Probing Anisotropic Interactions In Solid State NMR : Techniques And Applications

Abstract

The thesis aims at methodological developments in Nuclear Magnetic Resonance (NMR) and study of oriented samples like liquid crystals and single crystals and powder samples. Though methodological development in solid state NMR (ss-NMR) has gone far ahead, this work attempts to contribute some novel methods in this direction. The work presented here falls into two categories (i) methodological developments for obtaining information on anisotropic interactions and (ii) experiments which utilize the existing methodologies to study systems of interest under static condition and also under sample spinning at the Magic Angle. In the solid state, dipolar couplings play a crucial role. On the one hand these couplings could be used to transfer polarization from an abundant nucleus to a rare nucleus and increase the sensitivity of the rare nucleus. On the other hand, the measurement of dipolar couplings itself is crucial for extracting structural and dynamic information. A third aspect is that dipolar couplings could be used to obtain correlation, say between two different nuclear species or between the same kind of nuclei as in an exchange experiment. A major part of this thesis deals with all three aspects mentioned above. The thesis presents a new heteronuclear polarization transfer scheme which is devoid of some of the short comings of the existing and well-known polarization transfer schemes. This pulse sequence has been found to be useful in different contexts involving both spin ½ and spin 1 nucleus. The use of dipolar couplings for obtaining correlation in both static oriented systems and in powder samples has been illustrated. In the case of the powder sample, the study has been useful in obtaining useful orientation information. Finally, chemical shifts are known to be indicators of finer structural features of molecules in solution and solid state. 13 C MAS NMR studies have been exploited in understanding these structural features of short peptides containing prolines in the solid state and for comparing with their structures in solution. Chapter 1 covers the theoretical aspects required for the experimental work described in the thesis. A brief description of NMR has been followed by the explicit description of various interaction Hamiltonian’s in ss-NMR. Subsequently the experimental and the theoretical tools needed for ss-NMR study like Magic Angle Spinning (MAS), Cross-Polarization, Homo/Heteronuclear decoupling schemes have also been discussed. Chapter 2, describes a new heteronuclear polarization transfer scheme for oriented samples – named DAPT (Dipolar Assisted Polarization Transfer) and its application to different systems. DAPT uses a homonuclear decoupling sequence such as BLEW-12 for effecting heteronuclear polarization transfer. The chapter has been divided into five related parts. Section 2(A) starts with an introduction to the existing heteronuclear polarization transfer schemes. Subsequently the theoretical background of the new sequence is presented. Experimental implementation of the sequence in an oriented system, liquid crystal is presented and is compared with the well-known polarization transfer scheme, Hartmann-Hahn Cross Polarization (HH-CP). In 2(B) the implementation of the sequence as a local field spectroscopy for measuring heteronuclear dipolar couplings is presented. After initial discussion about local field spectroscopy and its relevance in ss-NMR, the improvements made in the earlier mentioned sequence along with its 2D implementation in a liquid crystal sample are described. A comparative study is also presented using DAPT with various other homonuclear decoupling sequences. Chapter 2(C) deals with the extension of DAPT to spin-1 systems. The difficulties in setting up the HH-CP in spin-1 systems are highlighted. Experimental demonstrations on a test sample of oriented CD3 I and also on a deuterated liquid crystal is described. The sequence has been incorporated as part of a 2D correlation experiment, where the F1 dimension provides the quadrupolar couplings of deuterium and the F2 the chemical shifts of the attached carbons. The comparison of the sequence with HH-CP, its merits and demerits are discussed and the potential applications are highlighted. Chapter 2(D) deals with the relatively less studied transition in 14N nucleus, known as the Overtone-Transition (OT). An introduction to OT and its relevance is provided in the beginning followed by the extension of DAPT in exciting and detecting OT. The experiments have been done on a single crystal of a model peptide, N-Acetyl-DL-Valine and are compared with the conventional method. Amide proton chemical shifts are also measured using DAPT in an indirect way. The advantages and the future application in studying OT are also discussed. Chapter 2(E) discusses the extension of DAPT to the single crystal of NAV and in identifying the molecules in the unit cell. The SLF spectrum of NAV is complicated due to the presence of two magnetically in-equivalent molecules in the unit cell and with pairs of splitting for each C - 1H and C - 1H pairs. The dipolar couplings are extracted from the experiment and with the aid of a MATLAB program and by incorporating the crystal coordinates, identification of C-1H and C-1H pairs belonging to a particular molecule have been carried out. Chapter 3 describes a novel and useful modification of the well-known Separated Local Field (SLF) sequence in solid state known as PISEMA (Polarization Spin Exchange at the Magic Angle). PISEMA is a popular technique for measuring heteronuclear dipolar couplings in oriented in oriented biological membranes and in liquid crystals. While it has several advantages such as a large dipolar scaling factor, narrow line-widths in the dipolar dimension and ease of setting up etc it suffers from a major problem. The technique is highly sensitive to the proton off-sets which affect the measured dipolar couplings. In the present chapter the origin of this problem has been analyzed in detail and a solution has been proposed. The modification to the experiment has been implemented on a liquid crystal and the off-set independence of the new sequence has been demonstrated. Further studies on a more rigid system such as a solid single crystal has been used to verify the effect of the modification on homonuclear decoupling efficiency and the consequent effects on the line widths in addition to off-set independence. The advantages of the proposed method over the existing one in terms of line-width and robustness in measuring heteronuclear dipolar couplings are demonstrated. Chapter 4 presents a study of deuterium exchange on a di-peptide. Deuterium as well as carbon-13 NMR spectroscopy has been extensively used earlier on static powder samples for studying exchange phenomena. In the present study we have applied the methodology for obtaining relative N-D vector orientation in a di-peptide. The magnetization exchange between deuterium nuclei through the dipolar couplings between them has been monitored. The need to match the quadrupolar split energy levels of two different deuterium’s differently oriented in the magnetic field requires that the sample be spun slowly. Characteristic exchange powder patterns were obtained which were used to infer relative orientation information. Comparison with the crystal structure indicates that the magnetization is likely to be inter-molecular rather than intra-molecular. The chapter follows the following sequence. A brief description about the importance of exchange studies in ss-NMR is presented. A theoretical approach is followed by a discussion of the angular dependence of the frequencies and the visualization of the mutual re-orientation angles. The motivation of the project followed by the experimental techniques, especially the use of slow MAS ~ 100 Hz in exchange studies are also presented. Initial studies have been carried out on di-methyl sulphone to check the reproducibility of the earlier reported results and later the sequence is extended to amide proton deuterated di-glycine. The 2D exchange spectrum recorded under slow MAS is then discussed in the context of the crystal structure and possible amide deuteriums involved in the exchange process are inferred. Chapter 5 discusses the natural abundant 13C and 15N NMR spectroscopy in the solid state in designed tri-peptides containing prolines. Proline is a unique amino acid because of it torsion angle values and is responsible for the turns and the globularity of the proteins. The well studied SH3 domain protein often binds to short peptides containing prolines and hence these study gains importance. Three peptides containing prolines were taken up for study. For peptide (1), the conformation was observed as cis/trans in the solution state and for the other two peptides it was all trans. The X-ray studies showed that peptide (1) has two molecules in the unit cell with both cis conformation. This motivated us to look at the solid state spectra of the peptides. Chemical shifts are signatures of conformers and it was established from the chemical shift differences that there exist two molecules in the unit cell for peptide (1), both in cis conformation. The conformers for the other two peptides predicted by NMR chemical shifts also matched with those obtained from X-ray studies. This opens up the possibility of using simple NMR measurements in the solid state as tools for secondary structure determination in larger peptides and proteins.