Person:

Nocera, Daniel

Enhanced synthetic control of the morphology, crystal structure, and composition of nanostructures can drive advances in nanoscale devices. Axial and radial semiconductor nanowires are examples of nanostructures with one and two structural degrees of freedom, respectively, and their synthetically tuned and modulated properties have led to advances in nanotransistor, nanophotonic, and thermoelectric devices. Similarly, developing methods that allow for synthetic control of greater than two degrees of freedom could enable new opportunities for functional nanostructures. Here we demonstrate the first regioselective nanowire shell synthesis in studies of Ge and Si growth on faceted Si nanowire surfaces. The selectively deposited Ge is crystalline, and its facet position can be synthetically controlled in situ. We use this synthesis to prepare electrically addressable nanocavities into which solution soluble species such as Au nanoparticles can be incorporated. The method furnishes multicomponent nanostructures with unique photonic properties and presents a more sophisticated nanodevice platform for future applications in catalysis and photodetection.

The observed water oxidation activity of the compound class Co4O4(OAc)4(Py–X)4 emanates from a Co(II) impurity. This impurity is oxidized to produce the well-known Co-OEC heterogeneous cobaltate catalyst, which is an active water oxidation catalyst. We present results from electron paramagnetic resonance spectroscopy, nuclear magnetic resonance line broadening analysis, and electrochemical titrations to establish the existence of the Co(II) impurity as the major source of water oxidation activity that has been reported for Co4O4 molecular cubanes. Differential electrochemical mass spectrometry is used to characterize the fate of glassy carbon at water oxidizing potentials and demonstrate that such electrode materials should be used with caution for the study of water oxidation catalysis.

Escherichia coli class Ia ribonucleotide reductase (RNR) converts ribonucleotides to deoxynucleotides. A diferric-tyrosyl radical (Y122•) in one subunit (β2) generates a transient thiyl radical in another subunit (α2) via long-range radical transport (RT) through aromatic amino acid residues (Y122 ⇆ [W48] ⇆ Y356 in β2 to Y731 ⇆ Y730 ⇆ C439 in α2). Equilibration of Y356•, Y731•, and Y730• was recently observed using site specifically incorporated unnatural tyrosine analogs; however, equilibration between Y122• and Y356• has not been detected. Our recent report of Y356• formation in a kinetically and chemically competent fashion in the reaction of β2 containing 2,3,5-trifluorotyrosine at Y122 (F3Y122•-β2) with α2, CDP (substrate), and ATP (effector) has now afforded the opportunity to investigate equilibration of F3Y122• and Y356•. Incubation of F3Y122•-β2, Y731F-α2 (or Y730F-α2), CDP, and ATP at different temperatures (2–37 °C) provides ΔE°′(F3Y122•–Y356•) of 20 ± 10 mV at 25 °C. The pH dependence of the F3Y122• ⇆ Y356• interconversion (pH 6.8–8.0) reveals that the proton from Y356 is in rapid exchange with solvent, in contrast to the proton from Y122. Insertion of 3,5-difluorotyrosine (F2Y) at Y356 and rapid freeze-quench EPR analysis of its reaction with Y731F-α2, CDP, and ATP at pH 8.2 and 25 °C shows F2Y356• generation by the native Y122•. FnY-RNRs (n = 2 and 3) together provide a model for the thermodynamic landscape of the RT pathway in which the reaction between Y122 and C439 is ∼200 meV uphill.

Acidity, hypoxia and glucose levels characterize the tumor microenvironment rendering pH, pO2 and pGlucose, respectively, important indicators of tumor health. To this end, understanding how these parameters change can be a powerful tool for the development of novel and effective therapeutics. We have designed optical chemosensors that feature a quantum dot and an analyte-responsive dye. These non-invasive chemosensors permit pH, oxygen, and glucose to be monitored dynamically within the tumor microenvironment by using multiphoton imaging.

Hemoprotein-based scaffolds containing phosphorescent ruthenium(II) CO mesoporphyrin IX (RuMP) are reported here for oxygen (O2) sensing in biological contexts. RuMP was incorporated into the protein scaffolds during protein expression utilizing a novel method that we have described previously. A high-resolution (2.00 Å) crystal structure revealed that the unnatural porphyrin binds to the proteins in a manner similar to the native heme and does not perturb the protein fold. The protein scaffolds were found to provide unique coordination environments for RuMP and modulate the porphyrin emission properties. Emission lifetime measurements demonstrate a linear O2 response within the physiological range and precision comparable to commercial O2 sensors. The RuMP proteins are robust, readily-modifiable platforms and display promising O2 sensing properties for future in vivo applications.

Supramolecular assemblies of a quantum dot (QD) associated to palladium(II) porphyrins have been developed to detect oxygen (pO2) in organic solvents. Palladium porphyrins are sensitive in the 0–160 torr range, making them ideal phosphors for in vivo biological oxygen quantification. Porphyrins with meso pyridyl substituents bind to the surface of the QD to produce self–assembled nanosensors. Appreciable overlap between QD emission and porphyrin absorption features results in efficient Förster resonance energy transfer (FRET) for signal transduction in these sensors. The QD serves as a photon antenna, enhancing porphyrin emission under both one– and two–photon excitation, demonstrating that QD–palladium porphyrin conjugates may be used for oxygen sensing over physiological oxygen ranges.

The E. coli class Ia ribonucleotide reductase (RNR) achieves forward and reverse proton-coupled electron transfer (PCET) over a pathway of redox-active amino acids (β-Y122 ⇌ β-Y356 ⇌ α-Y731 ⇌ α-Y730 ⇌ α-C439) spanning ~35 Å and two subunits every time it turns over. We have developed photoRNRs that allow radical transport to be phototriggered at tyrosine (Y) or fluorotyrosine (FnY) residues along the PCET pathway. We now report a new photoRNR in which photooxidation of a tryptophan (W) residue replacing Y356 within the α/β subunit interface proceeds by a stepwise ETPT (electron transfer then proton transfer) mechanism and provides an orthogonal spectroscopic handle with respect to radical pathway residues Y731/Y730 in α. This construct displays a ~3-fold enhancement in photochemical yield of W• relative to F3Y• and a ~7-fold enhancement relative to Y•. Photogeneration of the W• radical occurs with a rate constant of 4.4 ± 0.2 × 105 s−1, which obeys a Marcus correlation for radical generation at the RNR subunit interface. Despite the fact that the Y → W variant displays no enzymatic activity in the absence of light, photogeneration of W• within the subunit interface results in 20% activity for turnover relative to wt-RNR under the same conditions.

Substrate turnover in class Ia ribonucleotide reductase (RNR) requires reversible radical transport across two subunits over 35 A, which occurs by a multi-step proton-coupled electron transfer mechanism. Using a photooxidant-labeled β2 subunit of Escherichia coli class Ia RNR, we demonstrate photoinitiated oxidation of a tyrosine in an α2:β2 complex, which results in substrate turnover. Using site-directed mutations of the redox-active tyrosines at the subunit interface—Y356F(β) and Y731F(α)—this oxidation is identified to be localized on Y356. The rate of Y356 oxidation depends on the presence of Y731 across the interface. This observation supports the proposal that unidirectional PCET across the Y356(β)–Y731(α)–Y730(α) triad is crucial to radical transport in RNR.

We present a new class of polymeric ligands for quantum dot (QD) water solubilization to yield biocompatible and derivatizable QDs with compact size (~10-12 nm diameter), high quantum yields (>50%), excellent stability across a large pH range (pH 5-10.5), and low nonspecific binding. To address the fundamental problem of thiol instability in traditional ligand exchange systems, the polymers here employ a stable multidentate imidazole binding motif to the QD surface. The polymers are synthesized via reversible addition-fragmentation chain transfer (RAFT)-mediated polymerization to produce molecular weight controlled monodisperse random copolymers from three types of monomers that feature imidazole groups for QD binding, polyethylene glycol (PEG) groups for water solubilization, and either primary amines or biotin groups for derivatization. The polymer architecture can be tuned by the monomer ratios to yield aqueous QDs with targeted surface functionalities. By incorporating amino-PEG monomers, we demonstrate covalent conjugation of a dye to form a highly efficient QD-dye energy transfer pair as well as covalent conjugation to streptavidin for high-affinity single molecule imaging of biotinylated receptors on live cells with minimal non-specific binding. The small size and low serum binding of these polymer-coated QDs also allow us to demonstrate their utility for in-vivo imaging of the tumor microenvironment in live mice.

Ribonucleotide reductase (RNR) catalyzes the conversion of ribonucleotides to deoxyribonucleotides to provide the monomeric building blocks for DNA replication and repair. Nucleotide reduction occurs by way of multi-step proton-coupled electron transfer (PCET) over a pathway of redox active amino acids spanning ~ 35 Å and two subunits (α2 and β2). Despite the fact that PCET in RNR is rapid, slow conformational changes mask kinetic examination of these steps. As such, we have pioneered methodology in which site-specific incorporation of a [ReI] photooxidant on the surface of the β2 subunit (photoβ2) allows photochemical oxidation of the adjacent PCET pathway residue β-Y356 and time-resolved spectroscopic observation of the ensuing reactivity. A series of photoβ2s capable of performing photoinitiated substrate turnover have been prepared in which four different fluorotyrosines (FnYs) are incorporated in place of β-Y356. The FnYs are deprotonated under biological conditions, undergo oxidation by electron transfer (ET) and provide a means by which to vary the ET driving force (ΔG°) with minimal additional perturbations across the series. We have used these features to map the correlation between ΔG° and kET both with and without the fully assembled photoRNR complex. The photooxidation of FnY356 within the α/β subunit interface occurs within the Marcus inverted region with a reorganization energy of λ ≈ 1 eV. We also observe enhanced electronic coupling between donor and acceptor (HDA) in the presence of an intact PCET pathway. Additionally, we have investigated the dynamics of proton transfer (PT) by a variety of methods including dependencies on solvent isotopic composition, buffer concentration, and pH. We present evidence for the role of α2 in facilitating PT during β-Y356 photooxidation; PT occurs by way of readily exchangeable positions and within a relatively “tight” subunit interface. These findings show that RNR controls ET by lowering λ, raising HDA, and directing PT both within and between individual polypeptide subunits.