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Biological Chemistry

A Library Of Bright Ideas

Chemists look for new biological probes by synthesizing a library of more than 10,000 fluorescent molecules

by Grace Chua
March 23, 2015 | A version of this story appeared in Volume 93, Issue 12

A graphic showing several fluorescent molecules along a spectrum showing where they emit light.
Credit: Courtesy of Young-Tae Chang
The color palette of Chang’s library spans the visible spectrum.

Fluorescent small molecules are workhorses for scientists from many disciplines. Biologists use them to stain specific cell types, biochemists rely on them to monitor enzyme activity, and analytical chemists use them to detect molecules in the environment.

But the rational design of these fluorescent probes can be cumbersome. First, researchers design a way for the probe to selectively bind or attach to a given target. Then they have to synthesize the probe to execute that strategy.

One researcher in Singapore is developing probes in reverse, starting with synthesized fluorescent molecules and then finding the targets that they label. Young-Tae Chang of the National University of Singapore (NUS) has already built a library of more than 10,000 small molecules—among the world’s largest such collections, according to some in the field—that fluoresce in a wide array of colors. Now, he is determining the molecular and cellular targets that these compounds hit. Chang’s goal is a rainbow of probes that would provide researchers with different ways to image a target of choice, even allowing scientists to detect multiple targets at the same time.

Early this year for example, Chang’s group described a probe they call Neuron Orange, or NeuO. It can cross the blood-brain barrier and stain neurons in real time in live mice (Angew. Chem. Int. Ed. 2015, DOI: 10.1002/anie.201408614).

Dirk Trauner of Ludwig Maximilian University of Munich says neuroscientists could use NeuO to guide studies of neuronal circuits and image the brain. Such imaging is vital for mapping the brain and requires differentiating between the brain’s cell types, including neurons, microglia, oligodendrocytes, and astrocytes. NeuO stains only neurons. Other dyes can stain neurons but not selectively. Also, for bioimaging, chemical stains such as NeuO have an advantage over fluorescent proteins because scientists can easily inject them into an animal instead of genetically engineering the organism to express the protein.

Chang started work on his library when he was an assistant professor at New York University in the early 2000s. A colleague had shown him stunning images of cells stained by fluorescent dyes. The pictures inspired Chang to switch from screening combinatorial libraries for potential drugs to developing fluorescent compounds as imaging tools. “I started to dream of my own colorful toolboxes,” he says.

When he switched to synthesizing fluorescent probes, however, he found conventional design to be a time-consuming process with a high failure rate. So he decided to work in reverse, making the probes first through a diversity-oriented approach and then screening panels of them against targets.

Still, building the library took time. The sticky, planar, hydrophobic molecules aren’t easy to make and are costly to purify, Chang says. The key to lower synthesis costs, it turns out, was solid-phase chemistry.

Each library member is based on a known fluorescent scaffold, which Chang first attaches to a resin bead. A library of so-called rosamine compounds, for example, started with 12 xanthone derivatives as the scaffolds. Once loaded onto a bead, Chang modified the xanthones through reactions with each of 33 Grignard reagents. Cleaving the product from the bead yielded 396 rosamine compounds, of which 240 were selected for further study on the basis of their purity and spectroscopic properties.

Other researchers who develop fluorescent probes applaud the structural diversity of the compounds in Chang’s library. Seung Bum Park of Seoul National University, in South Korea, says the library is likely the world’s largest collection of organic fluorophores.

At a group picnic, members of the lab of Young-Tae Chang use t-shirts to show the color range of the fluorescent molecules in Chang’s library at the National University of Singapore.
Credit: Courtesy of Young-Tae Chang
Chang’s team at their annual party in 2011.

Some of the molecules Chang has designed have internal bonds that can rotate freely. This is a key feature to make the compounds good sensors. A freely rotating bond gives the molecule a high degree of conformational flexibility, which disrupts its fluorescence. When the probe latches onto its target, however, the rotation stops, its conformation becomes fixed, and it glows brightly. As a result, the sensor glows only when it has hit its target, rather than fluorescing as it wanders in a sample, cell, or body. For example, a rosamine-based probe barely glows in solution but shines bright when it finds zebrafish neurons.

After synthesizing potential probes, Chang’s team then screens the compounds against a range of cell types, proteins, metal ions, or small molecules. When they find a hit, they resynthesize the probe at scale for further in-depth tests. So far, they have run thousands of tests, yielding millions of data points about what dye reacts with what cell type or analyte and at what concentration.

NeuO is the latest molecule for which Chang has found a specific target. His group also has reported a molecule that glows alarmingly orange in the presence of the date-rape drug γ-hydroxybutyric acid. By adding the probe to a suspect beverage and irradiating it with a laser pointer, a person could spot the drug. Another molecule, the rosamine compound CDy1, stains embryonic and induced pluripotent stem cells.

Unfortunately, for the probes Chang develops to label cell types, the exact molecular target is sometimes unknown. For instance, when Markus Sauer of the University of Würzburg, in Germany, tried to hunt for NeuO’s cellular target, he couldn’t find it. It seems that whatever NeuO binds to is distributed evenly in neuronal membranes. Knowing the precise mechanism would enable researchers to better design applications, Sauer says.

In 2007, drawn by the promise of more funding and proximity to his native South Korea, Chang joined NUS. Concurrently, he leads bioimaging-probe development at the Singapore Bioimaging Consortium, a national body that oversees bioimaging activities in the city-state. The consortium was started in 2005 as part of Singapore’s multi-billion-dollar push to grow its biomedical research industry, and it coordinates efforts to develop diagnostic imaging capability.

A line structure and two micrographs.
Credit: Angew. Chem. Int. Ed.
NeuO stains only neurons (right) in a live cell culture containing several cell types (left).

Chang says he may license compounds developed for single applications. Already, the stem cell probe CDy1 is marketed by research supply firm Active Motif. But scientists worldwide can ask for samples or request a screen of the library for a particular target, absolutely free. To date, some 100 researchers or groups have taken the offer.

For instance, Yoichi Nakao, of Japan’s Waseda University, used a probe that labels neuronal stem cells. With the probe, Nakao could observe the cells as they differentiated, which is not possible with standard immunostaining methods. Nakao says the ability to watch this process could help researchers evaluate possible drug leads to treat neurodegenerative diseases.

And Itaru Hamachi, a Kyoto University bioorganic chemist studying neurotransmitter receptors in neurons, plans to request a sample of NeuO. He says that visualizing cultured neurons is common, but doing so for neurons in a brain slice or in a live animal has been difficult, in part because of the presence of other cell types.

Chang has slowed the synthesis phase of his project in favor of ramping up screening for more targets, such as cancer stem cells and bacteria. He’s excited about the potential applications on the horizon for his library. “We’re targeting very exciting goals,” he says. “I think this is just the beginning.”  

Grace Chua is a freelance writer based in Singapore.


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