Compounds useful for FRET and methods related thereto

Patent metadata

Field	Value
Publication number	US-9796746-B2
Application number	US-201514959280-A
Country	US
Kind code	B2
Filing date	Dec 4, 2015
Priority date	Jun 4, 2013
Publication date	Oct 24, 2017
Grant date	Oct 24, 2017

Abstract

The invention also relates to compounds, which are useful for intra-molecular fluorescence resonance energy transfer (FRET), comprising the oxidized form of a carbaNADH-based first fluorophore and a second fluorophore that is excitable at a wave-length of between 445 to 540 nm and that has an emission maximum of greater than 560 nm, and methods, kits and compositions related thereto.

First claim

The invention claimed is:

1. A compound comprising
(1) the oxidized form of a carbaNADH-based first fluorophore, and
(2) a second fluorophore that is excitable with light having a wave-length of between 445 to 540 nm, and that has an emission maximum of greater than 560 nm,
wherein the carbaNADH-based first fluorophore and the second fluorophore are covalently linked and the second fluorophore comprises, as a core, a chromophore selected from the group consisting of a 2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1-propen-1-yl]-1,3,3-trimethyl-3H-indolium chromophor moiety, a 5,6-dichloro-2-[3-(5,6-dichloro-1,3-dihydro-1,3-dimethyl-2H-benzimidazol-2-ylidene)-1-propen-1-yl]-1,3-dimethyl1H-benzimidazolium chromophor moiety, a 1-methyl-4-[3-(1-methyl-4(1H)-quinolinylidene-1-propen-1-yl]-quinolinium chromophore moiety, and a 2-(4-(Diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate chromophore moiety.

2. The compound of claim 1 , wherein
(a) the distance between the first and the second fluorophore is between 1.5 and 5 nm,
and/or

(b) the compound consists of
(i) the oxidized form of a carbaNADH-based first fluorophore, and
(ii) a second fluorophore that is excitable with light having a wave-length of between 445 to 540 nm, and that has an emission maximum of greater than 560 nm, and
(iii) a linker molecule with a length of 25 atoms or less,
and/or

(c) wherein the oxidized form of the first fluorophore is carbaNAD
and/or

(d) the oxidized form of the carbaNADH-based first fluorophore is a carbaNADH-based first fluorophore of formula II

wherein
Q is a NR 1 R 2 , wherein R 1 and R 2 are independently selected from H, a C 1 to C 12 alkyl group, a C 1 to C 12 alkenyl group and a C 1 to C 12 alkinyl group, optionally wherein one or more carbon atoms of the alkyl group, a alkenyl group and a alkinyl group is substituted with O, N or S and/or
optionally wherein said C 1 to C 12 alkyl, alkenyl or alkinyl group is substituted with ═O, —OH, —SH, ═S or a C 1 to C 4 alkyl, wherein optionally one or more carbon atoms of the alkyl group is substituted with O, N or S, and
J is selected from H, a C 1 to C 12 alkyl group, a C 1 to C 12 alkenyl group and a C 1 to C 12 alkinyl group, optionally wherein one or more carbon atoms of the alkyl group, a alkenyl group and a alkinyl group is substituted with O, N or S and/or optionally wherein said C 1 to C 12 alkyl, alkenyl or alkinyl group is substituted with ═O, —OH, —SH, ═S or a C 1 to C 4 alkyl, wherein optionally one or more carbon atoms of the alkyl group is substituted with O, N or S, particularly wherein one of J and Q is connected via a linker molecule with a length of 25 atoms or less to the second fluorophore, and
T is a hydrogen atom or phosphate group,
especially wherein Q is substituted with N-(6“cy3” aminohexyl)carbomyolmethyl.

3. The compound of claim 1 , wherein the second fluorophore comprises a “Cy3” (=2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1-propen-1-yl]-1,3,3-trimethyl-3H-indolium) moiety.

4. A compound of claim 1 , selected from the group consisting of
(i) N6-[N-(6-“Cy 3” aminohexyl)carbamoylmethyl] carbaNADH, or
(ii) N6-[N-(6-“Cy 3” aminohexyl)carbamoylmethyl] carbaNAD, or
(iii) a mixture of N6-[N-(6-“Cy 3” aminohexyl)carbamoylmethyl] carbaNADH and N6-[N-(6-“Cy 3” aminohexyl)carbamoylmethyl] carbaNAD.

5. A compound comprising
(1) the reduced form of a carbaNADH-based first fluorophore, and
(2) a second fluorophore that is excitable with light having a wave-length of between 445 to 540 nm, and that has an emission maximum of greater than 560 nm,
wherein the carbaNADH-based first fluorophore and the second fluorophore are covalently linked and the second fluorophore comprises, as a core, a chromophore selected from the group consisting of a 2-[3-(1,3-dihydro-1,3,3-trimethyl-2H-indol-2-ylidene)-1-propen-1-yl]-1,3,3-trimethyl-3H-indolium chromophor moiety, a 5,6-dichloro-2-[3-(5,6-dichloro-1,3-dihydro-1,3-dimethyl-2H-benzimidazol -2-ylidene)-1-propen-1-yl]-1,3-dimethyl1H-benzimidazolium chromophor moiety, a 1-methyl-4-[3-(1-methyl-4(1H)-quinolinylidene-1-propen-1-yl]-quinolinium chromophore moiety, and a 2-(4-(Diethylamino)-2-hydroxyphenyl)-4-(4-(diethyliminio)-2-hydroxycyclohexa-2,5-dienylidene)-3-oxocyclobut-1-enolate chromophore moiety.

6. The compound of claim 5 , wherein
(a) the distance between the first and the second fluorophore is between 1.5 and 5 nm,
and/or

(b) the compound consists of
(i) the reduced form of a carbaNADH-based first fluorophore, and
(ii) a second fluorophore that is excitable with light having a wave-length of between 445 to 540 nm, and that has an emission maximum of greater than 560 nm, and
(iii) a linker molecule with a length of 25 atoms or less,
and/or

(c) wherein the reduced form of the first fluorophore is carbaNADH.
(d) the reduced form of the carba a carbaNADH-based first fluorophore is a carbaNADH-based first fluorophore of formula III

wherein
Q is a NR 1 R 2 , wherein R 1 and R 2 are independently selected from H, a C 1 to C 12 alkyl group, a C 1 to C 12 alkenyl group and a C 1 to C 12 alkinyl group, optionally wherein one or more carbon atoms of the alkyl group, a alkenyl group and a alkinyl group is substituted with O, N or S and/or
optionally wherein said C 1 to C 12 alkyl, alkenyl or alkinyl group is substituted with ═O, —OH, —SH, ═S or a C 1 to C 4 alkyl, wherein optionally one or more carbon atoms of the alkyl group is substituted with O, N or S, and
J is selected from H, a C 1 to C 12 alkyl group, a C 1 to C 12 alkenyl group and a C 1 to C 12 alkinyl group, optionally wherein one or more carbon atoms of the alkyl group, a alkenyl group and a alkinyl group is substituted with O, N or S and/or optionally wherein said C 1 to C 12 alkyl, alkenyl or alkinyl group is substituted with ═O, —OH, —SH, ═S or a C 1 to C 4 alkyl, wherein optionally one or more carbon atoms of the alkyl group is substituted with O, N or S, particularly wherein one of J and Q is connected via a linker molecule with a length of 25 atoms or less to the second fluorophore, and
T is a hydrogen atom or phosphate group,
especially wherein Q is substituted with N-(6“cy3” aminohexyl)carbomyolmethyl.

7. A method for determining an analyte in a sample using an NAD(H)-dependent oxidoreductase and the oxidized form of a fluorogenic coenzyme, the method comprising following steps:
(a) mixing an NAD(H)-dependent oxidoreductase and the oxidized form of the fluorogenic coenzyme of claim 1 with the sample, thereby
(b) reacting the analyte with the reagent system comprising an NAD(H)-dependent oxidoreductase and the oxidized form of the fluorogenic coenzyme of claim 1 ,
(c) measuring the fluorescence emission of the reduced coenzyme.

8. The method according to claim 7 , wherein
(a) a change in the oxidized form of the fluorogenic coenzyme is generated in step (b) of claim 7
(i) wherein said change in the oxidized form of the fluorogenic coenzyme is an reduction, and/or
(ii) wherein said change in the coenzyme correlates with the amount and/or concentration of the analyte,

and/or
(b) wherein the method of claim 7 is followed by determining the amount and/or concentration of the analyte based on the fluorescence emission of the fluorogenic coenzyme.

Assignees

• Roche Diagnostics Operations Inc

Inventors

• Heindl Dieter
• Hoenes Joachim
• Horn Carina
• Meier Thomas
• Petrich Wolfgang

Classifications

• G01N33/581

  with enzyme label (including co-enzymes, co-factors, enzyme inhibitors or substrates) · CPC title

• G01N33/582

  with fluorescent label · CPC title

• G01N33/533

  with fluorescent label · CPC title

• G01N2201/062

  LED's · CPC title

• G01N33/5735

  co-enzymes or co-factors, e.g. NAD or ATP · CPC title