ANALYTE DETECTION USING NEAR-INFRARED FLUOROPHORES
Embodiments of compounds for selectively detecting an analyte are disclosed, along with methods and kits for detecting analytes with the compounds. The compounds are bridged viologen conjugates including at least one fluorophore according to the general structure At least one of R 1 /R 2 , R 2 /R 3 , R 3 /R 4 , R 5 /R 6 , R 6 /R 7 , and/or R 7 /R 8 together form a substituted or unsubstituted cycloalkyl or aryl.
1 . A compound according to Formula I, Formula II, or Formula III comprising a bridged viologen backbone and substituents R A and R B
where X − is any counteranion, R A and R B independently are substituted or unsubstituted aliphatic, or substituted or unsubstituted aryl or heteroaryl comprising one or more substituted or unsubstituted aromatic rings and/or heteroaromatic rings, and at least one of R A and R B is a fluorophore having a structure according to general formula (i)
where each bond depicted as “ ” is a single or double bond as needed to satisfy valence requirements;
X 1 is O, S, Se, Si(CH 3 ) 2 , Ge(CH 3 ) 2 , Sn(CH 3 ) 2 , CH 2 , C(CH 3 ) 2 , or NH;
R 1 , R 2 , R 4 independently are hydrogen, hydroxyl, oxygen, thiol, lower alkyl, carboxyalkyl, amino, substituted amino, alkoxy, halogen, or —NHR c where R c is
R 5 , R 7 , and R 8 independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, substituted amino, alkoxy, or halogen;
R 3 and R 6 independently are hydrogen, hydroxyl, halogen, oxygen, sulfur, thiol, amino, alkyl amino, imino, iminium, alkyl imino, alkyl iminium, cycloalkyl imino or —NHR c ;
at least one of R 1 and R 2 , R 2 and R 3 , R 3 and R 4 , R 5 and R 6 , R 6 and R 7 , and/or R 7 and R 8 together form a substituted or unsubstituted cycloalkyl or aryl;
R 9 -R 12 independently are hydrogen, alkyl, acyl, carboxyl, nitro, amino, alkyl amino, or —SO 3 H;
R 13 is hydrogen, hydroxyl, lower alkyl, lower alkoxy, —SO 3 H or —COOR 14 where R 14 is hydrogen or lower alkyl and the bond depicted as “ ” in ring B is a double bond, or R 13 is one or more atoms forming a ring system with rings B and D and the bond depicted as “ ” in ring B is a single bond; and
at least one of R 1 -R 13 is a linker covalently binding the fluorophore to the bridged viologen backbone,
wherein if R 3 and R 4 together form a substituted or unsubstituted cycloalkyl or aryl, then at least one of R 1 and R 8 is other than hydrogen, or R 13 is not —COOR 14 , or at least one additional substituted or unsubstituted cycloalkyl or aryl is formed by R 1 and R 2 , R 2 and R 3 , R 5 and R 6 , R 6 and R 7 , and/or R 7 and R 8 .
2 . The compound of claim 1 where at least one of R A and R B is a fluorophore having a structure according to general formula (iii), (iv), (v), (vi), (vii), (viii), (ix), (x), or (xi)
where R 15 -R 22 independently are hydrogen, halogen, hydroxyl, oxygen, thiol, amino, alkyl amino, alkoxy, sulfur, imino, iminium, alkyl imino, alkyl iminium, or —NHR c where R c is
and
at least one of R 1 -R 22 is a linker covalently binding the fluorophore to the viologen backbone,
wherein if the fluorophore has a structure according to general formula (iii) then at least one of R 1 and R 8 is other than hydrogen or R 13 is not —COOR 14 .
3 . The compound of claim 1 where R 13 is the linker and has the formula —COO—.
4 . The compound of claim 1 where both R A and R B are fluorophores, each fluorophore independently having a structure selected according to general formula (i).
5 . (canceled)
6 . The compound of claim 1 where one of R A and R B is a fluorophore according to general formula (i), and the other of R A and R B is a lower alkyl nitrile, lower alkyl substituted phenyl, alkenyl, alkynyl, substituted coumarin, acetal, or carboxylate group.
7 . The compound of claim 6 where the other of R A and R B is
8 . The compound of claim 1 where X 1 is oxygen.
9 . The compound of claim 1 where R 13 is —COO— and forms a lactone ring, and at least one of R 1 -R 12 is the linker.
10 . The compound of claim 2 where R 13 is —COO— and forms a lactone ring, and at least one of R 1 -R 12 or R 15 -R 22 is the linker.
11 - 15 . (canceled)
16 . The compound of claim 2 where the fluorophore has a structure according to:
(a) general formula (iii), (iv), (ix), or (x) where X 1 is oxygen and R 18 is halogen, hydroxyl, thiol, amino, alkyl amino, or alkoxy;
(b) general formula (iii) where X 1 is oxygen and R 6 and R 16 are —NHR c ;
(c) general formula (iv) where X 1 is oxygen and R 16 and R 21 are NHR c ;
(d) general formula (iv) where X 1 is oxygen and R 19 is hydroxyl, thiol, oxygen, imino, iminium, alkyl imino, alkyl iminium, amino, or alkyl amino;
(e) general formula (iv) where X 1 is oxygen, R 18 is halogen, hydroxyl, thiol, amino, alkyl amino, or alkoxy, and R 19 is hydroxyl, thiol, oxygen, imino, iminium, alkyl imino, alkyl iminium, amino, or alkyl amino; or
(f) general formula (vi) where R 17 is halogen, hydroxyl, thiol, amino, alkyl amino, or —NHR c , and R 20 is oxygen, sulfur, imino, iminium, alkyl iminium, or —NHR c .
17 - 20 . (canceled)
21 . A method for detecting an analyte, comprising:
forming a solution by combining a sample comprising an analyte with a compound according to claim 1 ;
allowing a reaction between the analyte and the compound in the solution to proceed for an effective period of time to a produce a detectable change in the solution's absorbance spectrum, emission spectrum, or both, where the change indicates that the analyte is present; and
detecting the change.
22 . The method of claim 21 where detecting the change comprises:
exposing the solution to a light source; and
detecting the analyte by detecting fluorescence from the compound at a wavelength corresponding to an emission spectrum maximum of the compound after the reaction has proceeded for the effective period of time.
23 - 30 . (canceled)
31 . The method of claim 21 where the analyte comprises cysteine, homocysteine, glutathione, succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, or a combination of succinyl-5-amino-4-imidazolecarboxamide riboside and succinyladenosine.
32 . The method of claim 31 where the compound comprises at least one fluorophore according to general formula (vi)
—where:
R 1 , R 4 , R 5 and R 8 independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, substituted amino, alkoxy, or halogen;
R 15 -R 18 independently are hydrogen, halogen, hydroxyl, thiol, amino, alkyl amino, alkoxy or —NHR c ;
R 19 -R 22 independently are hydrogen, hydroxyl, thiol, halogen, oxygen, imino, iminium, alkyl imino, alkyl iminium, amino, alkyl amino, or —NHR c ; and
at least one of R 1 , R 4 , R 5 , R 8 -R 13 , R 15 , R 16 , R 18 , R 19 , R 21 or R 22 is a linker covalently binding the fluorophore to the viologen backbone.
33 . (canceled)
34 . The method of claim 32 where:
(a) the analyte is glutathione, R 17 is hydroxyl, amino, or alkyl amino, and R 20 is oxygen, hydroxyl, amino, alkyl amino, imino, or alkyl iminium; or
(b) the analyte is succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, or a combination thereof, at least one of R 17 and R 20 is —NHR c , R 13 is —COOR 14 where R 14 is hydrogen or lower alkyl, and one of R 9 , R 10 , R 11 , or R 12 is the linker.
35 . (canceled)
36 . The method of claim 31 where the analyte is succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, or a combination thereof, and the compound comprises at least one fluorophore according to:
(a) general formula (iii):
where X 1 is oxygen and R 6 and R 16 are —NHR c ; or
(b) general formula (iv)
where X 1 is oxygen and R 16 and R 21 are —NHR c .
37 - 40 . (canceled)
41 . A kit for detecting an analyte, comprising at least one compound according to claim 1 , wherein the compound when combined with a sample comprising the analyte will undergo a change in its absorbance spectrum and/or emission spectrum compared to the compound in a sample that does not comprise the analyte.
42 . (canceled)
43 . The kit of claim 41 , further comprising at least one buffer solution in which the compound when combined with a sample comprising the analyte will undergo a change in its absorbance spectrum and/or emission spectrum compared to the compound combined with the buffer solution and a sample that does not comprise the analyte.
44 . The kit of claim 41 , where the analyte comprises glutathione, cysteine, homocysteine, succinyl-5-amino-4-imidazolecarboxamide riboside, succinyladenosine, or a combination of succinyl-5-amino-4-imidazolecarboxamide riboside and succinyladenosine.
45 . The kit of claim 44 where the compound comprises at least one fluorophore according to:
(a) general formula (vi)
where
R 1 , R 4 , R 5 and R 8 independently are hydrogen, hydroxyl, thiol, lower alkyl, carboxyalkyl, amino, substituted amino, alkoxy, or halogen,
R 15 -R 18 independently are hydrogen, halogen, hydroxyl, thiol, amino, alkyl amino, alkoxy or —NHR c ,
R 19 -R 22 independently are hydrogen, hydroxyl, thiol, halogen, oxygen, imino, iminium, alkyl imino, alkyl iminium, amino, alkyl amino, or —NHR c , and
at least one of R 1 , R 4 , R 5 , R 8 -R 13 , R 15 , R 16 , R 18 , R 19 , R 21 or R 22 is a linker covalently binding the fluorophore to the viologen backbone;
(b) general formula (iii)
where X 1 is oxygen and R 6 and R 16 are —NHR c ; or
(c) general formula (iv)
where X 1 is oxygen and R 16 and R 21 are —NHR c .
46 - 49 . (canceled)