Recently, surface tension has been shown to be important in emerging high technologies, such as in pumping and control of flow in microfluidic devices, in microchemical analysis of complex fluids, and in rapid DNA screening, etc. Advances in these new technologies will depend strongly on the availability of flexible methods for controlling surface tension. Photo-control using a photoresponsive surfactant is a potentially attractive route to accomplishing many of the tasks required in these processes. Photoresponsive surfactants typically incorporate an azobenzene group as the functional unit which experiences reversible trans-to-cis photoisomerization under different irradiation conditions. The photoisomerization usually causes a change in surface tension. Obviously, a large change in surface tension under different illumination conditions will be highly desirable in practical applications. However, the largest change in surface tension as reported in the literature is only 3 mN/m which is too small to generate any significant effect. In this presentation, we report a new class of photoresponsive surfactants which exhibit excellent performance in surface tension control. Under different illumination conditions, the change in surface tension can be as large as 11.0 mN/m. Experimental results are presented for two new photoresponsive surfactants. A discussion of experimental results follows. / Singapore-MIT Alliance (SMA)

An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented. A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light. The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils. Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation. A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated. Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.

An ability to manipulate the activity of a specific protein inside living cells offers exciting prospects for the study of protein function in vivo. Azobenzene derivatives introduced as intramolecular bridges have been demonstrated to reversibly photoregulate secondary structures and functions of peptides and proteins in vitro. My overall goal is to create a generally-applicable process for the reversible photocontrol of protein-protein interactions within the complex environment of a living cell. Results of studies toward this aim are presented. A blue-green absorbing (~480 nm) azobenzene derivative cross-linker was designed that reversibly controlled the helical content of attached peptides with a half-life of the cis state of ~50 ms. This rapid photoswitch may prove useful as a tool for probing dynamic processes in biochemical systems using light. The effect of cross-linker position (N-terminus, middle, C-terminus) on a photo-switchable 32-residue helical peptide was studied. Although the activation energies for thermal cis – trans relaxations were not the same, linker position did not affect the change in helix content. This work provides useful information for the effective photoregulation of much longer helices such as occur in coiled-coils. Fluorescently labeled, cross-linked, modified Fos/Jun peptides with and without cell-penetrating peptide (CPP) tags were prepared for the purpose of photocontrolling peptide-peptide interactions in vivo. One of the peptides showed a degree of photocontrol of helicity. Cell uptake of CPP-tagged peptides was demonstrated. However, overall peptide behavior was dominated by undesired aggregation. A simple reporter, a cross-linked peptide bearing an environmentally sensitive fluorophore at a key site, was designed for detecting photoswitching in vivo. Photoisomerization of the cross-linker caused changes in the local chemical environment and changes in fluorescence intensity of the environmentally sensitive dyes in vitro. However, no change in fluorescence was observed in the living systems we investigated. Conclusions and suggestions for further work aimed at achieving the overall goal stated above are discussed.

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.

Yes / Photoresponsive molecules that incorporate peptides capable of material-specific recognition provide a basis for biomolecule-mediated control of the nucleation, growth, organization, and activation of hybrid inorganic/organic nanostructures. These hybrid molecules interact with the inorganic surface through multiple noncovalent interactions which allow reconfiguration in response to optical stimuli. Here, we quantify the binding of azobenzene-peptide conjugates that exhibit optically triggered cis-trans isomerization on Ag surfaces and compare to their behavior on Au. These results demonstrate differences in binding and switching behavior between the Au and Ag surfaces. These molecules can also produce and stabilize Au and Ag nanoparticles in aqueous media where the biointerface can be reproducibly and reversibly switched by optically triggered azobenzene isomerization. Comparisons of switching rates and reversibility on the nanoparticles reveal differences that depend upon whether the azobenzene is attached at the peptide N- or C-terminus, its isomerization state, and the nanoparticle composition. Our integrated experimental and computational investigation shows that the number of ligand anchor sites strongly influences the nanoparticle size. As predicted by our molecular simulations, weaker contact between the hybrid biomolecules and the Ag surface, with fewer anchor residues compared with Au, gives rise to differences in switching kinetics on Ag versus Au. Our findings provide a pathway toward achieving new remotely actuatable nanomaterials for multiple applications from a single system, which remains difficult to achieve using conventional approaches. / Air Office of Scientific Research, grant number FA9550-12-1-0226.

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The photoviscosity effect in aqueous solutions of novel poly(4-methacryloyloxyazobenzene-co-N,N-dimethyl acrylamide) (MOAB-DMA) was demonstrated. The observed significant reduction in the zero-shear viscosity upon UV-irradiation of MOAB-DMA aqueous solutions was due to the dissociation of the interchain azobenzene aggregates. Such phenomena can be advantageously used in photoswitchable fluidic devices and in protein separation. Introduction of enzymatically degradable azo cross-links into Pluronic-PAA microgels allowed for control of swelling due to degradation of the cross-links by azoreductases from the rat intestinal cecum. Dynamic changes in the cross-link density of stimuli-responsive microgels enable novel opportunities for the control of gel swelling, of importance for drug delivery and microgel sensoric applications. / Singapore-MIT Alliance (SMA)

Azobenzene upon photochemical E/Z isomerization changes both its shape and size. The E-azobenzene moiety falls in the class of calamitic liquid crystalline mesogens, producing a wide variety of mesophases. Two series of linear bis(azobenzene) compounds, one with phenyl benzoate linkage and the other with benzyl benzoate linkage were synthesized. The termini of these molecules ranged from a dodecyloxy chain to hydrophobic amphiphilic dendrons up to first generation. We determined the effects of both the linkages and generation number on the mesogenic properties of these compounds. Our results show that the mesogenic behavior of these bis(azobenzene) compounds are highly dependent on the linkages between individual azobenzenes and that for the bis(azobenzene) compounds of the phenyl benzoate series, generation number had an effect on the liquid crystalline mesophase of the compounds

There are many different types of targeted therapy for cancer treatment. The method of light mediated targeted therapy that we have developed uses photocaged molecules and photoswitchable peptides. In photocaging, a biologically active molecule is made inactive by the attachment of a photocleavable blocking group. On exposure to UV radiation the photocleavable entity is removed and the biologically active molecule is released. Using this concept we have designed a prodrug that consists of a cell impermeable hydrophilic molecule attached to a photocaged doxorubicin. Upon irradiation with UV light the photosensitive group is removed and cytotoxic doxorubicin is released at the tumor site. This concept has been further modified by attaching receptor binding molecules to the photocaged entity to increase its specificity. A peptide which consists of an azobenzene photoswitch has been used which, in the dark state is randomly coiled and cell impermeable but upon illumination becomes helical and cell permeable and can be used to deliver drugs into the cells. Upon illumination with UV light of suitable wavelength the azobenzene linker will change from a trans to a cis form and this will convert the randomly coiled cell impermeable peptide into an α helical permeable form. Thus a series of peptides have been designed with different arginine mutations which develop an arginine patch in the helical form. This arginine patch would help in cell permeability by interacting with cell surface glycans. The method could potentially be used to deliver drugs into cells in presence of light.