Chemical Modification of Silk Sericin in Lithium ... - ACS Publications

Biomacromolecules 2004, 5, 1392-1398

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Chemical Modification of Silk Sericin in Lithium Chloride/ Dimethyl Sulfoxide Solvent with 4-Cyanophenyl Isocyanate Hidetoshi Teramoto,* Ken-ichi Nakajima, and Chiyuki Takabayashi New Silk Materials Laboratory, Insect Biotechnology and Sericology Department, National Institute of Agrobiological Sciences, 1-4-8 Gohda, Okaya, Nagano 394-0021, Japan Received December 19, 2003; Revised Manuscript Received April 7, 2004

This paper reports chemical modification of silk sericin in LiCl/dimethyl sulfoxide (DMSO) solvent with 4-cyanophenyl isocyanate. Sericin is a highly hydrophilic protein secreted by Bombyx mori, serving as a protein glue in a cocoon. LiCl/DMSO was found to be a good solvent of sericin and useful for homogeneous modification of its abundant hydroxyl groups under nonaqueous condition. Fourier transform infrared (FTIR) analysis of the modified sericins revealed that 4-cyanophenyl groups were incorporated into sericin molecules mainly through urethane linkages. Several characteristics of the modified sericins such as solubility characteristic, hygroscopic property, and thermal stability were investigated. Secondary structure analysis using FTIR spectra suggested that formation of strong intermolecular hydrogen bonds was inhibited by the modification that is probably attributable to the incorporation of bulky 4-cyanophenyl groups. These results demonstrate that chemical modification of sericin using LiCl/DMSO solvent markedly alters its characteristics. Introduction Sericin is a family of the adhesive silk proteins synthesized exclusively in the middle silk glands of silkworms, Bombyx mori1,2 and classified into at least six proteins of different lengths generated by alternatively splicing the primary transcripts of two sericin genes, Ser1 and Ser2.3-5 Sericin is characterized by an unusually high serine content (ca. 35%);6,7 it acts as a protein glue to fix fibroin fibers together in a cocoon. Although tons of sericin has been discarded during silk processing, sericin’s unique characteristics, including high hydrophilicity and affinity to skin and hair,8 have recently attracted much attention. Moreover, its applications to cosmetics and coatings are increasing.9 Takeuchi and colleagues recently reported that sericin has the ability to induce heterogeneous nucleation of apatite in a solution that mimics physiological conditions.10 These characteristics of sericin are probably attributable to its unique amino acid composition and sequence. Therefore, sericin is anticipated to be a promising natural resource offering specific properties for developing novel protein-based materials. Chemical modification is a powerful tool for adding new properties to natural polymers. For instance, various functional materials have been developed from polysaccharides, such as cellulose, starch, or chitin, by chemical modification at their hydroxyl groups. For protein, many studies have addressed fibroin, another component of Bombyx mori silk, to develop enzyme-immobilization support,11 biocompatible materials,12-14 anti-HIV reagents,15 anticoagulant materials,16 or inorganic-organic composites.17 Sericin contains a much larger amount of amino acids having nucleophilic groups * To whom correspondence should be addressed. E-mail: teramoto@ nias.affrc.go.jp.

than fibroin, which reaches 50% of all amino acids.6,7 This characteristic is a highly distinguishing feature of sericin from other proteins; it provides sericin’s high chemical reactivity. However, only a few attempts have been made at chemical modification of sericin.18-20 Another feature of sericin is that hydroxyl groups from Ser, Thr, and Tyr residues account for about 90% of the nucleophilic groups.6,7 This fact implies that modification of these hydroxyl groups is important for the efficient alteration of sericin characteristics. The present paper describes a novel technique for chemical modification of silk sericin using a LiCl/dimethyl sulfoxide (DMSO) solvent system. LiCl/DMSO solvent provides nonaqueous reaction conditions, which is preferred for the efficient modification of abundant hydroxyl groups in sericin. We performed reactions with 4-cyanophenyl isocyanate, which has high reactivity toward hydroxyl groups. The reaction proceeded as expected to form urethane linkages. We investigated effects of modification on sericin characteristics such as solubility characteristics, hygroscopic properties, thermal stability, and structural characteristics. Experimental Section Materials. Reagent grade dimethyl sulfoxide (DMSO) (99%) was distilled from CaH2 under reduced pressure and stored over molecular sieves of 4 Å. Anhydrous LiCl (99%) was dried overnight in a vacuum oven at 120 °C before use. Other reagents including 4-cyanophenyl isocyanate (Aldrich Chemical Co., Inc., Milwaukee, WI) were used without further purification. A new strain of Bombyx mori, Sericin-hope,21,22 which was developed using a naked pupa mutant strain, Nd,23,24 and a normal strain KCS83, was used in this study as the sericin source. Sericin-hope secretes approximately four times more

10.1021/bm034537r CCC: $27.50 © 2004 American Chemical Society Published on Web 05/14/2004

Chemical Modification of Sericin in LiCl/DMSO

sericin than the Nd mutant does. It produces thin cocoons consisting almost exclusively of sericin (98.5%).21,22 Therefore, Sericin-hope facilitates mass production of native sericin with high purity. In advance of this study, we verified that electrophoretic mobilities of the three main components of sericin are identical for both Sericin-hope and a normal strain. Sericin was purified from fresh cocoons of Sericin-hope silkworms by the following procedure: Sericin-hope cocoon shells (600 mg) were dissolved into a 8 M LiBr aqueous solution (24 mL) at 35 °C for 24 h. Centrifugation and filtration removed insoluble residue. The supernatant was adjusted to ca. pH 8 with 1 M Tris-HCl buffer of pH 9.0 (6.0 mL) to prevent precipitation of sericin during subsequent dialysis. The solution was thoroughly dialyzed to deionized water using a Spectra/Por membrane (MWCO 6-8000, Spectrum Laboratories Inc., Rancho Dominguez, CA) and then lyophilized to yield purified sericin (370 mg). For this study, the average molecular weight of the constituent amino acids of sericin was approximated to be 100. That figure was used to calculate the equivalent reagent and reaction yields. Preparation of Regenerated Sericins. The purified sericin, after vacuum-drying in an oven at 70 °C for 24 h, was dissolved in 100 volumes of 1 M LiCl/DMSO by heating at 60 °C for 45 min in an argon atmosphere. The solution was stirred for 5 h at 60 °C or at room temperature in an argon atmosphere; it was then poured into excess cold ethanol. The precipitate was collected by centrifugation, washed several times with ethanol and acetone, and then lyophilized to yield regenerated sericins. The regenerated sericin after 5 h of heating at 60 °C was used to investigate the stability of sericin in LiCl/DMSO. The regenerated sericin after 5 h stirring at room temperature was used as the reference of sericin treated under reaction conditions without modification reagents. SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE). The purified sericin and the regenerated sericin after 5 h heating at 60 °C were dissolved in 100 volumes of 8 M LiBr at 80 °C for 20 min. The solution was diluted 50 times with milli-Q water and then mixed with an equal amount of SDSPAGE sample buffer (0.1 M Tris-HCl of pH 6.8, 4% SDS, 12% 2-mercaptoethanol, 20% glycerol). SDS-PAGE was performed according to the method of Laemmli25 using a ready-made 3-10% gradient gel (ATTO Bioscience, Tokyo, Japan). After electrophoresis, the gel was stained with 2Dsilver stain II “Daiichi” (Daiichi Pure Chemicals Co. Ltd., Tokyo, Japan). Molecular weights of protein bands were estimated using Precision Protein Standards (Bio-Rad Laboratories Inc., Hercules, CA). Synthesis of 4-Cyanophenyl Isocyanate-Modified Sericin Derivatives (CPI-Src). The reactions were conducted after careful dehydration of sericin and its LiCl/DMSO solution because 4-cyanophenyl isocyanate easily reacts with water to be decomposed. The purified sericin (100 mg) was added to a round-bottom flask and dried in a vacuum oven at 70 °C for 24 h. The solvent, 1 M LiCl/DMSO (10 mL), was added to the flask. Then the mixture was heated at 60 °C for 45 min with stirring in an argon atmosphere to give a homogeneous solution. The solution was cooled to room

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temperature and vacuum-dried for another few hours to remove residual water. Then, 4-cyanophenyl isocyanate (72, 144, 216, or 288 mg; 0.5, 1.0, 1.5, or 2.0 equivalents to the constituent amino acids of sericin, respectively) was added to the solution. After stirring for 5 h at room temperature in an argon atmosphere, the reaction mixture was poured into excess cold ethanol. The precipitate was collected by centrifugation, washed several times with ethanol and acetone, and then lyophilized to give CPI-Src0.5, 1.0, 1.5, or 2.0, respectively. Characterization. Reaction yields of CPI-Src were determined by the following procedure. Each CPI-Src (10.0 mg) was hydrolyzed in 6 M HCl (2 mL) for 24 h at 110 °C. The hydrolysate was dried in vacuo and then dissolved in 0.1 M HCl (2 mL). The solution was diluted 10 times with 50 mM phosphate buffer of pH 2.6. After filtration through 0.45 µm Ultrafree-MC centrifugal filter devices (Millipore Corp., Billerica, MA), the concentration of 4-aminobenzoic acid hydrochloride in the solution was determined using a Shimadzu LC-VP HPLC system. The system consisted of a pump LC-10ADvp ((2% flow rate accuracy), a solvent degasser DGU-14A, a column oven CTO-10ACvp, and a photodiode array detector SPD-M10Avp. Separation was carried out at 40 °C on a STR ODS-II column, 150 × 4.6 mm (Shinwa Chemical Industries Ltd., Kyoto, Japan) in series preceded by a guard column STR ODS-II, 10 × 4.6 mm (Shinwa Chemical Industries Ltd., Kyoto, Japan). As the eluent, 50 mM phosphate buffer of pH 2.6 at a flow rate of 1.0 mL/min was used. The injection volume was 20 µL. The concentration of 4-aminobenzoic acid hydrochloride was calculated based on the peak area of the absorption at 280 nm using the calibration curve of the standard sample. Fourier transform infrared (FTIR) spectra were recorded at a resolution of 4 cm-1 in 64 scans using a Herschel FT/ IR-350 Fourier transform infrared spectrometer (Jasco Inc., Tokyo, Japan) equipped with a DuraSamplIR II singlereflection Diamond ATR attachment (SensIR Technologies, Danbury, CT). Gel permeation chromatography (GPC) analyses of CPISrc were performed using the LC-VP HPLC system described above. Separation was carried out on a hydrophilic vinyl polymer column TSKgel R-5000, 10 µm diameter particles, 300 × 7.8 mm (Tosoh Corp., Tokyo, Japan) in series preceded by a guard column TSKguardcolumn R, 40 × 6.0 mm (Tosoh Corp., Tokyo, Japan). The system was operated at 40 °C at a flow rate of 0.3 mL/min with 50 mM LiCl/DMSO as the eluent. Samples were dissolved in 100 volumes of 1 M LiCl/DMSO and then diluted five times with the eluent. The solution was filtered through 0.45 µm Ultrafree-MC centrifugal filter devices (Millipore Corp., Billerica, MA). Injection volume was 20 µL and absorption at 280 nm was monitored. A calibration curve for the molecular weight estimation was prepared using the standard MW-Marker for HPLC (Oriental Yeast Co., Ltd., Tokyo, Japan) containing glutamate dehydrogenase (290 kDa), lactate dehydrogenase (142 kDa), enolase (67 kDa), myokinase (32 kDa), and cytochrome c (12.4 kDa). Thermogravimetric (TG) analyses were performed in nitrogen flow (200 mL/min) from room temperature to 300

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Teramoto et al.

Figure 1. Solubility (%) of sericin in DMSO or DMF in the presence of LiCl or LiBr, which was measured by heating the purified sericin in 50 volumes of the solvents at 60 °C for 45 min. Solubility was calculated from UV absorption at 275 nm.

°C at a heating rate of 10 °C/min using a TG8120 TG-DTA apparatus (Rigaku Corp., Tokyo, Japan). Samples had been placed at 20 °C/65% RH for 48 h before measurement. Results and Discussion

Figure 2. SDS-PAGE patterns of the purified sericin (lane P) and the regenerated sericin after 5 h heating at 60 °C in LiCl/DMSO (lane R). Three protein bands (>250, 180, and 100 kDa) were observed for both purified and regenerated sericins.

Solubility and Stability of Sericin in LiCl/DMSO. We first examined the solubility of sericin in some polar aprotic solvents, N,N-dimethylacetamide (DMAc), N,N-dimethylformamide (DMF), and DMSO, to establish nonaqueous reaction condition. DMAc is used frequently as a solvent of natural polymers having strong hydrogen bonds such as Bombyx mori fibroin,26 cellulose,27 or chitin28 in the presence of LiCl. However, DMAc hardly dissolved sericin at 60 °C even in the presence of 1 M LiCl or LiBr. DMF, the same amide solvent as DMAc, dissolved a moderate amount of sericin (Figure 1; closed and open triangles). DMSO, in contrast, dissolved sericin much better than DMF (Figure 1; closed and open circles). Although DMSO alone did not dissolve sericin at 60 °C, the solubility rapidly increased with salt concentration especially when LiCl was used. Nearly complete dissolution of sericin with a concentration of about 2% was achieved at LiCl concentration above 0.5 M. In addition to dissolving many inorganic salts, DMSO dissolves a great variety of organic substances including synthetic polymers, carbohydrates, and proteins. Its high solvent power is probably related to its polar nature, capacity to accept hydrogen bonds, and relatively small and compact structure. Petrusˇ and colleagues reported dissolution of cellulose in LiCl/DMSO solvent and suggested its dissolution mechanism as the following: the unique ability of a solution of LiCl in a polar aprotic solvent to dissolve cellulose is attributable to the interaction of the lithium moieties of solvated, undissociated ion-pairs of the lithium chloride molecule with the hydroxyl group oxygen atoms of cellulose. At a sufficient concentration, these interacting species disrupt interchain hydrogen bonds of cellulose.29 We infer that a similar mechanism exists for dissolution of sericin in LiCl/ DMSO because sericin forms strong interchain hydrogen bonds along with cellulose. LiCl was more effective than LiBr (Figure 1), probably because of polarizability of anions: the larger anion, Br-, being more polarizable, is bound to the solvent more tightly. Thereby, Cl- is more active than Br- to interact with the hydroxyl hydrogen of sericin, disrupting its interchain hydrogen bonds.30 LiCl/ DMAc dissolves cellulose with a similar mechanism to the

one mentioned above;27 it is more frequently used as a solvent of cellulose than DMSO, whereas LiCl/DMAc hardly dissolves sericin. Different polarities or molecular sizes among solvents may play an important role. The dissolution mechanism and behavior of sericin in polar aprotic solvents should be studied in more detail. The purified sericin and the regenerated sericin after 5 h heating at 60 °C were examined by SDS-PAGE to investigate the stability of sericin in LiCl/DMSO. The purified sericin showed a broad and fuzzy band of over 250 kDa, two closely adjacent bands of around 180 kDa, and a faint band of about 100 kDa (Figure 2; lane P). We infer that the main component of over 250 kDa corresponds to Ser1C protein of 330 kDa deduced from Ser1 gene analysis.5 These protein bands were similarly identified for the regenerated sericin; no newly emerged bands were observed (Figure 2; lane R). This result demonstrated that no significant decomposition of sericin chains occurred during dissolution and heating at 60 °C for 5 h, indicating that LiCl/DMSO is a good solvent of sericin. In addition, the versatility of DMSO as the reaction solvent for natural polymers is apparent by the fact that it is used frequently for various chemical modifications of polysaccharides such as sulfation,31,32 silylation,33,34 acylation,35 esterification,36 or modifications with isocyanate compounds.37-39 Modification with 4-Cyanophenyl Isocyanate. We investigated the reaction of sericin with an isocyanate compound in LiCl/DMSO solvent. Isocyanates are frequently used as building blocks for agricultural and pharmaceutical chemicals because they undergo nucleophilic addition reactions with many substrates in a simple manner without side reactions.40 Isocyanates have also been used for chemical modification of proteins13,41,42 or polysaccharides.37-39,43,44 We used 4-cyanophenyl isocyanate for modification of sericin because aryl isocyanates have higher reactivity than alkyl isocyanates; also, it possesses a cyano substituent exhibiting an easily distinguishable infrared absorption. Scheme 1 illustrates an example of the reaction between sericin and 4-cyanophenyl isocyanate. A hydroxyl oxygen



Scheme 1. Reaction Scheme of Sericin Hydroxyl Group with 4-Cyanophenyl Isocyanate

of Ser residue, the most abundant amino acid in sericin, attaches to an electrophilic carbon of the isocyanate to form a urethane linkage. Amino groups from Lys and Arg residues, though they are much less than hydroxyl groups, will also react with the isocyanate to form urea linkages. When CPI-Src is subjected to acid hydrolysis, urethane (or urea) bonds and cyano groups are hydrolyzed to generate 4-aminobenzoic acid hydrochloride together with amino acids (Scheme 2). Concentration of 4-aminobenzoic acid hydrochloride in each hydrolysate from CPI-Src was measured by HPLC and each reaction yield was calculated because the molar amount of 4-aminobenzoic acid hydrochloride in hydrolysate represents the reaction yield. Reaction yields of CPI-Src increased with the increase of the isocyanate used (Figure 3). The reaction yield was 0.76 mmol/g when one equivalent of the isocyanate was used and 2.2 mmol/g when two equivalents of the isocyanate were used. These values correspond roughly to 8.5 and 31% modification of all of the amino acids in sericin. Reaction yields seemed to be low with respect to the amount of isocyanate used. Elongation of reaction times did not engender higher reaction yields. The cause of the low reaction yield will be discussed later. FTIR Analysis of CPI-Src. Dissolution and precipitation from LiCl/DMSO may cause structural changes of sericin that affect its FTIR spectra. The regenerated sericin after 5 h of stirring at room temperature was thus used as a reference to standardize the background. The regenerated sericin exhibited characteristic amide absorption bands of protein, amide I, II, and III, at 1620, 1518, and 1240 cm-1, respectively (Figure 4a). The other bands at 1400 and 1068 cm-1 were assignable to C-H and O-H bending and C-OH stretching vibrations, which largely arose from Ser residues. CPI-Src exhibited the characteristic absorption of CtN stretching vibration at 2225 cm-1 (Figure 4, parts b and c). Another new absorption appeared around 1733 cm-1 as a shoulder peak. We assigned this absorption to CdO stretching vibration of urethane bond based on a model urethane compound synthesized from 4-cyanophenyl isocyanate and ethanol. N-H bending and C-N and C-O stretching of urethane bond overlapped with amide II and III of sericin,

Figure 3. Reaction yields of CPI-Src, which were calculated from the concentration of 4-aminobenzoic acid hydrochloride in each hydrolysate.

Figure 4. Comparison of FTIR spectra before and after modification: the regenerated sericin after 5 h stirring at room temperature (a), CPI-Src1.0 (b), and CPI-Src2.0 (c).

which strengthened their apparent intensities. Two new absorptions at 1597 and 1410 cm-1 were assignable to aromatic ring stretching vibrations; the bands at 1315 and 1176 cm-1 were assigned to C (aromatic)-N stretching and C (aromatic)-H in-plane bending vibrations, respectively. Although amino groups in sericin were also capable of reacting with the isocyanate, we observed no notable peaks assignable to urea bonds. Hydroxyl groups must have reacted with the isocyanate preferentially because they were much more abundant than amino groups. We thus concluded from these analyses that 4-cyanophenyl groups were introduced into sericin molecules mainly through urethane linkages. Because it was reported that sericin contains several kinds of sugar chains,6,45 their hydroxyl groups may also have reacted with the isocyanate. GPC Analysis of CPI-Src. We have demonstrated by SDS-PAGE that heating at 60 °C for 5 h in LiCl/DMSO

Scheme 2. Acid Hydrolysis of CPI-Src, Which Gives 4-Aminobenzoic Acid Hydrochloride and Amino Acids

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Teramoto et al. Table 1. Solubility Characteristics of CPI-Src in 8 M LiBr Aqueous Solution, DMSO, and HFIPa CPI-Src solvent

additive

Srcb

0.5

1.0

1.5

2.0

water DMSO

8 M LiBr 0.25 M LiCl 0.1 M LiCl none none

S P × × ×

× P × × P

× S P × P

× S P P P

× S S S S

HFIP

a S: Soluble; P: partially soluble; ×: insoluble. Dissolution condition: 50 volumes of each solvent, 80 °C (water) or 60 °C (DMSO and HFIP), 45 min. b The purified sericin.

Figure 5. GPC diagrams of the purified sericin (a) and the modified sericins (b): CPI-Src1.0 (solid line) and CPI-Src2.0 (dotted line).

caused no significant decomposition of sericin molecules (Figure 2). Notwithstanding, the isocyanate may engender sericin decomposition. We then performed GPC analysis of CPI-Src to examine whether decomposition of sericin chains occurred during the reaction. A GPC diagram of the purified sericin exhibited a sharp peak at the retention time of 15.1 min, corresponding to 245 kDa according to the calibration curve of the standard proteins, and some broad peaks thereafter (Figure 5a). The sharp peak probably corresponds to the main component of over 250 kDa (Figure 2). CPI-Src1.0 showed a sharp peak at the retention time of 15.2 min (Figure 5b; solid line), which was almost the same position as that with the purified sericin. On the other hand, CPI-Src2.0 exhibited a remarkably different GPC pattern with a large broad band centered at the retention time of 21.9 min, corresponding to 86 kDa, and a shoulder peak at 15.5 min (Figure 5b; dotted line), showing the low molecular weight shift of the main chromatographic peak. These results suggest that the use of excess amounts of the isocyanate causes decomposition of sericin chains during the reaction. GPC diagrams also showed the drastic increase of UV absorption by the modification. This increase resulted from the introduction of aromatic substituents. CPI-Src exhibits the absorption maximum around 270 nm in DMSO. Isocyanate oligomers such as dimers or trimers40 are known to be formed in DMSO.38,39 A GPC diagram of 4-cyanophenyl isocyanate in DMSO exhibited two chromatographic peaks. The one with smaller elution volume probably indicates formation of some isocyanate oligomers. Oligomerization occurs in competition with the modification reaction, which may have caused the low reaction yield of CPI-Src. Sericin chain decomposition was observed when excess isocyanate was used. Such decomposition occurred more readily when reactions were conducted at 60 °C. We first assumed that the amine formed by the decomposition of the isocyanate with water was responsible for the decomposition. However, heating at 60 °C with the amine, 4-aminobenzonitrile, did not cause sericin chain decomposition. The cause

Figure 6. TG curves for the regenerated sericin after 5 h stirring at room temperature, CPI-Src1.0, and CPI-Src2.0, which had been placed at 20 °C/65% RH for 48 h before measurements.

of such decomposition remains unknown at present. Some impurities in the reagent or oligomerization of the isocyanate might be responsible for decomposition. Characteristics of CPI-Src. The solubility characteristic of sericin was also changed through modification. Sericin became insoluble in 8 M LiBr aqueous solution after the modification because of increased hydrophobicity (Table 1). In contrast, the solubility in DMSO increased by the modification. As the modification yield increased, a smaller amount of LiCl was required for the dissolution (Table 1). Additionally, sericin also became soluble in 1,1,1,3,3,3hexafluoro-2-propanol (HFIP) (Table 1), which is used as a solvent for many different types of polymers. CPI-Src2.0 exhibited the highest solubility in DMSO and HFIP, which corresponds to its high reaction yield and low molecular weight. Thin films of CPI-Src were prepared by casting their DMSO or HFIP solution. The TG curve of the regenerated sericin after 5 h stirring at room temperature exhibited the weight loss (-11.6%) up to about 120 °C (Figure 6), which resulted from water evaporation. The corresponding weight loss of CPI-Src1.0 and CPI-Src2.0 were -10.3 and -7.4%, respectively. This order showed that the modification lowered the hygroscopicity of sericin. However, even CPI-Src2.0 continued to exhibit high hygroscopicity, which demonstrates the highly hydrophilic character of sericin. The regenerated sericin underwent a second weight decrease from 215 °C, which is consistent with the reported decomposition temperature of sericin.19 Lower decomposition temperatures were observed as the reaction yield increased, indicating that the introduction of 4-cyanophenyl groups has a destabilizing effect on sericin. Changes of Structural Characteristics by Modification. Secondary structure analysis was performed using FTIR spectra to investigate the effects of the chemical modification



words, increased hydrophobicity and lower interaction between sericin chains probably facilitated the solvent accessibility. We have shown that the β-sheet-rich sericin film, prepared by alcohol treatment, exhibits higher decomposition temperature than random coil-rich film (unpublished data). This fact suggests the contribution of β-sheet formation to thermal stability. FTIR analysis suggested that the modification broke strong intermolecular β-sheets between extended chains, which may be responsible for the lower decomposition temperature of CPI-Src (Figure 6). At the same time, urethane linkages may be less stable and decompose earlier than main chains. Conclusions Figure 7. Amide I bands of the regenerated sericin after 5 h stirring at room temperature (a), CPI-Src0.5 (b), CPI-Src1.0 (c), CPI-Src1.5 (d), and CPI-Src2.0 (e). Increasing absorption at 1597 cm-1 (*) is not the amide I component but the aromatic ring stretching.

on structural characteristics of sericin. Amide I absorption, found in the 1700-1600 cm-1 region, is the most useful for estimating protein secondary structures because it arises predominantly from CdO stretching vibration. Because each secondary structure, such as β-sheet, R-helix, or turn, is associated with a characteristic pattern of hydrogen bonding between amide CdO and N-H groups, it is expected that individual secondary structures will have typical amide absorptions.46,47 The amide I band of the regenerated sericin after 5 h stirring at room temperature showed its absorption peak at 1620 cm-1 (Figure 7a). This absorption is a characteristic observed in proteins with strong intermolecular β-sheets between extended chains,47 indicating that the regenerated sericin was a β-sheet-rich structure. As the reaction yield increased, the peak top of amide I absorption shifted gradually to a higher wavenumber, and CPI-Src2.0 showed its peak top at 1630 cm-1 (Figure 7e), 10 cm-1 higher than the regenerated sericin. This peak is in the range typically seen for ordinary β-sheets. The stronger the hydrogen bond involving the amide CdO, the lower the electron density in the CdO group and the lower the amide I absorption appears.47 Therefore, the peak shift observed in Figure 7 suggested that strong intermolecular hydrogen bonds in the regenerated sericin were broken gradually and that weaker interactions increased as the reaction proceeded. Garel and colleagues reported that sericin contains many Ser-rich 38-amino acid repetitive motifs that exhibit mostly β-sheets.5 Huang and colleagues synthesized recombinant sericin proteins based on the repetitive motif and showed that recombinant sericin self-assembled during dialysis as a formation of β-sheets.48 We assume that this characteristic repetitive motif mostly accounts for the β-sheet-rich structure of the regenerated sericin. Because many Ser and Thr residues exist in the repetitive motif, 4-cyanophenyl groups must have been incorporated into the motif by the modification. Intrusion of bulky 4-cyanophenyl groups will increase the steric constraint of the motif and inhibit formation of strong intermolecular hydrogen bonds among sericin chains. Such structural changes may be responsible for higher solubility of CPI-Src in DMSO and HFIP (Table 1); in other

Chemical modification of silk sericin with 4-cyanophenyl isocyanate using the LiCl/DMSO solvent system was investigated. We found that LiCl/DMSO is a good solvent of silk sericin and is quite useful for modification of sericin hydroxyl groups under nonaqueous conditions. We synthesized 4-cyanophenyl isocyanate-modified sericin derivatives (CPI-Src) and analyzed their characteristics and structures. FTIR analysis indicated that most 4-cyanophenyl groups were connected with sericin molecules at hydroxyl side chains through urethane linkages. Solubility characteristics of sericin were changed remarkably: sericin became insoluble in 8 M LiBr aqueous solution and solubility in DMSO and HFIP increased. The modification rendered sericin less hygroscopic and thermally unstable than before modification. Secondary structure analysis using the amide I band showed that interaction between sericin chains became lower, probably as a result of the incorporation of bulky 4-cyanophenyl groups into the Ser-rich repetitive motif that mostly exhibits β-sheets. These results suggest that the chemical modification to the abundant hydroxyl groups in LiCl/DMSO is quite useful for adding new properties to sericin. A wide variety of isocyanate compounds have halogen atoms, sulfonyl and phosphoryl groups, alkyl chains, or silane groups.40 Use of these different isocyanates will lend sericin different properties. This new modification technique can be applied to various chemical modifications of silk sericin using other than isocyanates because DMSO has been used for a wide range of chemical reactions. Acknowledgment. We thank Dr. Mitsuhiro Miyazawa, Insect Biomaterial and Technology Department, National Institute of Agrobiological Sciences, for FTIR analysis and a thorough reading of the manuscript. We also thank Dr. Toshio Yamamoto and the staff of the Field Management Section, National Institute of Agrobiological Sciences, for providing Sericin-hope cocoons. This work was partially supported by the Insect Technology Project from the Ministry of Agriculture, Forestry and Fisheries of Japan. References and Notes (1) Fedicˇ, R.; Zˇ urovec, M.; Sehnal, F. J. Insect Biotechnol. Sericol. 2002, 71, 1. (2) Craig, C. L.; Riekel, C. Comp. Biochem. Physiol. B 2002, 133, 493.

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