Chemical biology is a rapidly evolving field that seeks to understand biological systems at the molecular level. One of the key challenges in this field is the ability to selectively modify or monitor biomolecules in complex biological environments. Recently, the concept of bioorthogonal handle has emerged as a powerful tool to address this challenge. In this article, we will explore what bioorthogonal handles are, how they work, their benefits, and their applications. We will also discuss the differences between bioorthogonal chemistry and click chemistry, a related but distinct concept. Finally, we will examine the incorporation of bioorthogonal handles into biomolecules.

What are Bioorthogonal Handles?

Bioorthogonal handles are small molecular motifs that can be selectively reacted with a complementary reagent in a biological context. The term "bioorthogonal" was coined by Carolyn Bertozzi and her colleagues to describe chemical reactions that can occur inside living systems without interfering with native biological processes. These handles are typically small, minimally perturbing, and inert until reacted with their corresponding partner. They serve as a kind of "chemical zip code" that allows for the specific modification or labeling of biomolecules in complex milieus.

How Do Bioorthogonal Handles Work?

Bioorthogonal handles work by providing a unique chemical functionality that can be selectively targeted by a complementary reagent. These handles are designed to be bioorthogonal, meaning they do not react with endogenous biomolecules or metabolic intermediates. Upon introduction of the corresponding reagent, the handle and reagent undergo a highly selective and efficient chemical reaction. This reaction can result in the covalent modification or labeling of the biomolecule of interest.

One of the most well-known examples of a bioorthogonal handle is the azide moiety. Azides are small, minimally perturbing, and bioorthogonal, meaning they do not react with endogenous biomolecules. They can be selectively reacted with cyclooctynes or strain-promoted alkyne tags in a process known as the Staudinger ligation or copper-free click chemistry. This reaction results in the covalent labeling of the azide-modified biomolecule.

Benefits of Bioorthogonal Handles

Bioorthogonal handles offer several key benefits that make them powerful tools in chemical biology. First, they allow for the selective modification or labeling of biomolecules in complex biological environments. This is in contrast to traditional biochemical methods that often rely on the spatial or temporal separation of biomolecules. Second, bioorthogonal handles are minimally perturbing, meaning they do not significantly alter the structure or function of the modified biomolecule. This allows for the study of biomolecules in a more native-like state. Finally, bioorthogonal handles can be selectively reacted with a variety of reagents, allowing for the multiplexed modification or labeling of biomolecules.

Applications of Bioorthogonal Handles

Bioorthogonal handles have found numerous applications in chemical biology. One of the most prominent applications is in the imaging of biomolecules in live cells or organisms. Bioorthogonal handles can be incorporated into biomolecules of interest, which can then be selectively labeled with fluorescent or affinity reagents. This allows for the real-time imaging of biomolecule dynamics and localization.

Bioorthogonal handles have also been used in the proteomic profiling of enzyme activity. Handles can be designed to selectively react with active enzymes, allowing for the enrichment and identification of active proteases or kinases. This can provide insights into enzyme function and regulation in complex biological contexts.

Bioorthogonal vs. Click Chemistry

Bioorthogonal chemistry and click chemistry are related but distinct concepts. Click chemistry refers to a set of highly efficient and selective chemical reactions that can occur under mild conditions. These reactions are often used to conjugate small molecules or tags to biomolecules. The Staudinger ligation and copper-free click chemistry, which involve the reaction of azides with cyclooctynes or strain-promoted alkynes, are examples of click chemistry reactions.

Bioorthogonal chemistry, on the other hand, refers to the design of chemical reactions that can occur inside living systems without interfering with native biological processes. These reactions involve the use of bioorthogonal handles that are inert until reacted with their corresponding partner. While there is some overlap between the two fields, not all click chemistry reactions are bioorthogonal. For example, the copper-catalyzed azide-alkyne cycloaddition is a click chemistry reaction, but it is not bioorthogonal due to the toxicity of copper to cells.

Incorporation of Bioorthogonal Handles

Bioorthogonal handles can be incorporated into biomolecules using a variety of strategies. One common approach is to use metabolic labeling, where biomolecules are fed analogues containing the bioorthogonal handle. For example, azidohomoalanine (AHA) is a methionine analogue containing an azide handle that can be incorporated into proteins during translation. Upon incorporation, the AHA-labeled proteins can be selectively reacted with cyclooctyne or strain-promoted alkyne reagents.

Another approach is to use genetic encoding, where the genetic code is expanded to incorporate non-canonical amino acids (ncAAs) containing the bioorthogonal handle. For example, the amber stop codon can be used to site-specifically incorporate azidolysine or alkynyllysine into proteins. These ncAAs can then be selectively reacted with complementary reagents.

Conclusion

Bioorthogonal handles are a powerful tool in the field of chemical biology. They allow for the selective modification or labeling of biomolecules in complex biological environments, providing insights into biomolecule dynamics and function. While related to click chemistry, bioorthogonal chemistry is a distinct field that seeks to design chemical reactions that can occur inside living systems without interfering with native biological processes. As the field continues to evolve, we can expect to see the development of new bioorthogonal handles and their application in a wide range of biological contexts.