US 12,221,649 B2
Fluorescent probe for detecting carboxypeptidase activity
Yasuteru Urano, Tokyo (JP); Mako Kamiya, Tokyo (JP); and Yugo Kuriki, Tokyo (JP)
Assigned to The University of Tokyo, Tokyo (JP)
Appl. No. 17/298,477
Filed by The University of Tokyo, Tokyo (JP)
PCT Filed Dec. 2, 2019, PCT No. PCT/JP2019/047000
§ 371(c)(1), (2) Date May 28, 2021,
PCT Pub. No. WO2020/111279, PCT Pub. Date Jun. 4, 2020.
Claims priority of application No. 2018-225735 (JP), filed on Nov. 30, 2018.
Prior Publication US 2022/0017940 A1, Jan. 20, 2022
Int. Cl. C12Q 1/37 (2006.01); C07F 9/655 (2006.01); C07F 9/6561 (2006.01); C09K 11/06 (2006.01); G01N 21/64 (2006.01); G01N 33/542 (2006.01)
CPC C12Q 1/37 (2013.01) [C09K 11/06 (2013.01); G01N 21/6428 (2013.01); G01N 33/542 (2013.01); C07F 9/655 (2013.01); C07F 9/6561 (2013.01); G01N 2021/6439 (2013.01); G01N 2333/948 (2013.01)] 5 Claims
OG exemplary drawing
 
1. A compound represented by the following general formula (I) or a salt thereof,

OG Complex Work Unit Chemistry
wherein R1 represents an alkyl group having 1 to 6 carbon atoms or an alkoxy group having 1 to 6 carbon atoms;
T represents an amino acid residue or a residue of an amino acid derivative,
wherein the amino acid residue is a residue of an amino acid selected from the group consisting of glycine, alanine, leucine, isoleucine, valine, lysine, cysteine, threonine, arginine, asparagine, aspartic acid, glutamine, glutamic acid, serine, histidine, phenylalanine, methionine, tryptophan, tyrosine, and proline,
wherein the amino acid derivative is selected from the group consisting of N-methylleucine, 2,3-diaminopropanoic acid, 2,4-diaminobutyric acid, ornithine, and α-hydroxyleucine;
S represents a C-terminal amino acid residue; and

OG Complex Work Unit Chemistry
represents a fluorophore, and
the fluorophore is represented by the following formulas (3) to (6);

OG Complex Work Unit Chemistry
wherein,
R1 represents hydrogen or an alkyl group having 1 to 6 carbon atoms;
R2 represents hydrogen, a carboxyl group, or an ester group of formula —COOR, wherein R represents an alkyl group having 1 to 6 carbon atoms;
* represents a bond to —O—P(═O)(—R1)-T-S,

OG Complex Work Unit Chemistry
wherein,
R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms, or a carboxyl group;
R2, when present, represents the same or different monovalent substituents present on the benzene ring, and said monovalent substituent is selected from an alkyl group having 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms or a carboxyl group;
m is an integer of 0 to 4, and when m is 2 or more, each R2 may be the same or different;
Y each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
R3 and R4 each independently represent an alkyl group or aryl group having 1 to 6 carbon atoms,
when R3 or R4 represents an alkyl group, one or more of halogen atoms, carboxy groups, sulfonyl groups, hydroxy groups, amino groups, or alkoxy groups may be present on the alkyl group,
when R3 or R4 represents an aryl group, the aryl group may be a monocyclic aromatic group or condensed aromatic group, and the aryl ring may include one or more ring-structured heteroatoms;
X is an oxygen atom of NR, wherein R represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms;
* represents a bond to —O—P(═O)(—R1)-T-S,

OG Complex Work Unit Chemistry
wherein,
R1, when present, represents the same or different monovalent substituents present on the benzene ring, and said monovalent substituent is selected from an alkyl group having 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms or a carboxyl group;
m is an integer of 0 to 4, and when m is 2 or more, each R1 may be the same or different;
R2 and R3 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom,
when R2 or R3 represents an alkyl group, one or more of halogen atoms, carboxy groups, sulfonyl groups, hydroxy groups, amino groups, or alkoxy groups may be present on the alkyl group;
R4 and R5 are each independently a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, or a halogen atom;
R6 is selected from NH2, NHR, NRR′, OH, or OR″, wherein R and R′ each independently represent an alkyl group having 1 to 5 carbon atoms or a fluoroalkyl group having 1 to 5 carbon atoms, R″ represents an alkyl group having 1 to 5 carbon atoms;
Y represents an oxygen atom, a silicon atom, a carbon atom, or a phosphorus atom;
when Y is a phosphorus atom, one of R7 and R8 is ═O;
when Y is an oxygen atom, R7 and R8 are not present;
R7 and R8, when present, each independently represent an alkyl group or aryl group having 1 to 6 carbon atoms,
when R7 or R8 represents an alkyl group, one or more substituents selected from halogen atoms, carboxy groups, sulfonyl groups, hydroxy groups, amino groups, or alkoxy groups may be present on the alkyl group,
when R7 or R8 represents an aryl group, the aryl group may be a monocyclic aromatic group or condensed aromatic group, the aryl ring may include one or more ring-structured heteroatoms, and one or more substituents selected from a halogen atom, carboxy group, sulfonyl group, hydroxy group, amino group, or alkoxy group may be present on the aryl ring;
X represents an oxygen atom or NRa, wherein Ra represents a hydrogen atom or an alkyl group having 1 to 5 carbon atoms;
n is an integer of 1 to 3;
* represents a bond to —O—P(═O)(—R1)-T-S,

OG Complex Work Unit Chemistry
wherein,
R1 represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms or a carboxyl group;
R2, when present, represents the same or different monovalent substituents present on the benzene ring, and said monovalent substituent is selected from an alkyl group having 1 to 6 carbon atoms, an alkoxyl group having 1 to 6 carbon atoms or a carboxyl group;
m is an integer of 0 to 4, and when m is 2 or more, each R2 may be the same or different;
Y each independently represents a hydrogen atom, an alkyl group having 1 to 6 carbon atoms or a halogen atom;
X is an oxygen atom or NR, wherein R represents a hydrogen atom or a substituted or unsubstituted alkyl group having 1 to 6 carbon atoms; and
* represents a bond to —O—P(═O)(—R1)-T-S.