Activated cysteine-directed polypeptide ligation technique

Inventors

Liu, Wenshe

Assignees

Texas A&M University System

Abstract

Embodiments of the present disclosure pertain to methods of conjugating a molecule to a polypeptide by (1) modifying one or more thiol residues on the polypeptide, where the modifying includes cyanylation of the one or more thiol residues; and (2) associating the polypeptide with the molecule, where the associating results in the conjugation of the molecule to the polypeptide through a reaction between a nucleophilic moiety on the molecule and the one or more modified thiol residues. The cyanylation may include attachment of cyano groups to sulfur atoms of the one or more thiol residues to form thiocyanato groups that undergo reversible intramolecular addition with a nearby N-amide group to generate a 1-acyl-2-iminothiazolidine intermediate. Thereafter, the nucleophilic moiety on the molecule reacts with the 1-acyl-2-iminothiazolidine intermediate to replace 2-iminothiazolidine in a nucleophilic acyl substitution reaction and result in the conjugation of the molecule to the polypeptide.

Core Innovation

The invention relates to an activated cysteine-directed protein ligation approach in which cyanylate thiol/cysteine residues on a polypeptide to generate thiocyanato groups. Formation of these thiocyanato groups enables reversible intramolecular addition to a nearby N-amide/cysteine N-amide, yielding a 1-acyl-2-iminothiazolidine intermediate. The intermediate supports subsequent conjugation by enabling a reaction with a nucleophilic moiety on a target molecule.

The conjugation is achieved by nucleophilic acyl substitution, where a nucleophilic moiety on the molecule reacts with the modified thiol residues. The described approach supports ligation under native and denatured conditions and is characterized as not requiring enzymatic catalysis. Thiol residues can be positioned near the C-terminus to promote the described intramolecular intermediate formation and downstream coupling.

The disclosed system is applied to conjugating molecules to polypeptides containing thiol residues within thiol-containing non-canonical amino acids, including cysteine-directed ligation workflows. The document further describes coupling examples including fluorogenic substrates for Ub/Ubl enzymes, construction of propargylamine conjugates, acetylated histone H2A-K129ac/nucleosome assembly, and installation of larger peptide/protein fragments via peptide hydrazide ligation. A therapeutic peptide application is also described involving exenatide procurement using an engineered fusion and SUMO processing.

Claims Coverage

The independent claim covers a method of conjugating a molecule to a polypeptide by cyanylation of thiol residues located in thiol-containing non-canonical amino acids, followed by conjugation through a nucleophilic-moiety reaction with the modified thiol residues. The main inventive features are further narrowed in dependent claims by specifying the cyanylation pathway and intermediates, the use of NTCB, the selected class of nucleophiles, constraints on the nucleophile options, and example polypeptide targets and non-enzymatic operation.

Overall claim coverage centers on cyanylation of thiol residues in thiol-containing non-canonical amino acids followed by conjugation via reaction between a nucleophilic moiety and the modified thiol residues. Dependent claims further specify a thiocyanato-to-reversible intramolecular addition pathway to a 1-acyl-2-iminothiazolidine intermediate, identify NTCB as a cyanylation reagent, constrain the nucleophile to specified amine/hydrazine/hydroxylamine classes, limit polypeptide targets to ubiquitin/Ubl-related proteins, and state that enzymatic catalysis is not required.

Stated Advantages

Documented Applications