Modified Nucleic Acid Binding Cyanine Dyes for the Detection of Reactive Oxygen Species

Abstract

Disclosed herein are compounds, compositions, methods, and kits for detecting reactive oxygen species (ROS) by conventional fluorescence microscopy, fluorescence spectroscopy, flow cytometry, and/or high content imaging. The compounds disclosed herein are novel reduced nucleic acid binding cyanine dyes, which dyes are probes for detecting ROS and measuring oxidative stress in cells either in vitro and/or in vivo. Also described herein are processes for preparing novel reduced dyes, i.e., ROS probes, for use in the disclosed compositions, methods and kits.

1 . A reduced dye compound having structural formula (I): 
       
         
           
           
               
               
           
         
         wherein 
         each R 1 , which may be the same or different, is independently H; C 1 -C 6  alkyl; trifluoromethyl; halogen; —OR 8 , —SR 8 , or —(NR 8 R 9 ), where R 8  and R 9 , which may be the same or different, are independently H; C 1 -C 6  alkyl; or 1-2 alicyclic hetroalicyclic, aromatic or heteroaromatic rings having a total of 3-16 ring atoms, wherein the heteroatoms are O, N, or S; or R 8  and R 9  taken in combination are —(CH 2 ) 2 -L-(CH 2 ) 2 — where L is a single bond; —CH 2 —; —O—; or —NR 10 , where R 10  is H or a C 1 -C 6  alkyl;
 t is 1, 2, 3 or 4; 
 
         R 2  is a saturated or unsaturated C 1 -C 6  alkyl; 
         X is O; S; Se; NR 15 , where R 15  is H or a C 1 -C 6  alkyl; or CR 16 R 17 , where R 16  and R 17 , which may be the same or different, are independently H or C 1 -C 6  alkyl, or the carbons of R 16  and R 17  taken in combination complete a five- or six-membered saturated ring; 
         Q has the formula Q1 or Q2: 
       
       
         
           
           
               
               
           
         
         
           wherein 
           Y is —CR 3 ═CR 4 —;
 p and m are 0 or 1, such that p+m=1; 
 
           R 5  is a C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  polyalkenyl, C 1 -C 6  alkynyl or C 1 -C 6  polyalkynyl group; or an OMEGA; 
           R 3 , R 4 , R 6  and R 7 , which may be the same or different, are each independently H; halogen; a C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  polyalkenyl, C 1 -C 6  alkynyl or C 1 -C 6  polyalkynyl group; —OR 8 ; —SR 8 ; —(NR 8 R 9 ), as defined previously; —OSO 2 R 19 , where R 19  is a C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, or aryl; or an OMEGA; or R 6  and R 7 , taken in combination are —(CH 2 ) v —, where v is 3 or 4, or R 6  and R 7  form a fused aromatic ring according to the formula Q2; 
           R 11 , R 12 , R 13  and R 14 , which may be the same or different, are each independently H; a C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  polyalkenyl, C 1 -C 6  alkynyl or C 1 -C 6  polyalkynyl group; a halogen; an OMEGA; or —OH, —OR 8 , —SR 8 , or —(NR 8 R 9 ), as defined previously; 
           OMEGA is a saturated or unsaturated, substituted or unsubstituted, cyclic substituent that has a total of 2-16 ring carbon atoms in 1-2 alicyclic, heteroalicyclic, aromatic, or heteroaromatic rings, containing 1-4 heteroatoms wherein the heteroatoms are O, N, or S, that is unsubstituted or optionally substituted one or more times, independently, by halogen, alkyl, perfluoroalkyl, amino, alkylamino, dialkylamino, alkoxy or carboxyalkyl, having 1-6 carbons, and that is attached as R 3 , R 4 , R 5 , R 11 , R 12 , R 13 , or R 14  by a single bond, and, where more than one of R 3 , R 4 , R 5 , R 11 , R 12 , R 13 , and R 14  is an OMEGA, each OMEGA is optionally the same or different; 
         
         R 20 , R 21  and R 22  are independently H, C 1 -C 6  alkyl, C 3 -C 10  cycloalkyl, aryl or heteroaryl; 
         W is H or D in either the R or S configuration; and 
         n is 0, 1, 2, or 3. 
       
     
     
         2 . (canceled) 
     
     
         3 . The compound according to  claim 1 , wherein the compound has structural formula (VIII): 
       
         
           
           
               
               
           
         
         wherein W is H or D and n is 0, 1 or 2. 
       
     
     
         4 . The compound according to  claim 1 , wherein the compound has structural formula (IX): 
       
         
           
           
               
               
           
         
         wherein W is H or D and n is 0, 1 or 2. 
       
     
     
         5 . The compound according to  claim 1 , wherein the compound has structural formula (X): 
       
         
           
           
               
               
           
         
         wherein W is H or D and n is 0, 1 or 2. 
       
     
     
         6 . The compound according to  claim 1 , wherein the compound has structural formula (XI): 
       
         
           
           
               
               
           
         
         wherein W is H or D and n is 0, 1 or 2. 
       
     
     
         7 . (canceled) 
     
     
         8 . (canceled) 
     
     
         9 . A composition for the detection of reactive oxygen species (ROS), the composition comprising:
 a) one or more reduced dyes; and   b) a carrier,   wherein said reduced dyes are compounds according to  claim 1  and are present in an amount effective to detect the presence of ROS upon reaction with ROS.   
     
     
         10 - 11 . (canceled) 
     
     
         12 . A method of detecting reactive oxygen species (ROS) in a sample, the method comprising the steps of:
 a) contacting the sample with an effective amount of one or more of the reduced dye compounds according to  claim 1 ; and   b) determining if the reduced dye compound has been oxidized.   
     
     
         13 . A method of detecting reactive oxygen species (ROS) in a sample, the method comprising the steps of:
 a) contacting the sample with an effective amount of the composition according to  claim 9 ; and   b) determining if the reduced dye compound has been oxidized.   
     
     
         14 . The method according to  claim 12 , wherein the sample comprises cells, tissues, biological fluids, or combinations thereof. 
     
     
         15 - 18 . (canceled) 
     
     
         19 . The method according to  claim 12 , wherein the detection of the reactive oxygen species (ROS) is used to diagnose a disease or disorder selected from the group consisting of carotid artery injuries, atherosclerosis, hypertension, cancers, diseases and disorders characterized by inflammation, radiation-induced late normal tissue damage; tissue damages due to chemotherapy, reperfusion after ischemia, or transplantation; diabetes, such as type 1 diabetes (T1D), neurodegenerative diseases, such as Alzheimer's disease, Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease; cerebrovascular disease, cystic fibrosis, chronic kidney disease, cardiovascular disease, preeclampsia, ophthalamic diseases, and combinations thereof. 
     
     
         20 . A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
 a) one or more reduced dye compounds according to  claim 1 ; and   b) one or more containers.   
     
     
         21 . A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
 a) the composition according to  claim 9 ; and   b) one or more containers.   
     
     
         22 . The kit according to  claim 20 , wherein the kit further comprises instructions for performing an assay for detecting one or more reactive oxygen species (ROS). 
     
     
         23 . (canceled) 
     
     
         24 . A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
 a) one or more unreacted cyanine dye compound having structural formula (III):   
       
         
           
           
               
               
           
         
         b) one or more reducing agents; and 
         c) one or more containers; 
         wherein R 1 , R 2 , R 20 , R 21 , R 22 , Q, n and t are as defined in  claim 1 . 
       
     
     
         25 . A kit for detecting reactive oxygen species (ROS) in a sample, the kit comprising:
 a) one or more unreacted cyanine dye compound having structural formula (IV):   
       
         
           
           
               
               
           
         
         b) one or more reducing agents; and 
         c) one or more containers; 
         wherein 
         each R 1  is H; 
         t is 4; 
         R 2  is a substituted or unsubstituted C 1 -C 6  alkyl; 
         R 5  is selected from the group consisting of C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  polyalkenyl, C 1 -C 6  alkynyl, C 1 -C 6  polyalkynyl and an OMEGA; 
         R 3 , R 4 , R 11 , R 12 , R 13  and R 14  are each independently selected from the group consisting of H, halogen, C 1 -C 6  alkyl, C 1 -C 6  alkenyl, C 1 -C 6  polyalkenyl, C 1 -C 6  alkynyl, C 1 -C 6  polyalkynyl, —OR 8 , —SR 8 , —(NR 8 R 9 ), —OSO 2 R 19 , and an OMEGA, wherein R 8  and R 9 , which may be the same or different, are independently selected from the group consisting of H and C 1 -C 6  alkyl; 
         R 19  is a C 1 -C 6  alkyl, C 1 -C 6  perfluoroalkyl, or aryl;
 OMEGA is a substituted or unsubstituted phenyl, 
 wherein the OMEGA is attached at R 3 , R 4 , R 5 , R 11 , R 12 , R 13 , or R 14  by a single bond, 
 wherein when more than one of R 3 , R 4 , R 5 , R 11 , R 12 , R 13 , and R 14  is an OMEGA, each OMEGA is optionally the same or different, and 
 wherein one or two of R 3 , R 4 , R 5 , R 6 , R 7 , R 11 , R 12  m R 13  and R 14  is an OMEGA; 
 
         X is O, S, Se, NR 15  or CR 16 R 17 ; 
         R 16  and R 17 , which may be the same or different, are independently selected from the group consisting of H and C 1 -C 6  alkyl; 
         R 15  is H or C 1 -C 6  alkyl; 
         R 20 , R 21  and R 22  are independently H, C 1 -C 6  alkyl, C 3 -C 10  cycloalkyl, aryl or heteroaryl; 
         W is H or D in either the R or S configuration; and 
         n is 0, 1, 2, or 3. 
       
     
     
         26 . The kit according to  claim 24 , wherein the kit further comprises instructions for performing an assay for detecting one or more reactive oxygen species (ROS). 
     
     
         27 . The kit according to  claim 24 , wherein the kit further comprises instructions for preparing one or more reduced dye compounds. 
     
     
         28 . The kit according to  claim 24 , wherein the one or more reducing agents is selected from the group consisting of LiAlH 4 , NaBH 4 , Zn(BH 4 ) 2 , NaD, LiAlD 4 , NaBD 4 , Zn(BD 4 ) 2 , compounds containing a Sn 2+  ion, sulfite compounds, diisobutyl aluminum hydride (DIBAH), oxalic acid, formic acid, ascorbic acid, phosphites, hypophosphate, hypophorphorous acid, dithiothreitol (DTT), and compounds containing a Fe 2+  ion. 
     
     
         29 . A process for preparing a reduced dye compound having structural formula (I) 
       
         
           
           
               
               
           
         
       
       the process comprising:
 a) reacting a cyanine compound having structural formula (III) 
 
       
         
           
           
               
               
           
         
       
       with a reducing agent, wherein: 
       Q, W, X, R 1 , R 2 , R 20 , R 21 , R 22 , n, and t are as defined in  claim 1 . 
     
     
         30 - 31 . (canceled) 
     
     
         32 . The process according to  claim 29 , wherein the reducing agent is selected from the group consisting of LiAlH 4 , NaBH 4 , Zn(BH 4 ) 2 , LiAlD 4 , NaBD 4 , Zn(BD 4 ) 2 , compounds containing a Sn 2+  ion, sulfite compounds, diisobutyl aluminum hydride (DIBAH), oxalic acid, formic acid, ascorbic acid, phosphites, hypophosphate, hypophorphorous acid, dithiothreitol (DTT), and compounds containing a Fe 2+  ion. 
     
     
         33 . (canceled)