G. Andrew Woolley

Azobenzene Photoswitching without Ultraviolet Light

Most azobenzene-based photoswitches use UV light for photoisomerization. This can limit their application in biological systems, where UV light can trigger unwanted responses, including cellular apoptosis. We have found that substitution of all four ortho positions with methoxy groups in an amidoazobenzene derivative leads to a substantial (~35 nm) red shift of the n-π* band of the trans isomer, separating it from the cis n-π* transition. This red shift makes trans-to-cis photoswitching possible using green light (530-560 nm). The cis state is thermally stable with a half-life of ~2.4 days in the dark in aqueous solution. Reverse (cis-to-trans) photoswitching can be accomplished with blue light (460 nm), so bidirectional photoswitching between thermally stable isomers is possible without using UV light at all.

Photoswitching Azo Compounds in Vivo with Red Light

The photoisomerization of azobenzenes provides a general means for the photocontrol of molecular structure and function. For applications in vivo, however, the wavelength of irradiation required for trans-to-cis isomerization of azobenzenes is critical since UV and most visible wavelengths are strongly scattered by cells and tissues. We report here that azobenzene compounds in which all four positions ortho to the azo group are substituted with bulky electron-rich substituents can be effectively isomerized with red light (630-660 nm), a wavelength range that is orders of magnitude more penetrating through tissue than other parts of the visible spectrum. When the ortho substituent is chloro, the compounds also exhibit stability to reduction by glutathione, enabling their use in intracellular environments in vivo.

Fluorescence Imaging of Azobenzene Photoswitching In Vivo

The photoisomerization of azobenzene has been used to control a wide variety of molecular processes. It has been applied to the photocontrol of biomolecular targets (peptides, proteins, and nucleic acids) in vitro and in cell extracts. Recently it has been applied to the photocontrol of coiled-coil proteins in living cells in culture and to the photocontrol of ion channels in vivo. 8] These studies highlight the promise of azobenzene-modified biomolecules as general agents for the remote control of biomolecular function using light. In order to function as agents for controlling molecular events in complex living systems, however, the azobenzene-based photoswitches must be chemically stable in a variety of intracellular environments. At least for certain azobenzene photoswitches used for conformational control, glutathione in the intracellular environment can reduce and inactivate the photoswitch. Enzyme-mediated reduction or other modification, as occurs with numerous azo dyes, 11] is also possible. Even if not inactivated, azobenzene photoswitches may exhibit different isomerization rates in a cellular environment. For instance glutathione has been found to catalyze thermal cis-to-trans isomerization of certain azobenzenes. Fluorescence imaging of azobenzene photoswitching would enable a direct test of the feasibility of using azobenzene for intracellular photocontrol in a living organism. Since azobenzenes used for conformational control are not intrinsically fluorescent, we developed a fluorescence reporter for azobenzene photoswitching. Peptides bearing pairs of Cys residues can be intramolecularly cross-linked with thiol reactive azobenzene-based photoswitches such as 1 (Figure 1). These p-amido substituted azobenzene derivatives are relatively electron-rich compounds among those that have been used for conformational control in vitro 8,9, 13] and are resistant to reduction by glutathione in vitro (see Supporting Information). The metabolism of azo dyes has been found to be sensitive to their redox potentials, but also to the pattern and nature of ring substituents. Photoisomerization of the azobenzene cross-linker 1 alters the conformation of the peptide depending on the location of the Cys residue attachment points (Figure 1). Since a substantial conformational change occurs (Figure 2a,b), we reasoned that attachment of a fluorescent dye near the photoswitch may result in a fluorescence change upon isomerization. We explored a variety of fluorescent dyes with both sulfonated and non-sulfonated photoswitches (see Supporting Information). Peptide 2, shown in Figure 1, produced the largest change (a 40% decrease, Figure 2c) in fluorescence emission intensity in vitro upon trans-to-cis isomerization. The dark-adapted reporter has the azobenzene switch in the trans state; incubation in the dark restores the trans isomer with a half life of 10.7 min at 25 8C. Irradiation in the UV range (350–390) causes trans-to-cis isomerization of the photoswitch as well as excitation of fluorescein. Irradiation with blue light (440–490) causes cis-to-trans isomerization as well as fluorescein excitation. Since the cis isomer of the peptide (2a-cis) has a lower quantum yield for fluorescence than the trans isomer (2a-trans), irradiation of a darkadapted solution of reporter peptide with UV light produces a time-dependent fluorescence decrease (Figure 2d). Conversely, irradiation of 2a-cis with blue light causes a timedependent fluorescence increase (Figure 2d). The rates of these switching processes depend on the intensity and the wavelength of irradiation; action spectra are shown in the Supporting Information, Figure S1. A variety of mechanisms for the decrease in fluorescence intensity observed upon trans-to-cis isomerization are possible. Partial protonation of the fluorescein moiety due to an increase in its pKa upon trans-to-cis isomerization is unlikely since the fluorescence response is unchanged between pH 7 Figure 1. Structure of the photoswitches and the fluorescent peptide reporter.

Using an Azobenzene Cross-Linker to Either Increase or Decrease Peptide Helix Content upon Trans-to-Cis Photoisomerization

Reversible photocontrol of peptide and protein conformation could prove to be a powerful tool for probing function in diverse biological systems. Here, we report reversible photoswitching of the helix content in short peptides containing an azobenzene cross-linker between cysteine residues at positions i, i + 4, or i, i + 11 in the sequence. Trans-to-cis photoisomerization significantly increases the helix content in the i, i + 4 case and significantly decreases the helix content in the i, i + 11 case. These cross-linker designs significantly expand the possibilities for photocontrol of peptide and protein structure.

Photocontrol of Coiled-Coil Proteins in Living Cells**

Light switching of the activity of a coiled-coil protein, the AP-1 transcription factor, in living cells was made possible by the introduction of a designed azobenzene-cross-linked dominant negative peptide, XAFosW (red and yellow in the picture). In the dark, XAFosW showed decreased helical content and decreased affinity for target Jun proteins (green); irradiation at 365 nm enhanced helicity and target affinity.

Bidirectional photocontrol of peptide conformation with a bridged azobenzene derivative.

It goes both ways: A thiol-reactive cross-linker based on a bridged azobenzene derivative permits photoreversible control of peptide conformation on irradiation with violet (407 nm) and green (500-550 nm) light (see picture) through isomerization of the cross-linker. The large separation of the absorbance bands of the cis (yellow) and trans (red) isomers enables complete bidirectional photoswitching.

Structure-based approach to the photocontrol of protein folding.

Photoswitchable proteins offer exciting prospects for remote control of biochemical processes. We propose a general approach to the design of photoswitchable proteins based on the introduction of a photoswitchable intramolecular cross-linker. We chose, as a model, a FynSH3 domain for which the free energy of folding is less than the energy available from photoisomerization of the cross-linker. Taking the experimentally determined structure of the folded protein as a starting point, mutations were made to introduce pairs of Cys residues so that the distance between Cys sulfur atoms matches the ideal length of the cis form, but not the trans form, of the cross-linker. When the trans cross-linker was introduced into this L3C-L29C-T47AFynSH3 mutant, the protein was destabilized so that folded and unfolded forms coexisted. Irradiation of the cross-linker to produce the cis isomer recovered the folded, active state of the protein. This work shows that structure-based introduction of switchable cross-linkers is a feasible approach for photocontrol of folding/unfolding of globular proteins.

Robust Visible Light Photoswitching with Ortho-Thiol Substituted Azobenzenes

Introduction of S-ethyl groups in all four ortho positions of azobenzene prevents reduction of the azo group by intracellular glutathione, while enhancing the absorptivity to ~10,000 M(-1) cm(-1) in the blue and green regions of the visible spectrum. cis-to-trans isomerization occurs thermally on the minutes timescale. Further, this substitution pattern permits switching with red light, a color that is more penetrating through biological tissues than other parts of the visible spectrum.

Photomodulation of ionic current through hemithioindigo-modified gramicidin channels

Incorporation of photo-switchable amino acids into peptides and proteins offers prospects for the control of complex biochemical processes using light. Currently, only a few photo-switchable amino acids are known. We report the design and synthesis of a novel hemithioindigo-based amino acid and its incorporation into the model ion channel gramicidin. Photoisomerization of the hemithioindigo moiety between E and Z isomeric forms is shown to modulate ionic current through the channel in a predictable way. This new amino acid thus expands the possibilities for photo-control in diverse systems.

Photoswitching of ortho‐Substituted Azonium Ions by Red Light in Whole Blood

Photo-control using red light is highly desirable for biological applications since red wavelengths are the only part of the visible spectrum that can effectively penetrate tissue.[1] Efforts to develop optogenetic and optochemical genetic tools that are red-shifted range from exploring natural biodiversity in the search for red-shifted opsins[2] to the conjugation of chemical photoswitches to upconverting nanoparticles.[3] We recently reported that azobenzenes with bulky polar substituents in all four positions ortho to the azo group could undergo red-light driven photoisomerization.[4] However, the compounds require intense red light or long irradiation times to reach the photostationary state because the absorption coefficients for wavelengths >600 nm are very small.[4]