Probing the dynamics and binding properties of fluorinated drugs, ionic liquides and biomolecules via NMR relaxation and diffusion experiments

Abstract

NMR has applications in several different fields such as drug discovery and design, structure and dynamics of biomolecules, drug delivery, medicine, material science, environmental science and metabolomics. One of the most important applications of NMR is to study drug dynamics and drug interactions with different molecules includ- ing proteins. This thesis is an attempt to study drug behavior in different environments, and drug interactions and dynamics by using NMR relaxation and diffusion experi- ments. Further, NMR spectroscopy is among the main analytical techniques that can be used for metabolomics studies as it can accurately quantify and detect metabolites in complex mixtures, with minimal sample preparation. The fast profiling of metabolite mixtures by using 2D NMR techniques and their chemometric analysis is a problem of current interest and several attempts have been made in recent years in this direc- tion. We have utilized a fast 2D heteronuclear correlation NMR experiment to perform full chemometric analysis of metabolites in a variety of model systems. Results were compared with standard 2D NMR correlation experiments and reported in the thesis. The contents of the thesis have been divided into six chapters whose brief account is sketched below: Chapter 1 The first chapter of the thesis begins with an introduction to the basic phenomenon of NMR, the various NMR interactions such as chemical shift and dipolar interactions. Fourier transform method is described to process digitized NMR data and the basic hardware components of an NMR spectrometer are described, including the supercon- ducting magnet, rf transmitter, probe, receiver, and workstation for signal acquisition and data processing. Nuclear spin relaxation including cross-correlated spin relaxation v0. Abstract is taken up next, followed by a basic overview of quantum chemical calculations of the chemical shift anisotropy tensor as well as diffusion NMR studies. The importance of 19 F NMR and its utility in drug delivery and analysis is discussed. Two-dimensional NMR methods for metabolomics studies are also discussed at the end of this chapter. Chapter 2 Due to its extensive antitumor activity and synergism with other anticancer medica- tions, 5-Fluorouracil (5FU) has become a prominent anticancer agent that has been used to treat numerous types of malignancies since its inception several decades ago. It is a major agent as a first line anti-cancer drug, commonly used to treat colorectal, gastrointestinal, and breast cancer. However many variables such as its tendency for inducing drug resistance in tumor cells, fast rate of metabolism in the body, cytotoxic- ity, a short biological half-life, and non-uniform oral adsorption restrict the efficacy of 5FU. Encapsulating 5FU in a host system with a long-term drug release could reduce the number of times the medicine needs to be given, as well as the negative side effects, and make oral administration possible. While there are several delivery systems that have been reported for 5FU, the efficacy of the drug and its delivery is affected by the solubility of drug in the medium as well as the local environment around the drug. In this chapter we describe the measurement of the fluorine NMR relaxation parameters of 5FU in different kinds of molecular environments, namely in D 2 O, in an ionic liquid, in a lipid bilayer and in a triblock copolymer. Due to different local environments, the fluorine cross-correlated spin relaxation rates obtained via measurement of differential line relaxation are significantly different for each environment. The longitudinal relax- ation rates and diffusion coefficients were also computed using NMR relaxation and diffusion experiments. The change in rotational correlation time τ c was also calculated and the effect of the environment on rotation and dynamics of the drug was quantified. Chapter 3 Drug delivery systems (DDS), which include particulate carriers made mostly of lipids and/or polymers, and their accompanying treatments can improve many of the pharma- cological features of conventional medications. Various materials such as liposomes, lipids, carbon nanotubes, and polymers have been utilized as nano-sized DDS. Due to vitheir versatility, polymers have been extensively researched for a variety of carriers, including micelles, nanogels, vesicles, and nanoparticles. Polymers have several ad- vantageous properties including ease of preparation, controlled release of drugs and high drug loading capacity. In this chapter, triblock copolymers in different polymer- ization states were studied for their effect on 5F-Tryptophan. 5F-Tryptophan is used as a model system and its interactions with triblock copolymers F127 and P123 stud- ied at different concentrations. The attenuation of signal amplitude in the pulse field gradient diffusion for 19 F spin of 5F-Tryptophan was studied and it was concluded that different polymerization states of both the polymers show significant differences in signal attenuation at different concentrations. The effect was also studied at three dif- ferent temperatures. Longitudinal spin-lattice relaxation rates and transverse spin-spin relaxation rates were also measured for all the samples. Chapter 4 The usage of ionic liquids (ILs) as a solvent has grown sharply over the last twenty years due to their unique properties and potential uses as novel reaction media. ILs are green alternatives to standard organic solvents because of their low volatility, non- flammability, and greater stability when exposed to thermal and chemical stressors. ILs can be highly flexible as a result of tuning their cations and anions as well as their substituents. Several studies have showed the interaction of ILs with drugs and demon- strated their utility as potential drug delivery systems. In this chapter, we quantified the interaction of an IL with 5-fluorouracil, which has been extensively used as an anti- tumor drug. Relaxation and diffusion NMR experiments can provide valuable insights into internal molecular dynamics and the overall hydrodynamic behavior of ILs as sol- vent media. We studied the interaction of 5-fluorouracil with the ionic liquid 1-butyl-3- methylimidazolium bromide (BMIMBr) via NMR relaxation experiments and quan- tum chemical calculations. It has been shown in previous studies that 5-fluorouracil is effective for treatment of skin cancer when it is dissolved in C4MIMBr as compared to water. Also, in the ionic liquid, the drug solubility increases 2.5 times as compared to water. In order to understand the dynamics of 5-Fluorouracil in C4MIMBr, we per- formed quantum chemistry calculations using the Gaussian package and were able to delineate a 5-fluorouracil-C4MIMBr interaction model. We have optimized and ar- vii0. Abstract ranged the stability order of structures and preferred interaction sites on the basis of energy value. It was found that the fluorine site in 5-fluorouracil is the third most preferable site for interaction of ionic liquid. These interaction sites were corrobo- rated via NMR relaxation rates measurements. Five samples at Drug:IL concentration ratios were prepared. Interaction sites were identified from longitudinal spin-lattice and transverse spin-spin relaxation rates as well as from diffusion coefficient measure- ments. The theoretical and experimental results were found to be in good agreement. Chapter 5 Due to ease of sample preparation and accurate metabolite identification and quan- tification, NMR spectroscopy is an important tool for metabolomics. 1D 1 H NMR spectra can be acquired easily with high sensitivity in few minutes, however due to the presence of several metabolites with severely overlapping peaks which are hard to differentiate and identify, 1D NMR has limited use. Two-dimensional (2D) NMR spec- troscopy has several advantages over 1D NMR and can provide detailed information about molecular structure via different correlation maps between atoms. However due to long experimental acquisition times, no direct quantification of peaks, pulse imper- fections and other experimental artifacts, 2D NMR has not been extensively used for metabolomics studies. In this chapter we performed chemometric analysis of green tea and black tea samples by using a fast 2D NMR pulse sequence to reduce experimental time. The ASAP-HSQC scheme (Acceleration by Sharing Adjacent Polarization Het- eronuclear Single Quantum Correlation spectroscopy) was used to record 2D 13 C– 1 H correlation spectra. The results in this chapter demonstrate the merits of directly using 2D NMR spectral peak intensities instead of the more traditional 1D 1 H NMR peaks as inputs for multivariate statistical analysis. Metabolites were identified solely from the 2D NMR spectra, and the NMR analysis was combined with multivariate pattern recognition to identify metabolites that contribute significantly to the metabolic differ- ences between green tea and black tea. The HSQC 0 approach was used to accurately quantify 2D cross-peak intensities. 2D NMR spectra were processed in MATLAB. The 2D ASAP-HSQC experiment is able to obtain the same two-dimensional car- bon–proton correlation information as the standard 2D HSQC experiment, in a frac- tion of the time as taken by normal HSQC. The results of performing multivariate data viiianalysis on 2D NMR datasets was compared with those obtained by using 1D NMR datasets and a good agreement was found between the two methods. Also, a multi- variate analysis was performed to identify metabolites that vary in concentration in Bougainvillea plant leaves collected at two different times during the day. For this model, there was no prior information about metabolites identified from 1D 1 H NMR data, and 2D NMR data alone was used to perform the entire analysis and identify the significant metabolites. Chapter 6 This chapter provides some general remarks on the problems covered in the thesis. Possible future applications of the projects in this thesis and the new avenues of re- search they open up are described. The overall contribution of this thesis in the context of drug delivery and 2D NMR-based metabolomics is summarized. ix