Genetically-encoded macrocyclic peptide libraries bearing a pharmacophore
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Solution Overview
Problem
Current methods for producing genetically-encoded macrocyclic peptides face challenges in introducing reactive groups under benign conditions, as they often require harsh chemicals or conditions that damage the integrity of the peptide or nucleic acid libraries, and existing functionalization methods are slow and inefficient.
Innovation Solution
A method involving the reaction of a polypeptide with reactive groups X1 and X2, using a compound with reactive groups Y1 and Y2 to form covalent bonds, allowing for the formation of macrocyclic peptides with diverse functionalities in biocompatible aqueous conditions, using diketones and hydrazines to introduce pharmacophores.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Adaptability or versatility
If traditional methods (oxime formation, azido/alkyne modification) are used to introduce reactive groups into macrocyclic peptides, then diverse functionalities can be introduced, but the integrity of the genetically-encoded library is destroyed due to harsh conditions (acidic conditions, toxic catalysts, redox active metals)
Solution Approach 1:
The patent uses a two-stage reaction system where a first reactive group (haloalkane) forms a stable intermediate macrocyclic structure, then a second reactive group (epoxide, oxirane, or aziridine) introduces the desired pharmacophore. This intermediary macrocyclic structure with embedded reactive groups allows functionalization without exposing the library to harsh conditions, thus preserving library integrity while achieving functional diversity.
Solution Approach 2:
The patent performs preliminary macrocyclization using haloalkane reagents under mild conditions to form stable cyclic structures with embedded reactive groups (epoxide, oxirane, aziridine). This preliminary action creates a protected intermediate that can subsequently be functionalized without damaging the library, resolving the contradiction between functional diversity and library integrity.
2Ease of manufacture
If prolonged incubation in acidic conditions is used to form oxime bonds, then macrocyclization can be achieved, but the integrity of the phage library is damaged
Solution Approach 1:
The patent changes the reaction parameters from traditional acidic conditions to mild physiological pH conditions by using haloalkane reagents that react with primary amines under neutral to slightly basic conditions. This parameter change enables efficient macrocyclization without damaging phage viability, resolving the contradiction between manufacturing ease and reliability.
3Adaptability or versatility
If redox active metals (copper) are used to modify azido or alkyne groups, then unnatural pharmacophores can be introduced, but nucleic acid integrity is destroyed via radical oxidative processes
Solution Approach 1:
The patent converts the harmful effect of requiring harsh modification conditions into a benefit by designing a system where the macrocyclization itself creates a protected structure with embedded reactive groups. The reactive groups (epoxide, oxirane, aziridine) are introduced during the benign macrocyclization process, eliminating the need for subsequent harsh metal-catalyzed modifications that would damage nucleic acids.
4Adaptability or versatility
If late-stage functionalization of ketone-macrocycle is performed, then diverse glycans can be introduced, but the process is slow (up to 24 hours) and requires acidic conditions detrimental to phage viability
Solution Approach 1:
The patent performs preliminary introduction of reactive groups (epoxide, oxirane, aziridine) during the macrocyclization step under mild conditions. This preliminary action creates a pre-functionalized macrocyclic structure that can undergo rapid subsequent functionalization without requiring prolonged acidic incubation, thus improving productivity while maintaining versatility.
Solution Approach 2:
The patent changes the reaction conditions from prolonged acidic incubation to rapid reactions at physiological pH by using epoxide/oxirane/aziridine reactive groups. These groups react rapidly with nucleophiles under mild conditions, eliminating the need for slow, acidic prolonged incubation and thereby improving functionalization speed while preserving phage viability.
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 rapid and stable functionalization of macrocyclic peptides in benign aqueous conditions, preserving the integrity of the peptides and nucleic acids, and allows for the creation of mixed libraries with identifiable pharmacophores for targeted binding.
Implementation Method 1
using a compound with reactive groups Y1 and Y2 to form covalent bonds
Implementation Method 2
using diketones and hydrazines to introduce pharmacophores
Data Source
AI summary
The present invention relates to a method of forming a macrocyclic peptide bearing a pharmacophore and said produced macrocyclic peptide, wherein the method comprises the steps of: reacting a peptide with two thiol groups of cysteine side chains with the reactive compound 1,5-dichloropentanedion-2,4. The reaction between the reactive compound and the peptide produces an 1,3-diketone-containing macrocyclic polypeptide. The macrocycle with a 1,3-diketone group is then modified by reaction of said macrocycle with an alkyl or aryl hydrazine group bearing a pharmacophore in benign aqueous conditions. The macrocycles may be displayed in a library, such as a phage display library, and used to biopan for affinity against a selected target.