Super-resolution fluorescent imaging probe

Patent metadata

Field	Value
Publication number	US-10837909-B2
Application number	US-201615551149-A
Country	US
Kind code	B2
Filing date	Feb 23, 2016
Priority date	Feb 27, 2015
Publication date	Nov 17, 2020
Grant date	Nov 17, 2020

Abstract

[Problem] To provide a novel fluorescent probe for super-resolution imaging that uses fluorescent light emission characteristics that originate from an intermolecular nucleophilic addition-dissociation equilibrium reaction, and to provide a super-resolution fluorescent imaging method that uses the probe. [Solution] A fluorescent probe for super-resolution imaging that comprises a compound represented by formula (I) or a salt thereof [in the formula, X represents an oxygen atom, C(Ra)(Rb), or Si(Ra)(Rb) (wherein Ra and Rb each independently represent a hydrogen atom or an alkyl group), R1 represents a hydrogen atom or an optionally substituted aryl (provided that, if R1 is a phenyl group, the benzene ring of the phenyl group does not have a substituent at position 2 or position 6), R2 and R3 each independently represent 1-3 identical or differing substituents that are independently selected from the group that consists of hydrogen atoms, hydroxyl groups, halogen atoms, optionally substituted alkyl groups, optionally substituted sulfo groups, optionally substituted carboxyl groups, optionally substituted ester groups, optionally substituted amide groups, and optionally substituted azide groups, R4 and R5 each independently represent a hydrogen atom or an optionally substituted alkyl group or N(R4)(R5) forms an amide group or a carbamate group (provided that, if R4 or R5 is an alkyl group, each may form, together with R2, a ring structure that contains the nitrogen atom that is bonded thereto), and R6 and R7 each independently represent a hydrogen atom or an optionally substituted alkyl group or N(R6)(R7) forms an amide group or a carbamate group (provided that, if R6 or R7 is an alkyl group, each may form, together with R3, a ring structure that contains the nitrogen atom that is bonded thereto)], the fluorescent probe for super-resolution imaging being characterized in that the compound represented by formula (I) or the salt thereof undergoes a nucleophilic addition-dissociation equilibrium reaction with a nucleophilic compound that contains an —SH group.

First claim

The invention claimed is:

1. A fluorescent probe for super-resolution imaging comprising a compound represented by formula (I) or a salt thereof

(in the formula, X represents C(R a )(R b ), or Si(R a )(R b ) (wherein R a and R b each are a methyl group);
R 1 represents a hydrogen atom;
R 2 and R 3 each independently represent one to three identical or differing substituents that are independently selected from the group consisting of a hydrogen atom, a hydroxyl group, a halogen atom, an optionally substituted alkyl group, an optionally substituted sulfo group, an optionally substituted carboxyl group, an optionally substituted ester group, an optionally substituted amide group, and an optionally substituted azide group;
R 4 and R 5 each independently represent a hydrogen atom or an optionally substituted alkyl group, or N(R 4 )(R 5 ) forms an amide group or a carbamate group (here, when R 4 or R 5 is an alkyl group, each may form, together with R 2 , a ring structure that contains a nitrogen atom that is bonded thereto); and
R 6 and R 7 each independently represent a hydrogen atom or an optionally substituted alkyl group, or N(R 6 )(R 7 ) forms an amide group or a carbamate group (here, when R 6 or R 7 is an alkyl group, each may form, together with R 3 , a ring structure that contains the nitrogen atom that is bonded thereto),
the fluorescent probe for super-resolution imaging being characterized in that the compound represented by formula (I) or the salt thereof undergoes a nucleophilic addition-dissociation equilibrium reaction with a nucleophilic compound that contains a —SH group,
wherein a dissociation constant in the nucleophilic addition-dissociation equilibrium reaction is in a range of 0.1 μM to 100 μM.

2. The fluorescent probe for super-resolution imaging according to claim 1 , wherein the nucleophilic addition-dissociation equilibrium reaction occurs on a carbon atom to which R 1 bonds.

3. The fluorescent probe for super-resolution imaging according to claim 1 , characterized in that a nucleophilic addition reaction rate constant in the nucleophilic addition-dissociation equilibrium reaction is 1 to 1.0×10 6 s −1 in an aqueous solution having neutral conditions.

4. The fluorescent probe for super-resolution imaging according to claim 1 , wherein R 4 , R 5 , R 6 , and R 7 are each a hydrogen atom.

5. The fluorescent probe for super-resolution imaging according to claim 1 , wherein R 4 , R 5 , R 6 , and R 7 are each a methyl group.

6. The fluorescent probe for super-resolution imaging according to claim 1 , wherein at least one of R 4 , R 5 , R 6 , and R 7 has a labeling substituent capable of covalently or non-covalently bonding to a biomolecule.

7. The fluorescent probe for super-resolution imaging according to claim 1 , wherein the foregoing compound containing the —SH group has a cysteine residue.

8. The fluorescent probe for super-resolution imaging according to claim 1 , wherein the foregoing compound containing the —SH group is glutathione.

9. The fluorescent probe for super-resolution imaging according to claim 1 , wherein the compound represented by formula (I) is selected from the following group.

10. A super-resolution fluorescent imaging method using the fluorescent probe for super-resolution imaging according to claim 1 , which comprises bonding a probe molecule to a biomolecule, irradiating laser light in the presence of a compound containing a —SH group to acquire image data that have captured fluorescent light emission from the probe molecule, and analyzing and thereafter superimposing a plurality of the image data obtained by repeating the foregoing at a constant time interval to thereby obtain a super-resolution image of a structure of the biomolecule.

Assignees

• Univ Tokyo

Inventors

• Urano Yasuteru
• Kamiya Mako
• MOROZUMI Akihiko
• Uno Shinnosuke
• Umezawa Keitaro

Classifications

• C09K2211/1011

  Condensed systems · CPC title

• A61K49/0019

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

• G01N33/533

  with fluorescent label · CPC title

• G02B27/58

  Optics for apodization or superresolution; Optical synthetic aperture systems · CPC title

• G02B21/0076

  arrangements using fluorescence or luminescence · CPC title