Preparation, Characterization, and Application of Optical Switch Probes

Chutima Petchprayoon1, Gerard Marriott1

1 Department of Bioengineering, University of California, Berkeley, California
Publication Name:  Current Protocols in Chemical Biology
Unit Number:   
DOI:  10.1002/9780470559277.ch100054
Online Posting Date:  August, 2010
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Abstract

Optical switches represent a new class of molecular probe with applications in high‐contrast imaging and optical manipulation of protein interactions. Small molecule, organic optical switches based on nitrospirobenzopyran (NitroBIPS) and their reactive derivatives and conjugates undergo efficient, rapid and reversible, orthogonal optically‐driven transitions between a colorless spiro (SP)‐state and a colored merocyanine (MC)‐state. The excited MC‐state also emits fluorescence, which serves as the readout of the state of the switch. Defined optical perturbations of SP and MC generate a defined waveform of MC‐fluorescence that can be isolated against unmodulated background signals by using a digital optical lock‐in detection approach or to control specific dipolar interactions on proteins. The protocols describe general procedures for the synthesis and spectroscopic characterization of NitroBIPS and specifically labeled conjugates along with methods for the manipulation of dipolar interactions on proteins and imaging of the MC‐state of NitroBIPS within living cells. Curr. Protoc. Chem. Biol. 2:153‐169 © 2010 by John Wiley & Sons, Inc.

Keywords: optical switch; nitrospiropyran; imaging

     
 
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Table of Contents

  • Introduction
  • Strategic Planning
  • Basic Protocol 1: Synthesis of 1′,3′,3′‐Trimethyl‐5′‐(N‐Succinimidyloxycarbonylpropyl)‐6‐Nitro‐Spiro(2H‐Benzopyran‐2,2′,3H‐Indole) (5′‐NHS Ester of NitroBIPS)
  • Basic Protocol 2: Synthesis of 1′,3′,3′‐Trimethyl‐6‐Nitro‐8‐Iodomethylspiro(2H‐Benzopyran‐2,2′,3H‐Indole) (8‐Iodo‐NitroBIPS, 7)
  • Basic Protocol 3: Absorption Characterization of NitroBIPS
  • Basic Protocol 4: Fluorescence Characterization of NitroBIPS
  • Basic Protocol 5: Microscopic Characterization of NitroBIPS
  • Basic Protocol 6: Actin‐Labeled 8‐Iodo‐NitroBIPS
  • Basic Protocol 7: Live‐Cell Imaging of NitroBIPS
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Synthesis of 1′,3′,3′‐Trimethyl‐5′‐(N‐Succinimidyloxycarbonylpropyl)‐6‐Nitro‐Spiro(2H‐Benzopyran‐2,2′,3H‐Indole) (5′‐NHS Ester of NitroBIPS)

  Materials
  • 4‐(4‐Aminophenyl)butanoic acid
  • Concentrated hydrochloric acid (HCl)
  • Sodium nitrite (NaNO 2)
  • Stannous chloride dihydrate (SnCl 2⋅2H 2O)
  • Anhydrous ethanol
  • 3‐Methyl‐2‐butanone
  • Sulfuric acid (H 2SO 4)
  • Saturated aqueous sodium carbonate
  • Diethyl ether
  • Anhydrous magnesium sulfate
  • Methanol
  • 1 M aqueous sodium hydroxide
  • 1 M aqueous hydrochloric acid (HCl)
  • Ethyl acetate
  • Dichloromethane (DCM)
  • Methyl iodide
  • 5‐Nitrosalicylaldehyde
  • Triethylamine
  • Silica gel 70 to 230 mesh
  • Hexane
  • N,N′‐Dicyclohexylcarbodiimide (DCC)
  • N‐Hydroxysuccinimide (NHS)
  • Anhydrous tetrahydrofuran (THF)
  • Nitrogen gas
  • 10‐ and 25‐ml round‐bottomed flasks
  • Magnetic stir bar
  • Stirring hotplate
  • 1.5‐ml microcentrifuge tubes
  • 4‐ml glass vials
  • Filter paper
  • Büchner funnel
  • Vacuum pump
  • Suction flask
  • Oil bath
  • Condenser
  • Rotary evaporator
  • 0.5‐in. diameter chromatography column with frit disc
  • Silica gel thin layer chromatography (250 µm, 60 Å with fluorescent indicator UV 254)
  • 254/365‐nm hand‐held UV lamp

Basic Protocol 2: Synthesis of 1′,3′,3′‐Trimethyl‐6‐Nitro‐8‐Iodomethylspiro(2H‐Benzopyran‐2,2′,3H‐Indole) (8‐Iodo‐NitroBIPS, 7)

  Materials
  • 1,3,3‐Trimethyl‐2‐methyleneindoline
  • 3‐Chloromethyl‐5‐nitrosalicylaldehyde
  • Anhydrous tetrahydrofuran (THF)
  • Acetone
  • Potassium iodide (KI)
  • Nitrogen gas
  • Dichloromethane (DCM)
  • Silica gel 70 to 230 mesh
  • Hexane
  • Ethyl acetate
  • 25‐ml round‐bottomed flasks
  • Magnetic bar
  • Stirring hotplate
  • Fume hood
  • Oil bath (70°C)
  • Rotary evaporator
  • Condenser
  • Filter paper
  • Büchner funnel
  • Suction flask
  • Vacuum pump
  • 0.5‐in. diameter chromatography column with frit disc
  • Silica gel thin‐layer chromatography (250 µm, 60 Å with fluorescent indicator
  • UV 254)
  • 254/365‐nm hand‐held UV lamp

Basic Protocol 3: Absorption Characterization of NitroBIPS

  Materials
  • 5 mM 1′,3′‐Dihydro‐1′,3′,3′‐trimethyl‐6‐nitrospiro(2H‐1‐benzopyran‐2,2′‐(2H)‐indole) (NitroBIPS) stock solution in methanol (Sigma‐Aldrich, see recipe)
  • Methanol
  • 1.5‐ml microcentrifuge tubes
  • 2‐ or 4‐window quartz cuvettes (Hellma)
  • Spectrophotometer (Shimadzu PC1601)
  • Hand‐held UV lamp (365 nm)
  • 530‐nm output of an LED or 546‐nm output of a 100‐Watt mercury arc lamp

Basic Protocol 4: Fluorescence Characterization of NitroBIPS

  Materials
  • 5 mM 1′,3′‐Dihydro‐1′,3′,3′‐trimethyl‐6‐nitrospiro(2H‐1‐benzopyran‐2,2′‐(2H)‐indole) (NitroBIPS) stock solution in methanol (Sigma‐Aldrich, see recipe)
  • Methanol
  • 4‐Window quartz cuvettes (Hellma)
  • Fluorescence spectrophotometer (SLM‐AB2)
  • Hand‐held UV lamp (365 nm)
  • 530‐nm output of an LED or 546‐nm output of a 100‐Watt mercury arc lamp

Basic Protocol 5: Microscopic Characterization of NitroBIPS

  Materials
  • 3‐µm Polybead amino microspheres (Polysciences)
  • 1× phosphate buffer saline (PBS; see recipe)
  • 5 mM 5′‐NHS ester of NitroBIPS stock solution in DMF (see recipe)
  • Immersion oil
  • 1.5‐ml microcentrifuge tube
  • Glass‐bottom petri dish: 35‐mm diameter incorporating a 14‐mm glass coverslip, no. 0 thickness (Mat Tek)
  • 100‐Watt mercury arc lamps
  • EMCCD camera (Hamamatsu)
  • Inverted fluorescence microscope (Zeiss) adapted for pulse probe microscopy (see Fig. )
  • Computer‐controlled mechanical shutter (Melles Griot)
  • Interference filter for excitation of Cy3 emission
  • UV‐pass filter (UG11, Schott glass)

Basic Protocol 6: Actin‐Labeled 8‐Iodo‐NitroBIPS

  Materials
  • Purified lyophilized actin (Cytoskeleton)
  • G‐buffer (see recipe)
  • G‐buffer with 1 mM DTT (see recipe)
  • 5 mM 8‐iodo‐NitroBIPS stock solution in DMF (see recipe)
  • 1.5‐ml microcentrifuge tubes
  • Vortexer
  • PD‐10 column (GE Healthcare)
  • Spectrophotometer

Basic Protocol 7: Live‐Cell Imaging of NitroBIPS

  Materials
  • HEK‐293 cells
  • Cell culture medium (see recipe)
  • 5 mM 8‐iodo‐NitroBIPS stock solution in DMF (see recipe)
  • Glass‐bottom petri dish: 35‐mm diameter incorporating a 14‐mm glass coverslip, no. 0 thickness (Mat Tek Corporation)
  • 2‐ml microcentrifuge tubes
  • 37°C, 5% CO 2 humidified incubator
  • Tissue culture hood
  • Inverted fluorescence microscope (Zeiss) adapted for pulse probe microscopy (see Fig. )
  • 100‐Watt mercury arc lamps or laser power source
  • Computer‐controlled mechanical shutter (Melles Griot)
  • Interference filter for excitation of Cy3 emission
  • UV‐pass filter (UG11, Schott glass)
  • EMCCD camera (Hamamatsu)
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Figures

  •   FigureFigure 1. Photochromic properties of benzospiropyran. The merocyanine (MC) state was generated by irradiation the spiropyran (SP) state with UV light (365 nm). While the excitation of the MC‐state with visible light (546 nm) generated the SP‐state.
  •   FigureFigure 2. Synthesis of 1′,3′,3′‐trimethyl‐5′‐( N‐succinimidyloxycarbonylpropyl)‐6‐nitro‐spiro(2 H‐benzopyran‐2,2′,3 H‐indole).
  •   FigureFigure 3. Synthesis of 1′,3′,3′‐trimethyl‐6‐nitro‐8‐iodomethylspiro(2 H‐benzopyran‐2,2′,3 H‐indole).
  •   FigureFigure 4. UV absorption spectra of NitroBIPS in the SP‐ and MC‐states in methanol during a single cycle of optical switching.
  •   FigureFigure 5. Fluorescence spectra of NitroBIPS in methanol in the SP‐ and MC‐states.
  •   FigureFigure 6. Schematic represent the inverted fluorescence microscope (Zeiss) adapted for pulse‐probe microscopy.
  •   FigureFigure 7. (A) Fluorescence intensity image of NitroBIPS‐labeled beads. (B) The MC‐fluorescence change corresponding to optical switch cycles of NitroBIPS in individual bead (red circle in A) over eight cycles of optical switching.
  •   FigureFigure 8. UV absorption spectra of NitroBIPS‐G‐actin conjugate in the SP‐ and MC‐state.
  •   FigureFigure 9. (A) Fluorescence intensity image of NitroBIPS in live HEK‐293 cell. (B) The MC‐fluorescence change of NitroBIPS in a region of interest (red circle in A) in live HEK‐293 cell during optical switch cycles.

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Literature Cited

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