Fluorinated Acetamide Reporter for Steric-Free Protein Labeling
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Solution Overview
Problem
Current chemical reporters for protein labeling are bulky, limiting their application due to steric hindrance, and existing bioorthogonal reactions are not effective for global profiling of molecular targets, particularly for enzymes with small active site pockets.
Innovation Solution
A novel bioorthogonal reaction method involving compounds of specific structures (Formula I and II) is used to staple amino acid sequences, allowing for steric-free labeling of protein substrates and global profiling of molecular targets.
Engineering Contradictions & Design Principles
Engineering Contradiction Analysis
1Reliability
If current chemical reporters (azide-alkyne cycloaddition, Staudinger ligation, tetrazine cycloaddition) are used for protein labeling, then successful development of chemical reporters is achieved, but the reporters are bulky in length and size causing steric hindrance that limits their application
Solution Approach 1:
The patent changes the chemical parameters of the reporter groups by using fluorinated acetamides and thiols instead of traditional azide-alkyne or tetrazine systems. This parameter change results in smaller molecular volume while maintaining bioorthogonal reaction capability, directly resolving the contradiction between reliability and bulkiness
Solution Approach 2:
The patent extracts the essential bioorthogonal reaction capability from the bulky traditional reporter systems and implements it through minimal fluorinated acetamide-thiol pairs. By taking out only the necessary functional groups and removing unnecessary bulk, the invention achieves reliable labeling with minimal steric hindrance
2Volume of moving object
If alkyne- or azide-based minimalist reporters for copper-catalyzed azide-alkyne cycloaddition are used, then reporter size is reduced, but they are still larger than the inherent carbon-hydrogen bond causing steric hindrance
Solution Approach 1:
The patent further reduces reporter size by changing from carbon-based alkyne/azide reporters to fluorinated acetamide-thiol systems. The fluorinated acetamide group is smaller than the inherent carbon-hydrogen bond, eliminating steric hindrance while maintaining reaction capability through fluorine's unique properties
3Reliability
If chemical reporters are used for metabolic incorporation by enzymes, then labeling of protein substrates is achieved, but the bulky reporters are too large to be incorporated by many cognate transferases with spacious active site pockets
Solution Approach 1:
The patent changes the size parameter of the metabolic reporter from bulky azide/alkyne groups to minimal fluorinated acetamide groups. This size reduction allows the reporters to fit into the active site pockets of cognate transferases, dramatically improving adaptability while maintaining reliable labeling through bioorthogonal chemistry
Solution Approach 2:
The fluorinated acetamide-thiol system serves multiple functions: it acts as a metabolic substrate for transferases, incorporates into proteins, and enables bioorthogonal labeling. This multi-functionality with minimal size enhances versatility across different enzyme systems while maintaining reliable labeling
4Adaptability or versatility
If "bump-hole" protein engineering strategy is used to accommodate bulky reporters, then incorporation by specific enzymes is achieved, but careful balances between mutations, structure folding, and function are required
Solution Approach 1:
The patent extracts the bulky reporter groups that necessitate bump-hole engineering and replaces them with minimal fluorinated acetamide reporters. By removing the bulk, the need for complex protein engineering is eliminated, reducing device complexity while maintaining enzyme adaptability
Solution Approach 2:
The patent changes the reporter size parameter to be smaller than the inherent carbon-hydrogen bond, which eliminates the need for bump-hole protein engineering. This parameter change allows direct incorporation by cognate transferases without mutations, simplifying the system while maintaining adaptability
5Adaptability or versatility
If mutations in the "hole" of protein engineering are made to accommodate bulky reporters, then incorporation is achieved, but substrate specificities are altered leading to results different from in vivo sub-acylome
Solution Approach 1:
The patent extracts and removes the bulky reporter groups that cause substrate specificity alterations. By using minimal fluorinated acetamide reporters, the natural substrate specificity of transferases is preserved, ensuring accurate in vivo sub-acylome results without requiring mutations that would alter specificity
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 efficient and precise labeling of protein substrates, overcoming the limitations of bulkiness and steric hindrance in existing reporters, facilitating the global profiling of molecular targets and potential therapeutic applications.
Implementation Method 1
The present invention provides, in part, methods of labeling, imaging, and/or detecting protein substrates using a fluorine-thiol displacement reaction
Data Source
AI summary
This invention provides compositions comprising linked amino acid sequences, pharmaceutical compositions comprising linked amino acid sequences, and methods of making thereof. This invention also provides methods of delivering said compositions to subjects and methods of treating various disorders and diseases using the said compositions.