Macrocyclic Peptide Display Using Non-Reducible Thioether Bridges
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Solution Overview
Problem
Existing methods for generating macrocyclic peptides are limited by the need for chemical or enzymatic cyclization, which can be unstable and result in undesired products, and do not allow for the display of genetically encoded macrocyclic peptides with non-reducible linkages on biological surfaces.
Innovation Solution
A method involving the use of non-canonical amino acids with side-chain functional groups that react with cysteine residues to form non-reducible thioether bridges, enabling the display of macrocyclic peptides on phage or cell surfaces through ribosomal synthesis, allowing for high-throughput screening of combinatorial libraries.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If chemical or enzymatic cyclization methods are used to generate macrocyclic peptides, then macrocyclic structures can be formed, but the linkages are unstable and result in undesired products
Solution Approach 1:
The patent incorporates non-canonical amino acids with reactive side-chain functional groups (such as halogens, epoxides, or aziridines) into the polypeptide chain during ribosomal synthesis. These pre-installed functional groups are positioned to react with cysteine residues, enabling intramolecular cyclization to form stable thioether linkages without requiring external chemical or enzymatic cyclization agents, thereby avoiding instability and undesired products
Solution Approach 2:
The patent uses non-canonical amino acids as intermediaries that carry reactive functional groups on their side chains. These intermediaries facilitate the formation of stable thioether linkages between cysteine residues through controlled intramolecular reactions, replacing unstable chemical or enzymatic cyclization methods and preventing the formation of undesired products
2Adaptability or versatility
If existing cyclization methods are used, then macrocyclic peptides can be generated, but they cannot be displayed on biological surfaces through ribosomal synthesis
Solution Approach 1:
The patent merges ribosomal synthesis with macrocyclic peptide generation by incorporating non-canonical amino acids directly into the polypeptide chain during translation. The reactive side-chain functional groups of these non-canonical amino acids enable intramolecular cyclization with cysteine residues, combining the advantages of genetic encoding with stable macrocyclic structure formation, and allowing display on biological surfaces such as phage or cell surfaces
Solution Approach 2:
The patent creates a universal system where non-canonical amino acids with reactive side chains serve multiple functions: they are incorporated during ribosomal synthesis, provide the reactive group for cyclization, and enable the resulting macrocyclic peptides to be displayed on various biological surfaces (phage, cell surfaces) without requiring separate chemical or enzymatic cyclization steps
3Productivity
If large libraries of macrocyclic peptides are to be screened, then high-throughput screening is needed, but existing methods lack efficient display systems
Solution Approach 1:
The patent enables macrocyclic peptides to self-cyclize through intramolecular reaction between the reactive side-chain functional group of the non-canonical amino acid and cysteine residues within the same polypeptide chain. This self-service cyclization occurs during or after ribosomal synthesis, eliminating the need for complex external cyclization apparatus or procedures, and facilitating high-throughput screening of large macrocyclic peptide libraries on biological surfaces
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
Enables the efficient production and screening of large libraries of macrocyclic peptides with desired properties, overcoming the limitations of existing methods by providing stable, non-reducible linkages and facilitating the identification of bioactive compounds.
Implementation Method 1
A method involving the use of non-canonical amino acids with side-chain functional groups that react with cysteine residues to form non-reducible thioether bridges
Data Source
AI summary
Methods and compositions are provided for the display of genetically encoded macrocyclic peptides on a biological surface. Also provided are nucleic acid molecules, polypeptides, and methods for generating combinatorial libraries of macrocyclic peptides displayed on a biological surface. These methods can be used to produce and screen vast libraries of conformationally constrained peptides in a high-throughput manner, from which macrocyclic peptides with a desired property can be selected and identified.