Detection of transmembrane potentials by optical methods

A compound of a formula Cou-PE wherein:

• Cou is a coumarin of formula:
• 21
• wherein:
• each R3, which may be the same or different, is independently selected from the group consisting of H, halogen, lower alkyl, CN, CF3, COOR5, CON(R5)2, OR5, and an attachment point;
• R4 is selected from the group consisting of OR5 and N(R5)2;
• Z is O, S or NR5;
• and
• each R5, which may be the same or different, is independently selected from the group consisting of H, lower alkyl and an alkylene attachment point;
• and
• PE is an N-linked phosphatidylethanolamine.

A compound of a formula

• 22
• wherein:
• Ln=Tb, Eu, or Sm;
• R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;
• X and Y are independently H, F, Cl, Br, I, NO2, CF3, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh;
• or X and Y together are —CH═CH—;
• and
• Z=alkylenediyl, heteroalkylenediyl or heterocyclodiyl.

A compound of a formula

• 23
• wherein:
• Ln=Tb, Eu, or Sm;
• R is independently H, C1-C8 alkyl, C1-C8 cycloalkyl or C1-C4 perfluoroalkyl;
• X′ and Y′ are independently H, F, Cl, Br, I, NO2, CF3, lower (C1-C4) alkyl, CN, Ph, O-(lower alkyl), or OPh;
• or X′ and Y′ together are —CH═CH—;
• and
• Z′ is independently a valence bond, CR2, pyridine-2,6-diyl or tetrahydofuran-2,5-diyl.

A polymethine oxonol of the formula

• 24
• wherein:
• X is oxygen or sulfur;
• R is a C4-12 hydrocarbyl;
• n is an integer from 1 to 3;
• provided that when n is 1, R is a C7-12 hydrocarbyl;
• and salts thereof.

A method of synthesizing a fluorescent tetraryl borate of formula

• [(Ar1)3B—Ar2—Y—FLU]—
• wherein:
• Ar1 is an aryl group;
• Ar2 is an arylene group;
• B is boron;
• Y is oxygen or sulfur;
• and
• FLU is a neutral fluorophore;
• said method comprising:
• (a) treating a triaryl borane (Ar1)3B with an organometallic reagent of formula (P—Y—Ar2)—M, wherein P is a protecting group and M is a metal to form a protected tetraaryl borate (Ar1)3B—Ar2—Y—P;
• (b) removing the protecting group from the protected tetraaryl borate to form (Ar1)3B—Ar2—YH;
• and
• (c) treating (Ar1)3B—Ar2—YH with a fluorophore FLU bearing a leaving group to form a fluorescent tetraryl borate.

A compound of the formula

• 26
• wherein:
• each R′ is independently H, hydrocarbyl, halogen, CF3 or a linker group;
• n is an integer from 0 to 5;
• each X is independently H, halogen or CF3;
• m is an integer from 0 to 4;
• Y is oxygen or sulfur;
• and
• FLU is a neutral fluorophore selected from the group consisting of xanthenes, coumarins, cyanines, bimanes and difluoroboradiazaindacenes.

A method for detecting electrical potential across cellular membranes in at least one cell, comprising;

• a) providing said at least one cell with a first reagent, comprising:
• a mobile hydrophobic molecule,
• wherein said mobile hydrophobic molecule is a FRET acceptor or FRET donor,
• b) providing said at least one cell with a second reagent, comprising:
• a luminescent or fluorescent compound,
• wherein said luminescent or fluorescent compound is targetable to a cellular membrane, and
• wherein said second reagent undergoes energy transfer with said first reagent or quenches light emission of said first reagent, and
• c) detecting light emission from said first reagent or said second reagent.

A method of monitoring subcellular organelle membrane potentials in a living cell, comprising:

• 1) providing a living cell with a first reagent, comprising:
• a mobile hydrophobic molecule,
• wherein said mobile hydrophobic molecule is a FRET acceptor or FRET donor,
• 2) providing said living cell with a second reagent, comprising:
• a luminescent or fluorescent compound,
• wherein said luminescent or fluorescent compound is targetable to said subcellular organelle membrane, and
• wherein said second reagent undergoes energy transfer with said first reagent or quenches light emission of said first reagent,
• 3) detecting light emission from said first reagent or said second reagent.

A transgenic organism, comprising:

• a) a first reagent, comprising:
• a mobil hydrophobic molecule,
• wherein said mobile hydrophobic molecule is a FRET acceptor or FRET donor,
• b) a second reagent, comprising:
• a bio-luminescent or naturally fluorescent protein,
• wherein said bio-luminescent or naturally fluorescent protein is targetable to a cellular membrane, and
• wherein said second reagent undergoes energy transfer with said first reagent or quenches light emission of said first reagent.

A living cell, comprising:

• a) a first reagent, comprising:
• a mobile hydrophobic molecule,
• wherein said mobile hydrophobic molecule is a FRET acceptor or donor,
• b) a second reagent, comprising:
• a bio-luminescent or naturally fluorescent protein,
• wherein said bio-luminescent or naturally fluorescent protein is targetable to a cellular membrane, and
• wherein said second reagent undergoes energy transfer with said first reagent or quenches light emission of said first reagent, and
• wherein the light emission of said first reagent, or said second reagent is dependent on the voltage across said cellular membrane.

A method for screening test chemicals that modulate the activity of a target ion channel, comprising;

• 1) providing a living cell, comprising
• i) a target ion channel,
• ii) a membrane potential modulator, wherein said membrane potential modulator sets the resting membrane potential of a cellular membrane in said living cell to a stable predefined value between about −150 mV and +100 mV,
• 2) contacting said living cell with a test chemical,
• 3) detecting the membrane potential across said cellular membrane.

A method for screening test chemicals that modulate the activity of a target ion channel, comprising;

• 1) providing a living cell, comprising
• i) a target ion channel,
• ii) a membrane potential modulator, wherein said membrane potential modulator stably maintains an otherwise unstable membrane potential of a cellular membrane in said living cell generated by the activation of said target ion channel,
• 2) contacting said living cell with a test chemical,
• 3) detecting the membrane potential across said cellular membrane.