Compositions and methods for delivering a substance to a biological target

Abstract

The present application provides compositions and methods using bioorthogonal inverse electron demand Diels-Alder cycloaddition reaction for rapid and specific covalent delivery of a “payload” to a ligand bound to a biological target.

1 . A composition for delivering an agent to a selected biological target, the composition comprising:
 a first component attached to a ligand that is specific for the biological target; and   a second component attached to the agent;   wherein the first and second components are each selected from either a diene or a dienophile and are reactants for the inverse electron demand Diels-Alder reaction.   
     
     
         2 . The composition of  claim 1 , wherein the agent is selected from either a detectable agent or therapeutic agent. 
     
     
         3 . The composition of  claim 2 , wherein the detectable agent is selected from the group consisting of organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, and contrast agents. 
     
     
         4 . The composition of  claim 2 , wherein the detectable agent is linked to a diene, and undergoes fluorogenic activation in the presence of the strained dienophile. 
     
     
         5 . The composition of  claim 2 , wherein the therapeutic agent is a small molecule, enzyme inhibitor, receptor protein inhibitor, small interfering RNA (siRNA), cytotoxin, radioactive ion, or other therapeutic agent. 
     
     
         6 . A method of delivering a detectable agent to a biological target, the method comprising:
 contacting a biological target with a ligand, wherein the ligand is linked to a first component selected from either a diene or a dienophile, to form a ligand-target conjugate;   contacting the ligand-target conjugate with a second component that is selected from either a diene or a dienophile and participates in an inverse electron demand Diels-Alder reaction with the first component, and wherein the conjugate is also linked to a detectable agent; under conditions and for a sufficient time for the first and second components to undergo an inverse electron demand Diels-Alder reaction, thereby delivering the detectable agent to the target.   
     
     
         7 . The method of  claim 6 , wherein the detectable agent is selected from the group consisting of organic small molecules, inorganic compounds, nanoparticles, enzymes or enzyme substrates, fluorescent materials, luminescent materials, bioluminescent materials, radioactive materials, and contrast agents. 
     
     
         8 . The method of  claim 6 , further comprising detecting the detectable agent. 
     
     
         9 . The method of  claim 8 , wherein detecting the detectable agent comprises using histochemistry, fluorescence detection, chemiluminescence detection, bioluminescence detection, magnetic resonance imaging, or nuclear magnetic resonance imaging, X-ray imaging, X-ray computed tomography, ultrasound imaging, or photoacoustic imaging. 
     
     
         10 . The method of  claim 6 , wherein the biological target is in or on a live or dead cell, tissue section, or organism 
     
     
         11 . The method of  claim 6 , wherein the biological target is in an in vitro assay. 
     
     
         12 . A method of delivering a therapeutic agent to a biological target, the method comprising:
 contacting a biological target with a ligand, wherein the ligand is linked to a first component selected from either a diene or a dienophile, to form a ligand-target conjugate;   contacting the ligand-target conjugate with a second component that is selected from either a diene or a dienophile and participates in an inverse electron demand Diels-Alder reaction with the first component, and wherein the conjugate is also linked to a therapeutic agent; under conditions and for a time sufficient for the first and second components to undergo an inverse electron demand Diels-Alder reaction, thereby delivering the therapeutic agent to the target.   
     
     
         13 . The method of  claim 12 , wherein the therapeutic agent is a small molecule, enzyme inhibitor, receptor protein inhibitor, small interfering RNA (siRNA), cytotoxin, radioactive ion, or other therapeutic agent. 
     
     
         14 . The composition of  claim 5 , wherein the cytotoxin is selected from the group consisting of taxol, cytochalasin B, gramicidin D, ethidium bromide, emetine, mitomycin, etoposide, tenoposide, vincristine, vinblastine, colchicin, doxorubicin, daunorubicin, dihydroxy anthracin dione, mitoxantrone, mithramycin, actinomycin D, 1-dehydrotestosterone, glucocorticoids, procaine, tetracaine, lidocaine, propranolol, puromycin, maytansinoids, and analogs or homologs thereof. 
     
     
         15 . The composition of  claim 5 , wherein the radioactive ion is selected from the group consisting of iodine 125, iodine 131, strontium 89, phosphorous, palladium, cesium, iridium, phosphate, cobalt, yttrium 90, Samarium 153, and praseodymium. 
     
     
         16 . The composition of  claim 5 , wherein the therapeutic agent is selected from the group consisting of antimetabolites, alkylating agents, anthracyclines, antibiotics, and anti-mitotic agents. 
     
     
         17 . A composition comprising a compound selected from the group consisting of 
       
         
           
           
               
               
           
         
       
     
     
         18 . A tetrazine comprising with a linker selected from the group consisting of an aromatic ring and unsaturated alkane; and a reactive group selected from the group consisting of amine and carboxylic acid. 
     
     
         19 . The tetrazine of  claim 18 , wherein the tetrazine further comprises H, CH 3 , or a π-electron-deficient aromatic ring.