Fluorogenic ph sensitive dyes and their method of use

Abstract

A new class of pH sensitive fluorescent dyes and assays relating thereto are described. The dyes and assays are particularly suited for biological applications including phagocytosis and monitoring intracellular processes. The pH sensitive fluorescent dyes of the present invention include compounds of Formula I: wherein the variables are described throughout the application.

1 .- 87 . (canceled) 
     
     
         88 . A compound for use as a fluorescent pH sensor, the compound having Formula I: 
       
         
           
           
               
               
           
         
       
       wherein,
 R 1 , R 2 , R 3  and R 6  are each independently H, Z, or an electron donating group (EDG), provided that R 1  and R 2  are not hydroxyl or thiol or their deprotonated forms, and that a Z moiety is present;
 Z is —NR b R c , wherein R b  is H, alkyl, or substituted alkyl, and R c  is alkyl, or substituted alkyl; 
 
 R 4  is H, alkyl, or substituted alkyl; 
 R 5  is substituted alkyl having a substituent selected from acyl, acylamino, amino, substituted amino, aminocarbonyl, arylthio, substituted arylthio, carboxyl, carboxyl ester, and (carboxyl ester)amino; 
 X is a xanthene;
 wherein alkyl is linear or branched C 1 -C 10 ; 
 
 or a stereoisomer, tautomer, or salt thereof; 
 
     
     
         89 . The compound of  claim 88 , wherein X is: 
       
         
           
           
               
               
           
         
         wherein, 
         R 7 , R 8 , R 9  and R 10  are each independently selected from the group consisting of alkyl, substituted alkyl, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted 20 cycloalkyl, heterocyclyl, and substituted heterocyclyl; and 
         R 11 , R 12 , R 13  and R 14  are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, —SO 3 H, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl; or 
         R 11  and R 14  are taken together with R 7  and R 8  to form a fused ring; and R 12  and R 13  are taken together with R 9  and R 10  to form a fused ring. 
       
     
     
         90 . The compound of  claim 89 , wherein:
 (i) R 7 , R 8 , R 9  and R 10  are alkyl; and/or   (ii) R 11 , R 12 , R 13  and R 14  are H; or   (iii) R 11  and R 14  are taken together with R and R 8  to form a fused ring; and R 12  and R 13  are taken together with R 9  and R 10  to form a fused ring; wherein each fused ring has the following structure:   
       
         
           
           
               
               
           
         
         wherein, 
         R 27  and R 28  are each independently selected from the group consisting of H, alkyl, substituted alkyl, alkoxy, substituted alkoxy, alkenyl, substituted alkenyl, alkynyl, substituted alkynyl, acyl, acylamino, acyloxy, amino, substituted amino, aminocarbonyl, aminothiocarbonyl, aminocarbonylamino, aminothiocarbonylamino, aminocarbonyloxy, aminosulfonyl, aminosulfonyloxy, aminosulfonylamino, amidino, carboxyl, carboxyl ester, (carboxyl ester)amino, (carboxyl ester)oxy, cyano, halo, hydroxy, nitro, S03, sulfonyl, substituted sulfonyl, sulfonyloxy, thioacyl, thiol, alkylthio, substituted alkylthio, aryl, substituted aryl, heteroaryl, substituted heteroaryl, cycloalkyl, substituted cycloalkyl, heterocyclyl, and substituted heterocyclyl. 
       
     
     
         91 . The compound of  claim 88  wherein R 5  is substituted by aminocarbonyl and the aminocarbonyl is —CONHR a  where R a  is C 1 -C 10  alkyl terminally substituted by a reactive group selected from carboxyl, carboxylester and maleimide. 
     
     
         92 . The compound of  claim 88  wherein R 5  is substituted by carboxyl ester. 
     
     
         93 . The compound of  claim 92  wherein R 5  is substituted by N-hydroxysuccinimide ester. 
     
     
         94 . The compound of  claim 88 , wherein the EDG is selected from the group consisting of alkoxy, substituted alkoxy, amino, substituted amino, thiol, alkylthio, hydroxy, acylamino, and (carboxyl ester)oxy. 
     
     
         95 . The compound of  claim 88 , wherein the EDG is selected from the group consisting of alkoxy, substituted alkoxy, amino, and substituted amino. 
     
     
         96 . The compound of  claim 88 , wherein R 1  and R 3  are —OCH 3  or —N(CH 3 ) 2  and/or R 2  and R 6  are H. 
     
     
         97 . A compound having Formula III: 
       
         
           
           
               
               
           
         
       
       wherein,
 R 3  is —NR b R c , wherein R b  is H, alkyl, or substituted alkyl, and R c  is alkyl, or substituted alkyl; 
 R 4  is H, alkyl, or substituted alkyl; and 
 R 5  is alkyl or substituted alkyl having a substituent selected from acyl, acylamino, amino, substituted amino, aminocarbonyl, arylthio, substituted arylthio, carboxyl, carboxyl ester, and (carboxyl ester)amino; 
 wherein alkyl is linear or branched C 1 -C 10 . 
 
     
     
         98 . A method for determining the pH of a sample, the method comprising:
 contacting the sample with a compound of  claim 88  to form a contacted sample;   incubating the contacted sample to form an incubated sample;   illuminating the incubated sample to form an illuminated sample; and   detecting fluorescent emissions from the illuminated sample; wherein the fluorescent emissions are used to determine the pH of the sample.   
     
     
         99 . The method of  claim 98 , wherein the sample comprises a cell, intracellular fluid, extracellular fluid, sera, biological fluid, biological fermentation media, an environmental sample, industrial sample, protein, peptide, buffer solution, chemical reactor, muscle tissue, extracellular vesicle, vascular tissue, blood fluid, saliva, urine, water, soil, waste water, sea water, pharmaceutical, foodstuff, or a beverage. 
     
     
         100 . The method of  claim 99  wherein the cell is a live cell, blood cell, immune cell, cultured cell or neuron. 
     
     
         101 . The method of  claim 98  wherein the sample is immobilized on a polymeric membrane, within a polymeric gel, on a microparticle, on a microarray, on a silicon chip, on a glass slide, on a microwell plate, or on a microfluidic chip. 
     
     
         102 . The method of  claim 98 , wherein the contacting step further comprises contacting the sample with a second dye and wherein the second dye is a pH sensitive dye having a different fluorescent emission spectrum from the compound and a different pKa from the pKa of the compound. 
     
     
         103 . A method for monitoring the pH inside a live cell, the method comprising:
 contacting the cell with a compound of  claim 88  to form a contacted cell;   incubating the contacted cell for a time to allow the compound to enter the cell to form a labelled cell;   illuminating the labelled cell with an appropriate wavelength whereby fluorescence is measured, thereby monitoring the pH inside the cell.   
     
     
         104 . The method of  claim 103 , wherein a change in the pH inside the cell corresponds to a cellular process. 
     
     
         105 . The method of  claim 103 , wherein the compound is conjugated to a protein, nucleic acid or lipid.