Polymer Biocatalysis and Biomaterials - American Chemical Society

Chapter 10

Downloaded by UNIV OF GUELPH LIBRARY on September 7, 2012 | http://pubs.acs.org Publication Date: February 15, 2005 | doi: 10.1021/bk-2005-0900.ch010

Synthesis of Zein Derivatives and Their Mechanical Properties Atanu Biswas, David J. Sessa, Sherald H. Gordon, John W. Lawton, and J. L. Willett Plant Polymer Research Unit, National Center for Agricultural Utilization Research, Agricultural Research Service, U.S. Department of Agriculture, 1815 North University Street, Peoria, IL 61604

Zein is a naturally occurring protein polymer, obtained as a product of industrial corn processing. It could be possibly used as a coating, ink, fiber, adhesive, textile, chewing gums, cosmetic and biodegradable plastics. Thus, we sought to develop a methodology to chemically modify the zein structure so that zein mechanical properties can be manipulated. A method to prepare acyl derivatives of zein was developed. Zein was dissolved in dimethyl formamide (DMF) and acylated with anhydrides and acid chlorides. The reactions were done by reacting zein DMF solution with anhydrides at 70C or at room temperature with acid chloride. The amine/ hydroxyl group of zein reacted to form ester/amide link. The structure was confirmed with proton NMR and IR spectra. These acetyl, benzoyl and butaryl amide/ester derivatives of zein were compression molded and their mechanical properties were measured. This study provided structure/ mechanical property relationships for these derivatives. It was found that chemical modifications by acetylation did have little impact on the mechanical properties. We also used di -anhydrides to crosslink zein. At low level, i.e. 2-5% of dianhydrides zein showed significant improvement in tensile strength. When dianhydrides were used at higher level the products were crosslinked and insoluble.

© 2005 American Chemical Society In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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142 Zein (1,2) the alcohol soluble protein from corn may well be a commercially valuable product because it can be spun into fibers and it possesses ability to form tough adherent films. In the past, zein fibers (3) were used for garments, hats and other commercial applications, such as coatings. However, zein's chemical inertness and globular structure make molding articles difficult. Yet, it has not been possible to manipulate the structure of zein to make it more moldable or obtain favorable properties. One reason is the alcohol water that is commonly used as solvent is reactive towards most reagents. There have been reports for crosslinking zein in alcohol/water solutions with cyanuric chloride, formaldehyde, carbodiimide—and others (4). However, DMF is one the very few non-reactive solvent for zein and reactions of zein in D M F solutions has not been studied. While our work was in progress Wu et. al. reported the synthesis of zein/nylon copolymer in DMF (5,6). A method to prepare zein acetate (7) was invented in order to increase the water resistance, strength, and flexibility of zein films, coating or other bodies. However, this method is useful for making acetyl derivative only i.e. zein acetate. It would be desirable to devise a general method to modify zein structure. Our objective was to develop a general method to prepare any amide or ester derivatives of zein. Zein has free alcohol and amine groups that are capable of reacting with anhydrides or acid chlorides. The common solvents for zein, such as alcohol/water mixtures prohibit such reactions because these solvents will react with anhydride and acid chloride. To circumvent this problem, we dissolved zein in dimethylformamide (8) and this clear solution was subjected to various reactions. Thus, our primary objective is to synthesize various esters/amides of zein by reacting with acid anhydrides and acid chlorides. Our second objective is to mold these derivatives into bars and the mechanical properties were determined.

Discussion of Results Reactions of zein: The reported chemical reactions of zein have been limited to zein dissolved in water or water/ alcohol mixture (9). As these solvents themselves are reactive towards electrophilic reagents, not many reactions have been reported. We found that at 50°C a solution of greater than 50% could be obtained in DMF. This opened up the possibilities of wide variety of known reactions of hydroxy and mostly secondary amine functional groups to be applied to zein (Figure 1).

In Polymer Biocatalysis and Biomaterials; Cheng, H., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 2005.

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Figure 1 Examples of potential chemical derivatives of zein We have made acyl, benzoyl and butyryl esters/amides of zein. These products were washed with ethyl acetate to remove unreacted acid chlorides or anhydrides. The NMR of these confirmed the acylation. When dianhydride, 1,2,4,5-benzenetetracarboxylic dianhydride (BTCD), was reacted in various levels, slightly crosslinked to highly crosslinked products were obtained. The highly crosslinked product gelled out of the DMF solution. However, the slightly crosslinked (10,11) zein was still soluble in DMF and we could make bars out of them by compression molding.

Aromatic protons at 7.5 to 8.00 ppm

9

8

7

6

5

4

3

2

1

0

ppm

Figure 2 NMR spectrum of a zein derivative. The arrow points out the aromatic protons of benzoate group.


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The NMR spectrum for zein in Figure 2 showed aromatic peaks in the region of 7.5-8.0 ppm., representing aromatic protons of benzoate group. The IR spectrum of the zein derivative in Figure 3 showed evidence of the ester linkage as a pronounced shoulder at 1731 cm" on the amide I peak of the zein. Evidence of the reaction of benzoic anhydride with the zein also appeared at 714 cm" from the mono-substituted benzene group in the derivative. 1


1

0.2 -,

1

Figure 3 IR spectrum of zein derivative. Shoulder at 1731 cm* of the amide peak of zein is evidence of an ester linkage.

Mechanical Properties of Native and Chemically-Modified Zein: Mechanical strength measurement was performed to evaluate the bulk properties of the tensile bars from both native and chemically-modified zeins. Triethylene glycol (TEG) is an excellent plasticizer (12) for zein. Native and chemically modified zeins were each blended with either 10 or 15% TEG in a Haake Rheocord 90 torque rheometer equipped with high shear roller blades to yield a taffy like matrix. The rheometer was stopped when the torque dropped and became constant.


145 Whether a longer residence time in the torque rheometer would impact on the mechanical properties of the compression-molded tensile bars has yet to be determined. The taffy-like blend was snipped into small pieces which were frozen with liquid nitrogen and ground in a Wiley mill. This ground mass was 3

Table 1: Tensile Properties of Native and Chemically Modified Zein


Treatment

%

%

Plasticizer

Moisture

IV Elongation ±SD 18.4 ±2.7 25.5 ± 12.5 25.7 ± 4.0 18.9 ±2.0 15.2 ± 2.5 46.6 ± 33.8 24.3 ± 2.8 27.2 ±6.1 23.1± 2.8 18.0 ±4.8 19.7 ± 3.3 19.7 ± 2.3

None 10 5.3 Benzoate 10 4.3 Benzoate 15 3.8 Benzoate 15 7.4 Acetate 15 8.2 Butyrylate 15 5.6 None 15 6.2 BTCD (0.25%) 15 5.0 BTCD (0.50%) 15 5.0 BTCD(1.00%) 15 5.2 BTCD (2.00%)" 15 5.0 BTCD (3.00%) 15 4.9 Samples stored at 50% RH for one week BTCD= 1,2,4,5-benzene tetracarboxylic dianhydride b b

b

b

Tensile Strength (MPa) ± SD 27.8 ±1.5 20.8 ± 6.8 28.3 ±2.1 21.6 ±3.3 17.3 ±0.1 21.1± 3.3 23.9 ± 0.2 37.4 ±0.9 37.6 ± 1.0 26.5 ± 1.7 31.5 ±0.8 32.1 ± 1.0

a b

sieved through 30 mesh screen to remove fines; samples retained on the sieve were compression molded into tensile bars. The grinding step may break down any network structure formed during the processing with the torque rheometer. Therefore, the mechanical properties of the tensile bars generated should reflect the properties related to the packing structure of the native derivatized zeins. Mechanical properties of the native and chemically-modified zeins are given in Table 1. The chemical modification used will either add a side chain or cross-link the zein, either of which should increase the free volume (i.e. space between free molecules) of the protein. In general, an increase in the free volume of the macromolecule should result in a plasticized mass with increased elongation and decreased tensile strength. Zein that was acetylated had lower elongation and tensile strength despite an increased moisture content of 8.2% when compared with unmodified zein similarly processed. The benzoate derivative possessed similar elongation and slightly higher tensile strength than did unmodified zein. When concentration of derivatizing agent was increased to 20% both mechanical properties dropped below the unmodified zein. When zein is cross-linked with 1,2,4,5-benzene tetracarboxylic dianhydride, labeled BTCD, the sample with 0.25% BTCD gave no change in the % elongation but did show


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a significant increase in tensile strength of 37.4MPa compared with 23.9MPa for unmodified zein. With BTCD, this reactant becomes part of the zein macromolecule. Amounts above 0.25% gave diminished % elongations as well as tensile strengths. Despite the diminished tensile strengths observed they were all significantly higher than those obtained with unmodified zeins. Crosslinking zein with BTCD gave significant changes in tensile strength that merit further investigation.

Experimental Zein was obtained from Freeman Industries. The acid chlorides, anhydrides and the solvents were obtained from Aldrich Chemical. NMR solutions were prepared by warming a mixture of 10 mg zein esters and 10 mg of 40% sodium deuteroxide in one ml of deuterium oxide (NaOD and D 0 were obtained from Cambridge Isotope Laboratories, Inc., Andover, MA). NMR spectra were obtained by using Bruker Instruments D R X 400 spectrometer. FTER spectra of samples pressed in KBr disks were measured on an FTS 6000 FTIR spectrometer (Digilab, Cambridge, CT) equipped with a DTGS detector. The absorbance spectra were measured at 4 cm" resolution, signal-averaged over 32 scans and baseline corrected. Mechanical Properties were measured with Haake Rheocord 90 equipped with high sheer roller blades; Carver Press, Model C with ASTM D 638 type V bar; Instron Model 4201. Reaction procedure: Six gms. of zein was dissolved in 18 gms. of DMF while stirring with a magnetic stirrer at room temperature. To this solution, 6.5 gms. of benzoic anhydride (28 mmole) was added. After 15 minutes, 2.2 gms. (28 mmole) of pyridine was added. The reaction mixture was heated to 50°C, stirred for 4 hours, subsequently cooled to room temperature and poured into 250 ml of water. The product, along with the excess reactants precipitated as a solid. It was filtered, washed with hot water and ground in a blender. As the DMF dissolved in water the pasty solid turned into a yellow powder, which was filtered and washed with dilute hydrochloric acid to remove pyridine. Finally the solid was washed three times with hot ethyl acetate to remove any organics such as benzoic acid or pyridine. We obtained 6.5 gms. of product as yellow solid. The other derivatives such as acetate, butyrate were prepared similarly. When acid chlorides were used they were added at 0°C and the reactions were done at room temperature. Preparation of Tensile Bars by Compression Molding: As stated in Discussion of Result section blends of TEG with either native or chemically modified zein were prepared with a Haake Rheocord 90 torque rheometer. The taffy like matrix were frozen with liquid nitrogen and ground in a Wiley mill. The powder was molded into tensile bars with a Carver Press, Model C at 2

1


147


temperatures 175°C for native zein and 240°C for chemically modified zein and pressures up to 10,000 lbs. for 20 minutes. The resulting tensile bars were conditioned for 1 week at 23°C and 50% relative humidity prior to testing mechanical properties with an Instron Universal tester of a crosshead speed of lcm/minute. Spent tensile bars were evaluated for moisture by heating at 105°C for 4 hours in a forced draft oven.

Conclusions We have developed a simple method to prepare amide/ester of zein. Zein was dissolved in dimethylformamide in 25% concentration and in this solution the free hydroxyl and amine groups of zein readily reacted with electrophilic reagents such as anhydrides. Thus, we were able to prepare acyl, benzoyl, butyroyl amides/esters of zein. We used di-anhydrides to crosslink zein, thus increasing the strength and stability of articles made from it. Crosslinked zein could be useful for coating, ink, adhesives (13) and cross-linking zein with other di functional organic molecules merit further investigation. In contrast, the mechanical properties of acylated zein derivatives did not show much improvement over zein itself.

Acknowledgements The authors would like to thank Janet Berfield, Luke Neal and Benjamin Rocke for their excellent technical assistance and David Weisleder for NMR analyses.

References 1. 2. 3. 4. 5. 6. 7.

Lawton, J.W. Cereal Chem. 2002, 79, 1-18. Cheryan. M . ; Shukla R. Ind. Crops Prod., 2001, 13, 171-192. Sturken, O.U.S. Patent 2,361,713, 1944. Veatch, C. U.S. Patent 2,236,768, 1941. Wu, Q.X.; Sakabe. H.; Isobe, S. Polymer, 2003, 44, 3901-3908. Wu, Q.X.; Yoshino,T.; Sakabe,H.; Zhang,H.K.; Isobe,S. Polymer, 2003, 44, 3909-3919. Veatch, C. U.S. Patent 2,236,768, 1941.


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Danzer, L. Α.; Rees, Ε. D. Can. J. Biochem., 1976, 54, 196-199. Hagemeyer, Jr., H. J. U.S. Patent 2,401,685, 1946. Howland, D.W.; Reiner, R. A. Paint and Varnish Prod., 1962 52, 31-35, 78. Pelosi, L F. U.S. Patent 5,596,080, 1997. Lawton, J.W. Cereal Chem., 2004, 81(l),l-5. Satow, S. U. S. Patents 1,245,976; 1,245,981; 1,245,977; 1917.


8. 9. 10. 11. 12. 13.


Polymer Biocatalysis and Biomaterials - American Chemical Society

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