.png)
TRACE allows chemistry to occur only in selected cells. Enzyme-activated tetrazine cages enable targeted cell death (left) and targeted fluorescent labeling (right).
Image Credit: Devaraj lab / UC San Diego
Scientific Frontline: Extended "At a Glance" Summary: Programmable Chemistry (TRACE Method)
The Core Concept: TRACE (tetrazine release and activation by cellular enzymes) is a novel bioorthogonal chemical method that locks reactive molecules inside protective cages until they are released by enzymes specific to diseased cells.
Key Distinction/Mechanism: Unlike traditional bioorthogonal "click chemistry," where tetrazine reactions can act indiscriminately across various cell types, TRACE uses molecular cages to keep the tetrazine chemically inert. The cage is strictly unlocked by encountering over-expressed cellular enzymes (such as alkaline phosphatase), ensuring that the chemical reaction—and subsequent drug delivery—happens exclusively in the targeted cells.
Major Frameworks/Components:
- Bioorthogonal Chemistry: Chemical reactions designed to occur inside living systems without disrupting or interfering with native biochemical processes.
- Tetrazine Cages: Engineered molecular enclosures that temporarily prevent tetrazines from indiscriminately reacting with other molecules.
- Enzyme Activation: A localized unlocking mechanism where target-specific cellular enzymes rapidly uncage the tetrazine to trigger a reaction.
- Reactive Scavengers: Competing tetrazine-reactive compounds introduced to suppress unwanted activation outside of target cells, drastically enhancing spatial precision.
Branch of Science: Chemical Biology, Biochemistry, Pharmacology, and Oncology.
Future Application: The highly targeted deployment of potent therapeutics (like the chemotherapy drug doxorubicin) directly into tumor cells to minimize healthy cell toxicity, alongside the creation of high-precision fluorescent probes for sharper diagnostic imaging of diseased tissues.
Why It Matters: Life-saving but potent drugs, such as chemotherapies, are often indiscriminate and kill healthy cells alongside diseased ones, causing life-threatening side effects. By programming chemical reactions to unfold only within specific target cells, TRACE maximizes on-target drug efficiency and diagnostic clarity, ultimately paving the way for safer and more effective clinical outcomes.
Potent drugs like chemotherapy can be lifesaving but often have life-threatening side effects. Notably, they can be indiscriminate, killing both cancer cells and healthy cells in one swoop. Increasing a drug’s on-target efficiency can reduce side effects and enable healthier outcomes for patients.
A new tool, developed in the lab of Neal K. Devaraj, professor of chemistry and biochemistry at the University of California, San Diego, promises to do just that. TRACE (tetrazine release and activation by cellular enzymes) locks tetrazine molecules in a cage that is released only when it comes into contact with a cell-specific enzyme. This work appears in Nature Chemical Biology.
What Is Bioorthogonal Chemistry?
Bioorthogonal chemistry is a process that allows researchers to perform chemical reactions in living systems to track and manipulate cells in real time without interfering with native biochemical processes. Two designed molecules exclusively seek each other out and “click” together to perform a chemical reaction. Many bioorthogonal reactions belong to a broader class of highly selective reactions often referred to as click chemistry. Scientists use bioorthogonal chemistry for coupling tags, drugs, or imaging dyes to biomolecules, even in cells.
A common tool for this is tetrazine, which reacts quickly with partner molecules. In 2008, Devaraj and Joseph M. Fox independently reported the use of tetrazine coupling for bioorthogonal chemistry, introducing one of the fastest bioorthogonal reactions available. Today, tetrazines are found in chemistry and materials science labs around the world, as well as in human clinical trials, where they can be used as a drug-delivery mechanism.
As exciting as this development has been, tetrazine reactions can be indiscriminate, reacting across cell types in complex biological systems. In humans, this means imaging may lack precision or drug therapeutics may act on healthy cells in addition to diseased ones.
To improve efficiency, Devaraj’s lab developed molecular cages that encase tetrazine, preventing it from “clicking” with other molecules. The tetrazine becomes activated only when it encounters a particular cellular enzyme that unlocks the cage. Once activated, the tetrazine can quickly trigger a chemical reaction inside target cells.
To achieve exquisite spatial control, where a reaction is happening in cell A but not cell B, activation must happen rapidly. The lab studied different tetrazine structures to determine which had the fastest uncaging rates and the quickest reaction times. The researchers also employed a competing tetrazine-reactive scavenger to suppress activation outside target cells, further improving spatial precision and essentially programming the chemistry to work in a single cell type.
“What we've shown is that you can, essentially, program the chemistry in specific cell types,” stated Devaraj, who is also the Murray Goodman Endowed Chair in Chemistry and Biochemistry. “You want this to work in a cell type that's overexpressing a particular enzyme, like a cancer cell, but not in other cells—that’s what we’ve figured out.”
After proof-of-concept testing, the researchers used real enzymes that are overexpressed in certain diseases in conjunction with doxorubicin (DOX), a potent drug used in cancer therapy with limited clinical applications due to its high cell toxicity. When the researchers compared the tetrazine cages to a control group, DOX was deployed only when the cages came into contact with a specific enzyme.
Beyond drug delivery, the team also built fluorescent probes that light up only after TRACE activation. The lab was able to show that only cells that expressed both the enzyme and the molecular tag fluoresced. Another probe was used to label the surface of cells that have high alkaline phosphatase (ALP) activity, a marker often elevated in certain tumors. The probe attached to a cell-surface “handle” and turned fluorescent only where ALP was active, allowing precise visualization of enzyme activity on live cells.
Devaraj has been researching tetrazines for nearly 20 years, and he shows no signs of stopping. Now that his lab has built the cages, he is looking for ways to improve selectivity, which may lead to increased drug efficacy with fewer side effects.
“I am very interested in the idea that you could rethink how you deliver drugs and imaging agents, and that you can do these things in the human body. That's what led us to develop tetrazine reagents a long time ago,” stated Devaraj. “It’s turned out to be a really rich space, and, all these years later, they're still offering surprises.”
Funding: Funding was provided, in part, by the National Institutes of Health (R35GM141939) and the German Research Foundation (KN 1447/1-1).
Published in journal: Nature Chemical Biology
Title: Achieving cell-type-specific bioorthogonal chemistry using enzyme-activated caged tetrazines
Authors: Caroline H. Knittel, Stormi R. Chadwick, Jacob A. Vance, Cedrik Kuehling, and Neal K. Devaraj
Source/Credit: University of California, San Diego | Michelle Franklin
Edited by: Scientific Frontline
Reference Number:
.jpg)