AsymPol-TEKs as efficient polarizing agents for MAS-DNP in glass matrices of non-aqueous solvents.

Two polarizing agents from the AsymPol family, AsymPol-TEK and cAsymPol-TEK (methyl-free version) are introduced for MAS-DNP applications in non-aqueous solvents. The performance of these new biradicals is rationalized in detail using a combination of electron paramagnetic resonance spectroscopy, density functional theory, molecular dynamics and quantitative MAS-DNP spin dynamics simulations. By slightly modifying the experimental protocol to keep the sample temperature low at insertion, we are able to obtain reproducable DNP-NMR data with 1,1,2,2-tetrachloroethane (TCE) at 100 K, which facilitates optimization and comparison of different polarizing agents. At intermediate magnetic fields, AsymPol-TEK and cAsymPol-TEK provide 1.5 to 3-fold improvement in sensitivity compared to TEKPol, one of the most widely used polarizing agents for organic solvents, with significantly shorter DNP build-up times of ∼1 s and ∼2 s at 9.4 and 14.1 T respectively. In the course of the work, we also isolated and characterized two diastereoisomers that can form during the synthesis of AsymPol-TEK; their difference in performance is described and discussed. Finally, the advantages of the AsymPol-TEKs are demonstrated by recording 2D 13C-13C correlation experiments at natural 13C-abundance of proton-dense microcrystals and by polarizing the surface of ZnO nanocrystals (NCs) coated with diphenyl phosphate ligands. For those experiments, cAsymPol-TEK yielded a three-fold increase in sensitivity compared to TEKPol, corresponding to a nine-fold time saving.

PyrroTriPol: a semi-rigid trityl-nitroxide for high field dynamic nuclear polarization.

Magic angle spinning (MAS) dynamic nuclear polarization (DNP) has significantly broadened the scope of solid-state NMR to study biomolecular systems and materials. In recent years, the advent of very high field DNP combined with fast MAS has brought new challenges in the design of polarizing agents (PA) used to enhance nuclear spin polarization. Here, we present a trityl-nitroxide PA family based on a piperazine linker, named PyrroTriPol, for both aqueous and organic solutions. These new radicals have similar properties to that of TEMTriPol-I and can be readily synthesized, and purified in large quantities thereby ensuring widespread application. The family relies on a rigid bridge connecting the trityl and the nitroxide offering a better control of the electron spin-spin interactions thus providing improved performance across a broad range of magnetic fields and MAS frequencies while requiring reduced microwave power compared to bis-nitroxides. We demonstrate the efficiency of the PyrroTriPol family under a magnetic field of 9.4, 14.1 and 18.8 T with respect to TEMTriPol-I. In particular, the superiority of PyrroTriPol was demonstrated on γ-Al2O3 nanoparticles which enabled the acquisition of a high signal-to-noise surface-selective 27Al multiple-quantum MAS experiment at 18.8 T and 40 kHz MAS frequency.

Fast magic angle spinning for the characterization of milligram quantities of organic and biological solids at natural isotopic abundance by 13C-13C correlation DNP-enhanced NMR.

We show that multidimensional solid-state NMR 13C-13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Highly Efficient Polarizing Agents for MAS-DNP of Proton-Dense Molecular Solids.

Efficiently hyperpolarizing proton-dense molecular solids through dynamic nuclear polarization (DNP) solid-state NMR is still an unmet challenge. Polarizing agents (PAs) developed so far do not perform well on proton-rich systems, such as organic microcrystals and biomolecular assemblies. Herein we introduce a new PA, cAsymPol-POK, and report outstanding hyperpolarization efficiency on 12.76 kDa U-13 C,15 N-labeled LecA protein and pharmaceutical drugs at high magnetic fields (up to 18.8 T) and fast magic angle spinning (MAS) frequencies (up to 40 kHz). The performance of cAsymPol-POK is rationalized by MAS-DNP simulations combined with electron paramagnetic resonance (EPR), density functional theory (DFT) and molecular dynamics (MD). This work shows that this new biradical is compatible with challenging biomolecular applications and unlocks the rapid acquisition of 13 C-13 C and 15 N-13 C correlations of pharmaceutical drugs at natural isotopic abundance, which are key experiments for structure determination.

Light-Induced Quantitative and Electrical-Field-Induced Barrierless Switching of Spiropyran Derivative on Graphite Surface.

A new class of pyridine-based spiropyran (SP) shows photoinduced reversible switching between the closed SP and ring-opened merocyanine (MC). We show that a condensed crystalline monolayer of SP on graphite can be quantitatively converted to MC upon UV irradiation. In solution only ∼10% of SP can be transformed to MC because of the establishment of a photostationary state. Using an electrical field applied by a scanning tunneling microscopy (STM) tip, single molecules are reversibly switched between SP and MC forms in their condensed phases without any threshold voltage at ambient conditions. The microscopic structure of submonolayer films of SP and MC are investigated using atomic force microscopy (AFM) and STM.

Proton-Transfer Dynamics of Photoacidic Merocyanines in Aqueous Solution.

Photoacids attract increasing scientific attention, as they are valuable tools to spatiotemporally control proton-release reactions and pH values of solutions. We present the first time-resolved spectroscopic study of the excited state and proton-release dynamics of prominent merocyanine representatives. Femtosecond transient absorption measurements of a pyridine merocyanine with two distinct protonation sites revealed dissimilar proton-release mechanisms: one site acts as a photoacid generator as its pKa value is modulated in the ground state after photoisomerization, while the other functions as an excited state photoacid which releases its proton within 1.1 ps. With a pKa drop of 8.7 units to -5.5 upon excitation, the latter phenolic site is regarded a super-photoacid. The 6-nitro derivative exhibits only a phenolic site with similar, yet slightly less photoacidic characteristics and both compounds transfer their proton to methanol and ethanol. In contrast, for the related 6,8-dinitro compound an intramolecular proton transfer to the ortho-nitro group is suggested that is involved in a rapid relaxation into the ground state.

Characterization of frequency-chirped dynamic nuclear polarization in rotating solids.

Continuous wave (CW) dynamic nuclear polarization (DNP) is used with magic angle spinning (MAS) to enhance the typically poor sensitivity of nuclear magnetic resonance (NMR) by orders of magnitude. In a recent publication we show that further enhancement is obtained by using a frequency-agile gyrotron to chirp incident microwave frequency through the electron resonance frequency during DNP transfer. Here we characterize the effect of chirped MAS DNP by investigating the sweep time, sweep width, center-frequency, and electron Rabi frequency of the chirps. We show the advantages of chirped DNP with a trityl-nitroxide biradical, and a lack of improvement with chirped DNP using AMUPol, a nitroxide biradical. Frequency-chirped DNP on a model system of urea in a cryoprotecting matrix yields an enhancement of 142, 21% greater than that obtained with CW DNP. We then go beyond this model system and apply chirped DNP to intact human cells. In human Jurkat cells, frequency-chirped DNP improves enhancement by 24% over CW DNP. The characterization of the chirped DNP effect reveals instrument limitations on sweep time and sweep width, promising even greater increases in sensitivity with further technology development. These improvements in gyrotron technology, frequency-agile methods, and in-cell applications are expected to play a significant role in the advancement of MAS DNP.

Dynamic Nuclear Polarization with Electron Decoupling in Intact Human Cells and Cell Lysates.

Dynamic nuclear polarization (DNP) is used to improve the inherently poor sensitivity of nuclear magnetic resonance spectroscopy by transferring spin polarization from electrons to nuclei. However, DNP radicals within the sample can have detrimental effects on nuclear spins close to the polarizing agent. Chirped microwave pulses and electron decoupling (eDEC) attenuate these effects in model systems, but this approach is yet to be applied to intact cells or cellular lysates. Herein, we demonstrate for the first time exceptionally fast 1H T1DNP times of just 200 and 300 ms at 90 and 6 K, respectively, using a newly synthesized methylated trityl radical within intact human cells. We further demonstrate that eDEC can also be applied to intact human cells and human and bacterial cell lysates. We investigate eDEC efficiency at different temperatures, with different solvents, and with two trityl radical derivatives. At 90 K, eDEC yields a 13C signal intensity increase of 8% in intact human cells and 10% in human and bacterial cell lysates. At 6 K, eDEC provides larger intensity increases of 15 and 39% in intact human cells and cell lysates, respectively. Combining the manipulation of electron spins with frequency-chirped pulses and sample temperatures approaching absolute zero is a promising avenue for executing rapid, high-sensitivity magic-angle spinning DNP in complex cellular environments.

Noncovalent Spin-Labeling of DNA and RNA Triplexes.

Studying nucleic acids often requires labeling. Many labeling approaches require covalent bonds between the nucleic acid and the label, which complicates experimental procedures. Noncovalent labeling avoids the need for highly specific reagents and reaction conditions, and the effort of purifying bioconjugates. Among the least invasive techniques for studying biomacromolecules are NMR and EPR. Here, we report noncovalent labeling of DNA and RNA triplexes with spin labels that are nucleobase derivatives. Spectroscopic signals indicating strong binding were detected in EPR experiments in the cold, and filtration assays showed micromolar dissociation constants for complexes between a guanine-derived label and triplex motifs containing a single-nucleotide gap in the oligopurine strand. The advantages and challenges of noncovalent labeling via this approach that complements techniques relying on covalent links are discussed.

Frequency-chirped dynamic nuclear polarization with magic angle spinning using a frequency-agile gyrotron.

We demonstrate that frequency-chirped dynamic nuclear polarization (DNP) with magic angle spinning (MAS) improves the enhancement of nuclear magnetic resonance (NMR) signal beyond that of continuous-wave (CW) DNP. Using a custom, frequency-agile gyrotron we implemented frequency-chirped DNP using the TEMTriPol-1 biradical, with MAS NMR at 7 T. Frequency-chirped microwaves yielded a DNP enhancement of 137, an increase of 19% compared to 115 recorded with CW. The chirps were 120 MHz-wide and centered over the trityl resonance, with 7 W microwave power incident on the sample (estimated 0.4 MHz electron spin Rabi frequency). We describe in detail the design and fabrication of the frequency-agile gyrotron used for frequency-chirped MAS DNP. Improvements to the interaction cavity and internal mode converter yielded efficient microwave generation and mode conversion, achieving >10 W output power over a 335 MHz bandwidth with >110 W peak power. Frequency-chirped DNP with MAS is expected to have a significant impact on the future of magnetic resonance.

Sensitivity analysis of magic angle spinning dynamic nuclear polarization below 6 K.

Dynamic nuclear polarization (DNP) improves signal-to-noise in nuclear magnetic resonance (NMR) spectroscopy. Signal-to-noise in NMR can be further improved with cryogenic sample cooling. Whereas MAS DNP is commonly performed between 25 and 110 K, sample temperatures below 6 K lead to further improvements in sensitivity. Here, we demonstrate that solid effect MAS DNP experiments at 6 K, using trityl, yield 3.2× more sensitivity compared to 90 K. Trityl with solid effect DNP at 6 K yields substantially more signal to noise than biradicals and cross effect DNP. We also characterize cross effect DNP with AMUPol and TEMTriPol-1 biradicals for DNP magic angle spinning at temperatures below 6 K and 7 Tesla. DNP enhancements determined from microwave on/off intensities are 253 from AMUPol and 49 from TEMTriPol-1. The higher thermal Boltzmann polarization at 6 K compared to 298 K, combined with these enhancements, should result in 10,000× signal gain for AMUPol and 2000× gain for TEMTriPol-1. However, we show that AMUPol reduces signal in the absence of microwaves by 90% compared to 41% by TEMTriPol-1 at 7 Tesla as the result of depolarization and other detrimental paramagnetic effects. AMUPol still yields the highest signal-to-noise improvement per unit time between the cross effect radicals due to faster polarization buildup (T1DNP = 4.3 s and 36 s for AMUPol and TEMTriPol-1, respectively). Overall, AMUPol results in 2.5× better sensitivity compared to TEMTriPol-1 in MAS DNP experiments performed below 6 K at 7 T. Trityl provides 6.0× more sensitivity than TEMTriPol-1 and 1.9× more than AMUPol at 6 K, thus yielding the greatest signal-to-noise per unit time among all three radicals. A DNP enhancement profile of TEMTriPol-1 recorded with a frequency-tunable custom-built gyrotron oscillator operating at 198 GHz is also included. It is determined that at 7 T below 6 K a microwave power level of 0.6 W incident on the sample is sufficient to saturate the cross effect mechanism using TEMTriPol-1, yet increasing the power level up to 5 W results in higher improvements in DNP sensitivity with AMUPol. These results indicate MAS DNP below 6 K will play a prominent role in ultra-sensitive NMR spectroscopy in the future.

A light-responsive RNA aptamer for an azobenzene derivative.

Regulation of complex biological networks has proven to be a key bottleneck in synthetic biology. Interactions between the structurally flexible RNA and various other molecules in the form of riboswitches have shown a high-regulation specificity and efficiency and synthetic riboswitches have filled the toolbox of devices in many synthetic biology applications. Here we report the development of a novel, small molecule binding RNA aptamer, whose binding is dependent on light-induced change of conformation of its small molecule ligand. As ligand we chose an azobenzene because of its reliable photoswitchability and modified it with chloramphenicol for a better interaction with RNA. The synthesis of the ligand 'azoCm' was followed by extensive biophysical analysis regarding its stability and photoswitchability. RNA aptamers were identified after several cycles of in vitro selection and then studied regarding their binding specificity and affinity toward the ligand. We show the successful development of an RNA aptamer that selectively binds to only the trans photoisomer of azoCm with a KD of 545 nM. As the aptamer cannot bind to the irradiated ligand (λ = 365 nm), a light-selective RNA binding system is provided. Further studies may now result in the engineering of a reliable, light-responsible riboswitch.

Low-Threshold Reversible Electron-Induced and Selective Photoinduced Switching of Azobenzene Derivatives under Ambient Conditions.

Mono-carboxyl-functionalized azobenzene and arylazopyrazole have been employed for electron-induced and photoinduced switching under ambient conditions. The microscopic structure and the switching behavior is understood using scanning tunneling microscopy. The carboxyl functional group in these molecules offers low threshold energy for the electron-induced reversible switching compared with nonfunctionalized azobenzene. The low threshold is understood using charged intermediate states during the switching. A selectivity has been observed for the photoinduced switching. Because of strong hydrogen bonding, only the free phenyl groups in the molecules change their configuration.

Computationally Assisted Design of Polarizing Agents for Dynamic Nuclear Polarization Enhanced NMR: The AsymPol Family.

We introduce a new family of highly efficient polarizing agents for dynamic nuclear polarization (DNP)-enhanced nuclear magnetic resonance (NMR) applications, composed of asymmetric bis-nitroxides, in which a piperidine-based radical and a pyrrolinoxyl or a proxyl radical are linked together. The design of the AsymPol family was guided by the use of advanced simulations that allow computation of the impact of the radical structure on DNP efficiency. These simulations suggested the use of a relatively short linker with the intention to generate a sizable intramolecular electron dipolar coupling/ J-exchange interaction, while avoiding parallel nitroxide orientations. The characteristics of AsymPol were further tuned, for instance with the addition of a conjugated carbon-carbon double bond in the 5-membered ring to improve the rigidity and provide a favorable relative orientation, the replacement of methyls by spirocyclohexanolyl groups to slow the electron spin relaxation, and the introduction of phosphate groups to yield highly water-soluble dopants. An in-depth experimental and theoretical study for two members of the family, AsymPol and AsymPolPOK, is presented here. We report substantial sensitivity gains at both 9.4 and 18.8 T. The robust efficiency of this new family is further demonstrated through high-resolution surface characterization of an important industrial catalyst using fast sample spinning at 18.8 T. This work highlights a new direction for polarizing agent design and the critical importance of computations in this process.

Pyridine-Spiropyran Derivative as a Persistent, Reversible Photoacid in Water.

A highly versatile water-soluble pyridine-spiropyran photoswitch is reported which functions as photoacid in a wide pH range. Under neutral conditions, the open-ring merocyanine (MC-) exists to 48% and closes quantitatively by irradiation with visible light, while the reverse reaction occurs rapidly in the dark or by irradiation at 340 nm. The different pKa of the pyridine nitrogen in the closed spiropyran (4.8) and open merocyanine form (6.8) leads to a reversible proton release in a pH range of 3-7. Only negligible hydrolytic decomposition was observed in the pH range from 1 to 12. The application of potentially harmful UV light can be circumvented due to the fast thermal ring-opening except for pH values below 3. Its photoacidic properties make this compound an effective pH-regulating photoswitch in water and enable controlled proton-transfer processes for diverse applications. Additionally, all of the involved protonated states of the compound exhibit discriminative fluorescence features within certain pH ranges, which even expands its utility to a light-controllable, pH-sensitive fluorophore.

Ultrafast Spectroscopy of Hydroxy-Substituted Azobenzenes in Water.

Ultrafast UV/Vis pump/probe experiments on ortho-, meta- and para-hydroxy-substituted azobenzenes (HO-ABs), as well as for sulfasalazine, an AB-based drug, were performed in aqueous solution. For meta-HO-AB, AB-like isomerisation behaviour can be observed, whereas, for ortho-HO-AB, fast proton transfer occurs, resulting in an excited keto species. For para-HO-AB, considerable keto/enol tautomerism proceeds in the ground state, so after excitation the trans-keto species isomerises into the cis form. Aided by TD-DFT calculations, insight is provided into different deactivation pathways for HO-AB, and reveals the role of hydroxy groups in the photochemistry of ABs, as well as their acetylation regarding sulfasalazine. Hydroxy groups are position-specific substituents for AB, which allow tuning of the timescale of thermal relaxation, as well as the amount and contribution of the keto species to photochemical processes.

Water-soluble Py-BIPS spiropyrans as photoswitches for biological applications.

The ultrafast photochemistry of a new spiropyran photoswitch (Py-BIPS) has been investigated, revealing many advantages in the application in water over the previously studied spiropyrans. Functionalized Py-BIPS derivatives are presented for the study of pH dependence, stability, toxicity, and the thermal and photochemical behavior on longer time scales in aqueous media using several spectroscopic methods. These investigations pave the way for the practical use of Py-BIPS derivatives as photoswitchable ligands of biomolecules.