New approaches to promote the use of magnets-free NMR for analysis in chemistry, biology, and medicine

The diverse work of Mainz-based physicists in the field of nuclear magnetic resonance is being boosted by a new highly application-oriented approach: In October 2020, Dr. Danila Barskiy will join Johannes Gutenberg University Mainz (JGU) to set up a group focusing on nuclear magnetic resonance (NMR) spectroscopy, the objective being to explore approaches that do not require magnetic fields for chemical, biological, and medical applications. To do so, he has been awarded a Sofja Kovalevskaja Award worth EUR 1.6 million by the Alexander von Humboldt Foundation. “We are very pleased that Danila Barskiy and his research have been honored in this way. His work not only covers new and previously unexplored aspects, but also excellently complements research we are already undertaking here in Mainz,” said Professor Dmitry Budker, Barskiy’s host at the Institute of Physics at JGU and at the Helmholtz Institute Mainz (HIM).

Nuclear magnetic resonance spectroscopy is a standard analytical technique used to study the structure and dynamics of materials and living objects. With its sister technology Magnetic Resonance Imaging (MRI), NMR spectroscopy is used in organic chemistry, biochemistry, and medicine with fluids being positioned particularly well to this type of analysis. However, NMR spectroscopy is reaching its limits: Due to the weak interaction between atomic nuclei and the applied magnetic field, signals produced by NMR-active nuclei are typically extremely low and, therefore, require high magnetic fields for detection. This rules out the possibility of developing portable point-of-care devices, among other things.

Researchers aim to develop compact and portable NMR devices

Sofja Kovalevskaja Award winner Dr. Danila Barskiy has been looking at ways of improving NMR spectroscopy for around ten years, most recently at the University of California, Berkeley, from where he will be making the transition to Mainz. He is pursuing various approaches with the aim of designing compact and portable NMR devices that will ultimately be as small as a chip and affordable for wide analytical markets. According to Barskiy, the problem is the following: “Despite improvements being made, most NMR systems are still not compact because they need field strengths of several Tesla in order to distinguish between the chemical signatures in an NMR spectrum.”

Barskiy’s new interdisciplinary group will focus on developing miniaturized, portable NMR sensors. These sensors would employ the principle of zero to ultra-low field magnetic resonance, or ZULF NMR for short, using optically pumped magnetometers which would not require any strong magnetic fields. In addition to applications in chemical and biomedical research, such sensors could find use for detecting metabolic disorders at an early stage.

By heading up a work group at Mainz, Barskiy also wants to develop hyperpolarizers for benchtop NMR spectrometers. Hyperpolarization improves the alignment of nuclear spins in a sample, thereby amplifying their NMR signals. The scientist predicts that application-specific hyperpolarizers for tabletop NMR devices may be soon available and that they will be about the size of a coffee machine. And with a tabletop NMR device, it will be possible to perform highly sensitive analyses of fuels, biofluids such as blood or urine, and food extracts. “This will democratize NMR spectroscopy by providing access to wider audiences and will accelerate technological progress in developing countries,” Barskiy emphasized.

Long-standing collaboration with experts from UC Berkeley paves the way for new research

This represents another positive result of the collaboration between UC Berkeley, in particular the laboratory of Professor Alexander Pines, and the Mainz group led by Professor Dmitry Budker. “We have been working very productively with Professor Pines and his team, including Dr. Barskiy, for many years and developed ZULF NMR together,” said Budker. “Danila Barskiy was one of the first to recognize the importance of this research in the biological and medical context.” Budker points out that Barskiy’s plans fit perfectly with the work being done at Mainz, which is also being pursued as part of the European Union’s ZULF NMR Marie Curie Innovative Training Network together with other European partners.

Danila Barskiy studied at Novosibirsk State University and earned a doctorate in physical chemistry for his research at the International Tomography Center (ITC SB RAS). In 2015, he began working as a postdoc at Vanderbilt University in Nashville, Tennessee, and subsequently joined Professor Alexander Pines’ team at the University of California, Berkeley in 2017. “The conditions in Mainz are unique for me. The planned collaborations and the resources available fit perfectly with the projects I am pursuing. Thanks to the Sofja Kovalevskaja Award, I can not only begin an independent research career, the multidisciplinary research in Germany is promoted as a whole,” emphasized Barskiy.

The Alexander von Humboldt Foundation gave the 2020 Sofja Kovalevskaja Award to eight international research talents aged between 29 and 36 years. It is one of the most richly endowed research awards in Germany and provides young researchers with venture capital for innovative projects at an early stage in their careers. They undertake research for up to five years at German universities and research institutions and build up their own work groups at their host institutes. The award is funded by the German Federal Ministry of Education and Research (BMBF).

Sources:

JOHANNES GUTENBERG UNIVERSITAET MAINZ

EurekAlert!

ChemEurope

We are, simply, chemical reactions

I have recently read this article and it was well discussed how scientists keep discovering new interesting and important facts about our gastrointestinal tract and its effect on our wellbeing (including decision making and brain function).

For example, it has been shown that if people eat more galactooligosaccharide, the fraction of bacteria Lactobacillus and Bifidobacteria in the gut increases among all other strains (because metabolism of these bacteria takes advantage of the excess of this chemical, a known prebiotic). At the same time, these particular strains of bacteria have been shown to produce certain neurotransmitters — chemicals that participate in our brain functioning (because neurotransmitters are responsible for the transmission of electrical signals between neurons). It is indeed possible that by eating certain types of food you can overproduce certain neurotransmitters and, therefore, influence your brain functioning — through bacteria in the gut (Figure 1).

Untitled-2-01
Figure 1. Feeding healthy volunteers with galactooligosaccharide resulted in the increased population of Lactobacillus and Bifidobacteria in the gut which, in turn, resulted in overproduction of neurotransmitters that affect anxiety, including one called brain-derived neurotrophic factor [1].

What is even cooler, some bacteria have shown to affect people’s mood (possibly, by increasing production of “happiness hormones”, in other words, chemicals responsible for our social behavior). This is reasonable because happy people are more social which leads to a big evolutionary advance for these bacteria: they would obviously tend to spread better between social humans than between loners. Interesting, isn’t it?

Let’s now think about it in an evolutionary context. Do bacteria in the gut understand that they change the social behavior of their hosts? The answer is – NO. They not only don’t know anything about social behavior, but they also don’t know that they have a host and even that they exist! Bacteria are simply self-sustaining chemical reactions capable of changing (mutating) their chemical dynamics. It is not that bacteria have goals to survive, they simply occur. One random mutation in their genome led to the overexpression of a random chemical which, by a myriad of other complicated chemical transformations led to the increase of a random neurotransmitter which, by accident, tended to affect social behavior of their hosts. Now, the bacteria have started to spread faster and still spread “happily” because these chemical dynamics help them to exist or, simply, to occur.

We, humans, are chemical reactions too. All hopes and dreams in our brains are interactions between atoms, molecules and their collections. We just tend to occur because it is evolutionary logical. Our mood is chemistry too: serotonin is happiness, dopamine is pleasure, noradrenaline is concentration (Figure 2).

tumblr_nsbas4f7Jj1trtj0xo1_1280
Figure 2. The structures of neurotransmitters and their common effects on the mood, concentration, and learning.

So, if all of this is chemistry, then how to study this? Are there techniques that would allow us to investigate gut microbiome and its effect on the brain non-invasively and in real time?

I believe the answer is yes. Non-invasive techniques like NMR and MRI will soon be able to help to answer important questions about gut metabolism with hyperpolarization being one of the main tools to achieve this. The main challenge – fast decay of polarization – will be overcome by using nuclear states that can preserve their “memory” on a timescale of hours. Remarkably, there are already reports on the long-lived hyperpolarized nuclear spin states [2-4]! Long-live the gut! 😉

[1] “When Gut Bacteria Change Brain Function” bDavid Kohn.

[2] https://onlinelibrary.wiley.com/doi/abs/10.1002/chem.201405063

[3] https://pubs.acs.org/doi/abs/10.1021/acs.jpclett.7b00987

[4] https://www.sciencedirect.com/science/article/abs/pii/S1090780717300216

My “Dream Research” Project

If I was asked to identify the most challenging biological question, I would answer immediately. What is the nature of memory and thought? This question always fascinated me as a child. For a long time, I thought only biologists can figure that out. It took me 10 years deeply studying physics and chemistry, becoming a specialist in nuclear magnetic resonance (NMR) and magnetic resonance imaging (MRI), to realize that actually now we are close to start answering the question which captivated my childish mind.

 

1260px-Metabolic_Metro_Map.svg
Metabolic Metro Map. The image is taken from Wikipedia.

 

With all its complexity, the end result of the genetic machinery is to affect the chemistry of the body. Small molecules, metabolites, serve as fingerprints of what is happening inside us. Studying metabolites is almost like looking at someone’s apartment and coming up with a story of their recent lives: we can make guesses about a lifestyle based on what we see! And the chemistry of thinking is not an exception – our thought processes are accompanied by myriads of chemical transformations, and leftover metabolites can tell us about the process behind them.

Studying metabolites is almost like looking at someone’s apartment and coming up with a story of their recent lives: we can make guesses about a lifestyle based on what we see!

Routinely, metabolites are measured through analytical techniques like NMR and mass spectrometry (MS). But a new astonishing era is emerging. With new sensitivity enhancement techniques (signals can be increased by more than 20,000 times [1-4]), MR imaging will become a new tool to study metabolism in vivo and will move beyond morphology onto a platform to visualize molecules. Being fundamentally a quantum mechanical technique, the full potential of MRI is yet to be discovered.

I see my “dream research” project as the development of a new experimental MRI-NMR/MS platform to study metabolomics of memory and thought in living creatures. By developing novel MRI pulse sequences (which will take into account quantum-mechanical nature of molecules) and by applying state-of-the-art signal enhancement techniques, we will be able to “light up” the regions of the brain to study chemistry in them with an unprecedented level of accuracy. I believe that once all new methodologies available today are combined, it will become possible to create functional MRI for metabolomics – a tool to study instant chemical changes in the brain associated with memory and thinking. This will not only revisit the known biochemical processes at a new quantitative level, it will allow unraveling unexpected secrets of metabolism. And it is not only a fun thing to do — understanding the biochemical reasons for making decisions will bring us much closer to a society in which everyone truly enjoys living.

 

[1] J. H. Ardenkjær-Larsen et al. Increase in signal-to-noise ratio of >10,000 times in liquid-state NMR. Proc. Nat. Acad. Sci., 2003, 100 (18), 10158-10163.

[2] R. W. Adams et al. Reversible Interactions with parahydrogen Enhance NMR Sensitivity by Polarization Transfer. Science, 2009, 323 (5922), 1708-1711.

[3] D. A. Barskiy et al. Over 20% 15N Hyperpolarization in Under One Minute for Metronidazole, an Antibiotic and Hypoxia Probe. J. Am. Chem. Soc., 2016, 138 (26), 8080–8083.

[4] D. A. Barskiy et al. NMR Hyperpolarization Techniques of Gases. Chem. Eur. J., 2017, 23 (4), 725-751.

Monday Morning Effect

Friday evening. While his friends had already met in the Pub on Shattuck Avenue to celebrate a happy hour, UC Berkeley’s Ph.D. student Henry Bryndza was still in the Lab. He wanted to finish preparation of his samples so that he could come over on Monday morning to focus on the NMR measurements, not worrying about sample preparations. In order to suppress chemical reactions which could have started in his samples over the weekend, Henry put them in the liquid nitrogen dewar (T=-196oC).

Henry was working in the Laboratory of Robert Bergman, a renowned UC Berkeley professor who has made a significant contribution to the organic and metallorganic chemistry. Bergman and Bryndza were studying Fischer–Tropsch reactions using exemplary Cobalt and Iridium catalysts [1].

When he came back on Monday, Henry started to observe very interesting phenomena. 1H NMR spectra of the samples he took in the morning showed very weird “negative” NMR peaks (Figure 1). Moreover, the intensity of these peaks decreased day after day during the week when Henry tried to repeat the experiments and completely disappeared by the end of the week [2]. Henry was confused and decided to repeat his measurements. Surprisingly, this phenomenon was not observed every single time but was definitely the strongest on Mondays. Bergman and Bryndza decided to jestingly call this a “Monday phenomenon”; this was the beginning of what was known later as Parahydrogen-Induced Polarization (PHIP).

Untitled-2-01
Figure 1. “Pseudo-CIDNP” observed in 1H NMR spectra during hydrogenolysis reactions of Cobalt alkylidyne complexes. Adapted from [3].

Bryndza and Bergman asked for help from many NMR specialists, including NMR expert Professor Alex Pines from UC Berkeley and Professor Joachim Bargon from the University of Bonn [2]. The last one was known for the discovery of so-called chemically-induced dynamic nuclear polarization (CIDNP). The CIDNP effect is usually manifested as positive and negative NMR signals (very similar to those observed in Henry’s experiments) for the reactions taking place via radical intermediates. After contacting Bargon and other CIDNP specialists, weird results were interpreted as “pseudo-CIDNP” in hydrogenation reactions [3]. However, it was clear that CIDNP-based explanation was at least not complete, first, because of the very unusual suggestion of radical pairs in the studied hydrogenation reactions and, second, because of the lack of convincing simulations supporting the observed phenomena. Moreover, it by no means explained why the effect was the strongest on Mondays and why it was only observed in the laboratory of Robert Bergman.

This “Monday morning” puzzle remained unresolved until the International Society of Magnetic Resonance meeting in Rio de Janeiro in June 1986. There, during an evening session, Professor Daniel Weitekamp from Caltech presented his “thought experiment” of using parahydrogen (para-H2) as a source of enhancing NMR signals. The concept and the expected results were immediately published in Physical Review Letters [4]. The experimental demonstration conducted by a Weitekamp’s Ph.D. student Russ Bowers followed in July, and brilliantly supported all theoretical predictions (Figure 2) [5].

2-01
Figure 2. First experimental demonstration of a PASADENA experiment. The characteristic PHIP multiplets are seen on the right. Adapted from [5].
Bowers and Weitekamp called their experiment PASADENA (Parahydrogen And Synthesis Allow Dramatically Enhanced Nuclear Alignment) to glorify the location of their institute (Caltech is located in Pasadena, CA). After their publication, it immediately became obvious that PASADENA is, in fact, a correct explanation of “Monday phenomenon” of Bryndza and Bergman. Indeed, the low-temperature storage of NMR tubes over the weekend partially converted normal hydrogen into para-H2. The conversion was not complete, but it was enough to observe antiphase lines in 1H NMR spectra (Figure 1). The PASADENA effect and discovered later effect ALTADENA (Adiabatic Longitudinal Transport After Dissociation Engenders Net Alignment) were collectively given the name PHIP (Parahydrogen-Induced Polarization) [6].

Now let’s talk about physical principles of this effect. As we discussed before, due to the absence of a net nuclear magnetic moment, para-H2 itself does not produce an NMR signal. However, this single nuclear spin state implies that, in a sense, it is cold. Indeed, a comparable degree of spin ordering is obtainable at equilibrium only at temperatures of a few mK and magnetic fields of several Tesla [7]. The brilliance of Wetekamp’s idea was to introduce magnetic inequivalence to release this potential signal by connecting the singlet to the triplet states. This would require chemistry, but simple bond cleavage would not suffice. A singlet state of two protons is a relationship of one spin relative to the other and this order would be dissipated if the pair were split and mixed with an ensemble of other such products. Rather, it is necessary that the pair have a special relationship even after being distinguished by magnetic inequivalence. This is called a “pairwise” hydrogen addition and can be realized in hydrogenation reactions in the presence of homogeneous catalysts. To see how it works, let’s take as an example the simplest situation and imagine that a chemical reaction leads to the association of para-H2 with a molecule not containing magnetic nuclei.

Figure_PASADENA-01
Figure 3. Schematic diagram of the PASADENA experiment: parahydrogen (para-H2) reacts with an unsaturated molecule forming a reaction product where two nascent para-H2 atoms occupy chemically inequivalent positions. Energy level diagrams and corresponding 1H NMR spectra are shown in the bottom. Thick blue rectangles indicate overpopulated energy levels.

The two-spin system of the hydrogen molecule gives rise to four nuclear spin energy levels. As we described before, three of these energy levels correspond to orthohydrogen, the state with total nuclear spin 1 (triplet state), whereas the remaining fourth energy level corresponds to parahydrogen (singlet state), the state with zero total nuclear spin (Figure 3). Transitions between singlet and triplet spin states are forbidden by symmetry and the spin 0 parahydrogen is NMR-silent.

Now, the incorporation of para-H2 into an asymmetric molecule breaks the symmetry of the singlet spin state. For simplicity, I will consider only the PASADENA experiment, the case where hydrogenation reaction is performed at a high magnetic field (wherein the chemical shift difference between the two para-H2-nascent protons is much greater than the spin-spin coupling J between them). In this situation, the population of the singlet spin state αββα (numerical factor is omitted) of para-H2 is immediately transferred to the population of spin states αβ and βα of the formed spin system.

This can be understood as follows. Because of the chemical reaction, two H atoms from para-Hsuddenly end up in a different molecular environment. This leads to a collapse of the nuclear spin wavefunction αββα into one of the two states, αβ or βα, each with 50% probability. Next, it is easy to deduce from the energy level diagram that the NMR spectrum of the produced in such a manner molecule will contain four peaks grouped in two antiphase multiplets (Figure 3), exactly what was observed in the experiments of Bryndza (Figure 1) and Bowers (Figure 2). The key requirement is that both hydrogen protons from the para-H2 molecule are added together without significant competition from exchange reactions. This is a property of many, but not all, hydrogenations.

Duckett_obsor-01.jpg
Figure 4. Hydride region of PHIP-enhanced 1H NMR spectra showing
negative-positive multiplets for products formed when RhI(CO)(PMe3)2 reacts with para-H2 in the absence (a) and presence (b) of styrene [8].
The assignment of the peaks to particular transitions depends on the sign of the J-coupling between the para-H2-nascent hydrogens. When J-coupling is positive, PASADENA multiplets are positive-negative; if J-coupling is negative, the spectral appearance is opposite. This feature is very useful for studying hydrogenation reaction intermediates. Normally, organic molecules possess positive J-couplings between protons; and J-couplings between them are negative in case of metal hydrides. Therefore, in a complex reaction involving many intermediates, it becomes possible to distinguish low-concentration hydrides (possessing negative-positive multiplets) from organic reaction products (Figure 4).

It is also important to realize that PHIP can lead to 100% nuclear spin polarization of the reaction product. In the case of PASADENA experiment, 100% population of para-His split into just two energy levels, making transitions from these levels enhanced by orders of magnitude compared to the thermal case. Theoretically, if all para-Hmolecules are transferred to products in a pairwise manner and relaxation loses are minimized, the reaction product can acquire 100% spin polarization. This would, of course, require an additional step to transfer spin order from αβ and βα into the state αα but this can be readily realized using a simple RF pulse sequence.

Enormous NMR signal enhancements and unique spectroscopic signatures made PHIP a very useful tool in chemistry for more than 25 years to elucidate hydrogenation reaction mechanisms, study metalorganic hydride complexes, and catalysis [6]. However, PHIP can be also used in a very different context. Imagine a suitable molecular precursor which can become a naturally occurring metabolite after hydrogenation. This metabolite can be produced in seconds, with a very high level of nuclear polarization, injected into a living organism and a metabolism of that organism can be monitored by magnetic resonance spectroscopy (MRS) and magnetic resonance imaging (MRI). Today PHIP, and its sister technology SABRE (Signal Amplification By Reversible Exchange) allow to efficiently hyperpolarize dozens of biologically relevant molecules on nuclei such as 1H, 13C, 15N, 19F, 29Si, 31P, 119Sn etc. But this is a story for a separate blog post! 🙂

It is important to emphasize that only the connection between nuclear spin and rotational degrees of freedom allows this unique situation to take place. Indeed, the fact that the nuclear spin state can be overpopulated simply by cooling is a remarkable quality inherent only to the small hydrogen molecule. Indeed, even though other molecules can have the similar connection between rotational and nuclear spin states (N2, F2 etc.), larger moments of inertia will make overpopulating these states much more challenging task (because of the lower temperature requirements). Moreover, it is very challenging to keep these molecules in the gas state at low temperatures, and the simple rule of making a total wavefunction be a product of individual wavefunctions will no longer hold true. So, it is more likely that hydrogen molecule is the only example when the rules of spin statistics and Pauli’s principle can lead to the nuclear spin hyperpolarization.

What excites me about this story is how a purely thought experiment, on the one hand, and a weird experimental phenomenon, on the other hand, emerged into a new discipline and a remarkable tool to study chemical reactions. Moreover, more exciting applications of the para-H2-based hyperpolarization techniques are expected to emerge in biomedicine. I really wish there were more Monday morning effects in science! Who knows but maybe someone today has come to a lab to look at a weird result which will form a new field of study tomorrow.

 

References

[1] J. Bargon. Chance Discoveries of Hyperpolarization Phenomena. eMagRes, 2007.

[2] Private conversations with Robert Bergman and Alex Pines.

[3] P. F. Seidler, H. E. Bryndza, J. E. Frommer, L. S. Stuhi, R. G. Bergman. Synthesis of Trinuclear Alkylidyne Complexes from Dinuclear Alkyne Complexes and Metal Hydrides. CIDNP Evidence for Vinyl Radical Intermediates In the Hydrogenolysis of These Clusters. Organometallics, 1983, 2 (11), 1701-1705.

[4] C. R. Bowers, D. P. Weitekamp. Transformation of Symmetrization Order to Nuclear-Spin Magnetization by Chemical Reaction and Nuclear Magnetic Resonance. Phys. Rev. Lett., 1986, 57 (21), 2645-2648.

[5] C. R. Bowers, D. P. Weitekamp. Parahydrogen and Synthesis Allow Dramatically Enhanced Nuclear Alignment. J. Am. Chem. Soc., 1987, 109 (18), 5541-5542.

[6] J. Natterer, J. Bargon. Parahydrogen-Induced Polarization. Prog. Nucl. Magn. Reson. Spect. 1997, 31, 293-315.

[7] D. Weitekamp. Sensitivity Enhancement Through Spin Statistics. Encyclopedia of Magnetic Resonance, 2007.

[8] S. Colebrooke, S. Duckett, J. Lohman, R. Eisenberg. Hydrogenation studies involving halobis(phosphine)-rhodium(I) dimers: Use of parahydrogen-induced polarisation to detect species present at low concentration. Chem. Eur. J., 2004, 10, 2459–2474.