Nuclear magnetic resonance is a robust tool in a broad range of scientific area from biochemistry, material science to MRI in medical application. In this thesis, two distinct works are presented in both solid sate and liquid state NMR. Firstly, benefiting from the fast MAS and cleverly designed devices, both resolution and sensitivity of solid state NMR spectra have been improved many folds, so that it is available to study complex biological solids, such as proteins. However there is always a need to increase resolution in order to work on more complicate systems. Here, we introduce a series of highly resolved scalar-based three-dimensional homonuclear correlation experiments for 13C sidechain correlation in solid-state proteins. These experiments are based on a sensitive constant-time format, in which homonuclear scalar couplings are utilized for polarization transfer, but decoupled during chemical shift evolution, to yield high resolution in indirect dimensions and band selectivity as desired. Together with the experiments designed to obtain backbone correlations, we could fully assign the backbone and aliphatic sidechain chemical shifts with the 3D spectra that are collected on 9.4T magnet (1H frequency 400MHz) for model protein GB1. We also discuss the method of chemical shift based structural refinement.

Another practice of NMR in my work is using 31P Dynamic NMR to characterize the backbone conformation and dynamics in DNA Dickerson Dodecamer. The results confirm solid-state 2H-NMR experiments showing that the C3pG4 and C9pG10 steps experience unique dynamics. And cytosine methylation has significant impact on the local dynamics. The results also show that 31P Dynamic NMR is an efficient way to extract DNA backbone dynamic information, and provide detail knowledge to study DNA-protein interactions.

NMR-assisted crystallography is the integrated use of solid-state NMR, X-ray crystallography, and first-principles calculations to define the protonation states in an enzyme active site to give a chemically detailed three-dimensional structure. Determining hydrogen positions is a notable feature of the technique, complementing neutron diffraction but here NMR-assisted crystallography applies to catalytically active samples. This integrated approach was applied to the intermediates of Tryptophan synthase and Toho-1-β-lactamase. To determine an experimental structure, an unbiased set of protonation states are modeled and their predicted chemical shifts are compared to experimental shifts using the reduced-2 statistics. The confidence in the experimental structure can be quantified using Bayesian probability. From the structure the positional uncertainties are determined for the shift-rich region as well as the surrounding surface. NMR crystallography gives a remarkably clear picture of the chemistry of the β-subunit active site in tryptophan synthase. This level of detail reveals why BZI, an indole analog, does not proceed with the bond formation step despite being the better nucleophile: BZI is held in the wrong orientation by hydrogen bonds to the charged βLys87 and βGlu109 residues. This chemically-detailed view also reveals water positioned for nucleophilic attack on Cβ of the substrate. Based on its position and alignment, we posit that it sits in the initial binding pocket for the β-hydroxyl leaving group.

ABSTRACT OF THE DISSERTATION

Theory and Applications of NMR to Problems in Material Science and Analytical Chemistry

by

Arun Agarwal

Doctor of Philosophy, Graduate Program in Chemistry

University of California, Riverside, August, 2010

Dr. Leonard J Mueller, Chairperson

Recent developments in NMR spectroscopy have seen an increase in its application for the study of structure and dynamics in the field of material science. NMR can be used as a complementary technique to X-ray diffraction and other structure probing techniques such as AFM and STM in the study of complex systems. Due to its non-invasive nature, measurements can be made on samples without altering their structural or functional properties.

In this thesis three different application of solid-state NMR will be highlighted. First, the butyl-substituted spiro-biphenalenyl radical will be studied. This molecule is a member of a new class of organic conductors which posses the highest conductivity of any neutral organic molecular solid via a magnetic phase transition. ¹³C chemical shift measurements allow us to track the transition between the diamagnetic and paramagnetic states, which we find builds in as a gradual increase in the spin-density at the aromatic sites and a decrease in the electron spin-spin coupling between adjacent radicals. Our model suggests that the electron is not delocalized over the entire molecule, but is in fast exchange between the phenalenyl rings.

Next, the photophysics of photomechanically responsive molecular crystal nanostructures based on anthracene esters is studied using solid-state NMR. A detailed study of these materials provides significant insight into the molecular-level dynamics that give rise to the micron-scale response of the nanorods. This work provides insight into how solid-state photochemistry within nanostructured molecular crystals can give rise to novel photomechanical behavior on micron lengthscales.

Finally, the last chapter discusses several examples of analytical applications of NMR. A combined approach of using solid as well as solution state NMR spectroscopy to study the stereo-chemical and functional properties of industrially useful compounds has been presented. The approach taken has the potential to be used for a wide variety of industrial applications.

Solid-state NMR (SSNMR) studies on biomolecules and organic molecular crystals are presented here. The biological part of the work is focused on improving resolution and sensitivity of SSNMR techniques for larger protein systems, while the studies on organic molecular crystals extend the application of SSNMR to determining the crystal structure of a photoreaction intermediate. First, a long-observation-window band-selective homonuclear decoupling scheme is introduced. The homonuclear decoupling technique decouples J-couplings during direct 13C acquisition between backbone alpha carbon and carbonyl carbon in U-13C-labeled proteins. The method can be directly incorporated into existing correlation methods that detect carbonyl resonances. Correlation spectroscopy examples are shown on the GB1 and the alpha-subunit of tryptophan synthase. Then, chemical shifts measured at within the beta-site of tryptophan synthase are shown, with indications of potential protonation states explained. After that, the setup of the SSNMR correlation methods are presented. 1H decoupling performance is interpreted using a spin echo experiment with varied decoupling power and magic angle spinning speeds with acquisition on 13C. After the 1H decoupling performance is mapped out, the distance measurements are performed using a perdeuterated Pf1 bacteriophage sample with exchangeable sites protonated. Effects of deuteration and radio frequency power on cross polarization are later discussed on GB1 samples at 50 kHz MAS. This part concludes the preparation of correlation methods to future applications on TS. In the second part of the thesis, NMR crystallography is used to determine the structure of the solid-state photoreaction intermediate of 9-tertbutyl anthracene. The work uses chemical shifts and chemical shift anisotropy parameters measured for microcrystalline solids to validate predicted structures from powder X-ray diffraction and computational data. The resulting structure for the photoreaction intermediate pointed to a possible mechanism for the elongation of 9-tertbutyl anthracene nanorods upon illumination. The studies within this work opens up new possibilities for the scope of the SSNMR technique.

The research in this thesis illustrates how chemical shift (CS) and chemical shift anisotropy (CSA) give a detailed view into atomic and molecular structure. Through the collaborative approach of NMR crystallography, utilizing solid-state NMR, x-ray diffraction, and computational methods a deeper understanding of crystal structure and systematic motion is formed. This thesis in constructed in a linear fashion as to shown how more detailed information can be obtained via more complex methods, culminating in the application of all the methods presented within to describe the structure and mechanism of expansion of a the novel anthracene system 9-tertbutylantrhacene ester (9TBAE).

Utilizing the CS, which provides information about the local electronic structure, directed questions such as the structure or arrangement of atoms can be answered. In this thesis problems in which the CS is used to provide detailed information about the location or inclusion of specific atoms in a molecule’s crystal structure will be presented. As crystallographic information for structures with greater complexity is required, the CS alone may be unable to differentiate between multiple similar structures, requiring a method that provides even greater detail of the local electronic environment.

The CSA is capable of providing this greater detail by quantifying a molecule’s discrete orientations in the magnetic field. Obtaining CSA values is non-trivial, requiring the application of pulse sequences with significantly greater complexity than those required to obtain the isotropic CS. Utilizing the approaches of slow spinning, a modified TOSS-t1-DeTOSS sequence, and xCSA, CSA principle components are obtained for increasingly complex systems.

The presentation of these methods culminates in their application on 9TBAE to elucidate not only the molecular structure, but also the mechanism of expansion when 9TBAE nanorods are photoreacted. The data obtained shows the generation of new lattice orientations within the nanorod, and based on these observations, the nanorods expand not due to a change in the volume of the unit cell, but rather due to a rotation of the unit cells. These results demonstrated while most photomechanical materials rely on the generation of a mixed phase bimorph structure, reconfiguration of the product phase can likewise generate a large mechanical response.

The combination of nuclear magnetic resonance (NMR) and ab initio calculations has become one of the most powerful tools to solve important structural and energetic problems in the field of chemistry. Chemical shifts observed in NMR present detailed information about local electronic structure while dynamic NMR allows us to extract kinetic and thermodynamic properties without perturbing equilibrium. Ab initio calculations are able to interpret the chemical shifts in terms of the chemical structure and provide detailed analysis of the energetic components that make up the process probed by dynamic NMR. This work involves three important applications of this powerful combination tool.

First, we present a combined X-ray, solid-state NMR, and ab initio study of the 140 kDa enzyme tryptophan synthase that allows for the determination of chemical structure for the key enzymatic intermediates in the enzyme cycle. The results support the ketoenamine and dipolar forms that are in fast exchange with each other in the quionoid intermediate, a result that we believe has mechanistic implications. The structural characterization of this enzymatic intermediate provides insight into the energetic and chemical transformations responsible for biological function.

Second, the combination of NMR and ab initio calculations has been applied to the field of catalysis. We find that specific position of the peripheral substitutions outside the central chiral pocket accounts for most of the discrepancies in fundamental physicochemical properties among related cinchona alkaloids via a combination of energetic and entropic effects. These differences in physicochemical properties are ultimately responsible for the differences in chemical reactivity that make each molecule so unique.

Finally, we make use of dynamic NMR and ab initio calculations to investigate the rotational barriers of a series of pyridine thiocarboxamides, and identify different electronic and structural factors that contribute to changes in the energetic processes. Comparison between thiocarboxamides and their corresponding amide analogues highlights the important role of the resonance interaction proposed by the canonical amide resonance model.

Understanding the dynamics of internal cavities of proteins large enough to host water molecules is an important step toward the analysis of the proton-transfer mechanism and water dynamics of PLP-dependent proteins. In the present study, the active site of the beta-subunit of Tryptophan Synthase was analyzed to determine the water molecule network present, its H-bond interactions with the amino acids as well as protonated states of cofactor and surrounding residues located in the α-aminoacrylate intermediate, and their roles in the proton-transfer mechanism. Here, we use method known as NMR crystallography – the synergistic combination of X-ray diffraction, solid-state NMR spectroscopy, and computational chemistry. NMR crystallography offers unprecedented insight into three-dimensional, chemically-detailed structure in biomolecules and revealing chemically-rich detail concerning interactions between enzyme site residues and the reacting substrate that is not achievable when X-ray, NMR, or computational methodologies are applied in isolation. We used NMR crystallography to create testable models for structure and function of enzyme active sites. Here, we employed this process to probe the active site in the β-subunit of tryptophan synthase with atomic-level resolution. NMR crystallography approach allows to perform cluster-based calculations to determine specific to the level of theory and basis sets magnetic shielding that can be converted later into the chemical shifts using rescaling parameters. The obtained calculated chemical shifts compared to the experimental values using chi-square statistical approach resulted in the detailed refinement of the structure and prediction of the proton exchange between tautomers. We determined that α-aminoacrylate intermediate formed during TS catalysis ensures a partial positive charge at Cβ of the serine substrate to facilitate nucleophilic attack by the incoming indole substrate. Reaction specificity is therefore accomplished through a combination of correct electrostatic environment (i.e. protonation states) and stereoelectronic control. The aminoacrylate structure also suggests the fate of the substrate’s β-hydroxyl group – it becomes the adjacent water molecule.

Nuclear Magnetic Resonance is the premier analytical tool for characterizing chemical structure and dynamics. Yet, it battles with the challenge of relatively low sensitivity. To combat this issue, various schemes have been devised over the years. One such strategy, Non-Uniform Sampling (NUS) refers to any method of collecting variable numbers of transients or irregularly spaced data points in the indirect dimension for signal acquisition in magnetic resonance. Non-Uniform Weighted Sampling (NUWS) is a specific embodiment of NUS that collects non-uniform numbers of repeated measurements on the uniformly sampled Nyquist grid, with the promise of increased sensitivity while retaining the ability to generate the frequency domain spectrum via Fourier transformation. However, there is no consensus on the best way to combine multiple samples at indirect time points. This objective of this study is to showcase the increased sensitivity of weighted non-uniform sampling.

Theoretical and experimental observations shows that the maximum signal-to-noise ratio for NUWS is found when the signal is constructed using consistent RMS noise (UCR) for the time domain signal. An alternate metric to signal-to-noise ratio, called Spectral Knowledge is used to quantify the spectral parameters, which is obtained using Monte Carlo Simulations and Fisher Information estimates. The experimental implementation of NUWS-UCR is demonstrated and the sensitivity gains of NUS are quantified. Another technique BFT-UCR is introduced which can work for acquisition that doesn’t cleave on to the uniform grid yet shows similar sensitivity gains to NUWS-UCR under the densely sampled regime. Multiple weighted and non-weighted sampling strategies have been employed to study the sensitivity advantages and trade-offs associated with weighted NUS, with focus being primarily on the variants of exponentially biased sampling schedules in the dense regime in indirect dimensions.

Both analytic theory and experiment suggest a re-examination of the NUS sensitivity theorem when comparing spectra acquired in the same overall experiment time. Ultimately, both SNR and spectral knowledge point to the efficacy of NUWS, with sensitivity increases of >50% for each indirect dimension under standard acquisition parameters.

It is posited that NUWS-UCR provides the ultimate threshold for sensitivity gains that can be attained under weighted NUS.

This dissertation aims to provide a full mechanistic understanding of enzyme catalysis by investigating the active site protonation states and chemical dynamics. Accurate knowledge of proton positions and sidechain orientations is crucial for establishing the mechanism of acid-base catalysis and allostery in enzymes These finite details are necessary for developing various computational techniques such as molecular dynamics simulations, molecular docking routines, and structure-based drug design. While high-resolution X-ray crystal structures provide information about protein residues and cofactors interacting with the substrate, they often fail to determine protonation states due to insufficient resolution. Currently, neutron crystallography is making strides in defining heavy and hydrogen atom locations but is limited by the need for large, preferably perdeuterated, crystals and multi-week-long acquisition times. Cryo-EM is also making progress in hydrogen atom detection but is far from routine.Nuclear magnetic resonance (NMR) spectroscopy has been proven to be a sensitive tool for detecting chemical environment in enzyme active sites and can be combined with Cryo-EM and diffraction methods for atomic-resolution descriptions of structure and function. However, to delineate the chemistry of the active site, NMR and diffraction are more powerful when combined with first-principles computational chemistry. This dissertation presents a comprehensive investigation of enzyme catalysis by combining X-ray Crystallography, NMR Spectroscopy and First Principles Calculations in a synergistic approach of NMR Crystallography to determine enzyme mechanism and establish a full understanding of the catalytic pathway.

Protonation and hybridization state are critical phenomena at the chemical-level that are vital in understanding an enzymes mechanism and function. Using high resolution X-ray crystallography unaccompanied is not enough to present a complete account of these details. On the other hand, the chemical shift from solid-state NMR spectroscopy is a remarkably insightful probe of the surrounding electro-chemical environment. The synergistic combination of NMR spectroscopy, X-ray crystallography and ab initio computational chemistry, also referred to as "NMR Crystallography", can be used as a unmatched tool for elucidating high resolution three-dimensional structures of diverse materials. This synergistic approach was used to determine the structures of the indoline-, 2-aminophenol (2AP)- quinonoid, and internal aldimine intermediates in the pyridoxal-5'-phosphate-dependent enzyme tryptophan synthase under conditions of active catalysis. In the presence of side-chain residues fixed at their crystallographically determined coordinates, models of the active site were created intending for the reactive substrate analogues to be optimized using ab initio computational chemistry. Experimentally measured chemical shifts at particular 13C - and 15N-labeled positions on the substrate are used to exclusively distinguish and emerge comparable species from the various computational model types; dictated by protonation state of the substrate analogues and nearby catalytic residues. The reduced chi squared analysis suggests the phenolic / phenolic-acid form to be the predominate protonated species for both the indoline and 2-aminophenol quinonoid intermediates, while the internal aldimine confirms the protonated Schiff base form.