COVER STORY
SENSITIZING NMR
DYNAMIC NUCLEAR POLARIZATION opens up new ways to illuminate biochemical processes
OF LATE, the nuclear magnetic resonance
community has been buzzing about a technique that can increase NMR sensitivity by 100-fold or more and is opening up new ways to follow biochemical reactions in vitro and in vivo. Called dynamic nuclear polarization (DNP), the technique can be used simply to speed up familiar experiments—data collection that historically took overnight can be done in mere seconds—but it is also making NMR a newly powerful tool for identifying reaction intermediates, probing enzyme kinetics, and imaging in vivo. The power of NMR lies in its high resolution; it enables researchers to see small differences in chemical environments. NMR
is also noninvasive and nonperturbing—it more nuclei to align with the magnetic field won’t harm whatever sample or organism so as to increase the spin polarization. you’re scanning. At the same time, NMR Dynamic nuclear polarization tackles is “horribly insensitive,” says Lucio Frydthat challenge by transferring the larger man, a chemistry professor at Weizmann polarization of electron spins, such as Institute of Science, those found in stable radical compounds, in Israel. NMR inO O sensitivity can be a to nuclear spins N N through irradiation particular problem for N O studies of biochemiwith high-frequency H cal systems, which microwaves. The OH are often limited to nuclei of the target TOTAPOL species then become low concentrations— typically, hundreds of dynamically polarscans must be averaged to bring a signal out ized, and their NMR signals are enhanced of the noise. anywhere from 50- to several hundredNMR basically involves putting a sample fold. Additional signal enhancements can in a magnetic field, where the spins of nube obtained by doing experiments at temclei with odd numbers of protons or neuperatures down to 1 K. trons will line up with or against the field Originally proposed by Albert W. Overdirection. Because of the energy difhauser in his 1951 doctoral thesis, DNP was ference between these states, first used experimentally by Charles P. Slichproportionately more nuclei ter in 1953 at very low magnetic fields. Interalign with the field. The popest in the technique is surging now because of the recent development of commercial ulation difference leads to a nuclear high-power gigahertz microwave sources. spin polarization, One area in which DNP-enhanced solidwhich is meastate NMR has made an impact is in the sured by NMR. study of bacteriorhodopsin, a membrane Because the difprotein that uses a retinylidene cofactor to ference in energy harness light energy for pumping protons out of cells. Chemistry professors Robert between spins aligned with or G. Griffin of Massachusetts Institute of Technology and Judith Herzfeld of Branagainst the magdeis University and colleagues used the netic field is very small, the difference technique to help distinguish and charbetween the two acterize intermediates in the pump cycle populations is typically (Proc. Nat. Acad. Sci. USA 2008, 105, 883). The research team combined the samples a mere one nucleus in 100,000 for hydrogen. of 15N-labeled, membrane-bound bacteFor other nuclei the ratio riorhodopsin with the nitroxide biradical TOTAPOL, irradiated the mixture with is even lower. Increasing the sensitivity of NMR light to produce desired pump cycle intermediates, and then cooled everything to 90 therefore entails coaxing K. The group then irradiated the samples with 250-GHz microwaves to transfer the POSITIVE PUMP DNPelectron polarization from TOTAPOL to enhanced NMR is being used to the nuclei of bacteriorhodopsin. elucidate how bacteriorhodopsin Focusing on the 15N NMR spectra of the uses the retinylidene cofactor (ballsecond and third intermediates of the bacand-stick structure) to pump protons teriorhodopsin cycle, dubbed K and L, the across a membrane. WWW.CE N-ONLI NE .ORG
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JYLLIAN KEMSLEY, C&EN WEST COAST NEWS BUREAU
researchers found indications that the protonated Schiff base of the retinylidene loses contact with its counterion in K and establishes contact with a new counterion in L. Concurrently, low-energy, single-bond torsion in the polyene of the retinylidene in K converts to high-energy, double-bond torsion in L. Thus, the researchers concluded, the chromophore initially stores the energy of absorbed photons electrostatically, then transforms it into torsional energy as the system moves from K to L. The energy is subsequently released when the Schiff base proton is transferred and the connectivity of the bacteriorhodopsin active site changes, although the original chromophore conformation is not regenerated until later in the cycle.
visible spectroscopy data. Having shown that the method works in principle, Hilty and colleagues are probing the mechanism of the uronate Hilty is now turning to enisomerase reaction by hyperpolarizing the substrate with the zymes that are less well underOX63 trityl radical and then tracking the reaction by NMR. stood. One project focuses on the enzyme uronate isomerase, OH O OH OH O O Uronate which catalyzes the isomerizaisomerase O HO tion of D-glucuronic acid to OH OH D-fructuronic acid in microbes. OH OH OH OH In collaboration with Texas A&M chemistry professor D-Glucuronic acid D-Fructuronic acid Frank M. Raushel, Hilty is trying to determine the reaction mechanism of the enzyme. The HO OH sugars complicate matters by OH HO converting into different forms spontaneously in solution, in S S addition to undergoing the S S COONa NaOOC catalyzed reaction. Because “there are several things goS S ing on at the same time,” Hilty • C S S says, it’s critical to do real-time measurements such as those HO OH enabled by his stopped-flow OH system. “It’s not sufficient HO to do the reaction and then THE GROUP also saw indicaHO OH measure after the fact what the tions that four different L S S states of the protein actually products are,” he adds. Although Hilty’s stoppedexist, and the researchers have S S further investigated them flow apparatus does enable HO OH COONa measurements to start quickly, through two-dimensional there is a delay time during NMR experiments. Their OX63 trityl radical results indicate that only one which the samples mix and of the L states is functional, stabilize in the tube before NMR data can be collected. Right now, the and the other three decay back to a resting and graduate student Sean Bowen initially state. Griffin says that the enhanced signals studied the hydrolysis of Nα-benzoyl-Ldelay time for Hilty’s system is about 200 milliseconds. Hilty is designing a new flow available through DNP are critical to the arginine ethyl ester by the well-known encell with the goal of reducing the delay to 10 studies because the researchers can trap zyme trypsin ((Angew. Chem. Int. Ed. 2008, or 20 milliseconds. Ultimately, Hilty would only 5 to 25% of the sample in the L state. 47, 5235). They were able to use the NMR spectra to calculate a catalytic rate con“We’re looking for small signals to distinlike to use the technique to observe protein guish between the species,” he says. stant of 12.1 s-1, which agrees well with the folding in real time. “Many proteins have Texas A&M University chemistry profesrate constant calculated from ultravioletfolding events that occur on a timescale sor Christian Hilty is also using DNP to investigate enzyme reactions. Hilty has coupled an NMR magnet with a stopped-flow system to study enzyme reaction kinetics and intermediates. His general approach is www.omnicaltech.com to freeze an enzyme substrate with a polarizing agent such as a trityl radical, irradiate the solution with microwaves to transfer polarization to the substrate, and then thaw the solution quickly by mixing with hot buffer before using the stopped-flow apparatus to mix the substrate and enzyme together in an NMR tube within the magnet. In contrast to Griffin’s bacteriorhodopSuperCRC An easy-to-use microcalorimeter for browsing reaction sin experiments, which dynamically polarconditions, kinetic profiles, and max energy release, used by ize protein nuclei, Hilty’s approach hypertop chemical and pharma companies worldwide. polarizes just the substrate nuclei. Hilty
HYPERPOLARIZATION AT WORK
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COVER STORY
NH2
N O• 4-Amino-TEMPO
IMAGING WITH WATER Water is injected into an 8-mm-diameter cell loaded
with molecular sieve beads (left panel). Normal water does not show up in NMR scans (center panel), but water hyperpolarized by 4-amino-TEMPO can be observed (yellow-green, right panel).
Meanwhile, Song-i Han, a chemistry professor at the University of California, Santa Barbara, is developing DNP methods to study interactions between biomolecules and macromolecules. By tethering a DNP reagent such as the nitroxide radical 4-amino-2,2,6,6-tetramethylpiperidine-Noxyl (4-amino-TEMPO) to surfactants or
water dynamics around such interactions should provide clues to how those interactions work. She is also using her technique to shed light on the mechanism of how water is passively transferred through membranes, in particular whether water is somehow dissolved in the lipid membrane or channeled through transient pores.
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DNP can also be used to enhance NMR or magnetic resonance imaging (MRI) in vivo. Although MRI can be used to visualize structure in the human body, positron emission tomography (PET) is typically used with radioactive tracers to visualize functional processes (C&EN, Sept. 8, page 13). Dynamically polarizing nonradioactive chemicals to serve as imaging agents could provide a way to use MRI to observe processes without the need for radioactive substances. Because living organisms cannot be irradiated with gigahertz microwaves, however, DNP imaging agents must be irradiated and then injected. But hyperpolarization does not last forever—30 seconds is a relatively long lifetime, whereas common radioactive agents can have half-lives of hours. Warren S. Warren, a chemistry professor at Duke University, is investigating DNP agents that would have a longer hyperpolarization lifetime made possible by the presence of two equivalent atoms. A molecule with a pair of equivalent atoms—C-2 and C-3 of CH3COCOCH3, for example—has four possible spin states. When the molecule is hyperpolarized, three of the spin states can relax easily, but because of symmetry the fourth is protected. “We LES TODD/DU KE
proteins, Han and colleagues can illuminate nearby water molecules to quantify local hydration dynamics (Langmuir 2008, 24, 10062). Whether one is interested in protein folding, protein-protein interactions, or protein-membrane interactions, “water exclusion is a general phenomenon that occurs when molecules come together and bind,” Han argues, so studying the
SO NG -I HA N/UCS B
that is observable with our method,” Hilty notes, and the information provided by NMR could provide key insights into folding mechanisms. Overall, Hilty says, “we’re trying to develop this application into a general way of measuring mechanisms and kinetics and intermediates in processes that are far from equilibrium. That is something that is rather new for NMR.”
SCAN WATCH Warren
and graduate student Elizabeth Jenista confer about MRI scans.
have shown that we can use this protected state to store polarization,” Warren says. “A reasonable estimate for the spin lifetime in such states is many minutes.” Warren and colleagues create the imaging agents by starting with a molecule that has inequivalent atoms, dynamically polarizing it, and then chemically converting it to the desired imaging agent in which the atoms are equivalent. (See how this works for diacetyl at www.cen-online.org.) The molecule maintains the hyperpolarization until another chemical reaction, such as a metabolic process, makes the atoms inequivalent again. DEVISING SUCH chemicals and reaction pathways is not difficult, Warren says, and there are several nontoxic options. Using DNP for in vivo imaging, therefore, could be “something more than a curiosity that’s cool in the lab,” he says. His group is working with several compounds that they’re packaging into temperature-sensitive liposomes that would protect the DNP agent from metabolic degradation. In theory, the liposomes could be injected into an organism and then broken open at a particular time or in a particular location. Some of the reagents Warren is developing are antidepressants that he envisions using to image metabolic pathways in the brain. Other groups are investigating dynamically polarizing pyruvate to monitor metabolic activity in different organs. UCSB’s Han is also experimenting with in vivo imaging. Her approach is to use perhaps the ultimate nontoxic imaging agent: dynamically polarized water. The idea is to anchor TEMPO or other free-radical compounds to a gel filtration matrix through which water flows continuously while being irradiated with microwaves; the resulting hyperpolarized, but radical-free, liquid can then be used for imaging (Proc. Natl. Acad. Sci. USA 2007, 104, 1754). So far, Han and colleagues have used the technique to image water flow in small sample vessels, but Han envisions using the technique with saline or another body-friendly fluid that
would cross the blood-brain barrier. “We could accurately monitor blood perfusion,” Han says, to pinpoint small strokes that are currently difficult to detect by MRI with imaging agents such as gadolinium. For his part, Weizmann Institute’s Frydman got his DNP instrumentation set up only in the past year and is specifically looking at coupling it to ultrafast multidi-
mensional NMR techniques (C&EN, May 7, 2007, page 61). One of his projects is looking at metabolic fluxes of cancer cells to observe the effects of hypoxia or drugs. “Much of the metabolism couldn’t be seen before because of the lack of sensitivity,” Frydman says. “Now we could have the sensitivity of PET with the spatial resolution of MRI. That is the promise of DNP.” ■
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