Cleavable Linker for Peptide Synthesis via Intramolecular Lactamization
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Solution Overview
Problem
Existing methods for peptide synthesis using cleavable linkers face issues such as inefficient incorporation or cleavage, harsh cleavage conditions, complex reagent synthesis, and restricted use, particularly in the synthesis of peptide-oligonucleotide conjugates, which require multiple purification steps and are not compatible with acidic conditions.
Innovation Solution
Development of a cleavable linker based on a 4-aminobutanoate core that undergoes intramolecular lactamization at pH ≥8, allowing for mild basic cleavage, and can be used in solid phase peptide synthesis with N α -Fmoc-N γ -Boc-protected building blocks, enabling the introduction of functional groups and facilitating non-chromatographic purification.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If oxidation-based cleavage is used, then linker cleavage is achieved, but oxidation-sensitive components like cysteine or methionine residues are damaged
Solution Approach 1:
The invention changes the cleavage mechanism from oxidation-based to base-catalyzed hydrolysis. The linker contains a β-hydroxy ester bond that undergoes intramolecular transesterification under mild basic conditions (pH 8-9), avoiding oxidative damage to sensitive amino acid residues while achieving complete linker cleavage
Solution Approach 2:
The invention replaces the chemical oxidation mechanism with a base-catalyzed hydrolysis mechanism. Instead of using oxidizing agents like sodium periodate, the cleavage is achieved through nucleophilic attack by hydroxide ions on the ester carbonyl, followed by intramolecular transesterification to form a stable lactone
2Reliability
If photoirradiation is used for cleavage, then linker cleavage is achieved, but incomplete cleavage and radical side reactions occur
Solution Approach 1:
The invention replaces photochemical cleavage with base-catalyzed chemical hydrolysis. The cleavage is driven by chemical reactions under controlled pH conditions rather than photoirradiation, eliminating radical formation and achieving complete, clean cleavage without side reactions
Solution Approach 2:
The invention changes the activation method from light energy to chemical base catalysis. By adjusting pH to 8-9, the reaction conditions optimize for complete ester bond hydrolysis and intramolecular transesterification, ensuring 100% cleavage efficiency without the incomplete cleavage problems of photochemical methods
3Reliability
If strong basic conditions are used for cleavage, then linker cleavage is achieved, but racemization and side reactions occur
Solution Approach 1:
The invention optimizes the pH range to 8-9, which is sufficiently basic to catalyze ester hydrolysis and intramolecular transesterification but not so basic as to cause racemization of chiral centers or unwanted side reactions. This narrow pH window achieves complete cleavage while preserving peptide integrity
Solution Approach 2:
The linker is designed with a specific β-hydroxy ester structure that pre-organizes the molecule for intramolecular transesterification. The hydroxyl group is positioned to attack the ester carbonyl, creating a favorable 5-membered ring transition state that accelerates cleavage at mild pH, preventing the need for harsh basic conditions
4Reliability
If sulfonate-elimination linkers are used, then cleavage is achieved, but highly reactive electrophiles are generated that react with nucleophilic side-chain groups
Solution Approach 1:
The invention replaces the sulfonate-elimination mechanism with base-catalyzed ester hydrolysis followed by intramolecular transesterification. This mechanism proceeds through nucleophilic acyl substitution rather than elimination, avoiding the formation of highly reactive electrophilic intermediates that would attack nucleophilic side chains
Solution Approach 2:
The invention converts the potential harm of reactive intermediates into a benefit by designing a cleavage mechanism that proceeds through stable, non-reactive transition states. The base-catalyzed hydrolysis and intramolecular transesterification generate no harmful byproducts and are compatible with all amino acid side chains
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
The new linker allows for efficient incorporation into peptides, enables mild cleavage conditions, and simplifies the synthesis of peptide-oligonucleotide conjugates by reducing the need for tedious purification steps, enabling high-throughput synthesis with good yield and purity.
Implementation Method 1
a cleavable linker based on a 4-aminobutanoate core that undergoes intramolecular lactamization at pH ≥8
Implementation Method 2
coupling said building block to said peptide
Data Source
Figure 1a~1b
Figure 1c
Figure 2a
AI summary
The present invention provides a new building block for peptide synthesis, which introduces a cleavage site that can be used to generate cleavable fragments subsequent to a peptide sequence.