Fluorescence Imaging of Azobenzene Photoswitching In Vivo

Beharry, Andrew A.; Wong, Leo; Tropepe, Vince; Woolley, G. Andrew

doi:10.1002/anie.201006506

Cited by 141 publications

(100 citation statements)

References 32 publications

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“…To address in vivo stability of photoswitching, we monitored photoswitching of a bis-p-amido substituted azobenzene using a fluorescent reporter assay and found photoswitching behavior was retained for days in developing zebrafish. 34 Bis-p-amido substituted azobenzene is the core of several photoswitches that have been developed to target biomolecules ( Figure 1). Newly developed switches are now tested for their stability to reduction by glutathione, the predominant intracellular reductant.…”

Section: ■ Azobenzenes In Vivomentioning

confidence: 99%

Red-Shifting Azobenzene Photoswitches for in Vivo Use

et al. 2015

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Recently, there has been a great deal of interest in using the photoisomerization of azobenzene compounds to control specific biological targets in vivo. These azo compounds can be used as research tools or, in principle, could act as optically controlled drugs. Such "photopharmaceuticals" offer the prospect of targeted drug action and an unprecedented degree of temporal control. A key feature of azo compounds designed to photoswitch in vivo is the wavelength of light required to cause the photoisomerization. To pass through tissue such as the human hand, wavelengths in the red, far-red, or ideally near infrared region are required. This Account describes our attempts to produce such azo compounds. Introducing electron-donating or push/pull substituents at the para positions delocalizes the azobenzene chromophore and leads to long wavelength absorption but usually also lowers the thermal barrier to interconversion of the isomers. Fast thermal relaxation means it is difficult to produce a large steady state fraction of the cis isomer. Thus, specifically activating or inhibiting a biological process with the cis isomer would require an impractically bright light source. We have found that introducing substituents at all four ortho positions leads to azo compounds with a number of unusual properties that are useful for in vivo photoswitching. When the para substituents are amide groups, these tetra-ortho substituted azo compounds show unusually slow thermal relaxation rates and enhanced separation of n-π* transitions of cis and trans isomers compared to analogues without ortho substituents. When para positions are substituted with amino groups, ortho methoxy groups greatly stabilize the azonium form of the compounds, in which the azo group is protonated. Azonium ions absorb strongly in the red region of the spectrum and can reach into the near-IR. These azonium ions can exhibit robust cis-trans isomerization in aqueous solutions at neutral pH. By varying the nature of ortho substituents, together with the number and nature of meta and para substituents, long wavelength switching, stability to photobleaching, stability to hydrolysis, and stability to reduction by thiols can all be crafted into a photoswitch. Some of these newly developed photoswitches can be used in whole blood and show promise for effective use in vivo. It is hoped they can be combined with appropriate bioactive targets to realize the potential of photopharmacology.

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Section: ■ Azobenzenes In Vivomentioning

confidence: 99%

Red-Shifting Azobenzene Photoswitches for in Vivo Use

et al. 2015

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“…In recent years, researches on stimulus-responsive polymers have gained increasing attention for their potential applications as biological sensors [1,2], fluorescence probes [3,4], and drug controlled delivery [5e7]. Environmental stimuli such as light [8], pH [9], temperature [10], humidity [11], CO 2 [12,13] and electric/ magnetic field [14] can induce conformational and/or soluble changes of segments of chains at molecular level.…”

Section: Introductionmentioning

confidence: 99%

Multi-responsive fluorescence of amphiphilic diblock copolymer containing carboxylate azobenzene and N-isopropylacrylamide

Ren

Chen

Shi

et al. 2016

Polymer

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“…For example, the incorporation of amino acids with an ortho-nitrobenzyl caging group into the active site of proteins allowed protein functions to become active only after the ortho-nitrobenzyl group had been removed by UV irradiation at 365 nm. Azo compounds undergo cis-to-trans isomerization by photoirradiation [11][12][13]. This isomerization is reversible, thus enabling the ''on-off'' photoswitching of protein functions, even in vivo.…”

Section: Introductionmentioning

confidence: 99%

Control of enzyme reaction by a designed metal-ion-dependent α-helical coiled-coil protein

Murase

Ishino

et al. 2012

J Biol Inorg Chem

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Regulation of protein function by external stimuli is a fascinating target for de novo design. We have constructed a peptide that assembles into a homotrimer in the presence of metal ions, such as Ni(2+), Cu(2+), and Zn(2+). We fused the peptide construct to the DNA-binding domain (DBD) of the heat shock factor from Saccharomyces cerevisiae, which binds tandem repeats of the heat shock element (HSE). However, the fusion protein bound to the natural three tandem HSEs even in the absence of metal ions, although mainly as the dimerized protein. Using "skipped" HSEs containing six additional nucleotides inserted between two adjacent HSEs, to prevent interactions between the DBDs, we found the fusion protein bound to the new DNA target in a metal-ion-dependent manner, as monitored by a HindIII protection assay. The fusion protein containing two metal binding sites in the metal-ion-controlled domain inhibited RNA transcription by T7 RNA polymerase in the presence of metal ions, in a template containing skipped HSEs downstream of the T7 promoter. The designed protein therefore regulates the functions of the enzyme in a metal-ion-dependent manner.

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Fluorescence Imaging of Azobenzene Photoswitching In Vivo

Abstract: Switching zebrafish: A fluorescent reporter peptide permits imaging of azobenzene photoisomerization in vivo (see picture), which indicates that azobenzene‐based photochemical switches may be generally useful for spatiotemporal control in living systems.

Cited by 141 publications

References 32 publications

Red-Shifting Azobenzene Photoswitches for in Vivo Use

Red-Shifting Azobenzene Photoswitches for in Vivo Use

Multi-responsive fluorescence of amphiphilic diblock copolymer containing carboxylate azobenzene and N-isopropylacrylamide

Control of enzyme reaction by a designed metal-ion-dependent α-helical coiled-coil protein

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