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

Dyes and Pigments 168 (2019) 300–310

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

T

Weiguo Chena,b, Pu Gaoa, Hua Jianga,b, Zhihua Cuia,b,∗ a b

Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, 310018, China Key Laboratory of Advanced Textile Materials and Manufacturing Technology, Ministry of Education of China, Hangzhou, 310018, China

A R T I C LE I N FO

A B S T R A C T

Keywords: Coloration characterization Silk fibroin Mannich reaction Aromatic primary amine dye Reactive Protein modification

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. 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 orthoposition 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. 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.

∗ Corresponding author. Engineering Research Center for Eco-Dyeing & Finishing of Textiles, Ministry of Education, Zhejiang Sci-Tech University, Hangzhou, 310018, China. E-mail address: [email protected] (Z. Cui).

https://doi.org/10.1016/j.dyepig.2019.04.061 Received 24 July 2018; Received in revised form 2 April 2019; Accepted 25 April 2019 Available online 27 April 2019 0143-7208/ © 2019 Elsevier Ltd. All rights reserved.

Dyes and Pigments 168 (2019) 300–310

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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, 1020 (-SO3H). 1H 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). 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. 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 spectrometer, using 32 scans at an effective resolution of 4 cm−1. 1H NMR spectra were determined on an Advance Bruker DMX400 MHz NMR spectrometer (using tetramethylsilane as internal 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.

2.2.3. 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, 6disulfonate, 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. 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 °C 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 °C 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 s. A solution of 0.07 mol of 4-(3-methyl-5-oxo-4,5-dihydropyrazol-1yl) 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 °C, 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 °C. 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. D1: λmax 434 nm. FTIR (ATR, cm−1): 3385 (NH2), 1164, 1028 (-SO3H). 1H 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, %): 372.2 (M-H, 100). D2: λmax 400 nm. FTIR (ATR, cm−1): 3385 (NH2), 1158, 1033(SO3H). 1H 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). 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).

2.2.4. Synthesis of H1, γ1 and J1 H1 (γ1 and J1) was prepared by coupling reaction of aniline diazonium salt and 1-naphthol-8-amino-3,6-disulfonic acid (or 2-amino-8naphthol-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(ArH), 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

2.2.2. Synthesis of X1, X2 and X3 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). 301

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Fig. 1. The Synthetic routes and structures of APAD.

2.4. Modification of silk fibroin without formaldehyde

out as controls from commercial dyes. Their structures were shown in Fig. 1.

The procedure was similar to that described in Section 2.3, except that there was no formaldehyde added. 2.3. Modification of silk fibroin with formaldehyde by the Mannich reaction 2.5. Measurement of color yield (K/S values) on silk fabric

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 M 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. Finally, the dyed fabric was washed with water, and the UV–visible absorbance of the residual solution was determined.

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 After the modification process, the absorbances of the original and 302

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Fig. 2. Effect of amino position in benzene ring on K/S values of the Mannich modifications. The K/S values curves of modified samples from D1-D3, X1-X3 and R1R3 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.

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.

residual dye solutions were measured, and the exhaustion rate (E) was calculated by Eq. (1). E /% = (A - A′) / A

(1)

Where E is the exhaustion rate, A and A′ are the absorbance at λmax of original and residual dye solutions, respectively.

2.8. Measurement of color fixation through dissolution and dialysis

2.7. Measurement of color fixation though extraction with DMF

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).

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 °C for 30 min, then 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

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.

(2) 303

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3. Results and discussion

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 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. 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 orthoamino 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.

3.1. 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.

3.2.2. 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 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. 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.

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, and that of D3, X3 and R3(hereinafter referred to as “para-position dyes”) is at para-position. 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

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 [23], D1, D2 and D3 usually exist in a hydrazone structure (see Figs. 5 and 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 they also exist in the hydrazone form [23]. 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. 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. 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

Table 1 Effect of amino position in benzene on color effect. Dyes

λmax/nm

E/%

K0

K1

F/%

D1 D2 D3 X1 X2 X3 R1 R2 R3

434 400 480 500 493 493 519 507 508

3.0 61.2 87.5 47.0 45.0 52.0 2.0 48.8 50.3

0.389 9.69 17.34 4.80 4.60 5.57 0.74 5.70 8.00

0.19 4.90 13.56 0.97 1.20 2.30 0.17 3.50 5.11

48.84 50.57 78.20 20.21 26.09 41.29 22.97 61.40 63.88

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Fig. 3. Reaction between ortho-position dyes and formaldehyde. 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.

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Fig. 4. Influence of amino position in naphthalene ring on K/S values of the Mannich modifications and 3a, 3b, 3c, 3d, 3e are the K/S values curve of H1, γ1, J1, H2 and H3 respectively. Samples before and after DMF extraction is displayed by H1, γ1, J1, H2, H3 and H1′, J1′, γ1′, H2′, H3′, respectively. 3f is the intramolecular reaction of H1 between Schiff base and hydroxyl group.

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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.

Table 2 Influence of amino position in naphthalene ring on color effect. Dyes

λmax/nm

E/%

K0

K1

F/%

H1 γ1 J1 H2 H3

540 410 480 630 620

45.04 75.80 70.29 82.38 78.30

9.12 15.14 13.67 16.98 14.68

0.88 9.03 3.62 0.72 7.00

9.65 59.64 26.48 4.24 47.68

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 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. The K/S values of samples dyed with D3 and H3 before and after DMF extraction are shown in Table 4. 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 withoutformaldehyde method dramatically decreased to near zero and the silk fabrics almost became white. For all modified 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 3 Mulliken charges of APAD. Dyes

Form

Mulliken charges of aniline

Mulliken charges of naphthylamine

D1 D2 D3 X1 X2 X3 R1 R2 R3 H1 γ1 J1 H2 H3

Hydrazone Hydrazone Hydrazone Hydrazone Hydrazone Hydrazone Azo Azo Azo Azo Hydrazone Hydrazone Azo Azo

−0.766 −0.726 −0.831 −0.718 −0.726 −0.722 −0.839 −0.987 −0.824 – – – – −0.823

– – – – – – – – – −0.864 −0.832 −0.83 −0.696 −0.885

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 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 paraposition 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. 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

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 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. It can be seen from Table 5 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 307

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Fig. 5. The Mulliken charges distribution of D1, D3, D3, 4a is the Azo-hydrazone tautomerism of azo dyes. 4b,4c,4d is the Mulliken charges distribution of D1, D3, D3 respectively.

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Table 4 K/S values and F of modified silk fabrics. Treatment Methods

a b

K/S values of samples modified with D3(λmax = 480 nm)

K/S values of samples modified with H3(λmax = 620 nm)

0

1

2

3

0

1

2

3

16.78 15.76

14.63 1.77

13.20 0.44

13.12 –

15.03 14.48

8.52 1.22

7.34 0.38

7.30 –

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. Table 5 Comparison of two methods of measuring color fixation.

Fig. 6. Relationship between Mulliken charges distribution and color fixation (F). (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Dyes

Fd/%

F/%

D3 H3

77.19 48.60

78.20 48.04

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 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

Fig. 7. Mechanism of the Mannich reaction.

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 [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] Jones Mathew W, Mantovani Giuseppe, Blindauer Claudia A, Ryan Sinead M, Wang Xuexuan, 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):15942–3. [7] Ban Hitoshi, Gavrilyuk Julia, Carlos F, Barbas III. Tyrosine bioconjugation through aqueous ene-type reactions: a click-like reaction for tyrosine. J Am Chem Soc 2010;132(5):1523–5. [8] Romanini Dante W, Francis Matthew B. Attachment of peptide building blocks to proteins through tyrosine bioconjugation. Bioconjug Chem 2008;19(1):153–7. [9] Guo Hai-Ming, Minakawa Maki, Tanaka Fujie. Fluorogenic imines for fluorescent detection of mannich-type reactions of phenols in water. J Org Chem 2008;73(10):3964–6. [10] Shetty GR, Rao BL, Gowda M, Shivananda CS, Asha S, Sangappa Y. The preparation

Fig. 8. The modification effect of samples dyed by D3 with and without formaldehyde (D3 and D3′ is the dyed samples with and without formaldehyde.)

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