Turn-on near infrared fluorescent probes for imaging lysosomal ROS in live cells at subcellular resolution

Patent metadata

Field	Value
Publication number	US-11422091-B2
Application number	US-202016891876-A
Country	US
Kind code	B2
Filing date	Jun 3, 2020
Priority date	Oct 9, 2015
Publication date	Aug 23, 2022
Grant date	Aug 23, 2022

Abstract

This invention is in the field of fluorescence imaging and relates to a new near infrared (NIR) reactive oxygen species (ROS) sensor designed with controlled fluorescence on-off switching mechanism.

First claim

I claim:

1. A compound with the structure:

wherein A and Z are each independently selected from the group consisting of C, Si, N, P, O, S, and Se; n=0, 1, 2, 3, or 4; R 1 to R 7 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, or a sulfur heterocycle; R 8 to R 10 are each independently selected from the group consisting of H or D, in either S or R, or cis- or trans-conformation/configuration; and R 11 to R 18 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amine, an imine, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, and sulfur heterocycle.

2. A method of monitoring reactive oxygen species, comprising:
a) providing
i) a fluid comprising at least one reactive oxygen species;
ii) a fluorescent sensor compound comprising the formula of:

wherein A and Z are each independently selected from the group consisting of C, Si, N, P, O, S, and Se; n=0, 1, 2, 3, or 4; R 1 to R 7 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, or a sulfur heterocycle; R 8 to R 10 are each independently selected from the group consisting of H or D, in either S or R, or cis- or trans-conformation/configuration; and R 11 to R 18 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amine, an imine, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, and sulfur heterocycle;

b) introducing said fluorescent sensor compound into said fluid;
c) collecting fluorescent light data from said fluorescent sensor compound; and
d) producing a superior spatial resolution photo image of said biological system using said fluorescent light data.

3. The method of claim 2 , wherein said fluid comprises a biological fluid system comprising basal levels of a plurality of reactive oxygen species.

4. A method for profiling the status of biologically reactive oxygen species in living cells or subcellular organelles, or both, said method comprising the steps of:
a) providing:
i) at least one sample of said living cells comprising a plurality of cellular organelles; and
ii) a selective reactive species indicator probe for detecting a plurality of biologically reactive species selected from the group consisting of specific reactive oxygen species and specific reactive nitrogen species, wherein said reactive indicator probe comprising the formula of:

wherein A and Z are each independently selected from the group consisting of C, Si, N, P, O, S, and Se; n=0, 1, 2, 3, or 4; R 1 to R 7 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, or a sulfur heterocycle; R 8 to R 10 are each independently selected from the group consisting of H or D, in either S or R, or cis- or trans-conformation/configuration; and R 11 to R 18 are each independently selected from the group consisting of H, F, Cl, Br, I, alkyl, arylalkyl, alkoxy, aryloxy, alkylamino, an amine, an imine, an amino, an azido, a carboxy, cyano, hydroxyl, nitro, phosphate, solfo, sulfate, a nitrogen heterocycle, an oxygen heterocycle, and sulfur heterocycle;

b) contacting said sample with said selective indicator probe, wherein said probe generates fluorescent signals; and
c) measuring said fluorescent signals, thereby providing a profile status of said plurality of biologically reactive species.

5. The method of claim 4 , wherein said living cells are isolated from a biological material selected from a tissue, an organ or an organism.

6. The method of claim 4 , wherein said subcellular organelles are selected from the group consisting of mitochondria, peroxisomes, cytosol, vesicles, lysosomes, plasma membranes, chloroplasts, nuclei, nucleoli, inner mitochondrial matrices, inner mitochondrial membranes, intermembrane spaces, outer mitochondrial membranes, secretory vesicles, endoplasmic reticuli, golgi bodies, phagosomes, endosomes, exosomes, plasma membranes, microtubules, microfilaments, intermediate filaments, filopodia, ruffles, lamellipodia, sarcomeres, focal contacts, podosomes, ribosomes, microsomes, lipid rafts, nuclear membranes, chloroplasts or cell walls, and combinations thereof.

7. The method of claim 4 , wherein said probe reacts with reactive oxygen species (ROS) selected from the group consisting of superoxide (O 2 .—), hydrogen peroxide (H 2 O 2 ), peroxynitrite (ONOO—), hydroxyl radical (HO.), tert-butoxide (TBO), tert-butyl hydroperoxide (TBHP), and combinations thereof.

8. The method of claim 4 , wherein said probe localizes within at least one of said subcellular organelle.

9. The method of claim 4 , wherein said profile status comprises a basal level profile of a plurality of biological reactive species levels.

10. The method of claim 4 , wherein said plurality of biological reactive species levels are subcellular organelle biological reactive species levels.

Assignees

• Univ Massachusetts

Inventors

• Guo Maolin

Classifications

• C07D405/06 Primary

  linked by a carbon chain containing only aliphatic carbon atoms · CPC title

• A61K49/0019

  characterised by the fluorescent group, e.g. oligomeric, polymeric or dendritic molecules · CPC title

• G01N2021/6439

  with indicators, stains, dyes, tags, labels, marks · CPC title

• G01N31/228

  for peroxides · CPC title

• G01N21/6428 Primary

  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) · CPC title