Compound Library and Method for Producing Compound Library
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Solution Overview
Problem
Existing methods do not effectively utilize cell-free translation systems to construct peptide compound libraries with diverse structures by linking them to thiopeptide-biosynthesis systems, particularly due to unclear substrate tolerance of thiopeptide-biosynthetic enzymes and the lack of functional determination of enzymes like LazB-F.
Innovation Solution
A method involving cell-free translation systems and thiopeptide-biosynthesis systems to synthesize peptide compounds with diverse structures using predetermined thiopeptide-biosynthetic enzymes, including steps for azole ring formation, α,β-unsaturated amino acid conversion, and macrocyclization to form nitrogen-containing 6-membered rings.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Productivity
If cell-free translation systems are used to synthesize peptides, then synthesis speed and convenience are improved, but the ability to construct diverse peptide compound libraries linked to thiopeptide-biosynthesis systems is insufficient
Solution Approach 1:
The invention creates a universal platform that integrates cell-free translation systems with thiopeptide-biosynthesis systems. The cell-free translation system is designed to accept diverse mRNA templates encoding various precursor peptides, while the thiopeptide-biosynthesis enzymes (LazB-F) process these diverse substrates to produce structurally diverse thiopeptide compounds. This multi-functional integration enables both rapid synthesis and library diversity construction simultaneously
Solution Approach 2:
The invention segments the peptide synthesis and modification processes into distinct functional modules: (1) cell-free translation system for rapid peptide synthesis from diverse mRNA templates, (2) LazB-F enzyme complex for post-translational modifications including azole ring formation and macrocyclization. This segmentation allows each module to be optimized independently while maintaining overall system versatility and productivity
2Adaptability or versatility
If thiopeptide-biosynthetic enzymes are used for peptide modification, then diverse thiopeptide structures can be produced, but substrate tolerance of these enzymes is unclear
Solution Approach 1:
The invention employs a dynamic screening approach where the LazB-F enzyme system is exposed to a diverse library of precursor peptides generated by the cell-free translation system. Through this dynamic interaction, the actual substrate tolerance and specificity of the enzymes are empirically determined. The system adapts to different substrates, and successful modifications are identified and characterized, thereby defining the reliable substrate range of the enzymes
Solution Approach 2:
The cell-free translation system automatically generates diverse precursor peptide substrates that are directly fed to the LazB-F enzyme system without requiring manual substrate preparation or selection. The system self-determines which substrates are suitable through the natural enzymatic reactions and subsequent activity screening, eliminating the need for prior knowledge of enzyme substrate preferences
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 rapid and convenient production of a diverse peptide compound library, facilitating screening for compounds binding to target substances and analysis of structure-activity relationships.
Implementation Method 1
Cell-free translation systems are systems for synthesizing peptides or proteins of interest in vitro through the use of protein-synthesis functions extracted from cells
Implementation Method 2
reacting an enzyme for forming azole ring(s) with the first peptide library, so that at least an azole ring is formed on Y′(11) in the precursor peptides
Implementation Method 3
reacting an enzyme for forming α,β-unsaturated amino acid(s) with the second peptide library, so that at least X′ and Y′(12) in the precursor peptides are converted to α,β-unsaturated amino acid residues
Implementation Method 4
reacting a macrocyclase with the third peptide library, so that a macrocycle is formed by macrocyclization to form the two or more cyclic compounds
Data Source
AI summary
Disclosed is a method for producing a compound library comprising two or more cyclic compounds represented by the formula (I), comprising a step of allowing a macrocyclase in vitro to act on two or more peptides represented by the formula (II): LP-X—(Xa)m-Y—Z (II) wherein X represents a group represented by the formula (1), Y is a peptide residue consisting of four amino acids and/or analogs thereof and contains a group represented by the formula (2) (wherein R1 and B1 are as defined above, and R3 represents a hydrogen or a hydrocarbon group), and LP is present or absent and, when present, represents a peptide residue consisting of 1 to 100 amino acids and/or analogs thereof, and forming the nitrogen-containing 6-membered ring A while eliminating LP, if present, to form the two or more cyclic compounds represented by the formula (I).