Genetically Encoded Bicyclic Peptide Libraries With Aqueous Linker Cyclization
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Solution Overview
Problem
Existing methods for generating bicyclic peptide libraries are limited by the use of specialized amino acids, protecting groups, organic solvents, and reaction conditions that are not compatible with biomolecules, leading to complex mixtures and susceptibility of N-terminal residues to proteolytic cleavage, and there is a lack of methods for producing genetically-encoded bicyclic peptides with blocked N-termini using natural amino acids.
Innovation Solution
The method involves modifying peptides with a two-fold symmetric linker that attaches to the peptide terminus and side chains of cysteine, lysine, or tyrosine residues, using reactive groups compatible with aqueous environments to form bicyclic structures without a free N-terminus, allowing for the synthesis of genetically-encoded bicyclic peptides.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Shape
If specialized amino acids, protecting groups, and organic synthesis methods are used to produce bicyclic peptide libraries, then bicyclic structures can be formed, but the N-terminal residues become susceptible to proteolytic cleavage and complex mixtures are generated
Solution Approach 1:
The invention removes the N-terminal residue from the peptide structure through selective degradation methods, eliminating the vulnerable site that causes proteolytic instability. This extraction of the problematic N-terminus while preserving the bicyclic core structure resolves the contradiction between forming bicyclic structures and maintaining proteolytic stability.
Solution Approach 2:
The method performs preliminary blocking of the N-terminal position by selective degradation before the peptide undergoes normal proteolytic degradation. By pre-removing or blocking the N-terminus through controlled chemical or enzymatic degradation, the peptide is protected against subsequent proteolytic cleavage events.
2Shape
If chemical post-translational modifications are used to produce bicyclic peptide libraries, then bicyclic structures can be formed, but the reaction conditions are not compatible with biomolecules
Solution Approach 1:
The invention changes the reaction parameters from harsh organic chemistry conditions to mild aqueous conditions that are compatible with biomolecules. By using aqueous buffers, physiological pH ranges, and temperature conditions compatible with life, the bicyclic structure formation can proceed without damaging associated biomolecules such as DNA, RNA, or proteins.
Solution Approach 2:
The method introduces intermediary substances such as water-soluble linkers and bioorthogonal functional groups that enable bicyclic structure formation in aqueous environments. These intermediaries facilitate the cyclization reaction while maintaining compatibility with biomolecules, acting as bridges between the peptide functional groups in a biocompatible medium.
3Quantity of substance
If traditional library generation methods are used, then libraries can be produced, but each library member must be separated into individual solutions and reaction vessels
Solution Approach 1:
The invention merges multiple library members into a single reaction vessel by using genetically encoded peptides displayed on phage particles or RNA molecules. Each phage or RNA molecule carries a unique genetic identifier that links the peptide sequence to its information template, allowing entire libraries to be processed, modified, and screened in bulk without separation into individual vessels.
Solution Approach 2:
The method uses genetic information templates (DNA or RNA sequences) as copies that encode each peptide member in the library. These genetic copies can be amplified and processed en masse, allowing the entire library to be manipulated through biochemical reactions while maintaining the ability to identify and retrieve individual members through their genetic sequences.
Applied Scientific Principles
This section explains which scientific principles are used to turn an abstract innovation direction into a practical engineering solution.
Function Achieved in This Case
This approach produces stable bicyclic peptides with enhanced binding interactions, avoiding proteolytic cleavage and enabling efficient screening of libraries for target molecules.
Implementation Method 1
a bicyclic structure resulting from attaching a linker having a first end reactive group (A) and a second end comprising two reactive groups (B2, B2) to the polypeptide, wherein the first end is ligated to a terminus of the polypeptide by a covalent bond, and wherein both reactive groups of the linker second end are attached to side chain residues of the polypeptide by covalent bonds
Data Source
AI summary
The invention relates to a bicyclic peptide complex comprising a peptide construct, said construct comprising (i) a polypeptide with free terminus (N or C); (ii) optionally, a nucleic acid encoding the polypeptide; (iii) a twofold-symmetric linker (TSL) compound attached to said polypeptide where the linker is attached to the terminus of polypeptide via a covalent bond and to at least two discrete side chains of the peptide. The invention also relates to libraries, and to methods for making complexes and to methods of screening using the same.