Our laboratory has developed and employed new instrumentation and methodologies to improve the sensitivity, performance, and applicability of dynamic nuclear polarization (DNP) enhanced magic angle spinning (MAS). We have designed, fabricated, and implemented a novel magnetic resonance spectrometer capable of performing: 1) the first electron decoupling experiments using chirped microwave pulses; 2) the first cryogenic MAS-DNP experiments <6 Kelvin; 3) the first MAS-DNP within intact human cells; 4) the first DNP using trimodal fluorescent polarizing agents; and 5) the first MAS experiments employing spherical rotors.

Similar to proton spin decoupling, we have demonstrated that electron spin decoupling improves NMR signal intensity and lengthens transverse relaxation (T2). We have performed electron decoupling in conjunction with DNP and CPMAS (at temperatures <6 K) in experiments leveraging enhanced Boltzmann spin polarization (42x signal increase compared to 298 K) and DNP (240x signal increase; ≥10,000x combined improvement), while retaining site-specific resolution from two-dimensional spectroscopy and short optimal recovery delays of <2 seconds. My laboratory has demonstrated this exquisite sensitivity both in model systems and within intact human embryonic kidney (HEK293) cells. Whereas we have shown that standard polarizing agents (AMUpol) yield >50x DNP enhancements in human cells, we also introduced a new class of fluorescent polarizing agent to confirm cellular uptake with optical localization of DNP-NMR signals. The cryogen consumption of our experiments is costly, a challenge shared by many MAS-DNP laboratories. Accordingly, we have implemented a novel counter-flow coil heat exchanger which recirculates exhausting nitrogen gas and performs cryogenic MAS at 85 K using less than 100 L N2(l) per day. Motivated by a desire to reduce liquid helium consumption in MAS <6 K while accessing spinning frequencies >100 KHz with DNP, we have also recently introduced MAS spheres. Spherical rotors improve microwave coupling, will scale well to diameters <2 mm, and require less cryogens to spin. We have demonstrated 79Br rotational echoes of KBr out to 10 ms, a spinning stability of ±1 Hz, and spinning frequencies of 10.4 KHz using 9.5 mm outside diameter spherical rotors.

With practical applicability at the forefront of our efforts, we have concurrently developed latency reversal agents (LRAs) for HIV cure research and performed structural NMR analysis of bryostatin, an LRA. Having a direct biological structural target has ensured that we develop the very best instrumentation to address relevant biological hypotheses rather then first inventing technology and subsequently seeking an application.

 

Barnes DNP Laboratory

Louderman Hall, Room 346
Washington University in St. Louis
Department of Chemistry
Campus Box 1134
One Brookings Drive
St. Louis, MO 63130-4899


Email: barnesab@wustl.edu