Hydrogels have been used for many applications, including as a mimic for the extracellular matrix (ECM) in cell culture. For example, a hydrogel containing protease-sensitive substrates can be used to create an environment that cells can modify via enzymatic degradation. In this study, we propose combining traditional hydrogels for cell culture with “cryptic” site that bury proteolytically cleavable peptide sequences using complex coacervation. Here, the goal is to take advantage of the phase separation of coacervates to protect the cleavable peptide against degradation until acted upon by a mechanical force, such as those generated by adherent cells. To this end, we studied the encapsulation of chymotrypsin as a model protease into our coacervate system and investigated the effect of incorporation into the coacervate on its activity. We have also synthesized a peptide containing cleavable site for both chymotrypsin as our model protease and more biologically relevant matrix metalloproteinases (MMPs). Future efforts will look to incorporate this peptide into both coacervate and hydrogel and test the level of cryptic response.

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Stuttgart, Univ., Diss., 2008.

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This thesis examines the design, synthesis and biological applications of a series of inhibitors which incorporate an azobenzene photoswitch, a peptidyl backbone and a trifluoromethyl ketone warhead. The photoswitch can be isomerised by irradiation with UV or visible light and has been employed to modulate the reactivity of the enzyme. Chapter one gives a brief outline of some of the important areas related to this work. Examples of previously utilised photoswitches as well as some background on serine protease and the uses of fluorine in medicine has been covered. Chapter two outlines the synthesis of the key trifluoromethyl carbinol 2.6 by two different methods. The condensation of a fluorinated aldehyde with a nitroalkane affords an α-nitro trifluoromethyl carbinol which can be reduced to give the desired amine 2.6. Treatment of oxazolones with trifluoroacetic anhydride via a modified Dakin-West reaction gives trifluoromethyl ketones which can be reduced to give trifluoromethyl carbinols. Chapter three investigate the synthesis of substituted stilbenes and phenanthrenes as alternative molecular switches to azobenzenes. Molecular modelling of phenanthrenes suggests they may be suitable mimics of E-azobenzenes. Chapter four outlines the synthesis of a series of mono and disubstituted azobenzenes by direct sulfonation of azobenzene or by condensation of nitroso arenes with aryl amines. The switches incorporate one or two peptidyl residues designed to improve specificity towards the enzyme. Chapter five examines the photoisomerisation of eight potential inhibitors by irradiating with UV or visible light. Irradiation with UV light enriches the sample to give 78-93 % of the Z-isomer. Irradiation with visible light gave photostationary states with 14-21 % Z-isomer. Ambient photostationary states are ca. 22 % Z-isomer. Chapter six looks at the testing of five trifluoromethyl ketones as potential inhibitors ofα-chymotrypsin. The inhibitors vary in substituents, substitution patterns and chain length. The inhibitors were tested at both ambient and Z-enriched photostationary states and were found to exhibit slow binding kinetics. In all cases the Z-enriched inhibitor solution was at least 3-fold more potent than the ambient solution. Chapter seven is an experimental chapter and outlines the synthesis of the compounds prepared in this thesis.

This thesis presents the first example of reversible photoregulation of the binding of a protease, α-chymotrypsin, to a surface. A modular approach is used involving the azobenzene photoswitch group, a surface linker and an enzyme binding group. This approach is designed to be easily extended to the photoregulation of binding of other proteases to surfaces by use of enzyme binding groups selective to these proteases. Chapter one gives a brief outline of some of the important areas involved in to this work, including molecular switches, proteases and surface modification. Chapter two describes the synthesis of azobenzene-containing boronate esters designed as photoswitch inhibitors of α-chymotrypsin. Boronate esters were prepared containing the aminophenylboronate group or the peptidomimetic borophenylalanine group for enzyme binding and a range of substituents designed for enzyme affinity and/or surface attachment. Syntheses primarily involved peptide coupling reactions and azobenzene formation by condensation of nitrosobenzenes and anilines. Coupling reactions were successfully carried out using EDCI or isobutyl chlorofomate in several cases where other reagents gave unacceptable decomposition. Chapter three describes the syntheses and HPLC stability studies of derivatives of a noncovalent α-chymotrypsin inhibitor. Several dipeptide-based compounds containing either an amide group for surface attachment or an azobenzene group for photoswitching were prepared, primarily using peptide coupling reactions. Each compound was incubated with α-chymotrypsin to assess its stability, and all were found by HPLC monitoring to be stable to α-chymotrypsin catalysed hydrolysis. Chapter four describes syntheses of azobenzene-containing trifluoromethylketones and α-ketoesters designed as photoswitch inhibitors of α-chymotrypsin. Trifluoromethylketones/α-ketoesters containing amine groups for surface attachment were prepared, primarily using peptide coupling reactions, but could not be isolated due to the incompatibility of the electrophilic ketone and primary amine groups. Trifluoromethylketones/α-ketoesters containing terminal alkynes for surface attachment were prepared either by the attachment of an alkyne substituent group to a symmetrical azobenzene core or by Pd-catalysed reaction of a protected alkyne with an azobenzene having a halide substitutent. Chapter five describes syntheses of sulfur-containing surface linkers for use in surface attachment of the photoswitch inhibitors described in chapters 2-4. A range of compounds containing disulfide or protected thiol groups for surface attachment and azide or carboxylic acid groups for inhibitor attachment were prepared. Syntheses primarily involved coupling of functionalised alcohols/amides to carboxylic acid-containing disulfides/thioacetates. Selected linkers were attached to azobenzenes by amide coupling or azide-alkyne cycloaddition for surface attachment, photoswitching and/or enzyme assay. Azide-alkyne cycloaddition yields were initially poor, but were improved by use of stoichiometric amounts of copper catalyst. Chapter six describes UV/vis photoisomerisation studies and enzyme assays carried out to assess enzyme photoswitching of the compounds described in chapters 2-5. The trifluoromethylketones and α-ketoesters described in chapter 4 gave the best results, with moderate inhibition of α-chymotrypsin (µM affinity constants) and up to 5.3 fold changes in inhibition on UV/vis irradiation. Many of the boronate esters described in chapter 2 were found to inhibit α-chymotrypsin, but were somewhat unstable to irradiation. The dipeptide-based compounds described in chapter 3 were inactive against α-chymotrypsin. Good photoisomerisation was obtained for an azobenzene containing a symmetrical disulfide surface linker and poor photoisomerisation was obtained for an azobenzene containing a lipoic acid surface linker. Chapter seven describes surface attachment of selected photoswitch inhibitors and studies of photoregulated enzyme binding to the resultant functional surfaces. Self assembled monolayers (SAMs) of disulfides were formed on gold surfaces and characterised by electrochemistry and contact angle measurements. Binding of α-chymotrypsin to SAMs containing a photoswitch inhibitor was detected by quartz crystal microbalance (QCM), but was found to be largely irreversible. An alkyne-containing photoswitch inhibitor was attached to a surface plasmon resonance (SPR) chip in a two step procedure involving generation of an azide modified surface followed by azide-alkyne cycloaddition. Binding of α-chymotrypsin to the resultant modified surface was detected by SPR and successfully regulated by UV/vis irradiation. Chapter eight provides conclusions for the work described in this thesis and suggests future directions. Chapter nine gives experimental details for the work described in this thesis.