Comparison of CLEC-2 and GPVI signaling in platelets: the role of adaptor proteins

GPVI activates platelets through an ITAM pathway by activation of Src and Syk kinases leading to activation of PLCy2. CLEC-2 has been shown to activate platelets using an ITAM-like sequence in its cytoplasmic tail that is also dependent on Src and Syk kinases, but shows a partial rather than an absolute dependence on adapter SLP-76 for activation of PLCy2. The aim of this thesis is to understand some of the key differences in these signalling pathways. GPVI is in complex with FcRwhich contains the ITAM sequence (Yxx(L/I)x6−12Yxx(L/I)). These two tyrosines provide a docking site for the tandem-SH2 domains of Syk. In this thesis I show that CLEC-2 signalling through Syk is mediated by phosphorylation of the CLEC-2 YxxL sequence, receptor dimerisation and cross-linking by the Syk SH2 domains. I also show that the differential requirement for SLP-76 is not mediated by Gads. Both signalling pathways also show partial dependency for LAT. I also show that a novel protein, G6f, is not able to substitute for LAT in this signalling pathway and also exclude the LAT-family proteins PAG, LIME, LAX and NTAL as potential LAT replacements in platelet activation by GPVI. These results extend our understanding of platelet activation by CLEC-2.

Tetraurea derivatives of calix[4]arenes and oligocalix[4]arenes, synthesis and self-assembly = Tetraharnstoff-Derivate von Calix[4 ]arenen und Oligocalix[4]arenen, Synthese und Selbstorganisierung

Wide rim tetraurea calix[4]arenes form hydrogen bonded dimeric capsules in apolar solvents in the presence of a suitable guest, which must be included in the cavity. The monomeric and dimeric form are never observed simultaneously under usual conditions. In general the combination of two different alkyl or aryl tetraurea derivatives results in the mixture of two homodimers and a heterodimer, however, only the heterodimeric species is observed in the 1:1 mixture of aryl and tosyl ureas. The (hetero)dimerization of oligourea calix[4]arenes (units) was used to construct larger structures via self-assembly of multiple calixarenes (building blocks) containing two (or more) covalently connected units. Among these self-assembled structures linear or branched polymers, cyclic oligomers and well-organized dendrimers were envisaged. The synthesis of the building blocks requires the preparation of calix[4]arene units possessing one (or more) functional group at the narrow or wide rim. Finally the oligourea units were covalently connected either directly or via suitable spacers within appropriate building blocks using amide bonds. Self-assembly properties of such building blocks were investigated.

Synthese eines 67-gliedrigen [2]Catenans und seines Konstitutionsisomeren als Bausteine für Polymere

Im Rahmen der Dissertation zum Thema „Synthese eines 67-gliedrigen [2]Catenans und seines Konstitutionsisomeren als Bausteine für Polymere“ ist die Darstellung eines neuen, funktionalisierten [2]Catenans mit 67-gliedrigen Ringen gelungen. Zum Aufbau der Catenanuntereinheiten wurde die Synthese einer langen Kette entwickelt, die zu einem 67-gliedrigen Makrozyklus führte. Die im Catenan eingebauten Ester-Funktionalitäten konnten in die Säure überführt werden, die entweder direkt oder auch nach einer nochmaligen Derivatisierung in einer Polymerisationsreaktion eingesetzt werden könnte. Durch die Darstellung des neuen [2]Catenans wurde die in der Arbeitsgruppe von A. Godt entwickelte Catenan-Synthesestrategie optimiert und gezeigt, dass sie auf ein Molekül mit kleineren Ringgrößen übertragen werden kann. Das Konstitutionsisomere des Catenans, das zyklische Dimer, wurde durch die Änderung der Abfolge einzelner Stufen der Synthesestrategie erhalten. Die Kombination beider Zyklisierungsschritte in einer Reaktion führte in hoher Ausbeute zum zyklischen Dimer. Die neu gefundene Syntheseroute lieferte das zyklische Dimer, ausgehend von der Zyklusvorstufe, in nur drei Stufen und löst die sehr aufwendige Templatsynthese, die zuvor entwickelt wurde, ab. Die Modellexperimente zur Derivatisierung der Catenandisäure mit einem Alkinol, Diol, Diamin oder Aminol wie auch zur Polymerisation mit oxidativer Alkindimerisierung führten nicht zu voll befriedigenden Ergebnissen, jedoch erweist sich die Methode der oxidativen Alkindimerisierung in den Modellversuchen als eine explizit geeignete Variante, ein [2]Catenan zu einem Polymer umzusetzen.

A theoretical study on the formation of iodine oxide aggregates and monohydrates

Biotic and abiotic emissions of molecular iodine and iodocarbons from the sea or the ice surface and the intertidal zone to the coastal/polar marine boundary layer lead to the formation of iodine oxides, which subsequently nucleate forming iodine oxide particles (IOPs). Although the link between coastal iodine emissions and ultrafine aerosol bursts is well established, the details of the nucleation mechanism have not yet been elucidated. In this paper, results of a theoretical study of a range of potentially relevant aggregation reactions of different iodine oxides, as well as complexation with water molecules, are reported. Thermochemical properties of these reactions are obtained from high level ab initio correlated calculations including spin–orbit corrections. The results show that the nucleation path most likely proceeds through dimerisation of I2O4. It is also shown that water can hinder gas-to-particle conversion to some extent, although complexation with key iodine oxides does not remove enough of these to stop IOP formation. A consistent picture of this process emerges from the theoretical study presented here and the findings of a new laboratory study reported in the accompanying paper (Gomez Martin et al., 2013).

Disulfide-linked dimers of human adrenaline synthesizing enzyme PNMT are catalytically active

The crystal structure of human phenylethanolamine N-methyltransferase (hPNMT) reveals a disulfide- linked dimer, despite the presence of reducing agent in the crystallisation conditions. By removing the reducing agent, hPNMT crystals grow more rapidly and at lower protein concentrations. However, it was unclear whether the disulfide bonds are only present in the crystal form or whether these affect enzyme activity. The solution oligomeric state of hPNMT was investigated using biochemical techniques and activity assays. We found that in the absence of reducing agent, hPNMT forms dimers in solution. Furthermore, the solution dimer of hPNMT incorporates disulfide bonds, since this form is sensitive to reducing agent. The C48A and C139A mutants of hPNMT, which are incapable of forming the disulfide bond observed in the crystal structure, have a decreased propensity to form dimer in solution. Those dimers that do form are also sensitive to reducing agent. Further, the C48A/C139A double mutant shows only monomeric behaviour. Both dimeric and monomeric hPNMT, as well as mutants have wildtype enzyme activity. These results show that a variety of disulfides, including those observed in the crystal structure, can form in solution. In addition, disulfide-linked dimers are as active as the monomeric enzyme indicating that the crystal structure of the protein is a valid target for inhibitor design. Crown Copyright (c) 2005 Published by Elsevier B.V. All rights reserved.

Reactions of directly related tellurium and selenium heterocycic compunds with triiron dodecacarbonyl

The reactions of directly related tellurium and selenium heterocyclic compounds with triiron dodecacarbonyl are described. The reaction of 2-telluraphthalide, C8H8OTe with [Fe3(CO)12 gave [Fe{C6H4(CH2)Te}(CO)3]2, (1). An iron atom has inserted into the telluracyclic ring, and it is probable that one co-ordinated CO ligand arises from the initially organic carbonyl group. X-ray analysis of compound (1) showed that the compound has a Fe2Te2 core, which is achieved by dimerisation. The reaction of telluraphthalic anhydride, C8H402Te with [Fe3(CO)12] gave a known, but unexpected, organic phthalide product, C8H602, which was confirmed by X-ray crystallography. Selenaphthalic anhydride,  C8H4O2Se gave intractable products on reaction with [Fe3(CO)12], 2-selenaphthalide, C8H6OSe, on reaction with [Fe3(CO)12] gave a major product [Fe2{C6H4(CH2)Se}(CO)6], (2) and a minor product [Fe3{C6H4(CH2)Se}(CO)8], (3) which is an intermediate in the formation of (2). X-ray analysis of (2) shows that compound (2) is very similar to (1) except that the 18 electron rule is satisfied by co-ordination of a Fe(CO)3 moiety, rather than dimerisation. Compound (3), also studied by X-ray crystallography, differs from (2) mainly in the addition of an Fe(CO)2 moiety. Telluraphtbalic anhydride, C8H402Te, and selenaphthalic anhydride, C8H402Se, are both monoclinic and crystallise in space group P21/n. 2-Selenaphthalide, C8H402Se, is also monoclinic, space group P21/C. The reactions of the following compounds (l,3-dihydrobenzo[c]selenophene, 1,3,7,9-tetrahydrobenzo[1,2c; 4,5c'] ditellurophene, dibenzoselenophene, phenoxselenine, 3, 5-naphtho-1-telluracyclohexane and 3,5-naphtho-1-selenacyclohexane) with [Fe3lCO)12] are reported. It is unfortunate that the above compounds do not react under the conditions employed; this may be due to differing degrees of ring strain. 1,8-bis(bromomethyl)naphthalene, C12H10Br2 is monoclinic and crystallises in space group C2/c. 1,1-diiodo-3,5-naphthotelluracyclohexane, C12H10TeI2 and 3,5-naphtho-l-telluracyclohexane, C12H10Te are monoclinic and crystallise in space group P21/c. 3,5-naphtho-l-selenacyclohexane, C12H10Se and 2,2,8,8-tetraiodo-1,3,7,9-tetrahydrobenzo[1,2c;4,5c']ditellurophene are also monoclinic, space group P21/a. The syntheses of intramolecular stabilised organo-tellurium and selenium compounds are reported, having a general formula of REX (where R = phenylazophenyl; E = Se, Te; X = electronegative group, for example C1, Br or I). The crystal structures of R'TeBr, RTeI, RSeCI, RSeCI/I and RSeI (where R = phenylazophenyl) are reported. The tellurium containing X-ray structures are triclinic and have a space group P-1. The selenium containing X-ray structures are monoclinic with space group P21/n. The inclusion of nitrogen in selenium heterocycles provides access to an entirely new area of organometallic chemistry. The reaction of 2-methylbenzoselenazole with [Fe3(CO)12] gave [Fe2{C6H4(NCH2CH3)Se}(CO)6]. The reactions of 2-(methyltelluro)benzanilide or 2-(methylseleno)benzanilide with [Fe3(CO)12] gave reaction products [Fe2(μTeMe)2(CO)6] and [Fe2 (μ-SeMe)2(CO)6] respectively, which were confmned by X-ray crystallography. The use of Mossbauer spectroscopy on the products obtained from the reactions of heterocyclic compounds with [Fe3(CO)12] can give useful information, for example the number of iron sites and the environments of these iron sites within the products.

The use of organotellurium heterocycles as precursors for novel organometallic compounds

The reactions of group 16 heterocycles with organometallic reagents are described. Thiophenes have been used as models for organic sulfur in coal and their reactivity towards triiron dodecacarbonyl has been investigated. Reaction of unsubstituted thiophene with Fe3(CO)12 results in desulfurisation of the heterocycle, with the organic fragment being recovered in the form of the ferrole, C4H4.Fe2(CO)6. In addition a novel organometallic compound of iron is isolated, the formula of which is shown to be C4H4.Fe3(CO)8. Bezothiophene reacts with Fe3(CO)12 to yield benzothiaferrole, C8H6S.Fe2(CO)6, in which the sulfur is retained in the heterocycle. Dibenzothiophene, a more accurate model for organic sulfur in coal, displays no reactivity towards the iron carbonyl, suggesting that the more condensed systems will desulfurise less readily. Microwave methodology has been successful in accelerating the reactions of thiophenes with Fe3(CO)12. However, reaction of benzothiophene does not proceed to the desulfurisation stage while dibenzothiophene is unreactive even under microwave conditions. Tellurophenes (Te analogues of thiophenes) are shown to mimic the behaviour of thiophenes towards certain organometallic reagents with the advantage that their greater reactivity enables recovery of products in higher yields. Hence, reaction of tellurophene with Fe3(CO)12 again affords the ferrole but with an almost ten-fold increase in yield over thiophene. More significantly, dibenzotellurophene is also detellurated by the iron carbonyl affording the previously inaccessible dibenzoferrole, C12H8.Fe2(CO)6, thereby demonstrating the mechanistic feasibility of dechalcogenation of the more condensed aromatic molecules. The potential of tellurium heterocycles to act as precursors for novel organometallics is also recognised owing to the relatively facile elimination of the heteroatom from these systems. Thus, 2-telluraindane reacts with Fe3(CO)12 to yield a novel organometallic compound of formula C16H16.Fe(CO)3, arising from the unsymmetric dimerisation of two organic fragments.

Strategic development and physicochemical analysis of oral preparations for unstable drugs

Oral liquid formulations are ideal dosage forms for paediatric, geriatric and patient with dysphagia. Dysphagia is prominent among patients suffering from stroke, motor neurone disease, advanced Alzheimer’s and Parkinson’s disease. However oral liquid preparations are particularly difficult to formulate for hydrophobic and unstable drugs. Therefore current methods employed in solving this issue include the use of ‘specials’ or extemporaneous preparations. In order to challenge this, the government has encouraged research into the field of oral liquid formulations, with the EMEA and MHRA publishing list of drugs of interest. The current work investigates strategic formulation development and characterisation of select API’s (captopril, gliclazide, melatonin, L-arginine and lansoprazole), each with unique obstacles to overcome during solubilisation, stabilisation and when developing a palatable dosage from. By preparing a validated calibration protocol for each of the drug candidates, the oral liquid formulations were assessed for stability, according to the ICH guidelines along with thorough physiochemical characterisation. The results showed that pH and polarity of the solvent had the greatest influence on the extent of drug solubilisation, with inclusion of antioxidants and molecular steric hindrance influencing the extent of drug stability. Captopril, a hydrophilic ACE inhibitor (160 mg.mL-1), undergoes dimerisation with another captopril molecule. It was found that with the addition of EDTA and HP-β-CD, the drug molecule was stabilised and prevented from initiating a thiol induced first order free radical oxidation. The cyclodextrin provided further steric hindrance (1:1 molar ratio) resulting in complete reduction of the intensity of sulphur like smell associated with captopril. Palatability is a crucial factor in patient compliance, particularly when developing a dosage form targeted towards paediatrics. L-arginine is extremely bitter in solution (148.7 g.L-1). The addition of tartaric acid into the 100 mg.mL-1 formulation was sufficient to mask the bitterness associated with its guanidium ions. The hydrophobicity of gliclazide (55 mg.L-1) was strategically challenged using a binary system of a co-solvent and surfactant to reduce the polarity of the medium and ultimately increase the solubility of the drug. A second simpler method was developed using pH modification with L-arginine. Melatonin has two major obstacles in formulation: solubility (100 μg.mL-1) and photosensitivity, which were both overcome by lowering the dielectric constant of the medium and by reversibly binding the drug within the cyclodextrin cup (1:1 ratio). The cyclodextrin acts by preventing UV rays from reaching the drug molecule and initiated the degradation pathway. Lansoprazole is an acid labile drug that could only be delivered orally via a delivery vehicle. In oral liquid preparations this involved nanoparticulate vesicles. The extent of drug loading was found to be influenced by the type of polymer, concentration of polymer, and the molecular weight. All of the formulations achieved relatively long shelf-lives with good preservative efficacy.