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Scholarly Works (8 results)

  • Thesis
  • Peer Reviewed
UC Berkeley Electronic Theses and Dissertations (2010)

Part one of this dissertation describes synthetic efforts toward two separate but structurally related groups of marine sesquiterpene quinone natural products, the smenochromene/likonide family and halenaquinone, and culminates in the total synthesis of smenochromene B and halenaquinone. The total synthesis of smenochromene B and efforts towards likonide A centered around the use of smenochromene D as a synthetic intermediate. A macrocyclic ring contraction and double bond isomerization was the key transformation necessary to produce smenochromene B. During the optimization of the synthesis of smenochromene D, two different and remarkable oxidative dimers were isolated, and the mechanism by which they are formed is discussed. Additionally, density functional theory calculations were used to investigate the oxa-6π electrocyclization used to synthesize smenochromene D and are believed to be biologically relevant to the markedly different levels of enantiomeric excess observed within members of the structural family.

Halenaquinone, a biologically active pentacyclic quinone isolated from the marine sponge Xestospongia exigua, is biosynthetically related to the smenochromene/likonide family. The total synthesis of halenaquinone reported here features two key steps: a diastereoselective intramolecular Heck reaction, which sets the all-carbon quaternary stereocenter, and an intramolecular inverse-electron-demand Diels-Alder cycloaddition using a vinyl-orthoquinone methide as a diene. This is the first application of such a Diels-Alder cycloaddition to total synthesis. In order to better understand the Diels-Alder reaction, density functional theory calculations were performed to analyze two relevant transition states.

Part two describes several projects focused around the synthesis of photoswitchable azobenzene ligands. In order to build on previous work that demonstrated photoregulation of wild-type voltage-gated potassium channels by membrane permeable blockers, a new series of compounds was synthesized with red-shifted absorption maxima. Two new azobenzene containing blockers were identified as being able to regulate voltage-gated potassium channels by toggling between darkness and irradiation with 472 nm light; one compound blocked voltage-gated potassium channels in the trans form, the other in the cis form. Additionally, azobenzene compounds based on the drugs lidocaine and retigabine were synthesized, and preliminary biological data are reported. Finally, the syntheses of azobenzene analogs of the anti-cancer and life-extending (in lower organisms) compound resveratrol and a related protein-tyrosine kinase inhibitor piceatannol are reported.

    Cover page: I. Total Synthesis of Smenochromene B and Halenaquinone and II. Studies on the Chemical and Biological Properties of Azobenzene Photoswitches
    • Article
    • Peer Reviewed
    UC Berkeley Previously Published Works (2018)
    Degenerative retinal diseases such as retinitis pigmentosa (RP) and age-related macular degeneration (AMD) affect millions of people around the world and lead to irreversible vision loss if left untreated. A number of therapeutic strategies have been developed over the years to treat these diseases or restore vision to already blind patients. In this Review, we describe the development and translational application of light-sensitive chemical photoswitches to restore visual function to the blind retina and compare the translational potential of photoswitches with other vision-restoring therapies. This therapeutic strategy is enabled by an efficient fusion of chemical synthesis, chemical biology, and molecular biology and is broadly applicable to other biological systems. We hope this Review will be of interest to chemists as well as neuroscientists and clinicians.
      Cover page: Restoring Vision to the Blind with Chemical Photoswitches
      • Article
      • Peer Reviewed
      UC Berkeley Previously Published Works (2013)
      The use of azobenzene photoswitches has become a dependable method for rapid and exact modulation of biological processes and material science systems. The requirement of ultraviolet light for azobenzene isomerization is not ideal for biological systems due to poor tissue penetration and potentially damaging effects. While modified azobenzene cores with a red-shifted cis-to-trans isomerization have been previously described, they have not yet been incorporated into a powerful method to control protein function: the photoswitchable tethered ligand (PTL) approach. We report the synthesis and characterization of a red-shifted PTL, L-MAG0460, for the light-gated ionotropic glutamate receptor LiGluR. In cultured mammalian cells, the LiGluR+L-MAG0460 system is activated rapidly by illumination with 400-520 nm light to generate a large ionic current. The current rapidly turns off in the dark as the PTL relaxes thermally back to the trans configuration. The visible light excitation and single-wavelength behavior considerably simplify use and should improve utilization in tissue.
        Cover page: A Red-Shifted, Fast-Relaxing Azobenzene Photoswitch for Visible Light Control of an Ionotropic Glutamate Receptor
        • Article
        • Peer Reviewed
        UC Berkeley Previously Published Works (2014)
        Membrane lipids serve as second messengers and docking sites for proteins and play central roles in cell signaling. A major question about lipid signaling is whether diffusible lipids can selectively target specific proteins. One family of lipid-regulated membrane proteins is the TWIK-related K channel (TREK) subfamily of K2P channels: TREK1, TREK2, and TWIK-related arachdonic acid stimulated K(+) channel (TRAAK). We investigated the regulation of TREK channels by phosphatidic acid (PA), which is generated by phospholipase D (PLD) via hydrolysis of phosphatidylcholine. Even though all three of the channels are sensitive to PA, we found that only TREK1 and TREK2 are potentiated by PLD2 and that none of these channels is modulated by PLD1, indicating surprising selectivity. We found that PLD2, but not PLD1, directly binds to the C terminus of TREK1 and TREK2, but not to TRAAK. The results have led to a model for selective lipid regulation by localization of phospholipid enzymes to specific effector proteins. Finally, we show that regulation of TREK channels by PLD2 occurs natively in hippocampal neurons.
          Cover page: Phospholipase D2 specifically regulates TREK potassium channels via direct interaction and local production of phosphatidic acid
          • Article
          • Peer Reviewed
          UC Berkeley Previously Published Works (2015)
          Mammalian neurotransmitter-gated receptors can be conjugated to photoswitchable tethered ligands (PTLs) to enable photoactivation, or photoantagonism, while preserving normal function at neuronal synapses. "MAG" PTLs for ionotropic and metabotropic glutamate receptors (GluRs) are based on an azobenzene photoswitch that is optimally switched into the liganding state by blue or near-UV light, wavelengths that penetrate poorly into the brain. To facilitate deep-tissue photoactivation with near-infrared light, we measured the efficacy of two-photon (2P) excitation for two MAG molecules using nonlinear spectroscopy. Based on quantitative characterization, we find a recently designed second generation PTL, L-MAG0460, to have a favorable 2P absorbance peak at 850 nm, enabling efficient 2P activation of the GluK2 kainate receptor, LiGluR. We also achieve 2P photoactivation of a metabotropic receptor, LimGluR3, with a new mGluR-specific PTL, D-MAG0460. 2P photoswitching is efficiently achieved using digital holography to shape illumination over single somata of cultured neurons. Simultaneous Ca(2+)-imaging reports on 2P photoswitching in multiple cells with high temporal resolution. The combination of electrophysiology or Ca(2+) imaging with 2P activation by optical wavefront shaping should make second generation PTL-controlled receptors suitable for studies of intact neural circuits.
            Cover page: Two-photon brightness of azobenzene photoswitches designed for glutamate receptor optogenetics
            • Article
            • Peer Reviewed
            UC Berkeley Previously Published Works (2016)
            NMDA receptors, which regulate synaptic strength and are implicated in learning and memory, consist of several subtypes with distinct subunit compositions and functional properties. To enable spatiotemporally defined, rapid and reproducible manipulation of function of specific subtypes, we engineered a set of photoswitchable GluN subunits ('LiGluNs'). Photo-agonism of GluN2A or GluN2B elicits an excitatory drive to hippocampal neurons that can be shaped in time to mimic synaptic activation. Photo-agonism of GluN2A at single dendritic spines evokes spine-specific calcium elevation and expansion, the morphological correlate of LTP. Photo-antagonism of GluN2A alone, or in combination with photo-antagonism of GluN1a, reversibly blocks excitatory synaptic currents, prevents the induction of long-term potentiation and prevents spine expansion. In addition, photo-antagonism in vivo disrupts synaptic pruning of developing retino-tectal projections in larval zebrafish. By providing precise and rapidly reversible optical control of NMDA receptor subtypes, LiGluNs should help unravel the contribution of specific NMDA receptors to synaptic transmission, integration and plasticity.
              Cover page: A family of photoswitchable NMDA receptors
              • Article
              • Peer Reviewed
              UC Berkeley Previously Published Works (2014)
              Most inherited forms of blindness are caused by mutations that lead to photoreceptor cell death but spare second- and third-order retinal neurons. Expression of the light-gated excitatory mammalian ion channel light-gated ionotropic glutamate receptor (LiGluR) in retinal ganglion cells (RGCs) of the retina degeneration (rd1) mouse model of blindness was previously shown to restore some visual functions when stimulated by UV light. Here, we report restored retinal function in visible light in rodent and canine models of blindness through the use of a second-generation photoswitch for LiGluR, maleimide-azobenzene-glutamate 0 with peak efficiency at 460 nm (MAG0(460)). In the blind rd1 mouse, multielectrode array recordings of retinal explants revealed robust and uniform light-evoked firing when LiGluR-MAG0(460) was targeted to RGCs and robust but diverse activity patterns in RGCs when LiGluR-MAG0(460) was targeted to ON-bipolar cells (ON-BCs). LiGluR-MAG0(460) in either RGCs or ON-BCs of the rd1 mouse reinstated innate light-avoidance behavior and enabled mice to distinguish between different temporal patterns of light in an associative learning task. In the rod-cone dystrophy dog model of blindness, LiGluR-MAG0(460) in RGCs restored robust light responses to retinal explants and intravitreal delivery of LiGluR and MAG0(460) was well tolerated in vivo. The results in both large and small animal models of photoreceptor degeneration provide a path to clinical translation.
                Cover page: Restoration of visual function by expression of a light-gated mammalian ion channel in retinal ganglion cells or ON-bipolar cells
                • Article
                • Peer Reviewed
                UC Berkeley Previously Published Works (2015)
                Chronic itch is a prevalent and debilitating condition for which few effective therapies are available. We harnessed the natural variation across genetically distinct mouse strains to identify transcripts co-regulated with itch behavior. This survey led to the discovery of the serotonin receptor HTR7 as a key mediator of serotonergic itch. Activation of HTR7 promoted opening of the ion channel TRPA1, which in turn triggered itch behaviors. In addition, acute itch triggered by serotonin or a selective serotonin reuptake inhibitor required both HTR7 and TRPA1. Aberrant serotonin signaling has long been linked to a variety of human chronic itch conditions, including atopic dermatitis. In a mouse model of atopic dermatitis, mice lacking HTR7 or TRPA1 displayed reduced scratching and skin lesion severity. These data highlight a role for HTR7 in acute and chronic itch and suggest that HTR7 antagonists may be useful for treating a variety of pathological itch conditions.
                  Cover page: HTR7 Mediates Serotonergic Acute and Chronic Itch
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