NMR for the masses
Take two aspirin and call me in the morning,” is the advice meted out late at night to an achy patient by the country doctor of fable. While it’s true that Aspirin has been the go-to remedy for much of humanity’s twinges and pangs, recent research indicates that the humble white pill may turn out to be something of a wonder drug.
Aspirin, or salicylic acid and its related salicylates, is naturally present in the bark of the willow tree; physicians like Hippocrates identified its efficacy at reducing pain and swelling millennia ago. In the 20th century, researchers discovered other useful qualities, such as the prevention of heart attacks and strokes by stopping clots from forming in blood vessels. More recently, an additional use for this modest pharmaceutical has been found, according to Carlos Velázquez, who teaches the chemistry of medicine as an assistant professor in the Faculty of Pharmacy and Pharmaceutical Sciences at the University of Alberta in Edmonton. When taken at a slightly higher dose than the daily 80 milligrams recommended by cardiologists for heart patients, aspirin has a chemo-preventative effect against cancer, particularly colon cancer, Velázquez says. But despite its broad therapeutic uses, the pill has a dark side. An irritant, it can cause gastrointestinal bleeding. Velázquez theorizes that, by altering the salicylic acid molecule slightly, this nasty trait can be eliminated. Of Velázquez’s three major research projects, one “relates to the structural modification of aspirin in order to create new molecules with the ability to prevent cancer — equally or even more effectively — but without this side effect.”
Nanalysis Corp. CEO Sean Krakiwsky, who was drafted by the LA Kings in the 1980s, says that being a high-tech entrepreneur is a bit like being a hockey player; you have to have perseverance as well as faith in your team. Photo credit: Nanalysis Corp.
Researchers like Velázquez want to characterize many molecular structures as quickly and accurately as possible. There are several ways, such as liquid chromatography and mass spectrometry, to determine new molecular structures and identify their reaction states and dynamics. Nuclear magnetic resonance (NMR) spectroscopy, however, is more effective at elucidating unknown molecules, is less ambiguous and provides more detail. Being large and publically funded, U of A can afford a $1 million, 600 MHz NMR instrument, which operates four days a week under the watchful eye of a specially trained technician. But faculty members from across campus use the machine, and experiments can stall for days at a time while samples sit in the NMR queue, says Velázquez. With three projects on the go, the wait can be frustrating, says Velázquez, whose students, from undergrad to post-graduate, assist with the experiments.
A small-scale solution to Velázquez’s problem lies just a few hours south along Highway 2 in Calgary at the offices of Nanalysis Corp., creator of the NMReady, a bench-top NMR spectroscopy instrument. Nanalysis is headed by CEO Sean Krakiwsky, a former NHL LA Kings draft pick who attended training camp with the likes of Wayne Gretzky. After hanging up his skates, Krakiwsky became an electrical engineer. He admits that he knew nothing about NMR spectroscopy when he began collaborating with two research partners to create a Lilliputian version of the hulking NMR machines found in big-budget laboratories.
At 20 kilograms and the size of a toaster, the NMReady is small enough to fit any laboratory. Photo credit: Nanalysis Corp.
The founders of Nanalysis got together almost by accident, Krakiwsky says. Through a friend, Krakiwsky met University of Calgary graduate student Greg McFeetors, who was developing Micro Electro Mechanical Systems (MEMS) chips to detect NMR signals, using Force Detection NMR (FDNMR) rather than the conventional Induction NMR (IDNMR). McFeetors, who is now the Director of Engineering at Nanalysis, suggested in late 2008 that Krakiwsky take a stab at commercializing the technology. When Krakiwsky began researching the opportunity, he found that a California Institute of Technology group had already patented MEMS chips to detect NMR signals. Krakiwsky purchased the option to license the technology and hired the inventor, Garett Leskowitz, as Nanalysis Chief Technology Officer. It was Leskowitz who pushed for development of a bench-top NMR spectrometer using a completely different approach that involved optimizing certain aspects of the already well-known and widely used IDNMR approach. Krakiwsky then met an NMR applications expert, Bruce Lix, who was managing the National High Field Nuclear Magnetic Resonance Centre NANUC at U of A. Lix joined the Nanalysis team as Director of Product Management and led a broad application development program for bench-top NMR. “These guys were my foundational technology and application team, and continue to be critical people in our organization that now has 20 people,” says Krakiwsky. It wasn’t all smooth sailing. Research and development, which began in 2009, was initially estimated to take two years and cost $4 million. In the end, it took 4 ½ years and $6 million to build a commercially viable product. Krakiwsky fell back on a key thing he learned in hockey: never lose “faith in the team. Even though we were behind and struggling we made progress and we were always honest with each other. So if we made a mistake and made incorrect assessments to do with the technology we got back on the right track.” In the end, “we developed 100 per cent of the technology ourselves,” using an innovative magnet design with powerful rare-earth magnets.
The NMReady is a fraction of the cost of a large, 600 MHz instrument, although Krakiwsky won’t reveal its price tag. However, it is cheap enough for small laboratories and education facilities with limited budgets, he says. A typical Nanalysis industry customer will run 40 samples per week: 30 on the NMReady while 10 would be sent to a large NMR machine, says Krakiwsky. By boosting the pace of research in laboratories, the NMReady allows scientists to immediately test whether their samples were prepared properly and see if the reaction worked as expected. “To give you an extreme case, our industry customers tell us that some times when they send their sample away and wait for the results, the response is: ‘you made a mistake preparing your sample.’ They would have avoided this mistake with an NMReady,” Krakiwsky says.
Bench-top NMR spectroscopy is an emerging but rapidly growing area of technology and companies like Massachusetts-based Thermo Scientific are elbowing into the market. But Nanalysis is confident in its ability to go toe-to-toe with its American counterparts. Recently, Nanalysis opened an office in Boston, Mass. with support from the Canadian Technology Accelerator (CTA) initiative. The CTA, which is managed by the Consulate General of Canada, helps Canadian companies access resources and contacts to help them compete internationally. “The Alberta and Canadian governments have been incredibly helpful in helping us export our products around the world.” Krakiwsky will be overseeing further expansion of offices along the American West Coast. Although he “expects to dominate” the bench-top NMR spectroscopy field, “there is room for many winners in the nascent bench-top NMR space.” NMR spectroscopy is the premier analytical technique for chemists and biochemists for use in research and quality control to determine not only the molecular structure of a sample but also its purity. While the term ‘nuclear’ often conjures up images of bombs and radioactive decay, the atomic nuclei in this case are actually quite stable; it’s not their destruction but their vibration that researchers are interested in.
Modern NMR spectroscopy is founded on more than half a century of research. American physicist Isidor Rabi is credited with the discovery of nuclear magnetic resonance, and won the 1944 Nobel Prize in physics for his research. The method of detection used in modern instrumentation is called nuclear induction, and this method also won the Nobel Prize in 1952 for physicists Felix Bloch and Edward Mills Purcell. To begin, samples are put into a magnetic field. Within this field, some atomic nuclei tend to align with the magnetic field, acting essentially like miniature magnets. As Krakiwsky explains, the weak magnetism produced by certain atomic nuclei is called nuclear paramagnetism. To have paramagnetism, the number of protons and/or neutrons must be odd, giving its nucleus a quantum mechanical property called spin. For example, simple hydrogen (1H) has only one proton, so it is paramagnetic and NMR active. However, 12C, the backbone of organic molecules, has an even number of protons and neutrons and is thus NMR inactive.
Derivatives of aspirin currently being synthesized in the University of Alberta pharmacy laboratory of Carlos Velázquez, who uses a Nanalysis NMReady to assess the new molecular structures. Here, R1 is a naturally occurring polyphenol while R2 and R3 are an organic nitrate or a hydrogen sulfide-releasing moiety. Photo credit: Carlos Velázquez
Under the influence of a pulsed magnetic field from a coil, the aligned nuclei can be made to undergo ‘coherent precession,’ where the direction of the nuclear magnetism circulates for a time around the main magnetic field direction. This precession produces an electric current in the coil. This current has a frequency typically in the tens to hundreds of MHz that depends on the strength of the magnetic field. Detecting and manipulating these signals with a computer provides a graph of frequency, sometimes called a chemical shift, versus intensity, forming a characteristic series of peaks — the classic NMR spectrum. Nuclei resonate at their own specific frequencies depending on the local chemical environment. The chemical shift tells which types of atoms are there, while the intensity speaks to how many there are. Of course, the picture is more complicated because atoms in close proximity can interact with each other, altering their signals slightly and splitting the peaks through a phenomenon called J-coupling. By analyzing the split peaks, researchers can gain more detailed information about the structure of the molecule than any other analytical technique. For any given molecule, NMR can infer a basic structure or match it against known compounds in a spectral library. This makes it useful in a wide variety of synthetic organic and inorganic chemistry fields, including petroleum, polymer, pharmaceutical and materials chemistry. “Consumer products companies are using our instrument to develop better cosmetics, automotive companies use NMR to help them develop better polymers, there are food quality applications, petrochemicals, atomic energy and many others,” says Krakiwsky. He points to one customer, a petroleum engineering professor, who uses the NMReady for analyzing bitumen and liquefied coal. In the food sector, it is being used to check the quality and purity of olive oil.
The NMReady is proving to be a boon to industry, but it is of equal importance to students, Krakiwsky says. Undergraduate chemistry students need to become proficient in NMR spectrometry, as it is becoming a main analytical tool for the modern chemist. Velázquez’s students use the NMReady to analyze aspirin derivatives that possess both nitric oxide (NO) and hydrogen sulfide (HS) moieties. “Most of the time, we have a preliminary idea as to whether or not we obtained a specific product, due to the simple nature of these compounds,” Velázquez says. The instrument also allows students to check for the presence of traces of solvents that are difficult to eliminate such as dimethylformamide (DMF), dimethyl sulfoxide (DMSO) and ethyl acetate before submitting samples to the university’s 600 MHz NMR machine. If there is one criticism to be made about the NMReady, says Velázquez, it is that its 60 MHz capability prevents it from scanning complicated molecules and it doesn’t have the required resolution to separate peaks that absorb in similar fields. Nonetheless, it allows students to run samples of simple molecules at different concentrations using various solvents to observe specific effects such as hydrogen bonding, solvent shielding and deuterium exchange, Velázquez says.
Carlos Velázquez of the University of Alberta is using the NMReady to assist his research into the structural modification of aspirin to create new molecules with a chemo-preventative effect against cancer. Photo credit: Carlos Velázquez
Velázquez is looking forward to even greater advancements within the field, such as increasing the sensitivity of the NMR instrument to 100 MHz or 200 MHz. This would ensure the instrument clearly records how many, for example, of each type of hydrogen is present in the molecule. “That would be terrific,” he says.
Krakiwsky says that Velázquez has correctly identified a limitation of the current model of NMReady, adding, “we have an exciting product road map that will yield new models to address this.” One example is the recently released dual-mode spectrometer, which adds Fluorine-19 (19F) capabilities. “The fact that we can have two nuclei in the same instrument with a software switch is a great addition to our product mix.” This is proving especially helpful in medicinal chemistry as well as agriculture related applications, says Krakiwsky, whose machine now sells throughout “all corners of the earth.” The NMReady has found markets inside the laboratories of both academia and industry. Just as importantly, its development affirms that Canadians can be leaders of the pack when it comes to high-tech innovation.