Revolutionizing X-ray crystallography with a sponge
Molecular structures, gigantic challenges
In the world of chemistry, molecular structure can be the difference between life and death – quite literally. Slightly altering the structure of a compound can transform it from an effective medicine to a dangerous poison. Knowing a compound’s three-dimensional architecture is therefore essential in terms of determining its biological effects, both good and bad.
Outside the world of drug development, determining detailed structures of molecules can help us do many things. From creating the ultra-pure chemicals needed to create liquid crystals and OLED displays, to determining what makes a rose smell the way it does.
But conventional molecular structure analysis techniques have their limitations. And this is where our groundbreaking crystalline sponge technology could change the game.
Did you know?
is the size of a crystalline sponge.
of a compound is usually needed for conventional X-ray crystallography.
of a compound was visualized via X-ray diffraction using crystalline sponges.
X-ray crystallography but without the crystals?
“With current technologies, there is a trade-off between the reliability and detail level you can achieve in the determination of molecular structure and the amount of material needed for the analysis,” says Clemens Kühn, technical lead of our crystalline sponges project based at our Innovation Center in Darmstadt.
At the moment, if a researcher has only a trace of the material for analysis (known as the analyte), their first port of call might be mass spectrometry, because this can be done using only tiny amounts of a compound.
“From doing that, you’ll get the mass of the molecule itself and some of the fragments, produced when the molecule breaks up,” continues Kühn. “And if you’re lucky, you can interpret the data in such a way that you can say the molecule has to have a particular structure. But it’s quite common in mass spectrometry that there are multiple possibilities for what the structure of the molecule could be.”
This means that often the result of mass spectrometry is that researchers understand what makes up a compound, but those fragments could fit together in multiple ways. So they can propose what the structure might be, but there’s no reliable way to confirm this.
At the other end of this trade-off is conventional X-ray crystallography which gives researchers the full structure of a compound and produces a 3D model of the actual molecule, with no ambiguities.
“But there is a big caveat on using this technique,” says Kühn. “You need to have your analyte in the form of a single crystal. And to get it to crystallize, you need larger amounts. Typically, you can only start doing that when you have a few milligrams or more available.”
X-ray crystallography is also very difficult to carry out on liquid or volatile compounds that can’t easily be grown into suitable crystals.
“Other techniques like nuclear magnetic resonance (NMR) spectrometry fall somewhere in between,” Kühn continues. “There’s no conventional method that offers a full three-dimensional structure with only small amounts of analyte available.”
The beginning of something small
In 2013, a team of scientists from Japan, led by Professor Makoto Fujita released a paper in the journal Nature  that caused an international stir. They reported the first-ever use of X-ray crystallography to determine a compound’s structure without growing the compound into a crystal.
Instead, Professor Fujita’s team had used crystals of a type of metal-organic framework (MOF) – an ultra-highly porous solid – as a ‘crystalline sponge’. Professor Fujita’s team ‘soaked’ these sponge crystals – tiny crystals of approximately a tenth of a millimeter across – with the compound they wanted to analyze, and the sponge absorbed the target molecules.
“This sponge crystal framework features large open pores and also a high degree of mechanical flexibility,” explains Kühn. “So if you expose it to a solution of unknown molecules then it will, as we say, ‘soak up’ those compounds and align them within the crystal structure. So what you form is a composite of that original sponge framework, with your unknown molecules placed in the void pattern of the lattice.”
Professor Fujita’s team showed that once the analyte was arranged in the crystal, its structure could be determined using conventional X-ray crystallography methods – all without the need to grow the compound itself into a crystal.
Crucially, they also found that only tiny amounts of the analyte were needed to determine structures using this method. The paper reports that they unambiguously determined the structure of a scarce marine natural product using only one microgram of the compound.
Two laboratories, one collaboration
After the 2013 publication, Professor Fujita’s team published several follow up papers, demonstrating different applications of the crystalline sponge technology. It wasn’t long before our technology scouts connected with the professor’s team and began to explore how to further develop crystalline sponge technology.
After an exploration phase, the Innovation Center set up a project team at the beginning of 2018 to turn the technology into a viable new business.
“This is the first time that somebody is actually pursuing this technology commercially,” says Wolfgang Hierse, commercial lead of the project. “We are the first company to start building a business around it.”
Our Innovation Center enables the project to operate in a startup-like environment, but with the backing of the company at large. Since the project formed, the team has been working to uncover a whole range of applications for crystalline sponge technology.
“It's always exciting to take something that is a raw idea or a method and then try to develop it into something that will have a real-world impact,” enthuses Hierse. “You don’t know exactly where the journey will lead you and you have the opportunity to shape and create something that could make a huge difference to so many different research and development areas.”
“We can now identify and visualize substances using much smaller amounts of analyte than was previously possible,” Hierse continues. “And this opens up totally new areas of exploration. So far, we know only a small part of the chemistry that’s all around us. And here we have a method that will help reveal this exciting world of molecules.”
The team is working in close collaboration with Japanese company Rigaku, a key player in scientific analytical instrumentation.
“We saw we’d need to get privileged access to the latest X-ray diffraction technology,” explains Hierse. “That’s not something we manufacture ourselves so Rigaku was the obvious choice of partner. And of course, they were also very interested because they saw the potential in the crystalline sponge technology but needed a partner to manufacture them. So, we joined forces and it's a very organic and very fruitful collaboration for both parties.”
Why a wasabi-molecule gives hope for great results
Hierse is not exaggerating when he says that the technology could have far-reaching implications for research. Perhaps most importantly it could have a big impact on the development of medicines.
A drug metabolite is a byproduct of the body breaking down, or metabolizing, a drug into a different substance. Sometimes, metabolites can be toxic and may cause adverse effects. So when researchers are developing a new drug, these metabolites must be explored thoroughly before the drug is tested on patients.
Previously this exploration of metabolites could only take place quite far down the development process, as there needed to be enough material to analyze using the conventional methods of structure determination.
“In close collaboration with colleagues from the Healthcare division, we’ve shown that we could identify the metabolites at early development stages, using the crystalline sponges,” describes Hierse.
These findings, reported in a paper published in early 2019 , and with further follow up research in 2020 , have industry-wide implications. Complete structure identification of human metabolites plays a critical role in early drug discovery. At this stage of development, only low amounts of a metabolite (at the nanogram scale) are available, making methods like NMR spectrometry unsuitable to use for analysis.
Because it requires only tiny amounts of metabolite to analyze, the crystalline sponge method has the potential to close this gap.
“I can see in maybe ten years or so that, using this method, pharmaceutical companies could shift the metabolite analysis and metabolic toxicology from the preclinical phase right to the much earlier drug discovery phase,” Hierse continues. “And this will have so much impact. It will lead to vastly improved risk management of pharmaceutical development and a reduction in the costs of developing new drugs.”
But it isn’t just drug development that could benefit from crystalline sponge technology.
“We’ve seen interest from a number of other areas, for example, companies that produce flavors and fragrances,” says Hierse. “They want to investigate the aromatic oils of plants, fruit, and so on, to identify what is behind a particular smell or flavor. These are complex mixtures that nature produces and each little component can contribute to a unique flavor. So identifying what's behind these natural flavors and identifying the compounds that are in there helps with developing and replicating them.”
Normally these types of compounds are too volatile to be crystallized and measured using conventional X-ray crystallography, but crystalline sponges are already proving their effectiveness in this area of research.
“Our partner Rigaku used crystalline sponge technology to visualize the molecule responsible for giving Japanese wasabi its unique taste,” Hierse explains.
“In simple terms, they put crystalline sponges into a small glass and added a piece of Japanese wasabi to them. Then they let it incubate a while. When they took the crystalline sponge out and put it on the X-ray diffraction machine, they were able to determine the pungent smelling compound. The identity of that compound was already known. But they were, for the first time, able to actually see it in the X-ray diffraction machine. Just like that.”
And the study of biological activity isn’t the only area where crystalline sponges could make an impact. In the high-performance electronics arena, there’s also been interest in the new technology.
Take liquid crystal displays. These contain several organic compounds in a mixture that varies depending on the type of display and its required performance levels. However, liquid crystal molecules can exist in different isomers which are difficult to distinguish by conventional methods of chemical structure determination, such as nuclear magnetic resonance (NMR).
Using crystalline sponge technology enables scientists to prove beyond doubt the chemical structure of the novel compounds used in liquid crystal mixtures.
The same is true for identifying chemical impurities, which can affect technological performance for displays and electronic equipment.
“We believe crystalline sponges could certainly have an impact in this area,” affirms Hierse. “For example, certain impurities in chemicals used in the manufacture of OLED screens or semiconductors can affect their technological performance. Crystalline sponge technology has the ability to identify these impurities, even when there are only trace amounts.”
Crystalline sponges: setting a new standard in research
The near-term future for the project team is about proving the technology’s application to a number of customer-relevant, real-world problems. Showing it can provide answers where other technologies fail.
The longer-term future, meanwhile, holds a number of exciting possibilities.
“Once we’ve shown we can address existing problems that cannot be addressed any other way, and we’ve been successful in establishing some initial business around that, that’s the point where we can actually start making meaningful change,” Hierse enthuses.
“We’ll be able to move research into areas that just haven’t been possible so far. As already mentioned, carrying metabolite analysis to earlier stages of pharmaceutical development. Or supporting discovery programs for nature-derived compounds, as we see in pharma, pesticides, and food. I believe such screening programs could be enabled on a much larger scale with our technology. This has the potential to change a number of different areas of research and maybe even entire industries.”
It’s hard not to be swept up in Kühn’s and Hierse’s excitement for where the project could lead. What’s clear is that crystalline sponges are one of those rare research findings that truly deserves the title of ‘game changer’.
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