For the first time, scientists are able to create new carbon-carbon bonds in proteins, opening a universe of possibilities for medicine and basic science.
Proteins are the workhorses of life, governing everything from how our cells communicate to how we fight disease. For decades, scientists have sought to engineer these molecular machines to enhance their natural abilities or赋予 them entirely new functions. Traditional methods, however, have been limited—like having a toolkit that only allows for minor adjustments rather than true redesign.
This isn't science fiction—it's the emerging frontier of radical-based protein modification, a transformative method that uses carbon-carbon bond formation to decorate proteins with an astonishing variety of chemical groups.
This breakthrough promises to unlock new understandings of biological processes and pave the way for next-generation protein therapeutics with precision previously thought impossible.
Protein modification is a practical strategy for enhancing functional characteristics and expanding commercial applications of proteins 2 . Before delving into radical methods, it's crucial to understand why scientists pursue protein modification in the first place.
Traditional approaches like pH-shifting and succinylation for protein modification.
Approaches such as sonication to alter protein structure and function.
Enzymatic cross-linking and other biological techniques for protein engineering.
The limitations of these conventional approaches become apparent when precision is required. Many traditional methods lack site-specificity, potentially modifying multiple locations on a protein and creating heterogeneous mixtures. This is where radical protein modifications represent a paradigm shift, offering unprecedented control and versatility.
In 2016, Benjamin G. Davis and his team at the University of Oxford unveiled a groundbreaking method called "chemical mutagenesis" 6 . This represented the first general technique for creating new carbon-carbon (C-C) bond linkages in proteins, allowing researchers to add a wide range of modifying groups with precision previously thought impossible.
Researchers first insert the unnatural amino acid dehydroalanine into the protein at the specific site they wish to modify. This can be achieved through genetic engineering, enzymatic methods, or chemical means 6 .
Dehydroalanine contains a reactive carbon-carbon double bond (C=C) that readily reacts with radicals. Scientists then introduce carbon-centered radicals from various donor molecules, which add across this double bond, forming stable new carbon-carbon bonds 6 .
This radical chemistry is "bioorthogonal"—meaning it tolerates aqueous biological conditions and remains unreactive with most natural functional groups found in biomolecules 6 . This compatibility with biological systems is crucial for studying proteins in their native environments.
The Davis group's use of dehydroalanine as a radical acceptor enables C–C bond-forming reactions with an enormous range of radical precursor substrates. It's a potential game changer for the field of protein chemistry.
The 2016 study published in Science demonstrated the remarkable versatility of this approach. The researchers successfully attached more than twenty-five different natural and unnatural sidechains and post-translational modifications to specific sites in eight different proteins 6 .
Specific serine or cysteine residues in the target protein were identified as modification sites.
These residues were converted to dehydroalanine through a selective elimination process.
Various radical precursors were prepared, including those containing sugars, fluorinated groups, and isotopic labels.
Radicals were generated and added to proteins under mild conditions, with modifications verified by mass spectrometry.
The results were staggering in their scope. The team demonstrated attachments including:
Lysozyme with Phosphonate group
Subtilisin with Glycosyl groups
Nanoluc Luciferase with Fluorescent tags
Histone Protein with Methylated lysine analog
| Modification Type | Examples | Potential Applications |
|---|---|---|
| Side Chain Replacements | Unnatural amino acids, fluorinated groups | Protein engineering, stability enhancement |
| Post-translational Modifications | Methyl groups, phosphates, acetyl groups | Studying cellular signaling pathways |
| Glycosylation | Single sugars, complex O-linked and N-linked glycans | Understanding protein-carbohydrate interactions |
| Labels and Tags | Isotopic labels, fluorescent probes | Protein tracking, imaging, and quantification |
Source: Davis et al., Science (2016) 6
Entering this field requires familiarity with both novel reagents and adapted classics. Below is a essential toolkit for radical-based protein modification approaches.
| Reagent/Category | Function | Role in Radical Modifications |
|---|---|---|
| Dehydroalanine | Unnatural amino acid handle | Serves as radical acceptor for C-C bond formation |
| Radical Precursors | Sources of carbon-centered radicals | Provide diverse functional groups for attachment |
| Transition Metal Catalysts | Generate radicals from precursors | Often used under mild, biocompatible conditions |
| Traut's Reagent | Adds sulfhydryl groups to primary amines | Traditional tool for introducing reactive handles 4 |
| SATA/SATP | Adds protected sulfhydryl groups to amines | Creates sites for subsequent conjugation chemistry 4 |
| PEGylation Reagents | Attaches polyethylene glycol chains | Improves solubility, stability; reduces immunogenicity 4 |
Since the initial breakthrough, the field has continued to evolve rapidly. Recent developments include more efficient radical generation systems and expanded applications.
A 2025 study from POSTECH demonstrates how the combination of deoxyoxanosine (dOxa) with aptamers enables precise modification of specific proteins in live biological environments .
Professor Seung Soo Oh highlighted the broad impact: "This technology will be widely utilized in fields such as protein-based therapeutics, bioimaging, and targeted drug delivery."
When combined with other methods, it could be particularly valuable for understanding the effects of modifying individual proteins with multiple groups at multiple sites.
Radical protein modification represents more than just a technical achievement—it fundamentally expands our ability to engineer biological systems at the molecular level.
Creating biotherapeutics with enhanced properties and specific targeting
Developing tools to decipher complex biological processes
Engineering enzymes with superior catalytic capabilities
The ability to rewrite protein structure with radical precision moves us closer to a future where we can not only understand life's molecular machinery but redesign it to overcome disease, probe biological mysteries, and create entirely new biological functions. The protein revolution is here, and it's being built one carbon-carbon bond at a time.