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.

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.

Engineering nucleic acid mechanisms for regulation and readout of gene expression : conditional Dicer substrate formation and sensitive multiplexed northern blots

Nucleic acids are most commonly associated with the genetic code, transcription and gene expression. Recently, interest has grown in engineering nucleic acids for biological applications such as controlling or detecting gene expression. The natural presence and functionality of nucleic acids within living organisms coupled with their thermodynamic properties of base-pairing make them ideal for interfacing (and possibly altering) biological systems. We use engineered small conditional RNA or DNA (scRNA, scDNA, respectively) molecules to control and detect gene expression. Three novel systems are presented: two for conditional down-regulation of gene expression via RNA interference (RNAi) and a third system for simultaneous sensitive detection of multiple RNAs using labeled scRNAs.

RNAi is a powerful tool to study genetic circuits by knocking down a gene of interest. RNAi executes the logic: If gene Y is detected, silence gene Y. The fact that detection and silencing are restricted to the same gene means that RNAi is constitutively on. This poses a significant limitation when spatiotemporal control is needed. In this work, we engineered small nucleic acid molecules that execute the logic: If mRNA X is detected, form a Dicer substrate that targets independent mRNA Y for silencing. This is a step towards implementing the logic of conditional RNAi: If gene X is detected, silence gene Y. We use scRNAs and scDNAs to engineer signal transduction cascades that produce an RNAi effector molecule in response to hybridization to a nucleic acid target X. The first mechanism is solely based on hybridization cascades and uses scRNAs to produce a double-stranded RNA (dsRNA) Dicer substrate against target gene Y. The second mechanism is based on hybridization of scDNAs to detect a nucleic acid target and produce a template for transcription of a short hairpin RNA (shRNA) Dicer substrate against target gene Y. Test-tube studies for both mechanisms demonstrate that the output Dicer substrate is produced predominantly in the presence of a correct input target and is cleaved by Dicer to produce a small interfering RNA (siRNA). Both output products can lead to gene knockdown in tissue culture. To date, signal transduction is not observed in cells; possible reasons are explored.

Signal transduction cascades are composed of multiple scRNAs (or scDNAs). The need to study multiple molecules simultaneously has motivated the development of a highly sensitive method for multiplexed northern blots. The core technology of our system is the utilization of a hybridization chain reaction (HCR) of scRNAs as the detection signal for a northern blot. To achieve multiplexing (simultaneous detection of multiple genes), we use fluorescently tagged scRNAs. Moreover, by using radioactive labeling of scRNAs, the system exhibits a five-fold increase, compared to the literature, in detection sensitivity. Sensitive multiplexed northern blot detection provides an avenue for exploring the fate of scRNAs and scDNAs in tissue culture.

The application of metallointercalators in recognition of and charge transport in nucleic acids

Metal complexes that utilize the 9,10-phenanthrene quinone diimine (phi) moiety bind to DNA through the major groove. These metallointercalators can recognize DNA sites and perform reactions on DNA as a substrate. The site-specific metallointercalator Λ-1-Rh(MGP)_2phi^(5+) competitively disrupts the major groove binding of a transcription factor, yAP-1, from an oligonucleotide that contains a common binding site. The demonstration that metal complexes can prevent transcription factor binding to DNA site-specifically is an important step in using metallointercalators as therapeutics.

The distinctive photochemistry of metallointercalators can also be applied to promote long range charge transport in DNA. Experiments using duplexes with regions 4 to 10 nucleotides long containing strictly adenine and thymine sequences of varying order showed that radical migration is more dependent on the sequence of bases, and less dependent on the distance between the guanine doublets. This result suggests that mechanistic proposals of long range charge transport must involve all the bases.

RNA/DNA hybrids show charge migration to guanines from a remote site, thus demonstrating that nucleic acid stacking other than B-form can serve as a radical bridge. Double crossover DNA assemblies also provide a medium for charge transport at distances up to 100 Å from the site of radical introduction by a tethered metal complex. This radical migration was found to be robust to mismatches, and limited to individual, electronically distinct base stacks. In single DNA crossover assemblies, which have considerably greater flexibility, charge migration proceeds to both base stacks due to conformational isomers not present in the rigid and tightly annealed double crossovers.

Finally, a rapid, efficient, gel-based technique was developed to investigate thymine dimer repair. Two oligonucleotides, one radioactively labeled, are photoligated via the bases of a thymine-thymine interface; reversal of this ligation is easily visualized by gel electrophoresis. This assay was used to show that the repair of thymine dimers from a distance through DNA charge transport can be accomplished with different photooxidants.

Thus, nucleic acids that support long range charge transport have been shown to include A-track DNA, RNA/DNA hybrids, and single and double crossovers, and a method for thymine dimer repair detection using charge transport was developed. These observations underscore and extend the remarkable finding that DNA can serve a medium for charge transport via the heteroaromatic base stack.

Design and analysis of nucleic acid reaction pathways

Nucleic acids are a useful substrate for engineering at the molecular level. Designing the detailed energetics and kinetics of interactions between nucleic acid strands remains a challenge. Building on previous algorithms to characterize the ensemble of dilute solutions of nucleic acids, we present a design algorithm that allows optimization of structural features and binding energetics of a test tube of interacting nucleic acid strands. We extend this formulation to handle multiple thermodynamic states and combinatorial constraints to allow optimization of pathways of interacting nucleic acids. In both design strategies, low-cost estimates to thermodynamic properties are calculated using hierarchical ensemble decomposition and test tube ensemble focusing. These algorithms are tested on randomized test sets and on example pathways drawn from the molecular programming literature. To analyze the kinetic properties of designed sequences, we describe algorithms to identify dominant species and kinetic rates using coarse-graining at the scale of a small box containing several strands or a large box containing a dilute solution of strands.

Functional evaluation of noncovalent interactions in neuroreceptors and progress toward the expansion of unnatural amino acid methodology

This dissertation primarily describes chemical-scale studies of G protein-coupled receptors and Cys-loop ligand-gated ion channels to better understand ligand binding interactions and the mechanism of channel activation using recently published crystal structures as a guide. These studies employ the use of unnatural amino acid mutagenesis and electrophysiology to measure subtle changes in receptor function.

In chapter 2, the role of a conserved aromatic microdomain predicted in the D3 dopamine receptor is probed in the closely related D2 and D4 dopamine receptors. This domain was found to act as a structural unit near the ligand binding site that is important for receptor function. The domain consists of several functionally important noncovalent interactions including hydrogen bond, aromatic-aromatic, and sulfur-π interactions that show strong couplings by mutant cycle analysis. We also assign an alternate interpretation for the linear fluorination plot observed at W6.48, a residue previously thought to participate in a cation-π interaction with dopamine.

Chapter 3 outlines attempts to incorporate chemically synthesized and in vitro acylated unnatural amino acids into mammalian cells. While our attempts were not successful, method optimizations and data for nonsense suppression with an in vivo acylated tRNA are included. This chapter is aimed to aid future researchers attempting unnatural amino acid mutagenesis in mammalian cells.

Chapter 4 identifies a cation-π interaction between glutamate and a tyrosine residue on loop C in the GluClβ receptor. Using the recently published crystal structure of the homologous GluClα receptor, other ligand-binding and protein-protein interactions are probed to determine the similarity between this invertebrate receptor and other more distantly related vertebrate Cys-loop receptors. We find that many of the interactions previously observed are conserved in the GluCl receptors, however care must be taken when extrapolating structural data.

Chapter 5 examines inherent properties of the GluClα receptor that are responsible for the observed glutamate insensitivity of the receptor. Chimera synthesis and mutagenesis reveal the C-terminal portion of the M4 helix and the C-terminus as contributing to formation of the decoupled state, where ligand binding is incapable of triggering channel gating. Receptor mutagenesis was unable to identify single residue mismatches or impaired protein-protein interactions within this domain. We conclude that M4 helix structure and/or membrane dynamics are likely the cause of ligand insensitivity in this receptor and that the M4 helix has an role important in the activation process.

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.

I. The crystal structure of trimesic acid. II. Topics in crystallographic calculations

I. Trimesic acid (1, 3, 5-benzenetricarboxylic acid) crystallizes with a monoclinic unit cell of dimensions a = 26.52 A, b = 16.42 A, c = 26.55 A, and β = 91.53° with 48 molecules /unit cell. Extinctions indicated a space group of Cc or C2/c; a satisfactory structure was obtained in the latter with 6 molecules/asymmetric unit - C₅₄O₃₆H₃₆ with a formula weight of 1261 g. Of approximately 12,000 independent reflections within the CuK_α sphere, intensities of 11,563 were recorded visually from equi-inclination Weissenberg photographs.

The structure was solved by packing considerations aided by molecular transforms and two- and three-dimensional Patterson functions. Hydrogen positions were found on difference maps. A total of 978 parameters were refined by least squares; these included hydrogen parameters and anisotropic temperature factors for the C and O atoms. The final R factor was 0.0675; the final "goodness of fit" was 1.49. All calculations were carried out on the Caltech IBM 7040-7094 computer using the CRYRM Crystallographic Computing System.

The six independent molecules fall into two groups of three nearly parallel molecules. All molecules are connected by carboxylto- carboxyl hydrogen bond pairs to form a continuous array of sixmolecule rings with a chicken-wire appearance. These arrays bend to assume two orientations, forming pleated sheets. Arrays in different orientations interpenetrate - three molecules in one orientation passing through the holes of three parallel arrays in the alternate orientation - to produce a completely interlocking network. One third of the carboxyl hydrogen atoms were found to be disordered.

II. Optical transforms as related to x-ray diffraction patterns are discussed with reference to the theory of Fraunhofer diffraction.

The use of a systems approach in crystallographic computing is discussed with special emphasis on the way in which this has been done at the California Institute of Technology.

An efficient manner of calculating Fourier and Patterson maps on a digital computer is presented. Expressions for the calculation of to-scale maps for standard sections and for general-plane sections are developed; space-group-specific expressions in a form suitable for computers are given for all space groups except the hexagonal ones.

Expressions for the calculation of settings for an Eulerian-cradle diffractometer are developed for both the general triclinic case and the orthogonal case.

Photographic materials on pp. 4, 6, 10, and 20 are essential and will not reproduce clearly on Xerox copies. Photographic copies should be ordered.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

A colorimetric determination of hydrocyanic acid

The object of this investigation is to devise a rapid, fairly accurate, colorimetric analysis for HCN to be used in field work for determining instantaneous concentrations of the gas under fumigating canvas. A large amount of money is expended yearly by the citrus industry of this state in attempting to control and to eradicate the scale pests. Although fumigation with HCN has been practiced tor many years, the progress made has been anything but satisfactory. The greater portion of the work has always been carried on by contractors, who in a large number of cases have been very unscrupulous. The materials and labor are very expensive and the growers have been satisfied to adhere to beaten paths and hope for the best results on scale kill with the least attendant foliage injury. One familiar with fumigating, either from the grower's or the operator's viewpoint, knows that very widely varying results are obtained, even under what are apparently identical condition. Even after discounting for the dishonesty of some operators and the prejudices of the grower, there is still a large variance between desired or expected results and those actually obtained.

Preparation and attempted resolution of benzoyl-malic acid

The final object of this research was to prepare m-nitrobenzoyl malic acid and to separate it, if possible, into the four stereoisomers predicted by the Huggins' theory of the benzene ring. Inasmuch as the quantity of m-nitro- benzoyl chloride available was limited it was thought better to first prepare i-benzoyl malic acid and then attempt to resolve it. The resolution of m-nitrobenzoyl malic acid could probably be accomplished by a similar method.

Part I. Synthesis of L-amino acid oxidase by a serine- or glycine-requiring strain of neurospora. Part II. Studies concerning multiple electrophoretic forms of tyrosinase in neurospora

Part I: Synthesis of L-Amino Acid Oxidase by a Serine- or Glycine-Requiring Strain of Neurospora

Wild-type cultures of Neurospora crassa growing on minimal medium contain low levels of L-amino acid oxidase, tyrosinase, and nicotinarnide adenine dinucleotide glycohydrase (NADase). The enzymes are derepressed by starvation and by a number of other conditions which are inhibitory to growth. L-amino acid oxidase is, in addition, induced by growth on amino acids. A mutant which produces large quantities of both L-amino acid oxidase and NADase when growing on minimal medium was investigated. Constitutive synthesis of L-amino acid oxidase was shown to be inherited as a single gene, called P110, which is separable from constitutive synthesis of NADase. P110 maps near the centromere on linkage group IV.

L-amino acid oxidase produced constitutively by P110 was partially purified and compared to partially purified L-amino acid oxidase produced by derepressed wild-type cultures. The enzymes are identical with respect to thermostability and molecular weight as judged by gel filtration.

The mutant P110 was shown to be an incompletely blocked auxotroph which requires serine or glycine. None of the enzymes involved in the synthesis of serine from 3-phosphoglyceric acid or glyceric acid was found to be deficient in the mutant, however. An investigation of the free intracellular amino acid pools of P110 indicated that the mutant is deficient in serine, glycine, and alanine, and accumulates threonine and homoserine.

The relationship between the amino acid requirement of P110 and its synthesis of L-amino acid oxidase is discussed.

Part II: Studies Concerning Multiple Electrophoretic Forms of Tyrosinase in Neurospora

Supernumerary bands shown by some crude tyrosinase preparations in paper electrophoresis were investigated. Genetic analysis indicated that the location of the extra bands is determined by the particular T allele present. The presence of supernumerary bands varies with the method used to derepress tyrosinase production, and with the duration of derepression. The extra bands are unstable and may convert to the major electrophoretic band, suggesting that they result from modification of a single protein. Attempts to isolate the supernumerary bands by continuous flow paper electrophoresis or density gradient zonal electrophoresis were unsuccessful.

The equivalent conductance of solutions of picric acid

Picric acid possesses the property, which is rare among strong electrolytes, of having a convenient distribution ratio between water and certain organic solvents such as benzene, chloroform, etc. Because of this property, picric acid offers peculiar advantages for studying the well known deviations of strong electrolytes from the law of mass action, for; by means of distribution experiments, the activities of picric acid in various aqueous solutions may be compared.

In order to interpret the results of such distribution experiments, it is necessary to know the degree of ionization of picric acid in aqueous solutions.

At least three series of determinations of the equivalent conductance of picric acid have been published, but the results are not concordant; and therefore, the degree of ionization cannot be calculated with any degree of certainty.

The object of the present investigation was to redetermine the conductance of picric acid solutions in order to obtain satisfactory data from which the degrees of ionization of its solutions might be calculated.

Studies of dinucleoside monophosphates and monomer-polynucleotide interactions by proton magnetic resonance

The nature of the intra- and intermolecular base-stacking interactions involving several dinucleoside monophosphates in aqueous solution have been investigated by proton magnetic resonance spectrosocopy, and this method has been applied to a study of the interaction of polyuridylic acid with purine and adenosine monomers.

The pmr spectra of adenylyl (3' → 5') cytidine (ApC) and cytidylyl (3' → 5') adenosine (CpA) have been studied as a function of concentration and temperature. The results of these studies indicate that the intramolecular base-stacking interactions between the adenine and cytosine bases of these dinucleoside monophosphates are rather strong, and that the stacking tendencies are comparable for the two sequence isomers. The chemical shifts of the cytosine H₅ and adenine H₂ protons, and their variations with temperature, were shown to be consistent with stacked conformations in which both bases of the dinucleoside monophosphates are preferentially oriented in the anti conformation as in similar dApdC, and dCpdA (dA = deoxyadenosine; dC = deoxycytidine) segments in double helical DNA. The intramolecular stacking interaction was found to have a pronounced effect on the conformations of the ribose moieties, and these conformational changes are discussed. The concentration studies indicate extensive self-association of these dinucleoside monophosphates, and analysis of the concentration data facilitated determination of the dimerization constant for the association process as well as the nature of the intermolecular complexes.

The dependence of the ribose conformation upon the extent of intramolecular base-stacking was used to demonstrate that the base-base interaction in cytidylyl (3' → 5') cytidine (CpC) is rather strong, while there appears to be little interaction between the two uracil bases of uridylyl (3' → 5') uridine (UpU).

Studies of the binding of purine to several ribose and deoxyribose dinucleoside monophosphates show that the mode of interaction is base-stacking, and evidence for the formation of a purine-dinucleoside monophosphate intercalated complex is presented. The purine proton resonances are markedly broadened in this complex, and estimates of the purine linewidths in the complex and the equilibrium constant for purine intercalation are obtained.

A study of the interaction of unsubstitued purine with polyuridylic acid at 29°C by pmr indicated that purine binds to the uracil bases of the polymer by base-stacking. The severe broadening of the purine proton resonances observed provides strong evidence for the intercalation of purine between adjacent uracil bases of poly U. This interaction does not result in a more rigid or ordered structure for the polymer.

Investigation of the interaction between adenosine and polyuridylic acid revealed two modes of interaction between the monomer and the polymer, depending on the temperature. At temperatures above 26°C or so, monomeric adenosine binds to poly U by noncooperative A-U base stacking. Below this temperature, a rigid triple-stranded 1A:2U complex is formed, presumably via cooperative hydrogen-bonding as has previously been reported.

These results clearly illustrate the importance of base-stacking in non-specific interactions between bases, nucleosides and nucleotides, and also reveal the important role of the base-stacking interactions in cooperatively for med structures involving specific base-pairing where both types of interaction are possible.

The structure elucidation of a proposed intermediate in the stereoselective synthesis of dl-desoxypodocarpic acid

The hydroxyketone C-3, an intermediate in the stereo-selective total synthesis of dl-Desoxypodocarpic acid (ii), has been shown by both degradative and synthetic pathways to rearrange in the presence of base to diosphenol E-1 (5-isoabietic acid series). The exact spatial arrangements of the systems represented by formulas C-3 and E-1 have been investigated (as the p-bromobenzoates) by single-crystal X-ray diffraction analyses. The hydroxyketone F-1, the proposed intermediate in the rearrangement, has been synthesized. Its conversion to diosphenol E-1 has been studied, and a single-crystal analysis of the p-bromobenzoate derivative has been performed. The initially desired diosphenol C-6 has been prepared, and has been shown to be stable to the potassium t-butoxide rearrangement conditions. Oxidative cleavage of diosphenol E-1 and subsequent cyclization with the aid of polyphosphoric acid has been shown to lead to keto acid I-2 (benzobicyclo [3.3.1] nonane series) rather than keto acid H-2 (5-isoabietic acid series).