Kevin McEleney

Modified mesoporous materials as Pd scavengers and catalyst supports

The use of mesoporous molecular sieves (MMSs) modified by mercaptopropyl trimethoxysilane (MPTMS) to scavenge Pd is described. The ordered mesoporous material displays excellent ability to remove Pd from organic and aqueous solutions. After only one treatment, a 500-ppm solution of PdCl2 in water can be reduced to 1 ppb. In addition, the resulting material is an effective, leach-proof catalyst for the Suzuki-Miyaura and Mizoroki-Heck reactions. Thus, the Suzuki-Miyaura reaction can be conducted in water at 80 °C with as little as 3 ppb Pd leaching. Hot filtrations and three-phase tests confirm that the catalyst acts without leaching Pd from the surface.

Designing electronic/ionic conducting membranes for artificial photosynthesis

We discuss the figures of merit for conducting membranes in artificial photosynthetic systems and describe an electronically and ionically conducting polymer composite with attractive performance characteristics.

Binding of G-quadruplexes to the N-terminal recognition domain of the RNA helicase associated with AU-rich element (RHAU).

Background: The helicase RHAU requires an N-terminal extension to bind quadruplex structures. Results: This extension adopts an elongated shape and interacts with the guanine tetrad face of quadruplexes. Conclusion: We provide a basis for the understanding of quadruplex binding by the N-terminal domain. Significance: The N-terminal region does not require the 2′-OH of the ribose to mediate the protein-quadruplex interaction. Polynucleotides containing consecutive tracts of guanines can adopt an intramolecular G-quadruplex structure where multiple planar tetrads of hydrogen-bound guanines stack on top of each other. Remodeling of G-quadruplexes impacts numerous aspects of nucleotide biology including transcriptional and translational control. RNA helicase associated with AU-rich element (RHAU), a member of the ATP-dependent DEX(H/D) family of RNA helicases, has been established as a major cellular quadruplex resolvase. RHAU contains a core helicase domain responsible for ATP binding/hydrolysis/helicase activity and is flanked on either side by N- and C-terminal extensions. The N-terminal extension is required for quadruplex recognition, and we have previously demonstrated complex formation between this domain and a quadruplex from human telomerase RNA. Here we used an integrated approach that includes small angle x-ray scattering, nuclear magnetic resonance spectroscopy, circular dichroism, and dynamic light scattering methods to demonstrate the recognition of G-quadruplexes by the N-terminal domain of RHAU. Based on our results, we conclude that (i) quadruplex from the human telomerase RNA and its DNA analog both adopt a disc shape in solution, (ii) RHAU53–105 adopts a defined and extended conformation in solution, and (iii) the N-terminal domain mediates an interaction with a guanine tetrad face of quadruplexes. Together, these data form the foundation for understanding the recognition of quadruplexes by the N-terminal domain of RHAU.

Enantioselective catalysis with a chiral, phosphane-containing PMO material

A novel bistriethoxysilyl-BINAP monomer was prepared and co-condensed with a biphenylene-bridged siloxane precursor in the presence of surfactant templates to give periodic mesoporous organosilicas (PMOs) functionalized with BINAP. Complexation of ruthenium followed by asymmetric catalytic hydrogenation and asymmetric transfer hydrogenation were carried out, and demonstrated that high levels of activity and selectivity are achievable with the chiral material.

Recognition of viral RNA stem-loops by the tandem double-stranded RNA binding domains of PKR.

In humans, the double-stranded RNA (dsRNA)-activated protein kinase (PKR) is expressed in late stages of the innate immune response to viral infection by the interferon pathway. PKR consists of tandem dsRNA binding motifs (dsRBMs) connected via a flexible linker to a Ser/Thr kinase domain. Upon interaction with viral dsRNA, PKR is converted into a catalytically active enzyme capable of phosphorylating a number of target proteins that often results in host cell translational repression. A number of high-resolution structural studies involving individual dsRBMs from proteins other than PKR have highlighted the key features required for interaction with perfectly duplexed RNA substrates. However, viral dsRNA molecules are highly structured and often contain deviations from perfect A-form RNA helices. By use of small-angle X-ray scattering (SAXS), we present solution conformations of the tandem dsRBMs of PKR in complex with two imperfectly base-paired viral dsRNA stem-loops; HIV-1 TAR and adenovirus VA(I)-AS. Both individual components and complexes were purified by size exclusion chromatography and characterized by dynamic light scattering at multiple concentrations to ensure monodispersity. SAXS ab initio solution conformations of the individual components and RNA-protein complexes were determined and highlight the potential of PKR to interact with both stem and loop regions of the RNA. Excellent agreement between experimental and model-based hydrodynamic parameter determination heightens our confidence in the obtained models. Taken together, these data support and provide a framework for the existing biochemical data regarding the tolerance of imperfectly base-paired viral dsRNA by PKR.

Activation of 2' 5'-oligoadenylate synthetase by stem loops at the 5'-end of the West Nile virus genome.

West Nile virus (WNV) has a positive sense RNA genome with conserved structural elements in the 5′ and 3′ -untranslated regions required for polyprotein production. Antiviral immunity to WNV is partially mediated through the production of a cluster of proteins known as the interferon stimulated genes (ISGs). The 2′ 5′-oligoadenylate synthetases (OAS) are key ISGs that help to amplify the innate immune response. Upon interaction with viral double stranded RNA, OAS enzymes become activated and enable the host cell to restrict viral propagation. Studies have linked mutations in the OAS1 gene to increased susceptibility to WNV infection, highlighting the importance of OAS1 enzyme. Here we report that the region at the 5′-end of the WNV genome comprising both the 5′-UTR and initial coding region is capable of OAS1 activation in vitro. This region contains three RNA stem loops (SLI, SLII, and SLIII), whose relative contribution to OAS1 binding affinity and activation were investigated using electrophoretic mobility shift assays and enzyme kinetics experiments. Stem loop I, comprising nucleotides 1-73, is dispensable for maximum OAS1 activation, as a construct containing only SLII and SLIII was capable of enzymatic activation. Mutations to the RNA binding site of OAS1 confirmed the specificity of the interaction. The purity, monodispersity and homogeneity of the 5′-end (SLI/II/III) and OAS1 were evaluated using dynamic light scattering and analytical ultra-centrifugation. Solution conformations of both the 5′-end RNA of WNV and OAS1 were then elucidated using small-angle x-ray scattering. In the context of purified components in vitro, these data demonstrate the recognition of conserved secondary structural elements of the WNV genome by a member of the interferon-mediated innate immune response.

Solution conformation of adenovirus virus associated RNA-I and its interaction with PKR

Adenovirus virus-associated RNA (VAI) provides protection against the host antiviral response in part by inhibiting the interferon-induced double stranded RNA-activated protein kinase (PKR). VAI consists of three base-paired regions; the apical stem responsible for the interaction with double-stranded RNA binding motifs (dsRBMs) of PKR, the central stem required for inhibition, and the terminal stem. The solution conformation of VAI and VAI lacking the terminal stem were determined using SAXS that suggested extended conformations that are in agreement with their secondary structures. Solution conformations of VAI lacking the terminal stem in complex with the dsRBMs of PKR indicated that the apical stem interacts with both dsRNA-binding motifs whereas the central stem does not. Hydrodynamic properties calculated from ab initio models were compared to experimentally determined parameters for model validation. Furthermore, SAXS envelopes were used as a constraint for the in silico modeling of tertiary structure for RNA and RNA-protein complex. Finally, full-length PKR was also studied, but concentration-dependent changes in hydrodynamic parameters prevented ab initio shape determination. Taken together, results provide an improved structural framework that further our understanding of the role VAI plays in evading host innate immune responses.

Cross‐Coupling in the Preparation of Pharmaceutically Relevant Substrates using Palladium Supported on Functionalized Mesoporous Silicas

Although the use of palladium and related transition metal catalysts in organic synthesis is ubiquitous, the presence of residual metals in the organic products is problematic, particularly in the case of pharmaceutical compounds, which are generally prepared without the benefit of chromatographic purification for reasons of scale. Interestingly, high toxicity of Pd in humans has not been widely documented, although with increasing environmental exposure being documented through the emission of Pd (along with other metals) from catalytic converters, an increasing number of researchers are studying uptake in plant and animal species living on roadsides. Despite the lack of human data, as noted by Prasad et al. , Pd is likely able to bind to many biomolecules and so caution in setting daily intake limits is prudent. The exact value of these limits depends on the dosage of a given pharmaceutical, with lower contamination levels desired if doses are higher. In general, levels of contamination at or below 10 ppm are desired. With this in mind, a variety of approaches have been presented in the literature to address Pd contamination, including the use of scavengers, specially designed catalysts or alternate reaction media, such as ionic liquids in which the catalyst is expected to reside, facilitating its removal. An additional approach to the problem of residual metals is to heterogenize the metal catalyst on an inorganic support that can be removed from the reaction by filtration. This approach faces two challenges. Firstly, the metal–ligand bond may not survive the reaction conditions, resulting in leaching and catalyst breakdown; and secondly, complex ligands must be synthesized with two binding sites, one for the metal and one for the support. In our attempt to address these issues, we and others have reported that simple, inexpensive linkers can be used to tether Pd to silica surfaces, and the resulting materials used in industrially significant reactions, such as the Suzuki–Miyaura coupling. Of those linkers examined, the optimum one for Pd appeared to be (EtO)3SiCH2CH2CH2SH (MPTMS), chosen because of the high affinity of sulfur for Pd, and the ready availability of the ligand. Inorganic supports functionalized with this ligand have been employed as scavengers, however, remarkably, the resulting material can be used to catalyze the Suzuki–Miyaura and Mizoroki–Heck reactions after the adsorption of Pd. Although alternative interpretations are possible, through a series of detailed studies we demonstrated that catalysis is likely affected by leached palladium, with the thiol groups acting to recapture the released palladium, minimizing metal contamination in the resulting solutions and products. As a support, we have employed mesoporous silica, as its high surface area ( 1000 m g ) and large pore size ( 70 ) have been shown to facilitate surface functionalization and access of surface groups to species dissolved in solution. In fact, the mesostructure was shown to play a pivotal role in catalytic applications by restricting the growth of palladium nanoparticles on palladium-loaded MPTMS-functionalized surfaces. f, 6a] This is critical, since agglomeration of Pd nanoparticles is a direct cause of catalyst decay. Thus, Pd nanoparticles formed on SBA-15-based supports were constrained by the size of the channels to approximately 6 nm, whereas amorphous silica or other forms of mesoporous silica with more open pore structures give agglomeration of Pd into large particles after use in catalytic reactions. 6a] Although the MPTMS-functionalized material was shown to be an excellent scavenger and the resulting Pd-loaded materials were active catalysts in test reactions, it is critical to challenge the use of this material in other coupling reactions, particularly of pharmaceutically relevant substrates. In particular, we chose substrates that are pharmaceutical intermediates and that contain a variety of heteroatoms. Herein, we report the results of this study, particularly directed towards the Suzuki–Miyaura and Sonogashira reactions. The catalyst was prepared as previously reported, by incorporation of the ligand in the walls of the material during its synthesis. 6] Thus MPTMS was co-condensed with Si(OEt)4 in the presence of a surfactant, as the structure directing agent and source of porosity in the final material. Removal of the surfactant was accomplished by repeated extraction with hot ethanol, such that the surfactant could be re-used if so desired. The sulfur content was determined by elemental analysis, and the material was treated with sufficient [Pd(OAc)2] to make a material with an S:Pd ratio of 2:1, designated SBA-15-SH·Pd. SBA-15-SH·Pd is able to catalyze the Suzuki–Miyaura reaction between bromoacetophenone (1) and the pinacol ester of phenyl boronic acid in high yields under both inert and air atmospheres (Scheme 1). However, turnover frequency (TOF) and turnover number (TON) were higher for reactions run in an inert atmosphere.

Structural elucidation of full-length nidogen and the laminin-nidogen complex in solution.

Nidogen-1 is a key basement membrane protein that is required for many biological activities. It is one of the central elements in organizing basal laminae including those in the skin, muscle, and the nervous system. The self-assembling extracellular matrix that also incorporates fibulins, fibronectin and integrins is clamped together by networks formed between nidogen, perlecan, laminin and collagen IV. To date, the full-length version of nidogen-1 has not been studied in detail in terms of its solution conformation and shape because of its susceptibility to proteolysis. In the current study, we have expressed and purified full-length nidogen-1 and have investigated its solution behavior using size-exclusion chromatography (SEC), dynamic light scattering (DLS) and small angle X-ray scattering (SAXS). The ab initio shape reconstruction of the complex between nidogen-1 and the laminin γ-1 short arm confirms that the interaction is mediated solely by the C-terminal domains: the rest of the domains of both proteins do not participate in complex formation.

Biophysical Characterization of G-Quadruplex Recognition in the PITX1 mRNA by the Specificity Domain of the Helicase RHAU

Nucleic acids rich in guanine are able to fold into unique structures known as G-quadruplexes. G-quadruplexes consist of four tracts of guanylates arranged in parallel or antiparallel strands that are aligned in stacked G-quartet planes. The structure is further stabilized by Hoogsteen hydrogen bonds and monovalent cations centered between the planes. RHAU (RNA helicase associated with AU-rich element) is a member of the ATP-dependent DExH/D family of RNA helicases and can bind and resolve G-quadruplexes. RHAU contains a core helicase domain with an N-terminal extension that enables recognition and full binding affinity to RNA and DNA G-quadruplexes. PITX1, a member of the bicoid class of homeobox proteins, is a transcriptional activator active during development of vertebrates, chiefly in the anterior pituitary gland and several other organs. We have previously demonstrated that RHAU regulates PITX1 levels through interaction with G-quadruplexes at the 3’-end of the PITX1 mRNA. To understand the structural basis of G-quadruplex recognition by RHAU, we characterize a purified minimal PITX1 G-quadruplex using a variety of biophysical techniques including electrophoretic mobility shift assays, UV-VIS spectroscopy, circular dichroism, dynamic light scattering, small angle X-ray scattering and nuclear magnetic resonance spectroscopy. Our biophysical analysis provides evidence that the RNA G-quadruplex, but not its DNA counterpart, can adopt a parallel orientation, and that only the RNA can interact with N-terminal domain of RHAU via the tetrad face of the G-quadruplex. This work extends our insight into how the N-terminal region of RHAU recognizes parallel G-quadruplexes.