Synthetic molecular machines for active self-assembly : prototype algorithms, designs, and experimental study

Computer science and electrical engineering have been the great success story of the twentieth century. The neat modularity and mapping of a language onto circuits has led to robots on Mars, desktop computers and smartphones. But these devices are not yet able to do some of the things that life takes for granted: repair a scratch, reproduce, regenerate, or grow exponentially fast–all while remaining functional.

This thesis explores and develops algorithms, molecular implementations, and theoretical proofs in the context of “active self-assembly” of molecular systems. The long-term vision of active self-assembly is the theoretical and physical implementation of materials that are composed of reconfigurable units with the programmability and adaptability of biology’s numerous molecular machines. En route to this goal, we must first find a way to overcome the memory limitations of molecular systems, and to discover the limits of complexity that can be achieved with individual molecules.

One of the main thrusts in molecular programming is to use computer science as a tool for figuring out what can be achieved. While molecular systems that are Turing-complete have been demonstrated [Winfree, 1996], these systems still cannot achieve some of the feats biology has achieved.

One might think that because a system is Turing-complete, capable of computing “anything,” that it can do any arbitrary task. But while it can simulate any digital computational problem, there are many behaviors that are not “computations” in a classical sense, and cannot be directly implemented. Examples include exponential growth and molecular motion relative to a surface.

Passive self-assembly systems cannot implement these behaviors because (a) molecular motion relative to a surface requires a source of fuel that is external to the system, and (b) passive systems are too slow to assemble exponentially-fast-growing structures. We call these behaviors “energetically incomplete” programmable behaviors. This class of behaviors includes any behavior where a passive physical system simply does not have enough physical energy to perform the specified tasks in the requisite amount of time.

As we will demonstrate and prove, a sufficiently expressive implementation of an “active” molecular self-assembly approach can achieve these behaviors. Using an external source of fuel solves part of the the problem, so the system is not “energetically incomplete.” But the programmable system also needs to have sufficient expressive power to achieve the specified behaviors. Perhaps surprisingly, some of these systems do not even require Turing completeness to be sufficiently expressive.

Building on a large variety of work by other scientists in the fields of DNA nanotechnology, chemistry and reconfigurable robotics, this thesis introduces several research contributions in the context of active self-assembly.

We show that simple primitives such as insertion and deletion are able to generate complex and interesting results such as the growth of a linear polymer in logarithmic time and the ability of a linear polymer to treadmill. To this end we developed a formal model for active-self assembly that is directly implementable with DNA molecules. We show that this model is computationally equivalent to a machine capable of producing strings that are stronger than regular languages and, at most, as strong as context-free grammars. This is a great advance in the theory of active self- assembly as prior models were either entirely theoretical or only implementable in the context of macro-scale robotics.

We developed a chain reaction method for the autonomous exponential growth of a linear DNA polymer. Our method is based on the insertion of molecules into the assembly, which generates two new insertion sites for every initial one employed. The building of a line in logarithmic time is a first step toward building a shape in logarithmic time. We demonstrate the first construction of a synthetic linear polymer that grows exponentially fast via insertion. We show that monomer molecules are converted into the polymer in logarithmic time via spectrofluorimetry and gel electrophoresis experiments. We also demonstrate the division of these polymers via the addition of a single DNA complex that competes with the insertion mechanism. This shows the growth of a population of polymers in logarithmic time. We characterize the DNA insertion mechanism that we utilize in Chapter 4. We experimentally demonstrate that we can control the kinetics of this re- action over at least seven orders of magnitude, by programming the sequences of DNA that initiate the reaction.

In addition, we review co-authored work on programming molecular robots using prescriptive landscapes of DNA origami; this was the first microscopic demonstration of programming a molec- ular robot to walk on a 2-dimensional surface. We developed a snapshot method for imaging these random walking molecular robots and a CAPTCHA-like analysis method for difficult-to-interpret imaging data.

Studying conscious and unconscious vision with functional Magnetic Resonance Imaging : the BOLD promise

Waking up from a dreamless sleep, I open my eyes, recognize my wife’s face and am filled with joy. In this thesis, I used functional Magnetic Resonance Imaging (fMRI) to gain insights into the mechanisms involved in this seemingly simple daily occurrence, which poses at least three great challenges to neuroscience: how does conscious experience arise from the activity of the brain? How does the brain process visual input to the point of recognizing individual faces? How does the brain store semantic knowledge about people that we know? To start tackling the first question, I studied the neural correlates of unconscious processing of invisible faces. I was unable to image significant activations related to the processing of completely invisible faces, despite existing reports in the literature. I thus moved on to the next question and studied how recognition of a familiar person was achieved in the brain; I focused on finding invariant representations of person identity – representations that would be activated any time we think of a familiar person, read their name, see their picture, hear them talk, etc. There again, I could not find significant evidence for such representations with fMRI, even in regions where they had previously been found with single unit recordings in human patients (the Jennifer Aniston neurons). Faced with these null outcomes, the scope of my investigations eventually turned back towards the technique that I had been using, fMRI, and the recently praised analytical tools that I had been trusting, Multivariate Pattern Analysis. After a mostly disappointing attempt at replicating a strong single unit finding of a categorical response to animals in the right human amygdala with fMRI, I put fMRI decoding to an ultimate test with a unique dataset acquired in the macaque monkey. There I showed a dissociation between the ability of fMRI to pick up face viewpoint information and its inability to pick up face identity information, which I mostly traced back to the poor clustering of identity selective units. Though fMRI decoding is a powerful new analytical tool, it does not rid fMRI of its inherent limitations as a hemodynamics-based measure.

Aqueous ring-opening metathesis polymerizations

Past workers in this group as well as in others have made considerable progress in the understanding and development of the ring-opening metathesis polymerization (ROMP) technique. Through these efforts, ROMP chemistry has become something of an organometallic success story. Extensive work was devoted to trying to identify the catalytically active species in classical reaction mixtures of early metal halides and alkyl aluminum compounds. Through this work, a mechanism involving the interconversion of metal carbenes and metallacyclobutanes was proposed. This preliminary work finally led to the isolation and characterization of stable metal carbene and metallacyclobutane complexes. As anticipated, these well-characterized complexes were shown to be active catalysts. In a select number of cases, these catalysts have been shown to catalyze the living polymerization of strained rings such as norbornene. The synthetic control offered by these living systems places them in a unique category of metal catalyzed reactions. To take full advantage of these new catalysts, two approaches should be explored. The first takes advantage of the unusual fact that all of the unsaturation present in the monomer is conserved in the polymer product. This makes ROMP techniques ideal for the synthesis of highly unsaturated, and fully conjugated polymers, which find uses in a variety of applications. This area is currently under intense investigation. The second aspect, which should lend itself to fruitful investigations, is expanding the utility of these catalysts through the living polymerization of monomers containing interesting functional groups. Polymer properties can be dramatically altered by the incorporation of functional groups. It is this latter aspect which will be addressed in this work.

After a general introduction to both the ring-opening metathesis reaction (Chapter 1) and the polymerization of fuctionalized monomers by transition metal catalysts (Chapter 2), the limits of the existing living ROMP catalysts with functionalized monomers are examined in Chapter 3. Because of the stringent limitations of these early metal catalysts, efforts were focused on catalysts based on ruthenium complexes. Although not living, and displaying unusually long induction periods, these catalysts show high promise for future investigations directed at the development of catalysts for the living polymerization of functionalized monomers. In an attempt to develop useful catalysts based on these ruthenium complexes, efforts to increase their initiation rates are presented in Chapter 4. This work eventually led to the discovery that these catalysts are highly active in aqueous solution, providing the opportunity to develop aqueous emulsion ROMP systems. Recycling the aqueous catalysts led to the discovery that the ruthenium complexes become more activated with use. Investigations of these recycled solutions uncovered new ruthenium-olefin complexes, which are implicated in the activation process. Although our original goal of developing living ROMP catalysts for the polymerization of fuctionalized monomers is yet to be realized, it is hoped that this work provides a foundation from which future investigations can be launched.

In the last chapter, the ionophoric properties of the poly(7-oxanobornene) materials is briefly discussed. Their limited use as acyclic host polymers led to investigations into the fabrication of ion-permeable membranes fashioned from these materials.

New materials for biological applications prepared by olefin metathesis reactions

With the advent of well-defined ruthenium olefin metathesis catalysts that are highly active and stable to a variety of functional groups, the synthesis of complex organic molecules and polymers is now possible; this is reviewed in Chapter 1. The majority of the rest of this thesis describes the application of these catalysts towards the synthesis of novel polymers that may be useful in biological applications and investigations into their efficacy.

A method was developed to produce polyethers by metathesis, and this is described in Chapters 2 and 3. An unsaturated 12-crown-4 analog was made by template- directed ring-closing metathesis (RCM) and utilized as a monomer for the synthesis of unsaturated polyethers by ring-opening metathesis polymerization (ROMP). The yields were high and a range of molecular weights was accessible. In a similar manner, substituted polyethers with various backbones were synthesized: polymers with benzo groups along the backbone and various concentrations of amino acids were prepared. The results from in vitro toxicity tests of the unsubstituted polyethers are considered.

The conditions necessary to synthesize polynorbornenes with pendent bioactive peptides were explored as illustrated in Chapter 4. First, the polymerization of various norbornenyl monomers substituted with glycine, alanine or penta(ethylene glycol) is described. Then, the syntheses of polymers substituted with peptides GRGD and SRN, components of a cell binding domain of fibronectin, using newly developed ruthenium initiators are discussed.

In Chapter 5, the syntheses of homopolymers and a copolymer containing GRGDS and PHSRN, the more active forms of the peptides, are described. The ability of the polymers to inhibit human dermal fibroblast cell adhesion to fibronectin was assayed using an in vitro competitive inhibition assay, and the results are discussed. It was discovered that the copoymer substituted with both GRGDS and PHSR peptides was more active than both the GRGDS-containing homopolymer and the GRGDS free peptide.

Historically, one of the drawbacks to using metathesis is the removal of the residual ruthenium at the completion of the reaction. Chapter 6 describes a method where the water soluble tris(hydroxymethyl)phosphine is utilized to facilitate the removal of residual ruthenium from RCM reaction products.

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.

Functional genomic studies of the structure and regulation of eukaryotic transcriptomes

The main focus of this thesis is the use of high-throughput sequencing technologies in functional genomics (in particular in the form of ChIP-seq, chromatin immunoprecipitation coupled with sequencing, and RNA-seq) and the study of the structure and regulation of transcriptomes. Some parts of it are of a more methodological nature while others describe the application of these functional genomic tools to address various biological problems. A significant part of the research presented here was conducted as part of the ENCODE (ENCyclopedia Of DNA Elements) Project.

The first part of the thesis focuses on the structure and diversity of the human transcriptome. Chapter 1 contains an analysis of the diversity of the human polyadenylated transcriptome based on RNA-seq data generated for the ENCODE Project. Chapter 2 presents a simulation-based examination of the performance of some of the most popular computational tools used to assemble and quantify transcriptomes. Chapter 3 includes a study of variation in gene expression, alternative splicing and allelic expression bias on the single-cell level and on a genome-wide scale in human lymphoblastoid cells; it also brings forward a number of critical to the practice of single-cell RNA-seq measurements methodological considerations.

The second part presents several studies applying functional genomic tools to the study of the regulatory biology of organellar genomes, primarily in mammals but also in plants. Chapter 5 contains an analysis of the occupancy of the human mitochondrial genome by TFAM, an important structural and regulatory protein in mitochondria, using ChIP-seq. In Chapter 6, the mitochondrial DNA occupancy of the TFB2M transcriptional regulator, the MTERF termination factor, and the mitochondrial RNA and DNA polymerases is characterized. Chapter 7 consists of an investigation into the curious phenomenon of the physical association of nuclear transcription factors with mitochondrial DNA, based on the diverse collections of transcription factor ChIP-seq datasets generated by the ENCODE, mouseENCODE and modENCODE consortia. In Chapter 8 this line of research is further extended to existing publicly available ChIP-seq datasets in plants and their mitochondrial and plastid genomes.

The third part is dedicated to the analytical and experimental practice of ChIP-seq. As part of the ENCODE Project, a set of metrics for assessing the quality of ChIP-seq experiments was developed, and the results of this activity are presented in Chapter 9. These metrics were later used to carry out a global analysis of ChIP-seq quality in the published literature (Chapter 10). In Chapter 11, the development and initial application of an automated robotic ChIP-seq (in which these metrics also played a major role) is presented.

The fourth part presents the results of some additional projects the author has been involved in, including the study of the role of the Piwi protein in the transcriptional regulation of transposon expression in Drosophila (Chapter 12), and the use of single-cell RNA-seq to characterize the heterogeneity of gene expression during cellular reprogramming (Chapter 13).

The last part of the thesis provides a review of the results of the ENCODE Project and the interpretation of the complexity of the biochemical activity exhibited by mammalian genomes that they have revealed (Chapters 15 and 16), an overview of the expected in the near future technical developments and their impact on the field of functional genomics (Chapter 14), and a discussion of some so far insufficiently explored research areas, the future study of which will, in the opinion of the author, provide deep insights into many fundamental but not yet completely answered questions about the transcriptional biology of eukaryotes and its regulation.

Density functional theory embedding for correlated wavefunctions

Methods that exploit the intrinsic locality of molecular interactions show significant promise in making tractable the electronic structure calculation of large-scale systems. In particular, embedded density functional theory (e-DFT) offers a formally exact approach to electronic structure calculations in which the interactions between subsystems are evaluated in terms of their electronic density. In the following dissertation, methodological advances of embedded density functional theory are described, numerically tested, and applied to real chemical systems.

First, we describe an e-DFT protocol in which the non-additive kinetic energy component of the embedding potential is treated exactly. Then, we present a general implementation of the exact calculation of the non-additive kinetic potential (NAKP) and apply it to molecular systems. We demonstrate that the implementation using the exact NAKP is in excellent agreement with reference Kohn-Sham calculations, whereas the approximate functionals lead to qualitative failures in the calculated energies and equilibrium structures.

Next, we introduce density-embedding techniques to enable the accurate and stable calculation of correlated wavefunction (CW) in complex environments. Embedding potentials calculated using e-DFT introduce the effect of the environment on a subsystem for CW calculations (WFT-in-DFT). We demonstrate that WFT-in-DFT calculations are in good agreement with CW calculations performed on the full complex.

We significantly improve the numerics of the algorithm by enforcing orthogonality between subsystems by introduction of a projection operator. Utilizing the projection-based embedding scheme, we rigorously analyze the sources of error in quantum embedding calculations in which an active subsystem is treated using CWs, and the remainder using density functional theory. We show that the embedding potential felt by the electrons in the active subsystem makes only a small contribution to the error of the method, whereas the error in the nonadditive exchange-correlation energy dominates. We develop an algorithm which corrects this term and demonstrate the accuracy of this corrected embedding scheme.

Advanced density functional theory methods for materials science

In this work we chiefly deal with two broad classes of problems in computational materials science, determining the doping mechanism in a semiconductor and developing an extreme condition equation of state. While solving certain aspects of these questions is well-trodden ground, both require extending the reach of existing methods to fully answer them. Here we choose to build upon the framework of density functional theory (DFT) which provides an efficient means to investigate a system from a quantum mechanics description.

Zinc Phosphide (Zn₃P₂) could be the basis for cheap and highly efficient solar cells. Its use in this regard is limited by the difficulty in n-type doping the material. In an effort to understand the mechanism behind this, the energetics and electronic structure of intrinsic point defects in zinc phosphide are studied using generalized Kohn-Sham theory and utilizing the Heyd, Scuseria, and Ernzerhof (HSE) hybrid functional for exchange and correlation. Novel 'perturbation extrapolation' is utilized to extend the use of the computationally expensive HSE functional to this large-scale defect system. According to calculations, the formation energy of charged phosphorus interstitial defects are very low in n-type Zn₃P₂ and act as 'electron sinks', nullifying the desired doping and lowering the fermi-level back towards the p-type regime. Going forward, this insight provides clues to fabricating useful zinc phosphide based devices. In addition, the methodology developed for this work can be applied to further doping studies in other systems.

Accurate determination of high pressure and temperature equations of state is fundamental in a variety of fields. However, it is often very difficult to cover a wide range of temperatures and pressures in an laboratory setting. Here we develop methods to determine a multi-phase equation of state for Ta through computation. The typical means of investigating thermodynamic properties is via ’classical’ molecular dynamics where the atomic motion is calculated from Newtonian mechanics with the electronic effects abstracted away into an interatomic potential function. For our purposes, a ’first principles’ approach such as DFT is useful as a classical potential is typically valid for only a portion of the phase diagram (i.e. whatever part it has been fit to). Furthermore, for extremes of temperature and pressure quantum effects become critical to accurately capture an equation of state and are very hard to capture in even complex model potentials. This requires extending the inherently zero temperature DFT to predict the finite temperature response of the system. Statistical modelling and thermodynamic integration is used to extend our results over all phases, as well as phase-coexistence regions which are at the limits of typical DFT validity. We deliver the most comprehensive and accurate equation of state that has been done for Ta. This work also lends insights that can be applied to further equation of state work in many other materials.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

A variational framework for spectral discretization of the density matrix in Kohn Sham density functional theory

Kohn-Sham density functional theory (KSDFT) is currently the main work-horse of quantum mechanical calculations in physics, chemistry, and materials science. From a mechanical engineering perspective, we are interested in studying the role of defects in the mechanical properties in materials. In real materials, defects are typically found at very small concentrations e.g., vacancies occur at parts per million, dislocation density in metals ranges from $10^{10} m^{-2}$ to $10^{15} m^{-2}$, and grain sizes vary from nanometers to micrometers in polycrystalline materials, etc. In order to model materials at realistic defect concentrations using DFT, we would need to work with system sizes beyond millions of atoms. Due to the cubic-scaling computational cost with respect to the number of atoms in conventional DFT implementations, such system sizes are unreachable. Since the early 1990s, there has been a huge interest in developing DFT implementations that have linear-scaling computational cost. A promising approach to achieving linear-scaling cost is to approximate the density matrix in KSDFT. The focus of this thesis is to provide a firm mathematical framework to study the convergence of these approximations. We reformulate the Kohn-Sham density functional theory as a nested variational problem in the density matrix, the electrostatic potential, and a field dual to the electron density. The corresponding functional is linear in the density matrix and thus amenable to spectral representation. Based on this reformulation, we introduce a new approximation scheme, called spectral binning, which does not require smoothing of the occupancy function and thus applies at arbitrarily low temperatures. We proof convergence of the approximate solutions with respect to spectral binning and with respect to an additional spatial discretization of the domain. For a standard one-dimensional benchmark problem, we present numerical experiments for which spectral binning exhibits excellent convergence characteristics and outperforms other linear-scaling methods.

Synthesis, Oxidation and Photophysics of Perfluoroborated Tetrakis(pyrophosphito)diplatinate (II) and Density Functional Theory (DFT) Study of Electrochemical CO2 Reduction by Mn Catalysts

In the first part of this thesis (Chapters I and II), the synthesis, characterization, reactivity and photophysics of per(difluoroborated) tetrakis(pyrophosphito)diplatinate(II) (Pt(POPBF₂)) are discussed. Pt(POP-BF₂) was obtained by reaction of [Pt₂(POP)₄]^4- with neat boron trifluoride diethyl etherate (BF₃·Et₂O). While Pt(POP-BF₂) and [Pt₂(POP)₄]^4- have similar structures and absorption spectra, they differ in significant ways. Firstly, as discussed in Chapter I, the former is less susceptible to oxidation, as evidenced by the reversibility of its oxidation by I2. Secondly, while the first excited triplet states (T₁) of both Pt(POP-BF₂) and [Pt₂(POP)₄]^4- exhibit long lifetimes (ca. 0.01 ms at room temperature) and substantial zero-field splitting (40 cm^-1), Pt(POP-BF₂) also has a remarkably long-lived (1.6 ns at room temperature) singlet excited state (S₁), indicating slow intersystem crossing (ISC). Fluorescence lifetime and quantum yield (QY) of Pt(POP-BF₂) were measured over a range of temperatures, providing insight into the slow ISC process. The remarkable spectroscopic and photophysical properties of Pt(POP-BF₂), both in solution and as a microcrystalline powder, form the theme of Chapter II.

In the second part of the thesis (Chapters III and IV), the electrochemical reduction of CO₂ to CO by [(L)Mn(CO)₃]^- catalysts is investigated using density functional theory (DFT). As discussed in Chapter III, the turnover frequency (TOF)-limiting step is the dehydroxylation of [(bpy)Mn(CO)₃(CO₂H)]^0/- (bpy = bipyridine) by trifluoroethanol (TFEH) to form [(bpy)Mn(CO)₄]^+/0. Because the dehydroxylation of [(bpy)Mn(CO)₃(CO₂H)]^- is faster, maximum TOF (TOF_max) is achieved at potentials sufficient to completely reduce [(bpy)Mn(CO)₃(CO₂H)]⁰ to [(bpy)Mn(CO)₃(CO₂H)]^-. Substitution of bipyridine with bipyrimidine reduces the overpotential needed, but at the expense of TOF_max. In Chapter IV, the decoration of the bipyrimidine ligand with a pendant alcohol is discussed as a strategy to increase CO₂ reduction activity. Our calculations predict that the pendant alcohol acts in concert with an external TFEH molecule, the latter acidifying the former, resulting in a ~ 80,000-fold improvement in the rate of TOF-limiting dehydroxylation of [(L)Mn(CO)₃(CO₂H)]^-.

An interesting strategy for the co-upgrading of light olefins and alkanes into heavier alkanes is the subject of Appendix B. The proposed scheme involves dimerization of the light olefin, operating in tandem with transfer hydrogenation between the olefin dimer and the light alkane. The work presented therein involved a Ta olefin dimerization catalyst and a silica-supported Ir transfer hydrogenation catalyst. Olefin dimer was formed under reaction conditions; however, this did not undergo transfer hydrogenation with the light alkane. A significant challenge is that the Ta catalyst selectively produces highly branched dimers, which are unable to undergo transfer hydrogenation.

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.