Development of a Cationic Mucic Acid Polymer-Based Nanoparticle siRNA Delivery System

Cancer chemotherapy has advanced from highly toxic drugs to more targeted treatments in the last 70 years. Chapter 1 opens with an introduction to targeted therapy for cancer. The benefits of using a nanoparticle to deliver therapeutics are discussed. We move on to siRNA in particular, and why it would be advantageous as a therapy. Specific to siRNA delivery are some challenges, such as nuclease degradation, quick clearance from circulation, needing to enter cells, and getting to the cytosol. We propose the development of a nanoparticle delivery system to tackle these challenges so that siRNA can be effective.

Chapter 2 of this thesis discusses the synthesis and analysis of a cationic mucic acid polymer (cMAP) which condenses siRNA to form a nanoparticle. Various methods to add polyethylene glycol (PEG) for stabilizing the nanoparticle in physiologic solutions, including using a boronic acid binding to diols on mucic acid, forming a copolymer of cMAP with PEG, and creating a triblock with mPEG on both ends of cMAP. The goal of these various pegylation strategies was to increase the circulation time of the siRNA nanoparticle in the bloodstream to allow more of the nanoparticle to reach tumor tissue by the enhanced permeation and retention effect. We found that the triblock mPEG-cMAP-PEGm polymer condensed siRNA to form very stable 30-40 nm particles that circulated for the longest time – almost 10% of the formulation remained in the bloodstream of mice 1 h after intravenous injection.

Chapter 3 explores the use of an antibody as a targeting agent for nanoparticles. Some antibodies of the IgG1 subtype are able to recruit natural killer cells that effect antibody dependent cellular cytotoxicity (ADCC) to kill the targeted cell to which the antibody is bound. There is evidence that the ADCC effect remains in antibody-drug conjugates, so we wanted to know whether the ADCC effect is preserved when the antibody is bound to a nanoparticle, which is a much larger and complex entity. We utilized antibodies against epidermal growth factor receptor with similar binding and pharmacokinetics, cetuximab and panitumumab, which differ in that cetuximab is an IgG1 and panitumumab is an IgG2 (which does not cause ADCC). Although a natural killer cell culture model showed that gold nanoparticles with a full antibody targeting agent can elicit target cell lysis, we found that this effect was not preserved in vivo. Whether this is due to the antibody not being accessible to immune cells or whether the natural killer cells are inactivated in a tumor xenograft remains unknown. It is possible that using a full antibody still has value if there are immune functions which are altered in a complex in vivo environment that are intact in an in vitro system, so the value of using a full antibody as a targeting agent versus using an antibody fragment or a protein such as transferrin is still open to further exploration.

In chapter 4, nanoparticle targeting and endosomal escape are further discussed with respect to the cMAP nanoparticle system. A diboronic acid entity, which gives an order of magnitude greater binding (than boronic acid) to cMAP due to the vicinal diols in mucic acid, was synthesized, attached to 5kD or 10kD PEG, and conjugated to either transferrin or cetuximab. A histidine was incorporated into the triblock polymer between cMAP and the PEG blocks to allow for siRNA endosomal escape. Nanoparticle size remained 30-40 nm with a slightly negative ca. -3 mV zeta potential with the triblock polymer containing histidine and when targeting agents were added. Greater mRNA knockdown was seen with the endosomal escape mechanism than without. The nanoparticle formulations were able to knock down the targeted mRNA in vitro. Mixed effects suggesting function were seen in vivo.

Chapter 5 summarizes the project and provides an outlook on siRNA delivery as well as targeted combination therapies for the future of personalized medicine in cancer treatment.

Synthesis of (±)-7,8-epoxy-4-basmen-6-one by a transannular cyclization strategy

The first synthesis of the cembranoid natural product (±)-7,8-epoxy-4-basmen-6- one (1) is described. Key steps of the synthetic route include the cationic cyclization of the acid chloride from 15 to provide the macrocycle 16, and the photochemical transannular radical cyclization of the ester 41 to form the tricyclic product 50. Product 50 was transformed into 1 in ten steps. Transition-state molecular modeling studies were found to provide accurate predictions of the structural and stereochemical outcomes of cyclization reactions explored experimentally in the development of the synthetic route to 1. These investigations should prove valuable in the development of transannular cyclization as a strategy for synthetic simplification.

Studies of Organic Molecule Recognition by Synthetic Cyclophane Receptors in Aqueous Media

The behaviors of six new cyclophane receptors for organic guest molecules in aqueous media are reported. These new hosts are modifications of more basic parent structures, and the main goal of their examination has been to determine how the modifications affect host selectivity for cationic guests. In particular, we have been interested in determining how additional non-covalent binding interactions can complement the cation-π interactions active in the parent systems. Three types of modifications were made to these systems. Firstly, neutral methoxy and bromine substituents were added to produce four of the six new macrocycles. Secondly, two additional aromatic rings (relative to the parent host) capable of making cation-π interactions with charged guest species were appended. Thirdly, a negatively charged carboxyl group was attached to produce a cavity in which electrostatic interactions should enhance cationic guest binding. ^1H-NMR and circular dichroic techniques were employed to determine the binding affinities of a wide variety of organic guests for the parent and modified structures in aqueous media.

Bromination of the parent host greatly enhances its binding in a general fashion, primarily as the result of hydrophobic interactions. The addition of methoxy groups does not enhance binding, apparently as a result of a collapse of the hosts into a conformation that is not suitable for binding. The appendage of extra aromatic rings enhances the binding of positively charged guests, most likely in response to more complete encapsulation of guest species. The addition of a negatively charged carboxylate enhances the binding to only selective groups of cationic guests. AM1 calculations of the electrostatic potentials of several guests molecules suggests that the enhancements seen with the modified receptor compared to the parent are most likely the result of close contact between regions of highest potential on the guest and the appended carboxylate.

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.

Capturing protein dynamics with time-resolved luminescence spectroscopy

The presented doctoral research utilizes time-resolved spectroscopy to characterize protein dynamics and folding mechanisms. We resolve millisecond-timescale folding by coupling time-resolved fluorescence energy transfer (trFRET) to a continuous flow microfluidic mixer to obtain intramolecular distance distributions throughout the folding process. We have elucidated the folding mechanisms of two cytochromes---one that exhibits two-state folding (cytochrome cb₅₆₂) and one that has both a kinetic refolding intermediate ensemble and a distinct equilibrium unfolding intermediate (cytochrome c₅₅₂). Our data reveal that the distinct structural features of cytochrome c₅₅₂ contribute to its thermostability.

We have also investigated intrachain contact dynamics in unfolded cytochrome cb₅₆₂ by monitoring electron transfer, which occurs as the heme collides with a ruthenium photosensitizer, covalently bound to residues along the polypeptide. Intrachain diffusion for chemically denatured proteins proceeds on the microsecond timescale with an upper limit of 0.1 microseconds. The power-law dependence (slope = -1.5) of the rate constants on the number of peptide bonds between the heme and Ru complex indicate that cytochrome cb₅₆₂ is minimally frustrated.

In addition, we have explored the pathway dependence of electron tunneling rates between metal sites in proteins. Our research group has converted cytochrome b₅₆₂ to a c-type cytochrome with the porphyrin covalently bound to cysteine sidechains. We have investigated the effects of the changes to the protein structure (i.e., increased rigidity and potential new equatorial tunneling pathways) on the electron transfer rates, measured by transient absorption, in a series of ruthenium photosensitizer-modified proteins.

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.

Mechanistic and reactivity studies of bent metallocene complexes of niobium and tantalum

A variety of olefin hydride complexes of niobium and tantalum has been prepared in order to study their reactivity and to gain insight into organometallic reaction mechanisms. Examination of a series of ethylene and propylene complexes of niobocene (CP_2Nb; Cp = η^5-C_5H_5), permethylniobocene (Cp*_2Nb; Cp* = η^5-C_5(CH_3)_5), tantalocene, and permethyltantalocene has indicated that there are both large electronic and steric effects deriving from the metal (and its ancillary ligands) in the olefin insertion (β-migratory insertion) process. Furthermore, a thermodynamic and kinetic analysis has been completed for a series of substituted styrene complexes of niobocene in order to better understand the important electronic properties of the olefin. The results are in accord with a concerted four-center process with only moderate charge development.

The special case of β-migratory insertion of a hydride ligand into coordinated benzyne has also been studied for the permethyltantalocene system. The coordinatively unsaturated (sixteen electron) phenyl tautomer, which is made accessible by the facile benzyne hydride insertion reaction, readily reacts with a variety of ligands, L, to afford Cp*_2 Ta(C_6H_5)L complexes (L = CO, O_2, NC≡R, :CH_2, H_2, etc.). This family of compounds exhibits interesting reactivity (a-migratory insertion, O_2 activation, and reductive elimination) which is discussed in some detail.

Finally a series of paramagnetic seventeen electron Cp*_2 TaX_2 (X = halide, alkyl, hydride) complexes, and the corresponding cationic and anionic species, have been prepared and studied. The odd electron neutral complexes exhibit surprising thermal stability and undergo very little reactivity. While the chemistry of the anionic compounds is almost completely dominated by their potent reducing power, that of the cations is quite diverse and amenable for study. Therefore the syntheses and reactivity (1 ,2-eliminations, ligand insertions, and deprotonation reactions) of these coordinatively unsaturated sixteen electron species are presented.

Catalytic conversion of nitrogen to ammonia by an iron model complex

Threefold symmetric Fe phosphine complexes have been used to model the structural and functional aspects of biological N₂ fixation by nitrogenases. Low-valent bridging Fe-S-Fe complexes in the formal oxidation states Fe(II)Fe(II), Fe(II)/Fe(I), and Fe(I)/Fe(I) have been synthesized which display rich spectroscopic and magnetic behavior. A series of cationic tris-phosphine borane (TPB) ligated Fe complexes have been synthesized and been shown to bind a variety of nitrogenous ligands including N₂H₄, NH₃, and NH₂-. These complexes are all high spin S = 3/2 and display EPR and magnetic characteristics typical of this spin state. Furthermore, a sequential protonation and reduction sequence of a terminal amide results in loss of NH₃ and uptake of N₂. These stoichiometric transformations represent the final steps in potential N₂ fixation schemes.

Treatment of an anionic FeN₂ complex with excess acid also results in the formation of some NH₃, suggesting the possibility of a catalytic cycle for the conversion of N₂ to NH₃ mediated by Fe. Indeed, use of excess acid and reductant results in the formation of seven equivalents of NH₃ per Fe center, demonstrating Fe mediated catalytic N₂ fixation with acids and protons for the first time. Numerous control experiments indicate that this catalysis is likely being mediated by a molecular species.

A number of other phosphine ligated Fe complexes have also been tested for catalysis and suggest that a hemi-labile Fe-B interaction may be critical for catalysis. Additionally, various conditions for the catalysis have been investigated. These studies further support the assignment of a molecular species and delineate some of the conditions required for catalysis.

Finally, combined spectroscopic studies have been performed on a putative intermediate for catalysis. These studies converge on an assignment of this new species as a hydrazido(2-) complex. Such species have been known on group 6 metals for some time, but this represents the first characterization of this ligand on Fe. Further spectroscopic studies suggest that this species is present in catalytic mixtures, which suggests that the first steps of a distal mechanism for N₂ fixation are feasible in this system.

Zirconocenes as models for homogeneous Ziegler-Natta olefin polymerization catalysts

Using density functional theory, we studied the fundamental steps of olefin polymerization for zwitterionic and cationic Group IV ansa-zirconocenes and a neutral ansa- yttrocene. Complexes [H₂E(C₅H₄)₂ZrMe]ⁿ (n = 0: E = BH₂ (1), BF₂ (2), AlH₂(3); n = +: E = CH₂(4), SiH₂(5)) and H₂Si(C₅H₄)₂YMe were used as computational models. The largest differences among these three classes of compounds were the strength of olefin binding and the stability of the β-agostic alkyl intermediate towards β-hydrogen elimination. We investigated the effect of solvent on the reaction energetics for land 5. We found that in benzene the energetics became very similar except that a higher olefin insertion barrier was calculated for 1. The calculated anion affinity of [CH₃BF₃]^- was weaker towards 1 than 5. The calculated olefin binding depended primarily on the charge of the ansa linker, and the olefin insertion barrier was found to decrease steadily in the following order: [H₂C(C₅H₄)₂ZrMe]⁺ > [F₂B(C₅H₄)₂ZrMe] ≈ [H₂B(C₅H₄)₂ZrMe] > [H₂Si(C₅H₄)₂ZrMe]⁺ > [H₂Al(C₅H₄)₂ZrMe].

We prepared ansa-zirconocene dicarbonyl complexes Me₂ECp₂Zr(CO)₂ (E = Si, C), and t-butyl substituted complexes (t-BuCp)₂Zr(CO)₂, Me₂E(t-BuCp)₂Zr(CO)₂ (E = Si, C), (Me₂Si)₂(t-BuCp)₂Zr(CO)₂ as well as analogous zirconocene complexes. Both the reduction potentials and carbonyl stretching frequencies follow the same order: Me₂SiCp₂ZrCl₂> Me₂CCp₂ZrCl₂> Cp₂ZrCl₂> (Me₂Si)₂Cp₂ZrCl₂. This ordering is a result of both the donating abilities of the cyclopentadienyl substituents and the orientation of the cyclopentadiene rings. Additionally, we prepared a series of analogous cationic zirconocene complexes [LZrOCMe₃][MeB(C₆F₅)₃] (L = CP₂, Me₂SiCp₂, Me₂CCP₂, (Me₂Si)₂Cp₂) and studied the kinetics of anion dissociation. We found that the enthalpy of anion dissociation increased from 10.3 kcal•mol^-1 to 17.6 kcal•mol^-1 as exposure of the zirconium center increased.

We also prepared series of zirconocene complexes bearing 2,2-dimethyl-2-sila-4-pentenyl substituents (and methyl-substituted olefin variants). Methide abstraction with B(C₆F₅) results in reversible coordination of the tethered olefin to the cationic zirconium center. The kinetics of olefin dissociation have been examined using NMR methods, and the effects of ligand variation for unlinked, singly [SiMe₂]-linked and doubly [SiMe₂]-linked bis(cyclopentadienyl) arrangements has been compared (ΔG‡ for olefin dissociation varies from 12.8 to 15.6 kcal•mol^-1). Methide abstraction from 1,2-(SiMe₂)₂(η⁵-C₅H₃)₂Zr(CH₃)-(CH₂CMe₂CH₂CH = CH₂) results in rapid β-allyl elimination with loss of isobutene yielding the allyl cation [{1,2-(SiMe₂)₂(η⁵-C₅H₃)₂Zr(η³-CH₂CH=CH₂)]⁺.

Reactions of platinum(II) complexes with dioxygen: progress toward alkane functionalization

Whereas stoichiometric activation of C-H bonds by complexes of transition metals is becoming increasingly common, selective functionalization of alkanes remains a formidable challenge in organometallic chemistry. The recent advances in catalytic alkane functionalization by transition-metal complexes are summarized in Chapter I.

The studies of the displacement of pentafluoropyridine in [(tmeda)Pt(CH_3)(NC_5F_5)][BAr^f_4] (1) with γ- tetrafluoropicoline, a very poor nucleophile, are reported in Chapter II. The ligand substitution occurs by a dissociative interchange mechanism. This result implies that dissociative loss of pentafluoropyridine is the rate-limiting step in the C-H activation reactions of 1.

Oxidation of dimethylplatinum(II) complexes (N-N)Pt(CH_3)_2 (N-N = tmeda(1), α-diimines) by dioxygen is described in Chapter III. Mechanistic studies suggest a two-step mechanism. First, a hydroperoxoplatinum(IV) complex is formed in a reaction between (N-N)Pt(CH_3)_2 and dioxygen. Next, the hydroperoxy complex reacts with a second equivalent of (N-N)Pt(CH_3)_2 to afford the final product, (N-N)Pt(OH)(OCH_3)(CH_3)_2. The hydroperoxy intermediate, (tmeda)Pt(OOH)(OCH_3)(CH_3)_2 (2), was isolated and characterized. The reactivity of 2 with several dime thylplatinum(II) complexes is reported.

The studies described in Chapter IV are directed toward the development of a platinum(II)-catalyzed oxidative alkane dehydrogenation. Stoichiometric conversion of alkanes (cyclohexane, ethane) to olefins (cyclohexene, ethylene) is achieved by C-H activation with [(N-N)Pt(CH_3)(CF_3CH_2OH)]BF_4 (1, N-N is N,N'-bis(3,5-di-t- butylphenyl)-1,4-diazabutadiene) which results in the formation of olefin hydride complexes. The first step in the C-H activation reaction is formation of a platinum(II) alkyl which undergoes β-hydrogen elimination to afford the olefin hydride complex. The cationic ethylplatinum(II) intermediate can be generated in situ by treating diethylplatinum(II) compounds with acids. Treatment of (phen)PtEt_2 with [H(OEt_2)_2]Bar^f_4 at low temperatures resulted in the formation of a mixture of [(phen)PtEt(OEt_2)]Bar^f_4 (8) and [(phen)Pt(C_2H_4)H] Bar^f_4 (7). The cationic olefin complexes are unreactive toward dioxygen or hydrogen peroxide. Since the success of the overall catalytic cycle depends on our ability to oxidize the olefin hydride complexes, a series of neutral olefin complexes of platinum(II) with monoanionic ligands (derivatives of pyrrole-2-carboxyaldehyde N-aryl imines) was prepared. Unfortunately, these are also stable to oxidation.

Ancillary ligand effects : from zirconium(IV)-catalyzed homogeneous propylene polymerization to platinum(II)-mediated C-H bond activation

A series of C_s- and C₁-symmetric doubly-linked ansa-metallocenes of the general formula {1,1'-SiMe₂-2,2'-E-('ƞ⁵-C₅H₂-4-R¹)-(ƞ⁵-C₅H-3',5'-(CHMe₂)₂)}ZrC₂ (E = SiMe₂ (1), SiPh₂ (2), SiMe₂ -SiMe₂ (3); R¹ = H, CHMe₂, C₅H₉, C₆H₁₁, C₆H₅) has been prepared. When activated by methylaluminoxane, these are active propylene polymerization catalysts. 1 and 2 produce syndiotactic polypropylenes, and 3 produces isotactic polypropylenes. Site epimerization is the major pathway for stereoerror formation for 1 and 2. In addition, the polymer chain has slightly stronger steric interaction with the diphenylsilylene linker than with the dimethylsilylene linker. This results in more frequent site epimerization and reduced syndiospecificity for 2 compared to 1.

C₁-Symmetric ansa-zirconocenes [1,1 '-SiMe₂-(C₅H₄)-(3-R-C₅H₃)]ZrCl₂ (4), [1,1 '-SiMe₂-(C₅H₄)-(2,4-R₂-C₅H₂)]ZrCl₂ (5) and [1,1 '-SiMe₂-2,2 '-(SiMe₂-SiMe₂)-(C₅H₃)-( 4-R-C₅H₂)]ZrCl₂ (6) have been prepared to probe the origin of isospecificity in 3. While 4 and 3 produce polymers with similar isospecificity, 5 and 6 give mostly hemi-isotactic-like polymers. It is proposed that the facile site epimerization via an associative pathway allows rapid equilibration of the polymer chain between the isospecific and aspecific insertion sites. This results in more frequent insertion from the isospecific site, which has a lower kinetic barrier for chain propagation. On the other hand, site epimerization for 5 and 6 is slow. This leads to mostly alternating insertion from the isospecific and aspecific sites, and consequently, a hemi-isotactic-like polymers. In comparison, site epimerization is even slower for 3, but enchainment from the aspecific site has an extremely high kinetic barrier for monomer coordination. Therefore, enchainment occurs preferentially from the isospecific site to produce isotactic polymers.

A series of cationic complexes [(ArN=CR-CR=NAr)PtMe(L)]⁺[BF₄]⁺ (Ar = aryl; R = H, CH₃; L = water, trifluoroethanol) has been prepared. They react smoothly with benzene at approximately room temperature in trifluoroethanol solvent to yield methane and the corresponding phenyl Pt(II) cations, via Pt(IV)-methyl-phenyl-hydride intermediates. The reaction products of methyl-substituted benzenes suggest an inherent reactivity preference for aromatic over benzylic C-H bond activation, which can however be overridden by steric effects. For the reaction of benzene with cationic Pt(II) complexes, in which the diimine ligands bear 3,5-disubstituted aryl groups at the nitrogen atoms, the rate-determining step is C-H bond activation. For the more sterically crowded analogs with 2,6-dimethyl-substituted aryl groups, benzene coordination becomes rate-determining. The more electron-rich the ligand, as reflected by the CO stretching frequency in the IR spectrum of the corresponding cationic carbonyl complex, the faster the rate of C-H bond activation. This finding, however, does not reflect the actual C-H bond activation process, but rather reflects only the relative ease of solvent molecules displacing water molecules to initiate the reaction. That is, the change in rates is mostly due to a ground state effect. Several lines of evidence suggest that associative substitution pathways operate to get the hydrocarbon substrate into, and out of, the coordination sphere; i.e., that benzene substitution proceeds by a solvent- (TFE-) assisted associative pathway.

The Flocculation of E. coli with Polyethyleneimine

A comprehensive study was made of the flocculation of dispersed E. coli bacterial cells by the cationic polymer polyethyleneimine (PEI). The three objectives of this study were to determine the primary mechanism involved in the flocculation of a colloid with an oppositely charged polymer, to determine quantitative correlations between four commonly-used measurements of the extent of flocculation, and to record the effect of varying selected system parameters on the degree of flocculation. The quantitative relationships derived for the four measurements of the extent of flocculation should be of direct assistance to the sanitary engineer in evaluating the effectiveness of specific coagulation processes.

A review of prior statistical mechanical treatments of absorbed polymer configuration revealed that at low degrees of surface site coverage, an oppositely- charged polymer molecule is strongly adsorbed to the colloidal surface, with only short loops or end sequences extending into the solution phase. Even for high molecular weight PEI species, these extensions from the surface are theorized to be less than 50 Å in length. Although the radii of gyration of the five PEI species investigated were found to be large enough to form interparticle bridges, the low surface site coverage at optimum flocculation doses indicates that the predominant mechanism of flocculation is adsorption coagulation.

The effectiveness of the high-molecular weight PEI species 1n producing rapid flocculation at small doses is attributed to the formation of a charge mosaic on the oppositely-charged E. coli surfaces. The large adsorbed PEI molecules not only neutralize the surface charge at the adsorption sites, but also cause charge reversal with excess cationic segments. The alignment of these positive surface patches with negative patches on approaching cells results in strong electrostatic attraction in addition to a reduction of the double-layer interaction energies. The comparative ineffectiveness of low-molecular weight PEI species in producing E. coli flocculation is caused by the size of the individual molecules, which is insufficient to both neutralize and reverse the negative E.coli surface charge. Consequently, coagulation produced by low molecular weight species is attributed solely to the reduction of double-layer interaction energies via adsorption.

Electrophoretic mobility experiments supported the above conclusions, since only the high-molecular weight species were able to reverse the mobility of the E. coli cells. In addition, electron microscope examination of the seam of agglutination between E. coli cells flocculation by PEI revealed tightly- bound cells, with intercellular separation distances of less than 100-200 Å in most instances. This intercellular separation is partially due to cell shrinkage in preparation of the electron micrographs.

The extent of flocculation was measured as a function of PEl molecular weight, PEl dose, and the intensity of reactor chamber mixing. Neither the intensity of mixing, within the common treatment practice limits, nor the time of mixing for up to four hours appeared to play any significant role in either the size or number of E.coli aggregates formed. The extent of flocculation was highly molecular weight dependent: the high-molecular-weight PEl species produce the larger aggregates, the greater turbidity reductions, and the higher filtration flow rates. The PEl dose required for optimum flocculation decreased as the species molecular weight increased. At large doses of high-molecular-weight species, redispersion of the macroflocs occurred, caused by excess adsorption of cationic molecules. The excess adsorption reversed the surface charge on the E.coli cells, as recorded by electrophoretic mobility measurements.

Successful quantitative comparisons were made between changes in suspension turbidity with flocculation and corresponding changes in aggregate size distribution. E. coli aggregates were treated as coalesced spheres, with Mie scattering coefficients determined for spheres in the anomalous diffraction regime. Good quantitative comparisons were also found to exist between the reduction in refiltration time and the reduction of the total colloid surface area caused by flocculation. As with turbidity measurements, a coalesced sphere model was used since the equivalent spherical volume is the only information available from the Coulter particle counter. However, the coalesced sphere model was not applicable to electrophoretic mobility measurements. The aggregates produced at each PEl dose moved at approximately the same vlocity, almost independently of particle size.

PEl was found to be an effective flocculant of E. coli cells at weight ratios of 1 mg PEl: 100 mg E. coli. While PEl itself is toxic to E.coli at these levels, similar cationic polymers could be effectively applied to water and wastewater treatment facilities to enhance sedimentation and filtration characteristics.

Electron transfer in chemically and genetically modified myoglobins

The temperature dependences of the reduction potentials (E^o') of wildtype human myoglobin (Mb) and three site-directed mutants have been measured by using thin-layer spectroelectrochemistry. Residue Val68, which is in van der Waals contact with the heme in Mb, has been replaced by Glu, Asp, and Asn. At pH 7.0, reduction of the heme iron (III) in the former two proteins is accompanied by uptake of a proton by the protein. The changes in E^o', and the standard entropy (ΔS^o') and enthalpy (ΔH^o') of reduction in the mutant proteins were determined relative to values for wild-type; the change in E^o' at 25°C was about -200 millivolts for the Glu and Asp mutants, and about -80 millivolts for the Asn mutant. Reduction of Fe(III) to Fe(II) in the Glu and Asp mutants is accompanied by uptake of a proton. These studies demonstrate that Mb can tolerate substitution of a buried hydrophobic group by potentially charged and polar residues, and that such amino acid replacements can lead to substantial changes in the redox thermodynamics of the protein.

Through analysis of the temperature dependence and shapes of NMR dispersion signals, it is determined that a water molecule is bound to the sixth coordination site of the ferric heme in the Val68Asp and in the Val68Asn recombinant proteins while the carboxyl group of the sidechain of Glu68 occupies this position in Val68Glu. The relative rhombic distortions in the ESR spectra of these mutant proteins combined with H₂¹⁷O and spin interconversion experiments performed on them confirm the conclusions of the NMRD study.

The rates of intramolecular electron transfer (ET) of (NH₃)₅Ru-His48 (Val68Asp, His81GIn, Cys110AIa)Mb and (NH₃)₅Ru-His48 (Val68GIu,His81GIn,Cys110Ala)Mb were measured to be .85(3)s^-1 and .30(2)s^-1, respectively. This data supports the hypothesis that entropy of 111 reduction and reorganization energy of ET are inversely related. The rates of forward and reverse ET for (NH₃)₅ Ru-His48 (Val68GIu, His81 GIn, Cys110AIa)ZnMb -7.2(5)•10⁴s^-1and 1.4(2)•10⁵s^-1, respectively- demonstrate that the placement of a highly polar residue nearby does not significantly change the reorganization energy of the photoactive Zn porphyrin.

The distal histidine imidazoles of (NH₃)₄isnRu-His48 SWMb and (NH₃)₅Ru-His48 SWMb were cyanated with BrCN. The intramolecular ET rates of these BrCN-modified Mb derivatives are 5.5(6)s^-1 and 3.2(5)s^-1, respectively. These respective rates are 20 and 10 times faster than those of their noncyanated counterparts after the differences in ET rate from driving force are scaled according to the Marcus equation. This increase in ET rate of the cyanated Mb derivatives is attributed to lower reorganization energy since the cyanated Mb heme is pentacoordinate in both oxidation states; whereas, the native Mb heme loses a water molecule upon reduction so that it changes from six to five coordinate. The reorganization energy from Fe-OH₂ dissociation is estimated to be .2eV. This conclusion is used to reconcile data from previous experiments in our lab. ET in photoactive porphyrin-substituted myoglobins proceed faster than predicted by Marcus Theory when it is assumed that the only difference in ET parameters between photoactive porphyrins and native heme systems is driving force. However, the data can be consistently fit to Marcus Theory if one corrects for the smaller reorganization in the photoactive porphyrin systems since they do not undergo a coordination change upon ET.

Finally, the intramolecular ET rate of (NH₃)₄isnRu-His48 SWMb was measured to be 3.0(4)s^-1. This rate is within experimental error of that for (NH₃)₄pyrRu-His48 SWMb even though the former has 80mV more driving force. One likely possibility for this observation is that the tetraamminepyridineruthenium group undergoes less reorganization upon ET than the tetraammineisonicotinamideruthenium group. Moreover, analysis of the (NH₃)₄isnRu-His48 SWMb experimental system gives a likely explanation of why ET was not observed previously in (NH₃)₄isnRu-Cytochrome C.

Electrocatalytic reduction of dioxygen at chemically modified electrodes

The kinetics of the reduction of O₂ by Ru(NH₃)₆⁺² as catalyzed by cobalt(II) tetrakis(4-N-methylpyridyl)porphyrin are described both in homogeneous solution and when the reactants are confined to Nafion coatings on graphite electrodes. The catalytic mechanism is determined and the factors that can control the total reduction currents at Nafion-coated electrodes are specified. A kinetic zone diagram for analyzing the behavior of catalyst-mediator-substrate systems at polymer coated electrodes is presented and utilized in identifying the current-limiting processes. Good agreement is demonstrated between calculated and measured reduction currents at rotating disk electrodes. The experimental conditions that will yield the optimum performance of coated electrodes are discussed, and a relationship is derived for the optimal coating thickness.

The relation between the reduction potentials of adsorbed and unadsorbed cobalt(III) tetrakis(4-N-methylpyridyl)porphyrin and those where it catalyzes the electroreduction of dioxygen is described. There is an unusually large change in the formal potential of the Co(III) couple upon the adsorption of the porphyrin on the graphite electrode surface. The mechanism in which the (inevitably) adsorbed porphyrin catalyzes the reduction of O₂ is in accord with a general mechanistic scheme proposed for most monomeric cobalt porphyrins.

Four new dimeric metalloporphyrins (prepared in the laboratory of Professor C. K. Chang) have the two porphyrin rings linked by an anthracene bridge attached to meso positions. The electrocatalytic behavior of the diporphyrins towards the reduction of O₂ at graphite electrodes has been examined for the following combination of metal centers: Co-Cu, Co-Fe, Fe-Fe, Fe-H₂. The Co-Cu diporphyrin catalyzes the reduction of O₂ to H₂O₂ but no further. The other three catalysts all exhibit mixed reduction pathways leading to both H₂O₂ and H₂O. However, the pathways that lead to H₂O do not involve H₂O₂ as an intermediate. A possible mechanistic scheme is offered to account for the observed behavior.

Photochemical electron transfer at fixed distance : a synthetic model of the photosynthetic primary process

A series of meso-phenyloctamethylporphyrins covalently bonded at the 4'phenyl position to quinones via rigid bicyclo[2.2.2]octane spacers were synthesized for the study of the dependence of electron transfer reaction rate on solvent, distance, temperature, and energy gap. A general and convergent synthesis was developed based on the condensation of ac-biladienes with masked quinonespacer-benzaldehydes. From picosecond fluorescence spectroscopy emission lifetimes were measured in seven solvents of varying polarity. Rate constants were determined to vary from 5.0x10⁹sec^-1 in N,N-dimethylformamide to 1.15x10¹⁰ Sec^-1 in benzene, and were observed to rise at most by about a factor of three with decreasing solvent polarity. Experiments at low temperature in 2-MTHF glass (77K) revealed fast, nearly temperature-independent electron transfer characterized by non-exponential fluorescence decays, in contrast to monophasic behavior in fluid solution at 298K. This example evidently represents the first photosynthetic model system not based on proteins to display nearly temperature-independent electron transfer at high temperatures (nuclear tunneling). Low temperatures appear to freeze out the rotational motion of the chromophores, and the observed nonexponential fluorescence decays may be explained as a result of electron transfer from an ensemble of rotational conformations. The nonexponentiality demonstrates the sensitivity of the electron transfer rate to the precise magnitude of the electronic matrix element, which supports the expectation that electron transfer is nonadiabatic in this system. The addition of a second bicyclooctane moiety (15 Å vs. 18 Å edge-to-edge between porphyrin and quinone) reduces the transfer rate by at least a factor of 500-1500. Porphyrinquinones with variously substituted quinones allowed an examination of the dependence of the electron transfer rate constant κ_ET on reaction driving force. The classical trend of increasing rate versus increasing exothermicity occurs from 0.7 eV≤ |ΔG^0'(R)| ≤ 1.0 eV until a maximum is reached (κ_ET = 3 x 10⁸ sec^-1 rising to 1.15 x 10¹⁰ sec^-1 in acetonitrile). The rate remains insensitive to ΔG⁰ for ~ 300 mV from 1.0 eV≤ |ΔG^0’(R)| ≤ 1.3 eV, and then slightly decreases in the most exothermic case studied (cyanoquinone, κ_ET = 5 x 10⁹ sec^-1).