# US 12,181,479 B2 · BODIPY-based dyes for digital spatial proteomics

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- Number: US 12,181,479 B2
- Document ID: US12181479B2
- Status: Granted
- Kind: B2
- Application number: 18/086,923
- Filed: 2022-12-22
- Published: 2023-06-22
- Granted: 2024-12-31
- Earliest claimed: 2021-12-22
- Assignees: Trustees of Boston University
- Applicants: Trustees of Boston University
- Inventors: Aaron Beaty Beeler; Andrew Emili; Jason Michael Lenihan; Nathaniel Ryan Hendrick; Ichun Anderson Chen
- Web page: https://patentgrep.com/patent/US12181479B2

## CPC

- G01N 33/6818: {Sequencing of polypeptides}
- C07F 5/022: {without C-boron linkages}
- G01N 33/582: {with fluorescent label}
- 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)
- C07F: ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM (metal-containing porphyrins C07D487/22)

## Abstract

The disclosure provides probes comprising dipyrromethane-BF2 derivatives which exhibits different fluorescent spectral properties when conjugated to the amino acids, compositions and kits comprising same. The disclosure also provides methods for detecting/identifying amino acids and sequencing polypeptide molecules by conjugating a dipyrromethane-BF2 derivative which exhibits different fluorescent spectral properties when conjugated to the amino acids.

## Claims

### Claim 1

A method of identifying amino acids in a plurality of amino acids, the method comprising:
(a) conjugating a probe to amino acids in a plurality of amino acids, wherein the amino acids in the plurality of amino acids are different, wherein the probe comprises a dipyrromethane-BF2 derivative, and wherein the dipyrromethane-BF2 derivative comprises a labile or reactive group and exhibits different fluorescent spectral properties when conjugated to the amino acids in the plurality of the amino acids, wherein each amino acid in the plurality of amino acids is at the N- or C-terminal of a polypeptide;
(b) detecting one or more fluorescent spectral properties of the probe conjugated to the different amino acids; and
(c) identifying the amino acids in the plurality of the amino acids by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different amino acid.

### Claim 2 (depends on 1)

The method of claim 1, wherein the probe is covalently conjugated to the amino acids.

### Claim 3 (depends on 1)

The method of claim 1, wherein the probe is conjugate to an amino, a carboxylic, a hydroxyl or a thiol group of the amino acids.

### Claim 4 (depends on 1)

The method of claim 1, wherein the plurality of the amino acids is in a sample, optionally, the sample is a biological sample, optionally the biological sample is a biological fluid, a tissue, an organ, or a cell.

### Claim 5 (depends on 1)

The method of claim 1, wherein said detecting one or more fluorescent spectral properties of the conjugated probe comprises super resolution microscopy, optionally, the super resolution microscopy comprises stochastic optical reconstruction microscopy (STORM).

### Claim 6 (depends on 1)

The method of claim 1, wherein detecting one or more fluorescent spectral properties comprises detecting fluorescence, fluorescence emission intensity, fluorescence polarity/anisotropy, or fluorescence lifetime of the conjugated probe.

### Claim 7 (depends on 6)

The method of claim 6, wherein said detecting the fluorescence emission intensity, fluorescence polarity/anisotropy or fluorescence lifetime is at a single wavelength.

### Claim 8 (depends on 6)

The method of claim 6, comprising said detecting the fluorescence emission intensity, fluorescence polarity/anisotropy or fluorescence lifetime is at a plurality of wavelengths.

### Claim 9 (depends on 1)

The method of claim 1, wherein the each amino acid in the plurality of amino acids is at the N-terminal of the polypeptide.

### Claim 10 (depends on 1)

The method of claim 1, wherein the dipyrromethane-BF2 derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

### Claim 11 (depends on 10)

The method of claim 10, wherein the probe is conjugated via position 8 (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

### Claim 12 (depends on 11)

The method of claim 11, wherein the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative comprises a labile or reactive group at position 8 (meso position).

### Claim 13 (depends on 1)

The method of claim 1, wherein the probe is of Formula (I):
wherein:
R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, provided that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is a labile or leaving group, and
any alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio, can be optionally substituted with one or more (e.g., 1, 2, 3, 4, 5 or 6) independently selected substituents from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.

### Claim 14 (depends on 13)

The method of claim 13, wherein R8 is a labile or leaving group.

### Claim 15 (depends on 14)

The method of claim 14, wherein R8 is optionally substituted alkylthio, halogen, optionally substituted alkoxyl, hydroxyl, optionally substituted acyloxy, tosylate, triflate, mesylate, nitrile, azide, carbamate, disulfide, thioester, and diazonium.

### Claim 16 (depends on 15)

The method of claim 15, wherein R8 is —SR8S or halogen, wherein R8S is optionally substituted alkyl, optionally substituted alkenyl, optionally cycloalkyl, optionally substituted heterocyclyl, optionally substituted aryl or optionally substituted heteroaryl.

### Claim 17 (depends on 16)

The method of claim 16, wherein R8 is —SR8S.

### Claim 18 (depends on 14)

The method of claim 14, wherein at least one of R1, R2, R3, R5, R6 and R7 is an electron withdrawing group (EWG).

### Claim 19 (depends on 14)

The method of claim 14, wherein at least one of R1, R2, R3, R5, R6 and R7 is an electron donating group (EDG).

### Claim 20 (depends on 14)

The method of claim 14, wherein:
a. at least two of R1, R2 and R3 are same;
b. at least two of R1, R2 and R3 are different;
c. R1 and R7 are different, or R2 and R6 are different, or R3 and R5 are different; or
d. R1 and R7 are same, R2 and R6 are same, and R3 and R5 are same.

### Claim 21 (depends on 14)

The method of claim 14, wherein:
a. at least one of R1, R2, R3, R5, R6 and R7 is not H,
b. R1 is H or optionally substituted C1-C6alkyl;
c. R3 is H, halogen, optionally substituted C1-C6alkyl, optionally substituted C1-C6alkenyl, optionally substituted phenyl or optionally substituted naphthalene;
d. R5 is H, halogen, optionally substituted C1-C6, optionally substituted C1-C6alkenyl, optionally substituted phenyl or optionally substituted naphthalene;
e. R6 is H, halogen, optionally substituted C1-C6 alkyl, —C(O)H, or —SO3H; or
f. R7 is H or optionally substituted C1-C6alkyl.

### Claim 22 (depends on 14)

The method of claim 14, wherein R1, R2, R3, R5, R6 and R7 are H.

### Claim 23

A method of sequencing a polypeptide, the method comprises:
(a) conjugating a probe to a terminal amino acid of a polypeptide, wherein the probe comprises a dipyrromethane-BF2 derivative, and wherein the dipyrromethane-BF2 derivative comprises a labile or reactive group and exhibits different fluorescent spectral properties when conjugated to different amino acids;
(b) detecting one or more fluorescent spectral properties of the probe conjugated to the amino acid; and
(c) identifying the amino acid by comparing the fluorescent spectral properties of the conjugated probe to a plurality of reference fluorescent spectral properties, wherein each reference fluorescent spectral property is representative of the probe conjugated to a different amino acid;
(d) cleaving the conjugated amino acid from the polypeptide; and
(e) sequentially repeating steps (a) to (d) one or more times to determine the sequence of at least a portion of the polypeptide.

### Claim 24 (depends on 23)

The method of claim 23, wherein the method comprises:
in step (a) conjugating a probe to a terminal amino acid of a plurality of polypeptides, wherein the probe comprises a dipyrromethane-BF2 derivative, and wherein the dipyrromethane-BF2 derivative comprises a labile or reactive group and exhibits different fluorescent spectral properties when conjugated to different terminal amino acids, and the method further comprises, after step (c), steps:
(d) cleaving the conjugated amino acid of each of the plurality of polypeptides; and
(e) sequentially repeating steps (a) to (d) one or more times to determine the sequence of at least a portion of each of the plurality of polypeptides.

### Claim 25 (depends on 24)

The method of claim 24, wherein the method comprises detecting one or more spectral properties for the probe conjugated to the terminal amino acid of each of the plurality of polypeptides at spatially resolved locations in a sample comprising the plurality of polypeptides, where the probe conjugated to the terminal amino acid of each of the plurality of polypeptides are from step (a) of claim 24.

### Claim 26 (depends on 23)

The method of claim 23, wherein the dipyrromethane-BF2 derivative comprises a 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

### Claim 27 (depends on 26)

The method of claim 26, wherein the probe is conjugated via position 8 (meso position) of the 4,4-difluoro-4-bora-3a,4a-diaza-s-indacene derivative.

### Claim 28 (depends on 23)

The method of claim 23, wherein the probe is of Formula (I):
wherein:
R1, R2, R3, R4, R5, R6, R7 and R8 are each independently selected from the group consisting of hydrogen, halogen, alkyl, alkenyl, alkynyl, optionally substituted alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, amino, alkylamino, dialkylamino, arylamino, heteroarylamino, hydroxyl, acyl, acyloxy, carbonyl, carboxyl, ester, alkoxyl, cyano, nitro, thiol, alkylthio, sulfinyl, sulfonyl, carbamoyl, isocyanato, thiocyanato, isothiocyanato, ureido, and a labile or leaving group, provided that at least one of R1, R2, R3, R4, R5, R6, R7 and R8 is a labile or leaving group, and
any alkyl, alkenyl, alkynyl, alkoxyl, aryl, heteroaryl, cycloalkyl, heterocyclyl, alkylamino, dialkylamino, arylamino, heteroarylamino, acyl, acyloxy, ester, alkoxyl, and alkylthio, can be optionally substituted with one or more independently selected substituents from the group consisting of halogen, hydroxy, carboxy, oxo, nitro, haloalkyl, alkyl, alkenyl, alkynyl, alkaryl, aryl, heteroaryl, cyclyl, heterocyclyl, aralkyl, alkoxy, aryloxy, amino, acylamino, alkylcarbanoyl, arylcarbanoyl, aminoalkyl, alkoxycarbonyl, carbonyl, carboxyl, hydroxyalkyl, alkanesulfonyl, arenesulfonyl, alkanesulfonamido, arenesulfonamido, aralkylsulfonamido, alkylcarbonyl, acyloxy, cyano or ureido.