Polydiacetylene Monolayers: Model Systems for Enzymatic Surface Modification
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POLYDIACETYLENE MONOLAYERS: MODEL SYSTEMS FOR ENZYMATIC SURFACE MODIFICATION Troy E. Wilson and Mark D. Bednarski* Department of Chemistry, University of California at Berkeley and the Center for Advanced Materials, Lawrence Berkeley Laboratory, Berkeley, CA 94720
We are exploring the requirements for enzyme-catalyzed reactions on small molecules tethered to the surfaces of organic monolayers. Despite considerable effort toward understanding enzymatic processes in solution1 , the chemistry of enzymes at interfaces has not been studied. Increasingly sophisticated methods of surface modification, including selfassembly 2 and photolithographic 3 techniques, raise intriguing prospects for enzymatic surface chemistry. This paper describes our initial investigations of the proteolysis of a dipeptide substrate covalently tethered to the surface of a polydiacetylene film using the enzyme, subtilisin BPN'. We have shown that complex molecules such as amino acids 4 and carbohydrates 5 can be readily presented at the surface of polydiacetylene monolayers. For these enzymatic studies, the phenylalanine-alanine (Phe-Ala) dipeptide was used because the subtilisin BPN' protease is specific for amide cleavage of substrates containing large, aromatic side chains such as phenylalanine. 6 The dipeptide was incorporated into a polymerizable lipid monomer 1 and mixed monolayers of 1 and 2 were constructed at an air-water interface using previously described methods (Figure 1).4,7 The lipid monolayers were cross-linked at the water surface by irradiation with ultraviolet light and then transferred horizontally to hydrophobic glass slides. 8 The structure of these monolayers has been confirmed by contact angle measurements, fluorescence microscopy, and X-ray photoelectron spectroscopy (XPS) depth profiling.
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2 The proteolysis reactions were accomplished by treatment of mixed polydiacetylene films containing 15% of 1 and 85% of 2 with 0.4 mM solutions of subtilisin BPN' (105.3 units, Mat. Res. Soc. Symp. Proc. Vol. 237. 01992 Materials Research Society
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Figure 1. A schematic representation of a polymerized, mixed monolayer of compounds 1 and 2. The Phe-Ala dipeptide is tethered to the film surface via a flexible, variable-length polyethylene glycol (PEG) linker. Cleavage of the Phe-Ala amide linkage using subtilisin BPN' will release compound 3 (F5 -Bz-N-Phe-OH) which is shown in the upper left comer of the figure. X-ray photoelectron spectroscopy (XPS) was used to monitor the progress of the reaction by observing the disappearance of the fluorine (F Is) signal with time. 1.5 mL of 0.1 M Tris, pH 8.6, 37 'C) with gentle agitation. 6 At successive time points, the surfaces were removed from the subtilisin BPN' solutions, rinsed with distilled water (5x2 mL), 0.1 M NaHCO 3 (5x2 mL), water (5x2 mL), methanol (5x2 mL) and then dried under flowing nitrogen gas. It was anticipated that successful enzymatic hydrolysis of the phenylalanine-alan
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