Dynamic nuclear polarization (DNP) is an emerging method for increasing signal-to-noise in solid-state NMR experiments by transferring polarization from electrons to nuclei. Stable nitroxide biradicals, the polarization source for the cross effect mechanism, are typically co-dissolved at millimolar concentrations with biomolecules of interest. There has been considerable recent interest, however, in co-localizing the paramagnetic polarizing agent with the analyte of interest through covalent or noncovalent interactions. In this thesis, we bound the biradical to the protein of interest through conjugation to a high affinity, non-covalent inhibitor using E.coli dihydrofolate reductase (DHFR) and its nanomolar inhibitor trimethoprim (TMP). Two different biradical affinity reagents (TMP-T and TMP-V-T) were created by covalently linking trimethoprim to the biradical polarizing agent TOTAPOL. These TMP-TOTAPOL compounds provided excellent enhancements of the DHFR NMR spectrum, comparable to when TOTAPOL is co-dissolved with the protein. In contrast to TOTAPOL, the specific interactions of these compounds enabled them to be added stoichiometrically to samples of DHFR at micromolar concentrations while retaining excellent enhancements. Benefits of lowered biradical concentration include reduced chemical perturbation of the sample and the ability to selectively enhance signals for the protein of interest in a perdeuterated solvent matrix. The binding of TMP-V-T to DHFR confines the paramagnetic TOTAPOL moiety to the protein binding pocket. We capitalized on this fact to site-specifically study paramagnet induced signal quenching in DNP samples. Using crystallography and solution NMR spectroscopy, we demonstrated that although the TMP fragment is well ordered the TOTAPOL moiety is disordered when bound to DHFR. We prepared site-specific isotpically enriched samples of DHFR and measured signal quenching in DNP samples. Intriguingly, the bleaching patterns seen in DNP samples are similar to those observed in room temperature studies. We argue that static disorder plays a role in this observation, among other contributions. Our results provide design principles for DNP experiments with affinity biradicals, and demonstrate the utility of affinity biradicals for DNP studies given the confined signal quenching. Affinity biradicals hold great promise for enabling selective DNP investigations of dilute proteins in whole cells or cellular lysates. We explored how to use TMP-T and TMP-V-T to study DHFR in whole cells and cellular lysates. We developed strategies to overcome fast chemical reduction of the nitroxide moiety in the cellular environment, and sample preparation protocols to obtain overexpressed DHFR with isotopic enrichment patterns that enable selective DNP enhancements. We demonstrate excellent enhancements of whole cell pellets by TOTAPOL, and also provide evidence for selective DHFR enhancement by TMP-V-T in cellular lysates. Our results form guidelines for future selective studies of DHFR in cellular contexts. These results collectively demonstrate the potential of the TMP-T(V-T) compounds and similar affinity biradical reagents both for mechanistic DNP studies and selective NMR studies in cellular contexts.

Nonadiabatic dynamical processes are ubiquitous in chemistry and biology. Such events are directly connected to the treatment of energetically close lying states which gives rise to strong vibronic interactions in which case the Born-Oppenheimer approximation tends to break down. In case of biradicals, nonadiabatic events are facilitated by conical intersections, as a result of symmetry lowering of degenerate electronic states due to Jahn-Teller distortion. A central problem in the treatment of the nonadiabatic molecular dynamics is posed by the representation of potential energy surfaces. A point by point calculation of a potential energy surface on a multi-dimensional grid is very cumbersome and in general does not provide with an analytical functional form of the potential. This becomes even more complicated when the adiabatic surfaces have cusps, where the function becomes non-differentiable. Vibronic model Hamiltonians, which represent the potential in the form of a potential matrix which contains the electronic energies as well as the couplings in a diabatic basis. A Taylor series expansion of the potential matrix can be done to get a smooth analytical functional form of the potential matrix elements. These models can then be used to perform nuclear dynamics using either exact diagonalization time-independent method or the wavepacket propagation based time-dependent methods. Thus, vibronic models provide a compact representation of complicated coupled potential energy surfaces, which can be used in conjunction with non-adiabatic nuclear dynamics Vibronic models have been constructed for selected biradicals, for which photodetachment spectra have been simulated using the time-independent (VIBRON) as well as time-dependent (MCTDH) methods. Consistent results have been obtained with both the approaches for small systems. This also assures the use of MCTDH program for larger systems, where the time-independent methods are not applicable. Moreover, for biradicals, the parent anionic state also undergoes a Jahn-Teller distortion, or often the ground state potential energy surface is highly anharmonic in nature. This requires the description of anionic ground state by a vibronic model. Therefore, in order to simulate the photodetachment spectra of biradicals, three vibronic models are constructed for each simulation. The first model describes the ground and excited states of the parent anionic (neutral) species. Two other vibronic models describe singlet and triplet states of the target neutral (cation) species, and the spectrum is simulated using the vibronic ground state(s) of the anion (neutral) as the absorbing state in VIBRON/MCTDH. The electronic states and vibronic model parameters are obtained using the IP-EOM-CCSD and DIP-STEOM-CCSD methodology as coded in the ACESII quantum chemistry program package. The photodetachment spectra of nitrate radical, cyclobutadiene negative ion and trimethylene negative ion have been studied using this methodology.

Thesis (Ph. D.)--West Virginia University, 2002. / Title from document title page. Document formatted into pages; contains xvi, 377 p. : ill. Includes abstract. Includes bibliographical references (p. 120-128).

Nonadiabatic dynamical processes are ubiquitous in chemistry and biology. Such events are directly connected to the treatment of energetically close lying states which gives rise to strong vibronic interactions in which case the Born-Oppenheimer approximation tends to break down. In case of biradicals, nonadiabatic events are facilitated by conical intersections, as a result of symmetry lowering of degenerate electronic states due to Jahn-Teller distortion. A central problem in the treatment of the nonadiabatic molecular dynamics is posed by the representation of potential energy surfaces. A point by point calculation of a potential energy surface on a multi-dimensional grid is very cumbersome and in general does not provide with an analytical functional form of the potential. This becomes even more complicated when the adiabatic surfaces have cusps, where the function becomes non-differentiable. Vibronic model Hamiltonians, which represent the potential in the form of a potential matrix which contains the electronic energies as well as the couplings in a diabatic basis. A Taylor series expansion of the potential matrix can be done to get a smooth analytical functional form of the potential matrix elements. These models can then be used to perform nuclear dynamics using either exact diagonalization time-independent method or the wavepacket propagation based time-dependent methods. Thus, vibronic models provide a compact representation of complicated coupled potential energy surfaces, which can be used in conjunction with non-adiabatic nuclear dynamics Vibronic models have been constructed for selected biradicals, for which photodetachment spectra have been simulated using the time-independent (VIBRON) as well as time-dependent (MCTDH) methods. Consistent results have been obtained with both the approaches for small systems. This also assures the use of MCTDH program for larger systems, where the time-independent methods are not applicable. Moreover, for biradicals, the parent anionic state also undergoes a Jahn-Teller distortion, or often the ground state potential energy surface is highly anharmonic in nature. This requires the description of anionic ground state by a vibronic model. Therefore, in order to simulate the photodetachment spectra of biradicals, three vibronic models are constructed for each simulation. The first model describes the ground and excited states of the parent anionic (neutral) species. Two other vibronic models describe singlet and triplet states of the target neutral (cation) species, and the spectrum is simulated using the vibronic ground state(s) of the anion (neutral) as the absorbing state in VIBRON/MCTDH. The electronic states and vibronic model parameters are obtained using the IP-EOM-CCSD and DIP-STEOM-CCSD methodology as coded in the ACESII quantum chemistry program package. The photodetachment spectra of nitrate radical, cyclobutadiene negative ion and trimethylene negative ion have been studied using this methodology.

Thesis (Ph. D.)--West Virginia University, 2005. / Title from document title page. Document formatted into pages; contains x, 165 p. : ill. Includes abstract. Includes bibliographical references (p. 65-71).

Doctor of Philosophy / Department of Chemistry / Sundeep Rayat / Sundeep Rayat / This dissertation focused on the synthesis of 1-(2-ethynylphenyl)-4-phenyl tetrazole-5-thione derivatives 1 and evaluating their potential as a new class of photoactivated DNA cleaving prodrugs. We hypothesized that light activation of 1 would cause the decomposition of the tetrazolethione ring system to enyne-carbodiimide 2 with simultaneous loss of dinitrogen and sulfur via 1,3-triplet biradicals 1′, which would be spontaneously followed by Schmittel cyclization to indoloquinolines 3 via benzofulvene type biradicals 2′. The biradicals 1′ and 2′ would have the potential to cause DNA cleavage by abstracting hydrogens from its sugar phosphate backbone, analogous to the mechanism of action of naturally occurring enediyne antitumor antibiotics. Note that our proposed prodrugs contained a 1,4-diaryl tetrazolethione functionality and a direct synthetic route for their construction was lacking in the literature despite their wide spread applications. Therefore, our initial efforts were directed towards developing a general strategy to obtain these ring systems. We employed a highly versatile and efficient copper mediated N-arylation to first obtain a series of 1,4-diaryl tetrazol-5-ones which were thionated with Lawesson’s reagent to afford the corresponding 1,4-diaryl tetrazole-5-thiones in moderate yields. Specifically, the synthesis of 1 involved Sonogashira coupling of the obtained 1-(2-bromophenyl)-4-phenyl-1H-tetrazole-5(4H)-thione with the appropriate ethynyl compounds (Chapter 2). Since the tetrazole ring is an important structural component in many biologically and medicinally relevant compounds, we were interested in evaluating the anticancer activity of these compounds in the absence of photochemical activation. The moderate IC50 values against leukemia and breast cancer cell lines showed that the anticancer activity of these compounds prior to photoirradiation was minimal (Chapter 3). Independent studies have shown that the photodecomposition of tetrazolethiones gives carbodiimides via biradicals, and photocyclization of enyne-carbodiimide forms indoloquinoline also via biradicals. However, it was not known whether these two photoreactions could happen sequentially in one pot with one light source from a substrate like 1, generating biradicals 1′ and 2′ which could later be employed for DNA photocleavage as hypothesized. Therefere, we photolysed 1 in acetonitrile, and our results show clean formation of a mixture of enyne-carbodiimides and indoloquinolines via biradicals 1′ and 2′ (Chapter 4). Finally, we investigated DNA photocleavage by 1 at 350 nm and our results showed significant DNA cleavage in concentrations as low as 100 μM (Chapter 5).

Univ., Diss.--Göteborg, 2007. / Enth. außerdem 5 Zeitschriftenaufsätze.

Thesis (Ph. D.)--West Virginia University, 2008. / Title from document title page. Document formatted into pages; contains xiv, 240 p. : ill. (some col.). Includes abstract. Includes bibliographical references (p. 95-104).

Thesis (Ph. D.)--University of Washington, 1999. / Vita. Includes bibliographical references (leaves 84-88).

Thesis (doctoral)--Göteborg University, 2007. / Includes bibliographical references.