react with formaldehyde and silk protein more
easily, then the highest color fixation could be achieved, and the color fixation is affected more
clearly by the change of Mulliken charges. In addition, the Mulliken charges of N atom of the same
series of meta- position dyes are higher than those of the para- position dyes, while the color fixation
of meta- position dyes was lower than that of para- position dyes.
For the naphthylamine dyes γ1 and J1, the Mulliken charges have only a slight difference, but
their color fixations are very different (γ1:59.64%, J1:26.48%). It is similar with the comparison of
aniline dyes D3 and R3. It can be concluded that there is also a most suitable range of Mulliken
charges for naphthylamine dyes, which might be slightly greater than the absolute value of γ1
(0.832), resulting in high Mannich reactivity and high color fixation.
From the Mannich reaction mechanism (see Fig. 7), it can be seen that the increase of electron
cloud density on the amino group could enhance the nucleophilicity of the amino group. The
improved reactivity of aromatic primary amine group is beneficial for attacking formaldehyde. Then
the positive ion intermediates are more likely to be formed to promote the Mannich reaction
efficiency, leading to the increase in color fixation.
21

ACCEPTED MANUSCRIPT
NH2
H
H
H
H+
N
-H+
H+
O
HO+
HO
H
H
H
H
H
H
H
-H2O
H2O+
H+
H
HO
N
N
N
H+
H
H
H
H
OH
OH
H
H
H
N
N
H+
H
H
350
351
Fig. 7. Mechanism of the Mannich reaction
352
353
354
355
356
357
358
359
360
361
362
363
364
365
366
On the other hand, when the electron cloud density increases, the alkalinity of the amino group
also increases, which gives priority to neutralization with acid to become positive ammonium ions,
thus losing the reactivity of the Mannich reaction. The final modification performance depends on
the combined effect of the two variables. Therefore, when the electron cloud density is lower, its
alkalinity is weak, and the ascending effect of reactivity occupies the dominant position, so the
increasing trend is generally presented. When the amino electron cloud density is higher, the
alkalinity enhancement factor is dominant, and amino groups prefer reaction with acid to form
ammonium salts instead of the Mannich reaction. This reduces the concentration of aromatic primary
amine, resulting in decrease on color fixation.
3.4 Confirmation of the Mannich modification on silk
3.4.1 Confirmation of the Mannich reaction on silk by DMF extracting method
Related research [6-9, 24, 25] showed that it is necessary to use formaldehyde to establish a
conjugation between the APAD and tyrosine residues in protein by the Mannich reaction. We have
compared the color fixation of samples with and without formaldehyde under the same modification
22

ACCEPTED MANUSCRIPT
367
368
369
370
371
372
373
374
conditions, and the DMF extraction method was used to measure the coloration effect.
Comparison of the Mannich modification performance between with-formaldehyde method and
without-formaldehyde method by using D3 are shown in Fig. 8. As can be seen, the K/S value of the
silk fabric dyed by with-formaldehyde method is much higher than that of without-formaldehyde
method. On the other hand, after extraction with DMF, the K/S value using without-formaldehyde
method was slightly decreased, while that of without-formaldehyde method plunged to near zero.
The comparison shows the formation of the covalent bonding between silk protein and dye
molecules after the Mannich modification.
16
D3 before extraction
D3 after extraction
D3' before extraction
14
D3' after extraction
12
10
8
6
4
2
0
350
400
450
500
550
600
650
700
750
wavelength / nm
375
376
D3 and D3′ is the dyed samples with and without formaldehyde.
377
378
379
380
381
382
383
Fig.8. The modification effect of samples dyed by D3 with and without formaldehyde
The K/S values of samples dyed with D3 and H3 before and after DMF extraction are shown in
Table 5. It can be seen that the K/S values of samples before extraction is high and the K/S values
clearly decrease after 3 times extraction with DMF. But K/S values of samples modified by
with-formaldehyde method were still higher, while that of without-formaldehyde method
dramatically decreased to near zero and the silk fabrics almost became white. For all modified
23

ACCEPTED MANUSCRIPT
384
385
386
387
388
389
390
391
392
samples, there was no change in λmax after extraction. It is known that there are only three possible
ways of attaching dyes to fibres: (a) by physical adsorption, (b) by mechanical retention, and (c) by
chemical reaction. Dyes attached to silk fibers by (a) and (b) can be disassociated by DMF [21]. In
our experiments, for without-formaldehyde modified samples, the dye molecule and silk fibroin have
no mechanism to form a covalent bond without formaldehyde, almost all the dye absorbed on silk
fibroin with non-covalent bond were extracted by DMF. So the K/S values measured on samples
modified in the Mannich reaction after DMF extraction can be reasonably assumed due to dye
molecules bound to the silk protein by covalent bonds.
Table 5. K/S values and F of modified silk fabrics
K/S values of samples modified with D3
（λmax=480nm）
K/S values of samples modified with H3
（λmax=620nm）
Treatment
Methods
0
1
2
3
13.12
--
0
1
2
3
7.30
--
a
16.78
15.76
14.63
1.77
13.20
0.44
15.03
14.48
8.52
1.22
7.34
0.38
b
393
394
395
396
397
398
399
400
E (D3)= 87.5%, E (H3)=78.3% .
(a-the Mannich modification with formaldehyde；b-Treatment without formaldehyde，samples 0 to 3 are
respectively colored samples without extracted, that extracted by DMF for one time, two times and three times.
3.4.2 Confirmation of the Mannich modification rate on silk by dialysis method
To verify the reliability of the easily-calculated color fixation by DMF extraction method, the
modification performance was also characterized by dialysis of dyed silk solution. Silk protein can
be dissolved in LiBr/H2O solution [26, 27], and any non-covalently bonded small dye molecules can
be separated from the dyed macromolecules of silk fibroin. Dialysis allows any small unbound dye
24

ACCEPTED MANUSCRIPT
401
402
403
404
405
molecules to escape with sufficient water flow, while the dissolved large protein macromolecules are
unable to pass through the semipermeable membrane. Therefore, the color fixation could be
calculated by the UV-vis absorbance of silk fibroin before and after dialysis.
Table 6. Comparison of two methods of measuring color fixation
Dyes
D3
Fd/%
77.19
48.60
F /%
78.20
48.04
H3
406
407
408
409
410
411
412
413
414
415
416
417
418
419
It can be seen from Table 6 that the results calculated from the two methods agree well with each
other, which indicates the convenient DMF extraction method for calculating the extent of covalent
bonding between dyes and silk fibroin has a high degree of credibility.
4. Conclusion
In this study, a series of dyestuffs containing aromatic primary amino groups as protein
modification agents was designed, synthesized and applied in the Mannich modification of silk
fibroin. Besides, a simple visual method has been established to determine the degree of modification.
Furthermore, the structure-performance relationship between the dye structure and degree of silk
modification was revealed. For aniline dyes, the meta- and para-position dyes were covalently bound
to silk fibroin, and the color depth of para-position dyes was much higher. The range of suitable
Mulliken charge numbers (not too high and not too low) is used to achieve higher color fixation. For
naphthylamine dyes, the amino group should not be close to the hydroxyl group on the naphthalene
ring to prevent the intramolecular reaction which decreases the modification efficiency with silk
25

ACCEPTED MANUSCRIPT
420
421
422
423
424
425
426
427
428
429
430
431
432
fibroin. Finally, the extent of modification of proteins using the Mannich reaction can be determined
visibly through the application of dyestuffs that contain an aromatic primary amino group. It can also
be concluded that suitable dyestuffs for Mannich modification of silk fibroin should meet the
following criteria: (1) suitable nucleophilic performance, (2) no high electron cloud density center
around the amino group. Hopefully this method may be developed further as a novel reactive dyeing
process for silk, providing good color yields and high color fastness.
Acknowledgements
This work was supported by National Natural Science Foundation of China (Nos. 51673176,
21808210), Zhejiang Provincial Natural Science Foundation of China (LY16B060006), and Public
Welfare Technology Research Project of Zhejiang Province (LGG18B060003).
References
433
434
435
436
437
438
439
440
441
442
443
444
445
446
447
448
449
450
[1] Kaplan ARM, David L. Biomedical applications of chemically-modified silk fibroin. J Mater
Chem. 2009;19(36):6443-50.
[2] Tamada Y. Sulfation of silk fibroin by chlorosulfonic acid and the anticoagulant activity.
Biomaterials. 2004;25(3):377-83.
[3] Oya T, Hattori N, Mizuno Y, Miyata S, Maeda S, Osawa T, et al. Methylglyoxal Modification of
Protein chemical and immunochemical characterization of methylglyoxal-arginine adducts. J
Biol Chem. 1999;274(26):18492-502.
[4] Murphy AR, St JP, Kaplan DL. Modification of silk fibroin using diazonium coupling chemistry
and the effects on hMSC proliferation and differentiation. Biomaterials. 2008;29(19):2829-38.
[5] Mathew W. Jones, Giuseppe Mantovani, Claudia A. Blindauer, Sinead M. Ryan, Xuexuan
Wang, David J. Brayden et al. Direct Peptide Bioconjugation/PEGylation at Tyrosine with
Linear and Branched Polymeric Diazonium Salts. J. Am. Chem. Soc. 2012, 134 (17): 7406–13.
[6] Joshi NS, Whitaker LR, Francis MB. A three-component Mannich-type reaction for selective
tyrosine bioconjugation. J Am Chem Soc. 2004;126(49):15942-43.
[7] Hitoshi Ban, Julia Gavrilyuk, Carlos F. Barbas III.Tyrosine Bioconjugation through Aqueous
Ene-Type Reactions: A Click-Like Reaction for Tyrosine. J. Am. Chem. Soc. 2010;132 (5):
1523–25.
[8] Dante W. Romanini, Matthew B. Francis. Attachment of Peptide Building Blocks to Proteins
26

ACCEPTED MANUSCRIPT
451
452
453
454
455
456
457
458
459
460
461
462
463
464
465
466
467
468
469
470
471
472
473
474
475
476
477
478
479
480
481
482
483
484
485
486
487
488
489
490
491
492
493
Through Tyrosine Bioconjugation. Bioconjugate Chem. 2008;19 (1): 153–157.
[9] Hai-Ming Guo, Maki Minakawa and Fujie Tanaka. Fluorogenic Imines for Fluorescent
Detection of Mannich-Type Reactions of Phenols in Water. J. Org. Chem. 2008, 73 (10):
3964–66.
[10] Shetty GR, Rao BL, Gowda M, Shivananda CS, Asha S, Sangappa Y. The preparation and
characterization of silk fibroin blended with low molecular weight hydroxypropyl
methylcellulose (HPMC). Proc Dae Solid State Physics Symposium2018. p. 070032.
[11] Inouye K, Kurokawa M, Nishikawa S, Tsukada M. Use of Bombyx mori silk fibroin as a
substratum for cultivation of animal cells. J Biochem Bioph Meth. 1998;37(3):159-64.
[12] Mottaghitalab F, Farokhi M, Shokrgozar MA, Atyabi F, Hosseinkhani H. Silk fibroin
nanoparticle as a novel drug delivery system. J control release. 2015;206:161-76.
[13] Dyakonov T, Yang CH, Bush D, Gosangari S, Majuru S, Fatmi A. Design and characterization
of a silk-fibroin-based drug delivery platform using naproxen as a model drug. J Drug Deliv.
2012;2012(4):490514.
[14] Seo YK, Choi GM, Kwon SY, Lee HS, Park YS, Song KY, et al. The Biocompatibility of Silk
Scaffold for Tissue Engineered Ligaments. Key Eng Mater. 2007;342-343:73-6.
[15] Shetty GR, Rao BL, Asha S, Wang Y, Sangappa Y. Preparation and characterization of silk
fibroin/hydroxypropyl methyl cellulose (HPMC) blend films. Fiber Polym. 2015;16(8):1734-41.
[16] Yin H, Ai S, Shi W, Zhu L. A novel hydrogen peroxide biosensor based on horseradish
peroxidase immobilized on gold nanoparticles–silk fibroin modified glassy carbon electrode and
direct electrochemistry of horseradish peroxidase. Sens Actuators B Chem. 2009;137(2):747-53.
[17] Murphy AR, Kaplan DL. Biomedical applications of chemically-modified silk fibroin. J Mater
Chem. 2009;19(36):6443-50.
[18] Pal RK, Kurland NE, Wang C, Kundu SC, Yadavalli VK. Biopatterning of silk proteins for soft
micro-optics. Acs Appl Mater Inter. 2015;7(16):8809-16.
[19] Li G, Liu H, Li TD, Wang J. Surface modification and functionalization of silk fibroin
fibers/fabric toward high performance applications. Mat Sci Eng C-Mater. 2012;32(4):627-36.
[20] Baltova S, Vassileva V, Valtcheva E. Photochemical behaviour of natural silk—I. Kinetic
investigation of photoyellowing. Polym Degrad Stabil. 1998;60(1):53-60.
[21] Bhate PM, Devi RV, Dugane R, Hande PR, Shaikh L, Vaidya S, et al. A novel reactive dye
system based on diazonium salts. Dyes Pigments. 2017;145:208-15.
[22] Hunger K. Industrial Dyes: Chemistry, Properties, Applications2004.
[23] Wu Z, Zhang R, Rong Z. Azo-hydrazone tautomerism of azo dyes. Ciesc Journal.
2015;66(1):52-59.
[24] Wang L, Gruzdys V, Pang N, Meng F, Sun XL. Primary arylamine-based tyrosine-targeted
protein modification. RSC Adv. 2014;4(74):39446-52.
[25] Zhao, XJ , Cui ZH, Wang, RL, Fan, SJ, et al. Synthesis of an electron-rich aniline-containing
dye and its dyeing behaviors on silk through a three-component Mannich-type reaction. Chinese
Chemical Letters. 2015;26(2):259-62.
[26] Khandker S. Hossain, Eiji Ohyama, Akie Ochi, Jun Magoshi and Norio Nemoto.
Dilute-Solution Properties of Regenerated Silk Fibroin. J Phys Chem B. 2003;107(32):8066-73.
[27] Cho HJ, Ki CS, Oh H, Lee KH, Um IC. Molecular weight distribution and solution properties of
silk fibroins with different dissolution conditions. Int J Biol Macromol. 2012;51(3):336-41.
27

ACCEPTED MANUSCRIPT
Highlights
1．The first study to apply Mannich reaction modification and reactive coloration in
fibrous proteins.
2．The degree of modification could be simply evaluated by visual characterization
according to the color yield and the color fixation on silk fibroin.
3．Some criteria that suitable dyes for both coloration characterization of protein
modification and dyeing of silk were revealed by studying of structure-performance
relationships of amine-containing dyes.