Photochemical cis-trans isomerization of β-styrylferrocene

The effect of metal atoms on photochemical transformations has been investigated by studies of the cis-trans isomerization of β-styrylferrocene.

The photostationary state lies entirely on the side of the trans isomer in the cases of direct irradiation at 3130 Å or at 3660 Å. The quantum yield at 3130 Å is 0.00650 and does not vary with concentration. In the presence of benzophenon as sensitizer the quantum yield is 0.00540. On the other hand, the quantum yield for direct irradiation at 3660 Å decreases with increasing concentration of cis β- styrylferrocene varying from 0.00365 to 0.00198.

These results lead to the suggestion that the isomerization takes place from a triplet state of β-styrylferrocene which probably has higher energy than the lowest triplet; reaction from the third triplet seems most likely.

Photosensitized CIS-TRANS isomerization of 2,3-diphenylbutene-2 investigation of anomalous sensitizers for substituted Stilbene isometrization

The behavior of the photosensitized cis-trans isomerization of 2,3-diphenylbutene-2 was studied as a function of sensitizer energy by previously established methods. In addition, certain sensitizers for which parameters other than energy transfer are operative in inducting isomerizations, were studied in more detail. Sensitization of various stilbenes and substituted stilbenes by triphenylene is discussed in terms of excited state complex formation with stilbene. Sensitization by quinones, halogen-containing aromatics and 1,2-diketones is discussed in terms of attack by photolytically produced free radicals, either by addition to and elimination from the double bond, or in the cases of 1,2-diphenylpropene and 2,3-diphenylbutene-2, by hydrogen abstraction from one of the methyl groups and reversible abstraction by the allylic radical to produce cis-trans isomerized substrate and the structurally isomerized products, 2,3-diphenylpropene and 2,3-diphenylbutene-1.

Beyond Watson and Crick : programming the self-assembly and reconfiguration of DNA nanostructures based on stacking interactions

Life is the result of the execution of molecular programs: like how an embryo is fated to become a human or a whale, or how a person’s appearance is inherited from their parents, many biological phenomena are governed by genetic programs written in DNA molecules. At the core of such programs is the highly reliable base pairing interaction between nucleic acids. DNA nanotechnology exploits the programming power of DNA to build artificial nanostructures, molecular computers, and nanomachines. In particular, DNA origami—which is a simple yet versatile technique that allows one to create various nanoscale shapes and patterns—is at the heart of the technology. In this thesis, I describe the development of programmable self-assembly and reconfiguration of DNA origami nanostructures based on a unique strategy: rather than relying on Watson-Crick base pairing, we developed programmable bonds via the geometric arrangement of stacking interactions, which we termed stacking bonds. We further demonstrated that such bonds can be dynamically reconfigurable.

The first part of this thesis describes the design and implementation of stacking bonds. Our work addresses the fundamental question of whether one can create diverse bond types out of a single kind of attractive interaction—a question first posed implicitly by Francis Crick while seeking a deeper understanding of the origin of life and primitive genetic code. For the creation of multiple specific bonds, we used two different approaches: binary coding and shape coding of geometric arrangement of stacking interaction units, which are called blunt ends. To construct a bond space for each approach, we performed a systematic search using a computer algorithm. We used orthogonal bonds to experimentally implement the connection of five distinct DNA origami nanostructures. We also programmed the bonds to control cis/trans configuration between asymmetric nanostructures.

The second part of this thesis describes the large-scale self-assembly of DNA origami into two-dimensional checkerboard-pattern crystals via surface diffusion. We developed a protocol where the diffusion of DNA origami occurs on a substrate and is dynamically controlled by changing the cationic condition of the system. We used stacking interactions to mediate connections between the origami, because of their potential for reconfiguring during the assembly process. Assembling DNA nanostructures directly on substrate surfaces can benefit nano/microfabrication processes by eliminating a pattern transfer step. At the same time, the use of DNA origami allows high complexity and unique addressability with six-nanometer resolution within each structural unit.

The third part of this thesis describes the use of stacking bonds as dynamically breakable bonds. To break the bonds, we used biological machinery called the ParMRC system extracted from bacteria. The system ensures that, when a cell divides, each daughter cell gets one copy of the cell’s DNA by actively pushing each copy to the opposite poles of the cell. We demonstrate dynamically expandable nanostructures, which makes stacking bonds a promising candidate for reconfigurable connectors for nanoscale machine parts.

Variable-angle electron spectroscopic studies of various polyatomic molecular systems

The complementary techniques of low-energy, variable-angle electron-impact spectroscopy and ultraviolet variable-angle photoelectron spectroscopy have been used to study the electronic spectroscopy and structure of several series of molecules. Electron-impact studies were performed at incident beam energies between 25 eV and 100 eV and at scattering angles ranging from 0° to 90°. The energy-loss regions from 0 eV to greater than 15 eV were studied. Photoelectron spectroscopic studies were conducted using a HeI radiation source and spectra were measured at scattering angles from 45° to 90°. The molecules studied were chosen because of their spectroscopic, chemical, and structural interest. The operation of a new electron-impact spectrometer with multiple-mode target source capability is described. This spectrometer has been used to investigate the spin-forbidden transitions in a number of molecular systems.

The electron-impact spectroscopy of the six chloro-substituted ethylenes has been studied over the energy-loss region from 0-15 eV. Spin-forbidden excitations corresponding to the π → π*, N → T transition have been observed at excitation energies ranging from 4.13 eV in vinyl chloride to 3.54 eV in tetrachloroethylene. Symmetry-forbidden transitions of the type π → np have been oberved in trans-dichloroethyene and tetrachlor oethylene. In addition, transitions to many states lying above the first ionization potential were observed for the first time. Many of these bands have been assigned to Rydberg series converging to higher ionization potentials. The trends observed in the measured transition energies for the π → π*, N → T, and N → V as well as the π → 3s excitation are discussed and compared to those observed in the methyl- and fluoro- substituted ethylenes.

The electron energy-loss spectra of the group VIb transition metal hexacarbonyls have been studied in the 0 eV to 15 eV region. The differential cross sections were obtained for several features in the 3-7 eV energy-loss region. The symmetry-forbidden nature of the ¹A_1g → ¹A_1g, 2t_2g(π) → 3t_2g(π*) transition in these compounds was confirmed by the high-energy, low-angle behavior of their relative intensities. Several low lying transitions have been assigned to ligand field transitions on the basis of the energy and angular behavior of the differential cross sections for these transitions. No transitions which could clearly be assigned to singlet → triplet excitations involving metal orbitals were located. A number of states lying above the first ionization potential have been observed for the first time. A number of features in the 6-14 eV energy-loss region of the spectra of these compounds correspond quite well to those observed in free CO.

A number of exploratory studies have been performed. The π → π*, N → T, singlet → triplet excitation has been located in vinyl bromide at 4.05 eV. We have also observed this transition at approximately 3.8 eV in a cis-/trans- mixture of the 1,2-dibromoethylenes. The low-angle spectrum of iron pentacarbonyl was measured over the energy-loss region extending from 2-12 eV. A number of transitions of 8 eV or greater excitation energy were observed for the first time. Cyclopropane was also studied at both high and low angles but no clear evidence for any spin- forbidden transitions was found. The electron-impact spectrum of the methyl radical resulting from the pyrolysis of tetramethyl tin was obtained at 100 eV incident energy and at 0° scattering angle. Transitions observed at 5.70 eV and 8.30 eV agree well with the previous optical results. In addition, a number of bands were observed in the 8-14 eV region which are most likely due to Rydberg transitions converging to the higher ionization potentials of this molecule. This is the first reported electron-impact spectrum of a polyatomic free radical.

Variable-angle photoelectron spectroscopic studies were performed on a series of three-membered-ring heterocyclic compounds. These compounds are of great interest due to their highly unusual structure. Photoelectron angular distributions using HeI radiation have been measured for the first time for ethylene oxide and ethyleneimine. The measured anisotropy parameters, β, along with those measured for cyclopropane were used to confirm the orbital correlations and photoelectron band assignments. No high values of β similar to those expected for alkene π orbitals were observed for the Walsh or Forster-Coulson-Moffit type orbitals.

Investigation of competitive antagonist binding to the nicotinic acetylcholine receptor using voltage-jump and light-flash techniques

1. The effect of 2,2’-bis-[α-(trimethylammonium)methyl]azobenzene (2BQ), a photoisomerizable competitive antagonist, was studied at the nicotinic acetycholine receptor of Electrophorus electroplaques using voltage-jump and light-flash techniques.

2. 2BQ, at concentrations below 3 μΜ, reduced the amplitude of voltage-jump relaxations but had little effect on the voltage-jump relaxation time constants under all experimental conditions. At higher concentrations and voltages more negative than -150 mV, 2BQ caused significant open channel blockade.

3. Dose-ratio studies showed that the cis and trans isomers of 2BQ have equilibrium binding constants (K _ᵢ) of .33 and 1.0 μΜ, respectively. The binding constants determined for both isomers are independent of temperature, voltage, agonist concentration, and the nature of the agonist.

4. In a solution of predominantly cis-2BQ, visible-light flashes led to a net cis→trans isomerization and caused an increase in the agonist-induced current. This increase had at least two exponential components; the larger amplitude component had the same time constant as a subsequent voltage-jump relaxation; the smaller amplitude component was investigated using ultraviolet light flashes.

5. In a solution of predominantly trans-2BQ, UV-light flashes led to a net trans→cis isomerization and caused a net decrease in the agonist-induced current. This effect had at least two exponential components. The smaller and faster component was an increase in agonist-induced current and had a similar time constant to the voltage-jump relaxation. The larger component was a slow decrease in the agonist-induced current with rate constant approximately an order of magnitude less than that of the voltage-jump relaxation. This slow component provided a measure of the rate constant for dissociation of cis-2BQ (k_ = 60/s at 20°C). Simple modelling of the slope of the dose-rate curves yields an association rate constant of 1.6 x 10⁸/M/s. This agrees with the association rate constant of 1.8 x 10⁸/M/s estimated from the binding constant (K_i). The Q₁₀ of the dissociation rate constant of cis-2BQ was 3.3 between 6° and 20°C. The rate constants for association and dissociation of cis-28Q at receptors are independent of voltage, agonist concentration, and the nature of the agonist.

6. We have measured the molecular rate constants of a competitive antagonist which has roughly the same K _ᵢ as d-tubocurarine but interacts more slowly with the receptor. This leads to the conclusion that curare itself has an association rate constant of 4 x 10⁹/M/s or roughly as fast as possible for an encounter-limited reaction.

Synthetic, structural and mechanistic studies of interactions of carbon, nitrogen, and oxygen nucleophiles with bis-pentamethylcyclopentadienyl zirconium and hafnium derivatives

The reactivity of permethylzirconocene and permethylhafnocene complexes with various nucleophiles has been investigated. Permethylzirconocene reacts with sterically hindered ketenes and allenes to afford metallacycle products. Reaction of these cummulenes with permethylzirconocene hydride complexes affords enolate and σ-allyl species, respectively. Reactions which afford enolate products are nonstereospecific, whereas reactions which afford allyl products initially give a cis-σ-allyl complex which rearranges to its trans isomer. The mechanism of these reactions is proposed to occur either by a Lewis Acid-Lewis Base interaction (ketenes) or by formation of a π-olefin intermediate (allenes).

Permethylzirconocene haloacyl complexes react with strong bases such as lithium diisopropylamide or methylene trimethylphosphorane to afford ketene compounds. Depending on the size of the alkyl ketene substituent, the hydrogenation of these compounds affords enolate-hydride products with varying degrees of stereoselectivity. The larger the substituent, the greater is the selectivity for cis hydrogenation products.

The reaction of permethylzirconocene dihydride and permethylhafnocene dihydride with methylene trimethylphosphorane affords methyl-hydride and dimethyl derivatives. Under appropriate conditions, the metallated-ylide complex 1, (η^5-C_5(CH_3)_5)_2 Zr(H)CH_2PMe_2CH_2, is also obtained and has been structurally characterized by X-ray diffraction techniques. Reaction of 1 with CO affords (η^5-C_5(CH_3)_5)_2 Zr(C,O-η^2 -(PMe_3)HC=CO)H which exists in solution as an equilibrium mixture of isomers. In one isomer (2), the η^2-acyl oxygen atom occupies a lateral equatorial coordination position about zirconium, whereas in the other isomer (3), the η-acyl oxygen atom occupies the central equatorial position. The equilibrium kinetics of the 2→3 isomerization have been studied and the structures of both complexes confirmed by X-ray diffraction methods. These studies suggest a mechanism for CO insertion into metal-carbon bonds of the early transition metals.

Permethylhafnocene dihydride and permethylzirconocene hydride complexes react with diazoalkanes to afford η^2-N, N' -hydrazonido species in which the terminal nitrogen atom of the diazoalkane molecule has inserted into a metal-hydride or metal-carbon bond. The structure of one of these compounds, Cp*_2Zr(NMeNCTol_2)OH, has been determined by X-ray diffraction techniques. Under appropriate conditions, the hydrazonido-hydride complexes react with a second equivalent of diazoalkene to afford η' -N-hydrazonido-η^2-N, N' -hydrazonido species.

Reactivity of titanocene methylidene with metal halides, alkene sulfides, and alkene oxides

Titanocene metallacyclobutanes show a wide variety of reactivites with organic and inorganic reagents. Their reactions include methylene transfer to organic carbonyls, formation of enolates, electron transfer from activated alkyl chlorides, olefin metathesis, ring opening polymerization. Recently, preparations of heterobinuclear µ-methylene complexes were reported. In this thesis, mechanistic, synthetic, and structural studies of the heterobinuclear µ-methylene complexes will be described. Also, the reaction of titanocene methylidene trimethylphosphine complex with alkene sulfide and styrene sulfide will be presented.

Heterobinuclear µ-methylene-µ-methyl complexes C_(p2)Ti(µ-CH_2)( µ-CH_3)M(1,5-COD) have been prepared (M = Rh, Ir). X-ray crystallography showed that the methyl group of the complex was bonded to the rhodium and bridges to the titanium through an agostic bond. The ^(1)H,^(13)CNMR, IR spectra along with partial deuteration studies supported the structure in both solution and solid state. Activation of the agostic bond is demonstrated by the equilibration of the µ-CH_3 and µ-CH_2 groups. A nonlinear Arrhenius plot, an unusually large kinetic isotope effect (24(5)), and a large negative activation entropy (-64(3)eu) can be explained by the quantum-mechanical tunneling. Calculated rate constants with Bell-type barrier fitted well with the observed one. This equilibration was best explained by a 4e-4c mechanism (or σ bond metathesis) with the character of quantum-mechanical tunneling.

Heterobinuclear µ-methylene-µ-phenyl complexes were synthesized. Structural study of C_(p2)Ti(µ-CH_(2))(µ-p-Me_(2)NC_(6)H_(4))Rh(l,5-COD) showed that the two metal atoms are bridged by the methylene carbon and the ipso carbon of the p-N,N-dimethylarninophenyl group. The analogous structure of C_(p2))Ti(µ-CH_(2))(µ-o-MeOC_(6)H_(4))Rh(1,5-COD) has been verified by the differential NOE. The aromaticity of the phenyl group observed by ^(1)H NMR, was confirmed by the comparison of the C-C bond lengths in the crystallographic structure. The unusual downfield shifts of the ipso carbon in the ^(13)C NMR are assumed to be an indication of the interaction between the ipso carbon and electron-deficient titanium.

Titanium-platinum heterobinuclear µ-methylene complexes C_(p2)Ti(µ-CH_(2))(µ -X)Pt(Me)(PM_(2)Ph) have been prepared (X= Cl, Me). Structural studies indicate the following:(1) the Ti-CH2 bond possesses residual double bond character, (2) there is a dative Pt→Ti interaction which may be regarded as a π back donation from the platinum atom to the 'Ti=CH_(2)'' group, and (3) the µ-CH_3 group is bound to the titanium atom through a three-center, two-electron agostic bond.

Titanocene (η^(2)-thioformaldehyde)•PMe_3 was prepared from C_(p2)Ti=CH_(2)•PMe_3 and sulfur-containing organic compounds (e.g. alkene sulfide, triphenylphosphine sulfide) including elemental sulfur. Mechanistic studies utilizing trans-styrene sulfide-d_1 suggested the stepwise reaction to explain equimolar mixture of trans- and cis-styrene-d_1 as by-products. The product reacted with methyl iodide to produce cationic titanocene (η_(2)-thiomethoxymethyl) complex. Complexes having less coordinating anion like BF_4 or BPh_4 could be obtained through metathesis. Together with structural analyses, the further reactivities of the complexes have been explored.

The complex C_(p2)TiOCH_(2)CH(Ph)CH_2 was prepared from the compound C_(p2)Ti=CH_(2)-PMe_3 and styrene oxide. The product was characterized with ^(1)H-^(1)H correlated 2-dimensional NMR, selective decoupling of ^(1)H NMR, and differential NOE. Stereospecificity of deuterium in the product was lost when trans-styrene oxide-d_1 was allowed to react. Relative rates of the reaction were measured with varying substituents on the phenyl ring. Better linearity (r = -0.98, p^(+) = -0.79) was observed with σ_(p)^(+)than σ(r = -0.87, p = -1.26). The small magnitude of p^+ value and stereospecificity loss during the formation of product were best explained by the generation of biradicals, but partial generation of charge cannot be excluded. Carbonylation of the product followed by exposure to iodine yields the corresponding β-phenyl γ-lactone.

Progress towards the total synthesis of a spermine-conjugated dynemicin analog

Progress towards the synthesis of the spermine-conjugated Dynemicin analog 4 is described. The synthetic route starts with the Michael addition of menthyl acetoacetate to trans-ethyl crotonate followed by a Dieckman condensation to form the cyclohexanedione 14 which, through a series of chemical reactions, is transformed into the quinone imine 6. Key features in the route include the Suzuki coupling reaction of the aryl boronic acid 11 and the enol triflate 12, thermal deprotection/internal amidation of the biaryl 19, cis addition of the (Z)-enediyne 33 to the quinoline 25, intramolecular acetylide addition to a carbonyl within the ketone 29, and an addition/elimination of the cyanophthalide to the quinone imine 6 to form the anthraquinone 36 utilizing the Kraus and Sugimoto methodology.

A genetic and biochemical analysis of pre-mRNA splicing in Saccharomyces cerevisiae

Pre-mRNA splicing requires interaction of cis- acting intron sequences with trans -acting factors: proteins and small nuclear ribonucleoproteins (snRNPs). The assembly of these factors into a large complex, the spliceosome, is essential for the subsequent two step splicing reaction. First, the 5' splice site is cleaved and free exon 1 and a lariat intermediate (intron- exon2) form. In the second reaction the 3' splice site is cleaved the exons ligated and lariat intron released. A combination of genetic and biochemical techniques have been used here to study pre-mRNA splicing in yeast.

Yeast introns have three highly conserved elements. We made point mutations within these elements and found that most of them affect splicing efficiency in vivo and in vitro, usually by inhibiting spliceosome assembly.

To study trans -acting splicing factors we generated and screened a bank of temperature- sensitive (ts) mutants. Eleven new complementation groups (prp17 to prp27) were isolated. The four phenotypic classes obtained affect different steps in splicing and accumulate either: 1) pre-mRNA, 2) lariat intermediate, 3) excised intron or 4) both pre-mRNA and intron. The latter three classes represent novel phenotypes. The excised intron observed in one mutant: prp26 is stabilized due to protection in a snRNP containing particle. Extracts from another mutant: prpl8 are heat labile and accumulate lariat intermediate and exon 1. This is especially interesting as it allows analysis of the second splicing reaction. In vitro complementation of inactivated prp18 extracts does not require intact snRNPs. These studies have also shown the mutation to be in a previously unknown splicing protein. A specific requirement for A TP is also observed for the second step of splicing. The PRP 18 gene has been cloned and its polyadenylated transcript identified.

Experimental and theoretical studies of Notch signaling-mediated spatial pattern

Notch signaling acts in many diverse developmental spatial patterning processes. To better understand why this particular pathway is employed where it is and how downstream feedbacks interact with the signaling system to drive patterning, we have pursued three aims: (i) to quantitatively measure the Notch system's signal input/output (I/O) relationship in cell culture, (ii) to use the quantitative I/O relationship to computationally predict patterning outcomes of downstream feedbacks, and (iii) to reconstitute a Notch-mediated lateral induction feedback (in which Notch signaling upregulates the expression of Delta) in cell culture. The quantitative Notch I/O relationship revealed that in addition to the trans-activation between Notch and Delta on neighboring cells there is also a strong, mutual cis-inactivation between Notch and Delta on the same cell. This feature tends to amplify small differences between cells. Incorporating our improved understanding of the signaling system into simulations of different types of downstream feedbacks and boundary conditions lent us several insights into their function. The Notch system converts a shallow gradient of Delta expression into a sharp band of Notch signaling without any sort of feedback at all, in a system motivated by the Drosophila wing vein. It also improves the robustness of lateral inhibition patterning, where signal downregulates ligand expression, by removing the requirement for explicit cooperativity in the feedback and permitting an exceptionally simple mechanism for the pattern. When coupled to a downstream lateral induction feedback, the Notch system supports the propagation of a signaling front across a tissue to convert a large area from one state to another with only a local source of initial stimulation. It is also capable of converting a slowly-varying gradient in parameters into a sharp delineation between high- and low-ligand populations of cells, a pattern reminiscent of smooth muscle specification around artery walls. Finally, by implementing a version of the lateral induction feedback architecture modified with the addition of an autoregulatory positive feedback loop, we were able to generate cells that produce enough cis ligand when stimulated by trans ligand to themselves transmit signal to neighboring cells, which is the hallmark of lateral induction.

Synthesis and characterization of 1,1-di-tert-butyldiazene

The synthesis and direct observation of 1,1-di-tert-butyldiazene (16) at -127°C is described. The absorption spectrum of a red solution of 1,1-diazene 16 reveals a structured absorption band with λ max at 506 run (Me_2O, -125°C). The vibrational spacing in S_1 is about 1200 cm^(-1). The excited state of 16 emits weakly with a single maximum at 715 run observed in the fluorescence spectrum (Me_2O:CD_2Cl_2, -196°C). The proton NMR spectrum of 16 occurs as a singlet at 1.41 ppm. Monitoring this NMR absorption at -94^0 ± 2°C shows that 1,1-diazene 16 decomposes with a first-order rate of 1.8 x 10^(-3) sec(-1) to form isobutane, isobutylene and hexarnethylethane. This rate is 10^8 and 10^(34) times faster than the thermal decomposition of the corresponding cis and trans 1,2-di-tert-butyldiazene isomers. The free energy of activation for decomposition of 1,1-diazene 16 is found to be 12.5 ± 0.2 kcal/mol at -94°C which is much lower than the values of 19.1 and 19.4 kcal/lmole calculated at -94°C for N-(2,2,6,6- tetramethylpiperidyl)nitrene (3) and N-(2,2,5,5- tetrarnethylpyrrolidyl)nitrene (4), respectively. This difference between 16 and the cyclic-1,1-diazenes 3 and 4 can be attributed to a large steric interaction between the tert-butyl groups in 1,1-diazene 16.

In order to investigate the nature of the singlet-triplet gap in 1,1-diazenes, 2,5-di-tert-butyl-N-pyrrolynitrene (22) was generated but was found to be too reactive towards dimerization to be persistent. In the presence of dimethylsulfoxide, however, N-pyrrolynitrene (22) can be trapped as N-(2,5-di-tert-butyl- N'-pyrrolyl)dimethylsulfoxirnine (38). N-(2,5-di-tert-butyl-N'-pyrrolyl)dimethylsulfoximine (38-d^6) exchanges with free dimethylsulfoxide at 50°C in solution, presumably by generation and retrapping of pyrrolynitrene 22.

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.

Cell-targeted regulation of gene expression through synthetic RNA devices

The ability to interface with and program cellular function remains a challenging research frontier in biotechnology. Although the emerging field of synthetic biology has recently generated a variety of gene-regulatory strategies based on synthetic RNA molecules, few strategies exist through which to control such regulatory effects in response to specific exogenous or endogenous molecular signals. Here, we present the development of an engineered RNA-based device platform to detect and act on endogenous protein signals, linking these signals to the regulation of genes and thus cellular function.

We describe efforts to develop an RNA-based device framework for regulating endogenous genes in human cells. Previously developed RNA control devices have demonstrated programmable ligand-responsive genetic regulation in diverse cell types, and we attempted to adapt this class of cis-acting control elements to function in trans. We divided the device into two strands that reconstitute activity upon hybridization. Device function was optimized using an in vivo model system, and we found that device sequence is not as flexible as previously reported. After verifying the in vitro activity of our optimized design, we attempted to establish gene regulation in a human cell line using additional elements to direct device stability, structure, and localization. The significant limitations of our platform prevented endogenous gene regulation.

We next describe the development of a protein-responsive RNA-based regulatory platform. Employing various design strategies, we demonstrated functional devices that both up- and downregulate gene expression in response to a heterologous protein in a human cell line. The activity of our platform exceeded that of a similar, small-molecule-responsive platform. We demonstrated the ability of our devices to respond to both cytoplasmic- and nuclear-localized protein, providing insight into the mechanism of action and distinguishing our platform from previously described devices with more restrictive ligand localization requirements. Finally, we demonstrated the versatility of our device platform by developing a regulatory device that responds to an endogenous signaling protein.

The foundational tool we present here possesses unique advantages over previously described RNA-based gene-regulatory platforms. This genetically encoded technology may find future applications in the development of more effective diagnostic tools and targeted molecular therapy strategies.

The logic of receptor-ligand interactions in the Notch signaling pathway

The Notch signaling pathway enables neighboring cells to coordinate developmental fates in diverse processes such as angiogenesis, neuronal differentiation, and immune system development. Although key components and interactions in the Notch pathway are known, it remains unclear how they work together to determine a cell's signaling state, defined as its quantitative ability to send and receive signals using particular Notch receptors and ligands. Recent work suggests that several aspects of the system can lead to complex signaling behaviors: First, receptors and ligands interact in two distinct ways, inhibiting each other in the same cell (in cis) while productively interacting between cells (in trans) to signal. The ability of a cell to send or receive signals depends strongly on both types of interactions. Second, mammals have multiple types of receptors and ligands, which interact with different strengths, and are frequently co-expressed in natural systems. Third, the three mammalian Fringe proteins can modify receptor-ligand interaction strengths in distinct and ligand-specific ways. Consequently, cells can exhibit non-intuitive signaling states even with relatively few components.

In order to understand what signaling states occur in natural processes, and what types of signaling behaviors they enable, this thesis puts forward a quantitative and predictive model of how the Notch signaling state is determined by the expression levels of receptors, ligands, and Fringe proteins. To specify the parameters of the model, we constructed a set of cell lines that allow control of ligand and Fringe expression level, and readout of the resulting Notch activity. We subjected these cell lines to an assay to quantitatively assess the levels of Notch ligands and receptors on the surface of individual cells. We further analyzed the dependence of these interactions on the level and type of Fringe expression. We developed a mathematical modeling framework that uses these data to predict the signaling states of individual cells from component expression levels. These methods allow us to reconstitute and analyze a diverse set of Notch signaling configurations from the bottom up, and provide a comprehensive view of the signaling repertoire of this major signaling pathway.

Synthesis and matrix isolated photolysis of 2,3-dimethylbicyclo [2.2.0]hexa-2,5-diene-1,4-dicarboxylic acid anhydride: a potential percursor to 2,3-dimethyl-1,4-dehydrobenzene

The synthesis of the first member of a new class of Dewar benzenes has been achieved. The synthesis of 2,3- dimethylbicyclo[2.2.0]hexa-2,5-diene-1, 4-dicarboxylic acid and its anhydride are described. Dibromomaleic anhydride and dichloroethylene were found to add efficiently in a photochemical [2+2] cycloaddition to produce 1,2-dibromo- 3,4-dichlorocyclobutane-1,2-dicarboxylic acid. Removal of the bromines with tin/copper couple yielded dichloro- cyclobutenes which added to 2-butyne under photochemical conditions to yield 5,6-dichloro-2,3-dimethylbicyclo [2.2.0] hex-2-ene dicarboxylic acids. One of the three possible isomers yielded a stable anhydride which could be dechlorinated using triphenyltin radicals generated by the photolysis of hexaphenylditin.

Photolysis of argon matrix isolated 2,3-dimethylbicyclo [2.2.0]hexa-2, 5-diene-1,4-dicarboxylic acid anhydride produced traces whose strongest bands in the infrared were at 3350 and 600 cm^(-1). This suggested the formation of terminal acetylenes. The spectra of argon matrix isolated E- and Z- 3,4-dimethylhexa-1,5-diyne-3-ene and cis-and trans-octa- 2,6-diyne-4-ene were compared with the spectrum of the photolysis products. Possibly all four diethynylethylenes were present in the anhydride photolysis products. Gas chromatograph-mass spectral analysis of the volatiles from the anhydride photolysis again suggested, but did not confirm, the presence of the diethynylethylenes.