Ligands and methods for labeling biomolecules in vivo

A method of labeling a biomolecule in a living system or in a subject, the method comprising:

• (i) administering to the living system or subject a substrate specific to the biomolecule of interest wherein the substrate comprises a reporter;
• and
• (ii) administering to the living system or subject
• (a) a detectable marker that reacts covalently with the reporter,
• (b) a tris(triazolylmethyeamine ligand of formula (I), and
• (c) Cu(I) or Cu(II) in the presence of a reducing agent;
• wherein the biomolecule of interest is detectably marked, and where the ligand of formula (I) has the structure:
• wherein R1 and R2 are independently
• wherein R3 is
• wherein R4 and R5 are independently
• wherein n1, n2 and n3 are independently 2-3, and n4 is 1-2;
• and wherein ( ) is the point of attachment of the R1, R2, R3, R4 or R5 group to the ring structure.

A kit for labeling and/or detecting a biomolecule in vivo, the kit comprising a tris(triazolylmethyl)amine ligand of formula (I), wherein the ligand of formula (I) has the structure:

• wherein R1 and R2 are independently
• wherein R3 is
• wherein R4 and R5 are independently
• wherein n1, n2 and n3 are independently 2-3, and n4 is 1-2;
• and wherein ( ) is the point of attachment of the R1, R2, R3, R4 or R5 group to the ring structure.

A method of synthesizing 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanesulfonic acid (BTTES) comprising:

• i) reacting 3,3-diethoxy-1-propyne and tert-butyl azide in a mixture of tert-butyl alcohol and water in the presence of sodium bicarbonate, copper(II) sulfate pentahydrate, and sodium ascorbate to produce 1-tert-butyl-4-(diethoxymethyl)-1H-1,2,3-triazole;
• ii) combining 1-tert-butyl-4-(diethoxymethyl)-1H-1,2,3-triazole with dichloromethane followed by addition of water and trifluoroacetic acid to produce 1-tert-butyl-1H-1,2,3-triazole-4-carbaldehyde;
• iii) dissolving 1-tert-butyl-1H-1,2,3-triazole-4-carbaldehyde in dichloroethane or THF followed by addition of propargyl amine and sodium triacetoxyborohydride to produce N,N-bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl) prop-2-yn-1-amine;
• iv) combining N,N-bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl) prop-2-yn-1-amine with 2-azidoethanol in tetrahydrofuran followed by copper(I) acetate and sodium ascorbate to produce 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanol;
• and
• v) combining 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanol in pyridine with sulfur trioxide pyridine complex to produce 2-(4-((bis((1-tert-butyl-1H-1,2,3-triazol-4-yl)methyl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethanesulfonic acid.