On the active site in H3PW12O40/SiO2 catalysts for fine chemical synthesis

The surface environment and structural evolution of silica supported phosphotungstic acid (H3PW12O40) catalysts have been investigated as a function of acid loading. H3PW12O40 clusters are deposited intact upon the silica surface, adopting a Stranksi-Krastanov growth mode forming a two-dimensional adlayer which saturates at 45wt% acid. Intimate contact with the silica support perturbs the local chemical environment of three tungstate centres, which become inequivalent with those in the remaining cluster, suggesting an adsorption mode involving three terminal W==O groups. Above the monolayer, H3PW12O40 clusters form three-dimensional crystallites with physico-chemical properties indistinguishable from those in the bulk heteropoly acid. These H3PW12O40/SiO2 materials are efficient for the solventless isomerisation of α-pinene under mild reaction conditions. Activity scales directly with the number of accessible perturbed tungstate sites at the silica interface; these are the active species.

Nanoengineering the active site in heterogeneous catalysis

Here we demonstrate the first application of time-resolved synchrotron X-ray absorption spectroscopy to simultaneously follow dynamic nanoparticle surface restructuring and the evolution of surface and gas-phase products during an organic reaction. Surface palladium oxide, and not metal, is identified as the catalytic species responsible for the selective oxidation (selox) of crotyl alcohol to crotonaldehyde. Elevated reaction temperatures facilitate reversible nanoparticle redox processes, and concomitant catalytic selectivity loss, in response to reaction conditions. These discoveries highlight the importance of stabilizing surface palladium oxide and minimizing catalyst reducibility in order to achieve high selox yields, and will aid the future design of Pd-derived selox catalysts. This discovery has important implications for the design of future liquid and vapor phase selox catalysts, and the thermochemical behavior of Pd nanostructures in general.

On the active site in heterogeneous palladium selox catalysts

The nature of the active site in the Pd-catalysed aerobic selective oxidation of cinnamyl and crotyl alcohols has been directly probed by bulk and surface X-ray techniques. The importance of high metal dispersions and the crucial role of surface palladium oxide have been identified. © The Royal Society of Chemistry 2006.

Effect of preparation conditions on the formation of the active phase of carbon fiber catalytic systems for the low-temperature oxidation of carbon monoxide

We studied the effects of the composition of impregnating solution and heat treatment conditions on the activity of catalytic systems for the low-temperature oxidation of CO obtained by the impregnation of Busofit carbon-fiber cloth with aqueous solutions of palladium, copper, and iron salts. The formation of an active phase in the synthesized catalysts at different stages of their preparation was examined with the use of differential thermal and thermogravimetric analyses, X-ray diffraction analysis, X-ray photoelectron spectroscopy, and elemental spectral analysis. The catalytic system prepared by the impregnation of electrochemically treated Busofit with the solutions of PdCl, FeCl, CuBr, and Cu(NO ) and activated under optimum conditions ensured 100% CO conversion under a respiratory regime at both low (0.03%) and high (0.5%) carbon monoxide contents of air. It was found that the activation of a catalytic system at elevated temperatures (170-180°C) leads to the conversion of Pd(II) into Pd(I), which was predominantly localized in a near-surface layer. The promoting action of copper nitrate consists in the formation of a crystalline phase of the rhombic atacamite CuCl(OH). The catalyst surface is finally formed under the conditions of a catalytic reaction, when a joint Pd(I)-Cu(I) active site is formed. © 2014 Pleiades Publishing, Ltd.

Design and synthesis of potential antimicrobial agents

Chorismate mutase is one of the essential enzymes in the shikimate pathway and is key to the survival of the organism Mycobacterium tuberculosis. The x-ray crystal structure of this enzyme from Mycobacterium tuberculosis was manipulated to prepare an initial set of in silico protein models of the active site. Known inhibitors of the enzyme were docked into the active site using the flexible ligand / flexible active site side chains approach implemented in CAChe Worksystem (Fujitsu Ltd). The resulting complexes were refined by molecular dynamics studies in explicit water using Amber 9. This yielded a further set of protein models that were used for additional rounds of ligand docking. A binding hypothesis was established for the enzyme and this was used to screen a database of commercially available drug-like compounds. From these results new potential ligands were designed that fitted appropriately into the active site and matched the functional groups and binding motifs founds therein. Some of these compounds and close analogues were then synthesized and submitted for biological evaluation. As a separate part of this thesis, analogues of very active anti-tuberculosis pyridylcarboxamidrazone were also prepared. This was carried out by the addition and the deletion of the substitutions from the lead compound thereby preparing heteroaryl carboxamidrazone derivatives and related compounds. All these compounds were initially evaluated for biological activity against various gram positive organisms and then sent to the TAACF (USA) for screening against Mycobacterium tuberculosis. Some of the new compounds proved to be at least as potent as the original lead compound but less toxic.

Purification and crystallization of the hydroxylase component of the methanesulfonate monooxygenase from Methylosulfonomonas methylovora strain M2

The aim of the work described in this paper was two-fold: (1) the purification of the hydroxylase component of the MSAMO to electrophoretic homogeneity using a four-step chromatographic strategy and (2) the crystallization of the two-component hydroxylase of the MSAMO in order to enhance our understanding of the precise three-dimensional structure of the MSAMO, thus yielding an insight into the nature of the active site of this enzyme. Optimised crystallization conditions were identified allowing growth of crystals of the hydroxylase component of the MSAMO within five days. Crystals exhibited a brown colour suggesting the presence on an intact Rieske-iron sulfur centre and diffracted to 7.0 Å when a few degrees of data were evaluated on a beam line X11. © 2006 Elsevier Inc. All rights reserved.

Thermodynamics of binding of regulatory ligands to tissue transglutaminase

The transamidating activity of tissue transglutaminase is regulated by the ligands calcium and GTP, via conformational changes which facilitate or interfere with interaction with the peptidyl-glutamine substrate. We have analysed binding of these ligands by calorimetric and computational approaches. In the case of GTP we have detected a single high affinity site (K (D) approximately 1 muM), with moderate thermal effects suggestive that binding GTP involves replacement of GDP, normally bound to the protein. On line with this possibility no significant binding was observed during titration with GDP and computational studies support this view. Titration with calcium at a high cation molar excess yielded a complex binding isotherm with a number of "apparent binding sites" in large excess over those detectable by equilibrium dialysis (6 sites). This binding pattern is ascribed to occurrence of additional thermal contributions, beyond those of binding, due to the occurrence of conformational changes and to catalysis itself (with protein self-crosslinking). In contrast only one site for binding calcium with high affinity (K (D) approximately 0.15 muM) is observed with samples of enzyme inactivated by alkylation at the active site (to prevent enzyme crosslinkage and thermal effects of catalysis). These results indicate an intrinsic ability of tissue transglutaminase to bind calcium with high affinity and the necessity of careful reassessment of the enzyme regulatory pattern in relation to the concentrations of ligands in living cells, taking also in account effects of ligands on protein subcellular compartimentation.

Characterization of tissue transglutaminase 2 in macrophage function

Programmed cell death, apoptosis, is a highly regulated process that removes damaged or unwanted cells in vivo and has significant immunological implications. Defective clearance of apoptotic cells by macrophages (professional phagocytes) is known to result in chronic inflammatory and autoimmune disease. Tissue transglutaminase 2 (TG2) is a Ca2+-dependent protein cross linking enzyme known to play an important role in a number of cell functions. Up-regulation of TG2 is thought to be involved in monocyte to macrophage differentiation and defective clearance of apoptotic cells by TG2 null mice has been described though in this context the role of TG2 is yet to be fully elucidated. Cell surface-associated TG2 is now recognized as being important in regulating cell adhesion and migration, via its association with cell surface receptors such as syndecan-4, ß1 and ß3 integrin, but its extracellular role in the clearance of apoptotic cells is still not fully explored. Our work aims to characterize the role of TG2 and its partners (e.g. syndecan-4 and ß3 integrin) in macrophage function within the framework of apoptotic cell clearance. Both THP-1 cell-derived macrophage-like cells and primary human macrophages were analyzed for the expression and function of TG2. Macrophage-apoptotic cell interaction studies in the presence of TG2 inhibitors (both cell permeable and impermeable, irreversible and active site directed) resulted in significant inhibition of interaction indicating a possible role for TG2 in apoptotic cell clearance. Macrophage cell surface TG2 and, in particular, its cell surface crosslinking activity was found to be crucial in dictating apoptotic cell clearance. Our further studies demonstrate syndecan-4 association with TG2 and imply possible cooperation of these proteins in apoptotic cell clearance. Knockdown studies of syndecan-4 reveal its importance in apoptotic cell clearance. Our current findings suggest that TG2 has a crucial but yet to be fully defined role in apoptotic cell clearance which seems to involve protein cross linking and interaction with other cell surface receptors.

The synthesis of biodegradable polymers using aluminium alkoxide initiators

Alkyl aluminium alkoxides have been used as initiators for the ring opening polymerisation of ε-caprolactone and δ-valerolactone. The effect of the reaction solvent on the kinetics of the polymerisation of ε-caprolactone has been studied. The rate of polymerisation was found to be faster in solvents of lower polarity and donor nature such as toluene. In general solvents of higher polarity resulted in a decreased rate of polymerisation. However solvents such as THF or DMF with a lone pair of electrons capable of forming a complex with the aluminium centre slowed the polymerisation further. The size of the monomer also proved to be an important factor in the kinetics of the reaction. The six membered ring, δ-valerolactone has less ring strain than the seven membered ring ε-caprolactone and thus the polymerisation of δ-valerolactone is slower than the corresponding polymerisation of ε-caprolactone. Both the alkoxide and alkyl group structures have an effect on the polymerisation. In general bulkier alkoxide groups provide greater steric hindrance around the active site at the beginning of the reaction. This causes an induction or a build up period that is related to the both the steric hindrance and also the electronic effects provided by the alkoxide group. The alkyl group structure has an effect throughout the polymerisation because it remains adjacent to the active centre. The number of alkoxide groups on the aluminium centre is also important, using a dialkoxide as an initiator yields polymers with molecular weights approximately half that of the corresponding reactions using a mono alkoxide. Transesterification reactions have also been found to occur after most of the monomer has been consumed. These transesterification reactions are exaggerated as temperature increases. A method of producing tri-block co-polymers has also been developed. A di-hydroxy functional pre-polymer, PHBV, was reacted with an aluminium alkyl to form a di-alkoxide macroinitiator which was subsequently used as an initiator for the polymerisation of ε-caprolactone to form an ABA type tri-block co-polymer. The molecular weight and other properties were predictable from the initial monomer/initiator ratios.

Neurite outgrowth by the alternatively spliced region of human tenascin-C is mediated by neuronal alpha7beta1 integrin

The region of tenascin-C containing only alternately spliced fibronectin type-III repeat D (fnD) increases neurite outgrowth by itself and also as part of tenascin-C. We previously localized the active site within fnD to an eight amino acid sequence unique to tenascin-C, VFDNFVLK, and showed that the amino acids FD and FV are required for activity. The purpose of this study was to identify the neuronal receptor that interacts with VFDNFVLK and to investigate the hypothesis that FD and FV are important for receptor binding. Function-blocking antibodies against both alpha7 and beta1 integrin subunits were found to abolish VFDNFVLK-mediated process extension from cerebellar granule neurons. VFDNFVLK but not its mutant, VSPNGSLK, induced clustering of neuronal beta1 integrin immunoreactivity. This strongly implicates FD and FV as important structural elements for receptor activation. Moreover, biochemical experiments revealed an association of the alpha7beta1 integrin with tenascin-C peptides containing the VFDNFVLK sequence but not with peptides with alterations in FD and/or FV. These findings are the first to provide evidence that the alpha7beta1 integrin mediates a response to tenascin-C and the first to demonstrate a functional role for the alpha7beta1 integrin receptor in CNS neurons.

Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.

The percutaneous absorption of ionisable compounds

The effects of ionisation on transdermal drug delivery using excised human epidermis (HS) and silastic rubber (SR) as model permeation barriers were investigated in vitro using Franz-type absorption cells. Suspensions and solutions of salicylic acid (SA), the model ionogenic permeant, were used as donors and the variables studied were vehicle pH and trans-membrane pH-gradients. For solutions, the pH effect was related to the level of ionisation of the drug and the degree of saturation of the solution. With suspensions, the observed permeation rate was unaffected by pH. The penetration profiles through HS and SR were similar, although the overall flux through HS was about 70% of that observed through SR. Pretreatment of the membranes with various enhancer regimens, including oleic acid, Azone and N, N-dimethylamides in propylene glycol (PG) and isopropyl myristate (IPM) promoted the penetration of SA. SR was not a suitable model for enhancer pretreatment using IPM as a vehicle as the membrane was significantly disrupted by this vehicle. The results from comparable experiments with and without a trans-membrane pH-gradient did not have a significant effect upon flux or flux enhancement after pretreatment with the above enhancers. A theoretical model for the extraction coefficients of weak acids was derived using the partition coefficients of the ionised and unionised species, pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. This model was shown to account for the variation in overall partition of salicylic acid dependent upon pH and pKa. The distribution of this solute between aqueous and oily phases, with and without added enhancer, was measured as a function of pH. The extraction coefficients determined were consistent with the model and showed that the behaviour of the system can be explained without referral to ion-pair mechanisms. Phosphonoacetate is an effective antiviral agent. However, as it is charged at physiological pH, its permeation across cell membranes is limited. To assess the improvement of the transport properties of this molecule, mono-, di- and tri-ester prodrugs were examined. These were assessed for stability and subsequent breakdown with respect to pH by HPLC. In vitro percutaneous absorption was observed using the triester, but not the ionic mono- or di-esters. The triester absorption could be potentiated using a range of enhancers with oleic acid being the most effective. Cyclodextrins (CD) have a role as absorption enhancers for peptide compounds across nasal epithelium. One potential mode of action is that CDs include these compounds, protect them from enzymic attack and thereby increase their residence time in the nasal epithelium. This study investigated the potential of CDs to protect ester prodrugs from enzymatic breakdown and prevent production of poorly transportable ionic species. Using a range of CD to ester molar ratios (10:1 to 2500:1) a small, but measurable, protection for the model esters (parabens) against esterase attack was observed. Possible mechanisms for this phenomenon are that CDs include the ester, making it unavailable for hydrolysis, the CDs may also affect the esterase in some way preventing access for the ester into the active site.

Structural studies on antifolate drugs

Single crystal X-ray structure determinations are reported for eleven compounds all of which are either biologically active or potentially biologically important. The compounds fall into two distinct classes:- 1. Substituted diaminopyrimidines 2. Substituted aminopyrimidinones The first class of compounds were all selected on the basis of their common diaminopyrimidine nucleus which has been demonstrated to be a vital requirement for antifolate activity. They may all be described as non-classical or small molecule lipophilic dihydrofolate reductase (DHFR) inhibitors, as opposed to the classical folate analogues, having the ability to cross the blood-brain barrier, enter cells via a rapid passive diffusion process, and achieve high intracellular concentrations. Thus they are an excellent choice in the search for crystallography in the solid state, providing geometrical and distance data not available from any other analytical techniques to date; supporting and enhancing data obtained in the lower resolution studies of protein crystallography. The biological importance of these compounds is discussed and an attempt is made to relate/predict their pharmacological activity to observed structural features in the crystalline environment. Special attention is focussed on hydrogen bonding, confirmational flexibility and hydrophobicity of substituents; each of which appear to make contributions to tight binding in the enzyme active site. Chapter 9 describes the use of data from the literature and the solid state modelling of an observed enzyme-substrate interaction in an attempt to define it more accurately in terms of its geometric flexibility. Of the second class, one compound (ABPP) is reported; studies in two different crystal forms. In demonstrating both antiviral and high interferon inducing activity it is possible that this compound could be useful against cancer and also viral infections.

The design of novel antifolates against pneumocystis carinii

The pneumonia caused by Pneumocystis carinii is ultimately responsible for the death of many acquired immunodeficiency syndrome (AIDS) patients. Large doses of trimethoprim and pyrimethamine in combination with a sulphonamide and/or pentamidine suppress the infection but produce serious side-effects and seldom prevent recurrence after treatment withdrawal. However, the partial success of the aforementioned antifolates, and also trimetrexate used alone, does suggest dihydrofolate reductase (DHFR) as a target for the development of antipneumocystis agents. From the DHFR inhibitory activities of 3'-substituted pyrimethamine analogues it was suggested that the 3'-(3'',3''-dimethyltriazen-1''-yl) substituent may be responsible for the greater activity for the P.carinii over the mammalian enzyme. Crystallographic and molecular modeling studies revealed considerable geometrical and electronic differences between the triazene and the chemically related formamidine functions that may account for the differences in DHFR inhibitory profiles. Structural and electronic parameters calculated for a series of 3'-(3'',3''-disubstitutedtriazen-1''-yl) pyrimethamine analogues did not correlate with the DHFR inhibitory activities. However, the in vitro screening against P.carinii DHFR revealed that the 3''-hydroxyethyl-3''-benzyl analogue was the most active and selective. Models of the active sites of human and P.carinii DHFRs were constructed using DHFR sequence and structural homology data which had identified key residues involved in substrate and cofactor binding. Low energy conformations of the 3'',3''-dimethyl and 3''-hydroxyethyl-3''-benzyle analogues, determined from nuclear magnetic resonance studies and theoretical calculations, were docked by superimposing the diaminopyrimidine fragment onto a previously docked pyrimethamine analogue. Enzyme kinetic data supported the 3''-hydroxyethyl-3''-benzyl moiety being located in the NADPH binding groove. The 3''-benzyl substituent was able to locate to within 3 AA of a valine residue in the active site of P.carinii DHFR thereby producing a hydrophobic contact. The equivalent residue in human DHFR is threonine, more hydrophilic and less likely to be involved in such a contact. This difference may account for the greater inhibitory activity this analogue has for P.carinii DHFR and provide a basis for future drug design. From an in vivo model of PCP in immunosuppressed rats it was established that the 3"-hydroxyethyl-3"-benzyl analogue was able to reduce the.P.carinii burden more effectively with increasing doses, without causmg any visible signs of toxicity. However, equivalent doses were not as effective as pentamidine, a current treatment of choice for Pneumocystis carinii pneumonia.

DNA methylation at cytosine position 5

DNA methylation appears to be involved in the regulation of gene expression. Transcriptionally inactive (silenced) genes normally contain a high proportion of 5-methyl-2'-deoxycytosine residues whereas transcriptionally active genes show much reduced levels. There appears good reason to believe that chemical agents capable of methylating 2'-deoxycytosine might affect gene expression and as a result of hypermethylating promoter regions of cytosine-guanine rich oncogenic sequences, cancer related genes may be silenced. This thesis describes the synthesis of a number of `electrophilic' S-methylsulphonium compounds and assesses their ability to act as molecules capable of methylating cytosine at position 5 and also considers their potential as cytotoxic agents. DNA is methylated in vivo by DNA methyltransferase utilising S-adenoxylmethionine as the methyl donor. This thesis addresses the theory that S-adenoxylmethionine may be replaced as the methyl donor for DNA methytransferase by other sulphonium compounds. S-[3H-methyl]methionine sulphonium iodide was synthesised and experiments to assess the ability of this compounds to transfer methyl groups to cytosine in the presence of DNA methyltransferase were unsuccessful. A proline residue adjacent to a cysteine residue has been identified to a highly conserved feature of the active site region of a large number of prokaryotic DNA methyltransferases. The thesis examines the possibility that short peptides containing the Pro-Cys fragment may be able to facilitate the alkylation of cytosine position 5 by sulphonium compounds. Peptides were synthesised up to 9 amino acids in length but none were shown to exhibit significant activity. Molecular modelling techniques, including Chem-X, Quanta, BIPED and protein structure prediction programs were used to assess any structural similarities that may exist between short peptides containing a Pro-Cys fragment and similar sequences present in proteins. A number of similar structural features were observed.