# US 10,370,351 B2 · Photoinduced electron transfer voltage-sensitive compounds

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- Number: US 10,370,351 B2
- Document ID: US10370351B2
- Status: Granted
- Kind: B2
- Application number: 15/496,897
- Filed: 2017-04-25
- Published: 2017-11-09
- Granted: 2019-08-06
- Earliest claimed: 2016-04-25
- Assignees: The Regents of the University of California
- Applicants: The Regents of the University of California
- Inventors: Evan W. Miller; Rishikesh Upendra Kulkarni
- Web page: https://patentgrep.com/patent/US10370351B2

## CPC

- C07D 311/82: Xanthenes
- C07D 219/04: with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the ring system
- G01N 21/6428: {Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" (in vivo A61B5/00; immunoassay G01N33/53)}
- G01N 33/5008: {for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics}
- G01N 33/502: {for testing non-proliferative effects}
- G01N 33/542: with steric inhibition or signal modification, e.g. fluorescent quenching
- G01N 2500/10: involving cells
- C07D: HETEROCYCLIC COMPOUNDS (macromolecular compounds C08)
- G01N: INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES (measuring or testing apparatus or processes other than immunoassay, involving enzymes or microorganisms C12M, C12Q)

## Abstract

The disclosure provides methods to synthesize photoinduced electron transfer voltage-sensitive compounds, the compounds made therefrom, and uses of the compounds thereof.

## Claims

### Claim 1

A compound comprising the structure of Formula II:
wherein,
L1 is selected from the group consisting of:
and any combination of the foregoing;
A1, A4-A10 are each independently selected from CH2, CHR′, CR′2, NH, O, S, Se, Te, SiH2, SiHR′, SiR′2, GeH2, GeHR′, GeR′2, SnH2, SnHR′, SnR′2, PbH2, PbHR′, or PbHR′2, wherein R′ is selected from the group consisting of D, optionally substituted FG, optionally substituted (C1-C12)alkyl, optionally substituted (C1-C11)heteroalkyl, optionally substituted (C1-C12)alkenyl, optionally substituted (C1-C11)heteroalkenyl, optionally substituted (C1-C12)alkynyl, and optionally substituted (C1-C11)heteroalkynyl;
A2 is selected from NH2, S or O;
A3 is selected from NH2, OH, SH and methoxy;
X1-X39 are independently selected from N or C, wherein when an X group is an N, then the R group is absent; and
R1-R3, R5-R6, R8-R10, R12, and R14-R47 are independently selected from H, D, optionally substituted FG, optionally substituted (C1-C12)alkyl, optionally substituted (C2-C10)heteroalkyl, optionally substituted (C1-C12)alkenyl, optionally substituted (C2-C10)heteroalkenyl, optionally substituted (C1-C12)alkynyl, optionally substituted (C2-C10)heteroalkynyl, optionally substituted (C3-C12)cycloalkyl, optionally substituted (C5-C12)cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising (C3-C12)cycloalkyl, (C3-C12)cycloalkenyl, heterocycle, aryl, and mixed ring system;
wherein R4 and R7 are F or Cl;
R11 is selected from NH2, NH(R50), N(R50)2 wherein R50 is a (C1-C3)alkyl;
wherein R13 is an alkoxy; and
n1-n8 are independently an integer selected from 0 to 10.

### Claim 2 (depends on 1)

The compound of claim 1, wherein the compound comprises the structure of Formula III:
wherein,
A1 is selected from CH2, CHR′, CR′2, NH, O, S, Se, Te, SiH2, SiHR′, SiR′2, GeH2, GeHR′, GeR′2, SnH2, SnHR′, SnR′2, PbH2, PbHR′, or PbHR′2, wherein R′ is selected from the group consisting of D, optionally substituted FG, optionally substituted (C1-C12)alkyl, optionally substituted (C2-C10)heteroalkyl, optionally substituted (C1-C12)alkenyl, optionally substituted (C2-C10)heteroalkenyl, optionally substituted (C1-C12)alkynyl, and optionally substituted (C2-C10)heteroalkynyl,
A2 is NH2 or O;
A3 is selected from NH2, OH and methoxy;
X1-X17 are independently selected from N or C, wherein when an X group is an N, then the R group is absent; and
R1-R3, R5-R6, R8-R10, R12, and R14-R21 are independently selected from H, D, optionally substituted FG, optionally substituted (C1-C12)alkyl, optionally substituted (C2-C10)heteroalkyl, optionally substituted (C1-C12)alkenyl, optionally substituted (C2-C10)heteroalkenyl, optionally substituted (C1-C12)alkynyl, optionally substituted (C2-C10)heteroalkynyl, optionally substituted (C3-C12)cycloalkyl, optionally substituted (C5-C12)cycloalkenyl, optionally substituted aryl, optionally substituted heterocycle, optionally substituted mixed ring system, wherein one or more adjacent R groups can be linked together to form one or more substituted rings selected from the group comprising (C3-C12)cycloalkyl, (C3-C12)cycloalkenyl, heterocycle, aryl, and mixed ring system;
wherein R4 and R7 are F or Cl;
R11 is selected from NH2, NH(R50), N(R50)2 wherein R50 is a (C1-C3)alkyl;
wherein R13 is an alkoxy; and
n1 is an integer selected from 0 to 10.

### Claim 3 (depends on 2)

The compound of claim 2, wherein the compound comprises the structure of Formula III(a):
wherein,
A1 is selected from CH2, CHR′, CR′2, NH, O, S, Se, Te, SiH2, SiHR′, SiR′2, GeH2, GeHR′, GeR′2, SnH2, SnHR′, SnR′2, PbH2, PbHR′, or PbHR′2, wherein R′ is selected from the group consisting of D, optionally substituted FG, optionally substituted (C1-C6)alkyl, optionally substituted (C2-C5)heteroalkyl, optionally substituted (C1-C6)alkenyl, optionally substituted (C2-C5)heteroalkenyl, optionally substituted (C1-C6)alkynyl, and optionally substituted (C2-C5)heteroalkynyl,
A2 is NH2 or O;
A3 is NH2 or OH;
R4 and R7 are independently F, and/or Cl;
R11 is selected from NH2, NH(R50), N(R50)2, wherein R50 is a (C1-C3)alkyl;
R13 is an alkoxy; and
n1 is an integer selected from 0 to 5.

### Claim 4 (depends on 3)

The compound of claim 3, wherein the compound comprises the structure of Formula III(b):
wherein,
R4 and R7 are independently F, and/or Cl;
R11 is selected from NH2, NH(R50), N(R50)2, wherein R50 is a (C1-C3)alkyl; and
R13 is a methoxy.

### Claim 5 (depends on 1)

The compound of claim 1, wherein the compound has one or more of the following characteristics:
the compound is water soluble;
the compound exhibits a tilt angle from 0° to 20°;
the compound exhibits a voltage sensitivity of 63% ΔF/F per 100 mV or greater; and
the compound can undergo photoinduced electron transfer.

### Claim 6 (depends on 5)

The compound of claim 5, wherein the compound is characterized by being water soluble; exhibits a tilt angle of about 0°; exhibits a voltage sensitivity of 63% ΔF/F per 100 mV or greater; and can undergo photoinduced electron transfer.

### Claim 7 (depends on 1)

A method to image cells, comprising:
contacting the cell with a compound of claim 1;
illuminating the cells with light having a first wavelength;
imaging the cells by detecting light having a second wavelength,
wherein the first wavelength and second wavelength of light have different wavelengths, and wherein the light having the second wavelength is in the far red to near infrared region.

### Claim 8 (depends on 7)

The method of claim 7, wherein the method further comprises:
contacting the cells with one or more additional optogenetic tools; and
imaging the cells be detecting light emissions at one or more additional wavelengths.

### Claim 9 (depends on 8)

The method of claim 8, wherein the one or more optogenetic tools are selected from GFP, Ca2+ indicators, voltage sensors based on cpGFP, and ChannelRhodopsin2 (ChR2).

### Claim 10 (depends on 1)

A method to measure changes in membrane potential in an excitable cell comprising:
contacting the excitable cell with a compound of claim 1;
stimulating the cell to evoke action potentials; and
measuring action potential firing by optical or electrical sampling.

### Claim 11 (depends on 10)

The method of claim 10, wherein the optical sampling is measured using an electron multiplying charge couple device.

### Claim 12 (depends on 11)

The method of claim 11, wherein the excitable cell is stimulated using a whole-cell current clamp or by field stimulation.

### Claim 13 (depends on 10)

The method of claim 10, wherein the excitable cell is a neuron, cardiomyocyte, myocyte, or a secretory cell.

### Claim 14 (depends on 13)

The method of claim 13, wherein the method interrogates membrane potentials of a neuron.

### Claim 15 (depends on 1)

A kit comprising:
a plurality of compartments which comprise a compound of claim 1 in a buffered solution, or a concentrated solution.