Structure-, stereochemistry-, and metal-regulated DNA binding/cleaving molecules

In the cell, the binding of proteins to specific sequences of double helical DNA is essential for controlling the processes of protein synthesis (at the level of DNA transcription) and cell proliferation (at the level of DNA replication). In the laboratory, the sequence-specific DNA binding/cleaving properties of restriction endonuclease enzymes (secreted by microorganisms to protect them from foreign DNA molecules) have helped to fuel a revolution in molecular biology. The strength and specificity of a protein:DNA interaction depend upon structural features inherent to the protein and DNA sequences, but it is now appreciated that these features (and therefore protein:DNA complexation) may be altered (regulated) by other protein:DNA complexes, or by environmental factors such as temperature or the presence of specific organic molecules or inorganic ions. It is also now appreciated that molecules much smaller than proteins (including antibiotics of molecular weight less than 2000 and oligonucleotides) can bind to double-helical DNA in sequence-specific fashion. Elucidation of structural motifs and microscopic interactions responsible for the specific molecular recognition of DNA leads to greater understanding of natural processes and provides a basis for the design of novel sequence-specific DNA binding molecules. This thesis describes the synthesis and DNA binding/cleaving characteristics of molecules designed to probe structural, stereochemical, and environmental factors that regulate sequence-specific DNA recognition.

Chapter One introduces the DNA minor groove binding antibiotics Netropsin and Distamycin A, which are di- and tri(N-methylpyrrolecarboxamide) peptides, respectively. The method of DNA affinity cleaving, which has been employed to determine DNA binding properties of designed synthetic molecules is described. The design and synthesis of a series of Netropsin dimers linked in tail-to-tail fashion (by oxalic, malonic, succinic, or fumaric acid), or in head-to-tail fashion (by glycine, β-alanine, and γ-aminobutanoic acid (Gaba)) are presented. These Bis(Netropsin)s were appended with the iron-chelating functionality EDTA in order to make use of the technique of DNA affinity cleaving. Bis(Netropsin)-EDTA compounds are analogs of penta(N-methylpyrrolecarboxamide)-EDTA (P5E), which may be considered a head-to-tail Netropsin dimer linked by Nmethylpyrrolecarboxamide. Low- and high-resolution analysis of pBR322 DNA affinity cleaving by the iron complexes of these molecules indicated that small changes in the length and nature of the linker had significant effects on DNA binding/cleaving efficiency (a measure of DNA binding affinity). DNA binding/cleaving efficiency was found to decrease with changes in the linker in the order β-alanine > succinamide > fumaramide > N-methylpyrrolecarboxamide > malonamide >glycine, γ-aminobutanamide > oxalamide. In general, the Bis(Netropsin)-EDTA:Fe compounds retained the specificity for seven contiguous A:T base pairs characteristic of P5E:Fe binding. However, Bis(Netropsin)Oxalamide- EDTA:Fe exhibited decreased specificity for A:T base pairs, and Bis(Netropsin)-Gaba-EDT A:Fe exhibited some DNA binding sites of less than seven base pairs. Bis(Netropsin)s linked with diacids have C₂-symmmetrical DNA binding subunits and exhibited little DNA binding orientation preference. Bis(Netropsin)s linked with amino acids lack C₂-symmetrical DNA binding subunits and exhibited higher orientation preferences. A model for the high DNA binding orientation preferences observed with head-to-tail DNA minor groove binding molecules is presented.

Chapter Two describes the design, synthesis, and DNA binding properties of a series of chiral molecules: Bis(Netropsin)-EDTA compounds with linkers derived from (R,R)-, (S,S)-, and (RS,SR)-tartaric acids, (R,R)-, (S,S)-, and (RS,SR)-tartaric acid acetonides, (R)- and (S)-malic acids, N ,N-dimethylaminoaspartic acid, and (R)- and (S)-alanine, as well as three constitutional isomers in which an N-methylpyrrolecarboxamide (P1) subunit and a tri(N-methylpyrrolecarboxamide)-EDTA (P3-EDTA) subunit were linked by succinic acid, (R ,R)-, and (S ,S)-tartaric acids. DNA binding/cleaving efficiencies among this series of molecules and the Bis(Netropsin)s described in Chapter One were found to decrease with changes in the linker in the order β-alanine > succinamide > P1-succinamide-P3 > fumaramide > (S)-malicamide > N-methylpyrrolecarboxamide > (R)-malicamide > malonamide > N ,N-dimethylaminoaspanamide > glycine = Gaba = (S,S)-tartaramide = P1-(S,S)-tanaramide-P3 > oxalamide > (RS,SR)-tartaramide = P1- (R,R)-tanaramide-P3 > (R,R)-tartaramide (no sequence-specific DNA binding was detected for Bis(Netropsin)s linked by (R)- or (S)-alanine or by tartaric acid acetonides). The chiral molecules retained DNA binding specificity for seven contiguous A:T base pairs. From the DNA affinity cleaving data it could be determined that: 1) Addition of one or two substituents to the linker of Bis(Netropsin)-Succinamide resulted in stepwise decreases in DNA binding affinity; 2) molecules with single hydroxyl substituents bound DNA more strongly than molecules with single dimethylamino substituents; 3) hydroxyl-substituted molecules of (S) configuration bound more strongly to DNA than molecules of (R) configuration. This stereochemical regulation of DNA binding is proposed to arise from the inherent right-handed twist of (S)-enantiomeric Bis(Netropsin)s versus the inherent lefthanded twist of (R)-enantiomeric Bis(Netropsin)s, which makes the (S)-enantiomers more complementary to the right-handed twist of B form DNA.

Chapter Three describes the design and synthesis of molecules for the study of metalloregulated DNA binding phenomena. Among a series of Bis(Netropsin)-EDTA compounds linked by homologous tethers bearing four, five, or six oxygen atoms, the Bis(Netropsin) linked by a pentaether tether exhibited strongly enhanced DNA binding/cleaving in the presence of strontium or barium cations. The observed metallospecificity was consistent with the known affinities of metal cations for the cyclic hexaether 18-crown-6 in water. High-resolution DNA affinity cleaving analysis indicated that DNA binding by this molecule in the presence of strontium or barium was not only stronger but of different sequence-specificity than the (weak) binding observed in the absence of metal cations. The metalloregulated binding sites were consistent with A:T binding by the Netropsin subunits and G:C binding by a strontium or barium:pentaether complex. A model for the observed positive metalloregulation and novel sequence-specificity is presented. The effects of 44 different cations on DNA affinity cleaving by P5E:Fe were examined. A series of Bis(Netropsin)-EDTA compounds linked by tethers bearing two, three, four, or five amino groups was also synthesized. These molecules exhibited strong and specific binding to A:T rich regions of DNA. It was found that the iron complexes of these molecules bound and cleaved DNA most efficiently at pH 6.0-6.5, while P5E:Fe bound and cleaved most efficiently at pH 7.5-8.0. Incubating the Bis(Netropsin) Polyamine-EDTA:Fe molecules with K₂PdCl₄ abolished their DNA binding/cleaving activity. It is proposed that the observed negative metalloregulation arises from kinetically inert Bis(Netropsin) Polyamine:Pd(II) complexes or aggregates, which are sterically unsuitable for DNA complexation. Finally, attempts to produce a synthetic metalloregulated DNA binding protein are described. For this study, five derivatives of a synthetic 52 amino acid residue DNA binding/cleaving protein were produced. The synthetic mutant proteins carried a novel pentaether ionophoric amino acid residue at different positions within the primary sequence. The proteins did not exhibit significant DNA binding/cleaving activity, but they served to illustrate the potential for introducing novel amino acid residues within DNA binding protein sequences, and for the development of the tricyclohexyl ester of EDTA as a superior reagent for the introduction of EDT A into synthetic proteins.

Chapter Four describes the discovery and characterization of a new DNA binding/cleaving agent, [SalenMn(III)]OAc. This metal complex produces single- and double-strand cleavage of DNA, with specificity for A:T rich regions, in the presence of oxygen atom donors such as iodosyl benzene, hydrogen peroxide, or peracids. Maximal cleavage by [SalenMn(III)]OAc was produced at pH 6-7. A comparison of DNA singleand double-strand cleavage by [SalenMn(III)]⁺ and other small molecules (Methidiumpropyl-EDTA:Fe, Distamycin-EDTA:Fe, Neocarzinostatin, Bleomycin:Fe) is presented. It was found that DNA cleavage by [SalenMn(III)]⁺ did not require the presence of dioxygen, and that base treatment of DNA subsequent to cleavage by [SalenMn(III)]⁺ afforded greater cleavage and alterations in the cleavage patterns. Analysis of DNA products formed upon DNA cleavage by [SalenMn(III)] indicated that cleavage was due to oxidation of the sugar-phosphate backbone of DNA. Several mechanisms consistent with the observed products and reaction requirements are discussed.

Chapter Five describes progress on some additional studies. In one study, the DNA binding/cleaving specificities of Distamycin-EDTA derivatives bearing pyrrole N-isopropyl substituents were found to be the same as those of derivatives bearing pyrrole N-methyl substituents. In a second study, the design of and synthetic progress towards a series of nucleopeptide activators of transcription are presented. Five synthetic plasmids designed to test for activation of in vitro run-off transcription by DNA triple helix-forming oligonucleotides or nucleopeptides are described.

Chapter Six contains the experimental documentation of the thesis work.

The needle in the hundred square degree haystack: The hunt for binary neutron star mergers with LIGO and Palomar Transient Factory

The Advanced LIGO and Virgo experiments are poised to detect gravitational waves (GWs) directly for the first time this decade. The ultimate prize will be joint observation of a compact binary merger in both gravitational and electromagnetic channels. However, GW sky locations that are uncertain by hundreds of square degrees will pose a challenge. I describe a real-time detection pipeline and a rapid Bayesian parameter estimation code that will make it possible to search promptly for optical counterparts in Advanced LIGO. Having analyzed a comprehensive population of simulated GW sources, we describe the sky localization accuracy that the GW detector network will achieve as each detector comes online and progresses toward design sensitivity. Next, in preparation for the optical search with the intermediate Palomar Transient Factory (iPTF), we have developed a unique capability to detect optical afterglows of gamma-ray bursts (GRBs) detected by the Fermi Gamma-ray Burst Monitor (GBM). Its comparable error regions offer a close parallel to the Advanced LIGO problem, but Fermi's unique access to MeV-GeV photons and its near all-sky coverage may allow us to look at optical afterglows in a relatively unexplored part of the GRB parameter space. We present the discovery and broadband follow-up observations (X-ray, UV, optical, millimeter, and radio) of eight GBM-IPTF afterglows. Two of the bursts (GRB 130702A / iPTF13bxl and GRB 140606B / iPTF14bfu) are at low redshift (z=0.145 and z = 0.384, respectively), are sub-luminous with respect to "standard" cosmological bursts, and have spectroscopically confirmed broad-line type Ic supernovae. These two bursts are possibly consistent with mildly relativistic shocks breaking out from the progenitor envelopes rather than the standard mechanism of internal shocks within an ultra-relativistic jet. On a technical level, the GBM--IPTF effort is a prototype for locating and observing optical counterparts of GW events in Advanced LIGO with the Zwicky Transient Facility.

Light-matter Interactions in Semiconductors and Metals: From Nitride Optoelectronics to Quantum Plasmonics

This thesis puts forth a theory-directed approach coupled with spectroscopy aimed at the discovery and understanding of light-matter interactions in semiconductors and metals.

The first part of the thesis presents the discovery and development of Zn-IV nitride materials.The commercial prominence in the optoelectronics industry of tunable semiconductor alloy materials based on nitride semiconductor devices, specifically InGaN, motivates the search for earth-abundant alternatives for use in efficient, high-quality optoelectronic devices. II-IV-N2 compounds, which are closely related to the wurtzite-structured III-N semiconductors, have similar electronic and optical properties to InGaN namely direct band gaps, high quantum efficiencies and large optical absorption coefficients. The choice of different group II and group IV elements provides chemical diversity that can be exploited to tune the structural and electronic properties through the series of alloys. The first theoretical and experimental investigation of the ZnSnxGe1−xN2 series as a replacement for III-nitrides is discussed here.

The second half of the thesis shows ab−initio calculations for surface plasmons and plasmonic hot carrier dynamics. Surface plasmons, electromagnetic modes confined to the surface of a conductor-dielectric interface, have sparked renewed interest because of their quantum nature and their broad range of applications. The decay of surface plasmons is usually a detriment in the field of plasmonics, but the possibility to capture the energy normally lost to heat would open new opportunities in photon sensors, energy conversion devices and switching. A theoretical understanding of plasmon-driven hot carrier generation and relaxation dynamics in the ultrafast regime is presented here. Additionally calculations for plasmon-mediated upconversion as well as an energy-dependent transport model for these non-equilibrium carriers are shown.

Finally, this thesis gives an outlook on the potential of non-equilibrium phenomena in metals and semiconductors for future light-based technologies.

Z-selective olefin metathesis using chelating ruthenium alkylidene catalysts

Publications about olefin metathesis will generally discuss how the discovery and development of well-defined catalysts to carry out this unique transformation have revolutionized many fields, from natural product and materials chemistry, to green chemistry and biology. However, until recently, an entire manifestation of this methodology had been inaccessible. Except for a few select examples, metathesis catalysts favor the thermodynamic trans- or E-olefin products in cross metathesis (CM), macrocyclic ring closing metathesis (mRCM), ring opening metathesis polymerization (ROMP), and many other types of reactions. Judicious choice of substrates had allowed for the direct synthesis of cis- or Z-olefins or species that could be converted upon further reaction, however the catalyst controlled synthesis of Z-olefins was not possible until very recently.

Research into the structure and stability of metallacyclobutane intermediates has led to the proposal of models to impart Z-selectivity in metathesis reactions. Having the ability to influence the orientation of metallacyclobutane substituents to cause productive formation of Z- double bonds using steric and electronic effects was highly desired. The first successful realization of this concept was by Schrock and Hoveyda et al. who synthesized monoaryloxide pyrolidine (MAP) complexes of tungsten and molybdenum that promoted Z-selective CM. The Z-selectivity of these catalysts was attributed to the difference in the size of the two axial ligands. This size difference influences the orientation of the substituents on the forming/incipient metallacyclobutane intermediate to a cis-geometry and leads to productive formation of Z-olefins. These catalysts have shown great utility in the synthesis of complicated natural product precursors and stereoregular polymers. More recently, ruthenium catalysts capable of promoting Z-selective metathesis have been reported by our group and others. This thesis will discuss the development of ruthenium-based NHC chelated Z-selective catalysts, studies probing their unique metathesis mechanism, and synthetic applications that have been investigated thus far.

Chapter 1 will focus on studies into the stability of NHC chelated complexes and the synthesis of new and improved stable chelating architectures. Chapter 2 will discuss applications of the highly active and Z-selective developed in Chapter 1, including the formation of lepidopteran female sex pheromones using olefin cross metathesis and highly Z- and highly E-macrocycles using macrocyclic ring closing metathesis and Z-selective ethenolysis. Chapter 3 will explore studies into the unique mechanism of olefin metathesis reactions catalyzed by these NHC chelated, highly Z-selective catalysts, explaining observed trends by investigating the stability of relevant, substituted metallacyclobutane intermediates.