Binding-site modified lectins and uses thereof

Abstract

In one aspect, the disclosure provides cross-linked materials that include multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one covalently linked affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites; and conjugates that include two or more separate affinity ligands bound to a conjugate framework, wherein the two or more affinity ligands compete with glucose for binding with the lectins at said binding sites and wherein conjugates are cross-linked within the material as a result of non-covalent interactions between lectins and affinity ligands on different conjugates. These materials are designed to release amounts of conjugate in response to desired concentrations of glucose. Depending on the end application, in various embodiments, the conjugates may also include a drug and/or a detectable label.

1 . A cross-linked material comprising:
 multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one covalently linked affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites; and   conjugates that include two or more separate affinity ligands bound to a conjugate framework, wherein the two or more affinity ligands compete with glucose for binding with the lectins at said binding sites and wherein conjugates are cross-linked within the material as a result of non-covalent interactions between lectins and affinity ligands on different conjugates.   
     
     
         2 . The material of  claim 1 , wherein the conjugates further comprise a drug bound to the conjugate framework. 
     
     
         3 - 6 . (canceled) 
     
     
         7 . The material of  claim 1 , wherein the lectins are Con A lectins. 
     
     
         8 . (canceled) 
     
     
         9 . The material of  claim 1 , wherein the affinity ligands that are covalently bound to the lectins include a saccharide. 
     
     
         10 . The material of  claim 9 , wherein the saccharide is glucose. 
     
     
         11 . (canceled) 
     
     
         12 . The material of  claim 9 , wherein the affinity ligands that are covalently bound to the lectins include a saccharide and a linker and the saccharide is covalently bound to the linker via an anomeric carbon. 
     
     
         13 - 17 . (canceled) 
     
     
         18 . The material of  claim 12 , wherein the affinity ligands were covalently bound to the lectins using a photoactivatable linker of the formula: 
       
         
           
           
               
               
           
         
         wherein: 
         R 3  is independently selected from the group consisting of hydrogen, —OH, —NO 2 , and halogen; 
         X L  is a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-20  hydrocarbon chain wherein one or more methylene units of X L  are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R′)C(O)—, —C(O)N(R′)—, —S(O)—, —S(O) 2 —, —N(R′)SO 2 —, —SO 2 N(R′)—, a heterocyclic group, an aryl group, or a heteroaryl group; and 
         each occurrence of R′ is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety. 
       
     
     
         19 - 34 . (canceled) 
     
     
         35 . The material of  claim 12 , wherein the affinity ligands were covalently bound to the lectins using a photoactivatable linker of the formula: 
       
         
           
           
               
               
           
         
         R 4  is hydrogen, C 1 -C 6  alkyl or —CF 3 ; 
         X L  is a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-20  hydrocarbon chain wherein one or more methylene units of X L  are optionally and independently replaced by —O—, —S—, —N(R′)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R′)C(O)—, —C(O)N(R′)—, —S(O)—, —S(O) 2 —, —N(R′)SO 2 —, —SO 2 N(R′)—, a heterocyclic group, an aryl group, or a heteroaryl group; and 
         each occurrence of R′ is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety. 
       
     
     
         36 - 53 . (canceled) 
     
     
         54 . The material of  claim 1 , wherein the affinity ligands of the conjugates include a saccharide. 
     
     
         55 . The material of  claim 54 , wherein the affinity ligands of the conjugates include a saccharide selected from glucose, mannose, glucosamine, mannosamine, methylglucose, methylmannose, ethylglucose, and ethylmannose. 
     
     
         56 . The material of  claim 54 , wherein the affinity ligands of the conjugates include a bimmanose or trimannose. 
     
     
         57 . The material of  claim 54 , wherein the affinity ligands of the conjugates include aminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose (AEBM) or aminoethyltrimannose (AETM). 
     
     
         58 - 67 . (canceled) 
     
     
         68 . The material of  claim 1 , wherein the conjugate has the general formula: 
       
         
           
           
               
               
           
         
         wherein: 
         R x  is hydrogen or optionally substituted C 1-6  alkyl; 
         Z 1  is an optionally substituted bivalent C 1-10  hydrocarbon chain, wherein 1, 2, 3, 4 or 5 methylene units of Z 1  are optionally and independently replaced with one or more groups selected from —S—, —O—, —NR a —, —(C═NR a )—, —(C═O)—, —(S═O)—, —S(═O) 2 —, —(CR b ═CR b )—, —(N═N)—, an optionally substituted arylene moiety or an optionally substituted heteroarylene moiety, wherein R a  is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, or a suitable amino protecting group; and R b  is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, or optionally substituted heteroaryl; 
         each occurrence of X 1  is independently —OR c  or —N(R d ) 2 , wherein R c  is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, a suitable hydroxyl protecting group, a cation group, or an affinity ligand, and each R d  is, independently, hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, a suitable amino protecting group, or an affinity ligand, with the proviso that at least two occurrences of X 1  include an affinity ligand; 
         Y 1  is hydrogen, halogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, optionally substituted heteroaryl, —OR e  or —SR e  wherein R e  is hydrogen, optionally substituted aliphatic, optionally substituted heteroaliphatic, optionally substituted aryl, or optionally substituted heteroaryl; 
         r is an integer between 5-25, inclusive; 
         W 1  is a drug or detectable label; and 
            corresponds to a single or double covalent bond. 
       
     
     
         69 . The material of  claim 1 , wherein the conjugate has the general formula: 
       
         
           
           
               
               
           
         
         wherein: 
         each occurrence of 
       
                         
 represents a potential branch within the conjugate;
 each occurrence of 
 
                         
 represents a potential repeat within a branch of the conjugate;
 each occurrence of   is independently a covalent bond, a carbon atom, a heteroatom, or an optionally substituted group selected from the group consisting of acyl, aliphatic, heteroaliphatic, aryl, heteroaryl, and heterocyclic; 
 each occurrence of T is independently a covalent bond or a bivalent, straight or branched, saturated or unsaturated, optionally substituted C 1-30  hydrocarbon chain wherein one or more methylene units of T are optionally and independently replaced by —O—, —S—, —N(R)—, —C(O)—, —C(O)O—, —OC(O)—, —N(R)C(O)—, —C(O)N(R)—, —S(O)—, —S(O) 2 —, —N(R)SO 2 —, —SO 2 N(R)—, a heterocyclic group, an aryl group, or a heteroaryl group; 
 each occurrence of R is independently hydrogen, a suitable protecting group, or an acyl moiety, arylalkyl moiety, aliphatic moiety, aryl moiety, heteroaryl moiety, or heteroaliphatic moiety; 
 —B is -T-L B -X; 
 each occurrence of X is independently an affinity ligand; 
 each occurrence of L B  is independently a covalent bond or a group derived from the covalent conjugation of a T with an X; 
 -D is -T-L D -W; 
 each occurrence of W is independently a drug or a detectable label; 
 each occurrence of L D  is independently a covalent bond or a group derived from the covalent conjugation of a T with a W; 
 k is an integer from 2 to 11, inclusive, defining at least two k-branches within the conjugate; 
 q is an integer from 1 to 4, inclusive; 
 k+q is an integer from 3 to 12, inclusive; 
 each occurrence of p is independently an integer from 1 to 5, inclusive; and 
 each occurrence of n is independently an integer from 0 to 5, inclusive; and 
 each occurrence of m is independently an integer from 1 to 5, inclusive; and 
 each occurrence of v is independently an integer from 0 to 5, inclusive, with the proviso that within each k-branch at least one occurrence of n is ≧1 and at least one occurrence of v is ≧1. 
 
     
     
         70 - 72 . (canceled) 
     
     
         73 . The material of  claim 69 , wherein at least two occurrences of X include an affinity ligand that comprises a saccharide. 
     
     
         74 . The material of  claim 69 , wherein at least two occurrences of X include an affinity ligand that comprises a saccharide selected from the group consisting of glucose, mannose, glucosamine, mannosamine, methylglucose, methylmannose, ethylglucose, and ethylmannose. 
     
     
         75 . The material of  claim 69 , wherein at least two occurrences of X include an affinity ligand that comprises a bimmanose or a trimannose. 
     
     
         76 . The material of  claim 69 , wherein at least two occurrences of X include an affinity ligand selected from aminoethylglucose (AEG), aminoethylmannose (AEM), aminoethylbimannose (AEBM) and aminoethyltrimannose (AETM). 
     
     
         77 - 84 . (canceled) 
     
     
         85 . A method comprising administering a material of  claim 1  to a patient. 
     
     
         86 . (canceled) 
     
     
         87 . The method of  claim 85 , wherein the conjugates comprise an insulin molecule bound to the framework. 
     
     
         88 - 94 . (canceled) 
     
     
         95 . A method comprising steps of:
 (I) mixing:
 (a) multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one covalently linked affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites and the lectins include a first label which generates a measurable response when in close proximity to a second label, and 
 (b) conjugates that comprise an affinity ligand and the second label; 
   (II) exposing a sample to the mixture of multivalent lectins and conjugates, wherein:
 (a) if glucose is absent from the sample, the conjugates form a cross-linked material with the lectins through affinity binding to the multivalent lectins to produce a measurable response, and 
 (b) if glucose is present in the sample, the response is reduced because formation of cross-linked material is inhibited as a result of glucose from the sample competing with the conjugates for the binding sites on the multivalent lectins; and 
   (III) detecting and optionally measuring the response with a sensor to determine the presence and optionally the amount of glucose in the sample.   
     
     
         96 . A method comprising steps of:
 (I) mixing:
 (a) multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one covalently linked affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites, 
 (b) a first group of molecules that comprise an affinity ligand and a first label which generates a measurable response when in close proximity to a second label, and 
 (c) a second group of molecules that comprise an affinity ligand and the second label; 
   (II) exposing a sample to the mixture of multivalent lectins, and first and second groups of molecules, wherein:
 (a) if glucose is absent from the sample, members of the first and second group of molecules are brought in close proximity through affinity binding to the multivalent lectins to produce a binding complex and a measurable response, and 
 (b) if glucose is present in the sample, the response is reduced because fewer of said binding complexes form as a result of glucose from the sample competing with the first and second molecules for the binding sites on the multivalent lectins; and 
   (III) detecting and optionally measuring the response with a sensor to determine the presence and optionally the amount of glucose in the sample.   
     
     
         97 . A method comprising steps of:
 (I) providing:
 (a) conjugates that comprise a plurality of affinity ligands, and 
 (b) multivalent lectins with at least two binding sites for glucose, wherein the lectins include at least one covalently linked affinity ligand which is capable of competing with glucose for binding with at least one of said binding sites; 
   (II) mixing the conjugates and lectins, wherein the viscosity of the resulting mixture is due to the binding between the conjugates and lectins;   (III) contacting the mixture with a sample containing glucose which displaces conjugates from the lectins and causes a concentration dependent reduction in viscosity; and   (IV) detecting and optionally measuring the resulting change in viscosity to determine the presence and optionally the amount of glucose in the sample.