The effect of PEO length on the self-assembly of poly(ethylene oxide)−tetrapeptide conjugates prepared by “click” chemistry

Two series of poly(ethylene oxide)-tetrapeptide conjugates have been prepared using a “Click” reaction between an alkyne-modified tetra(phenylalanine) or tetra(valine) and various azide-terminated poly(ethylene oxide) (PEO) oligomers. Three different PEO precursors were used to prepare these conjugates, with number-average molecular weights of 350, 1200, and 1800 Da. Assembly of mPEO-F4-OEt and mPEO-V4-OEt conjugates was achieved by dialysis of a THF solution of the conjugate against water or by direct aqueous rehydration of a thin film. The PEO length has a profound effect on the outcome of the self-assembly, with the F4 conjugates giving rise to nanotubes, fibers, and wormlike micelles, respectively, as the length of the PEO block is increased. For the V4 series, the propensity to form ß-sheets dominates, and hence, the self-assembled structures are reminiscent of those formed by peptides alone, even at the longer PEO lengths. Thus, this systematic study demonstrates that the self-assembly of PEO-peptides depends on both the nature of the peptides and the relative PEO block length.

Living cationic polymerization of isobutene

The polymerization of isobutene initiated by 1-chloro-1-phenylethane has been investigated, and molecular weight studies conducted using size exclusion chromatography. Polymerizations carried out in a 40/60 (v/v) mixture of dichloromethaneIcyclohexane, using titanium (IV) chloride as a catalyst in the presence of pyridine at -30 °C were found to be controlled and living. The number average molecular weights of the polymers increased linearly with monomer conversion, and the molecular weight distributions were between 1.15 and 1.20. Efficiencies of initiation were between 80 and 100%, and evidence was found to suggest that backbiting to the initiator had occurred, resulting in the formation of cyclic oligomers during the early stages of polymerization. The kinetics of polymerization can be explained in terms of active species in. equilibrium with dormant species. The effects of temperature. and dielectric constant on this equilibrium were studied and a model based upon the Fuoss equation was developed. Pyridine was found to behave as proton trap in the system, and when it was used in excess the rate of polymerization was retarded. By assuming that the catalyst and pyridine formed a one to one complex, it was possible to show that the reaction was second order with respect to the catalyst. The synthesis of low molecular weight polyisobutenes was studied. When the concentration of initiator was increased relative to that of the isobutene, such that the theoretical degree of polymerization was 20 or less, the rate of initiation was slow compared to propagation. The efficiency of initiation in these polymerizations was typically between 30 and 40 %. Optimal conditions of temperature. and.catalyst concentration were established, leading to a 60 % efficiency of initiation. A one-pot synthesis of phenol end-capped polyisobutene was attempted by adding phenol at the end of a living polymerization. Evidence to substantiate the existence of capped polymer chains in the resultant product was inconclusive. Block copolymerizations of oxetane and isobutene were conducted using 1-chloro-1phenylethane/TiCl4, but no copolymer or oxetane homopolymer could be isolated.

Atom transfer radical polymerisation - towards the synthesis of a fully-functional photorefractive polymer

Atom transfer radical polymerisation (ATRP) of styrene in xylene solution initiated with 1-phenylethyl bromide and mediated by CuBr/N-propyl-2- pyridinemethanimine catalyst complex was studied. The polymerisation was ill-controlled, yielding polymers with broad molecular weight distributions and values of number average molecular weight considerably higher than the theoretical values calculated from 100% initiator efficiency. The degree of control afforded over the polymerisation was enhanced by use of a more soluble catalyst complex, CuBr/N-octyl-2-pyridinemethanimine. Furthermore, the use of a more polar solvent, diglyme, generated a homogeneous catalyst complex that facilitated the production of polymers having narrow molecular weight distributions (1.10 < PDi < 1.20). The kinetics of the atom transfer radical polymerisation of methyl methacrylate at 90°C in diglyme solution initiated with ethyl-2-bromoisobutyrate and mediated by CuBr/N-octyl-2-pyridinemethanimine was studied and the orders of the reaction were established. The effect on the rate of polymerisation of the ratio of CuBr:N-octyl-2-pyridinemethanimine was also determined. The temperature dependencies of the rate of polymerisation of methyl methacrylate in diglyme solution and xylene solution were studied, and were found to be non-linear and dependent upon the polarity of the solvent. The use of highly polar aprotic solvents, such as N,N-dimethylformamide and dimethylsulphoxide, was found to be detrimental to the degree of control afforded over the polymerisation of methyl methacrylate. This was circumvented by use of a 5-fold excess, over that conventionally used, of catalyst complex. The atom transfer radical polymerisation of (4-nitrophenyl)-[3-[N-[2- (methacryloyloxy)ethyl]carbazolyl]]diazene in dimethyl sulphoxide solution was studied. Although homopolymerisation yielded only oligomers, copolymerisation of this monomer with methyl methacrylate was found to be readily achievable. Keywords: ATRP, Styrene; Methyl methacrylate; Polar solvents; Fully-functional photorefractive polymer. 2

The cationic ring-opening polymerization of cyclic ethers

The kinetics and mechanisms of ring opening polymerization and copolymerizntion of different cyclic ethers were studied using mainly a cationic system of iinitiation. BF30Et2/ethanediol. The cyclic ethers reacted differently showing that ring strain and basicity are the main driving forces in cationic ring opening polymerizaion. In most cases it was found that the degree of polymerization is controlled kinetically via terminations with the counterion and the monomers, and that the contribution of each type of reaction to the overall termination differs markedly. The Gel permeation chromatography studies showed that the molecular weight distribution of the samples of polyoxetanes were bimodal. This was in accordance with previous work establishing that the cyclic tetramer is found in much higher proportions than any of the other cyclic oligomers. However the molecular weight distribution of the copolymers made from oxetane and THF or from oxetane and oxepane were shown to be unimodal. These observations could be explained by a change in the structure of the growing end involved in the cationic polymerization. In addition crown ethers like dibenzo-crown-6 and compounds such as veratrole are believed to stabilise the propagating end and promote the formation of living polymers from oxetane.

The synthesis, characterisation and electrical conductivity of poly(styrene-co-acetylene)

A novel metathesis catalyst for the polymerisation of acetylene has been developed. The polyacetylene produced by this new catalyst has been characterised by infra-red and NMR spectroscopy. The conductivity of the pristine material has been studied as a function of temperature, pressure and frequency. The effect on the conductivity of doping the material has also been investigated. The new metathesis catalyst has been incorporated into an anionic-to-metathesis transformation reaction. This novel reaction has been used to prepare samples of poly(styrene-co-acetylene). The copolymer has been characterised using U.V./Visible, NMR, infra-red spectroscopy and the surface morphology looked at using scanning electron microscopy. GPC was also used to give some idea of the molecular weights of the materials prepared. The conductivity of the copolymer has been studied as a function of temperature, pressure and frequency. The effect of doping on the conductivity the material has also been investigated. The conductivity results obtained from both materials have been used to try and gain an insight into the mechanism of the conduction processes occurring within the materials. An attempt has also been made to synthesise polyacetylene oligomers (polyenes) by modifying the Ziegler/Natta type catalysts commonly used to synthesise polyacetylene. The polyenes were characterised using U.V./Visible and infra-red spectroscopy together with GPC and GCMS.

Chemical synthesis of DNA and RNA containing thio-substituted bases and their post-synthetic modifications

Modified oligonucleotides containing sulphur group have been useful tools for studies of carcinogenesis, protein or nucleic acid structures and functions, protein-nucleic acid interactions, and for antisense modulation of gene expression. One successful example has been the synthesis and study of oligodeoxynucleotides containing 6-thio-2'-deoxyguanine. 6-Thio-2-deoxyguanosine was first discovered as metabolic compound of 6- mercaptopurine (6-MP). Later, it was applied as drug to cure leukaemia. During the research of its toxicity, a method was developed to use the sulphur group as a versatile position for post-synthetic modification. The advantage of application of post-synthetic modification lies in its convenience. Synthesis of oligomers with normal sequences has become routine work in most laboratories. However, design and synthesis of a proper phosphoramidite monomer for a new modified nucleoside are always difficult tasks even for a skilful chemist. Thus an alternative method (post-synthetic method) has been invented to overcome the difficulties. This was achieved by incorporation of versatile nucleotides into oligomers which contain a leaving group, that is sufficiently stable to withstand the conditions of synthesis but can be substituted by nucleophiles after synthesis, to produce, a series of oligomers each containing a different modified base. In the current project, a phosphoramidite monomer with 6-thioguanine has been successfully synthesised and incorporated into RNA. A deprotection procedure, which is specific for RNA was designed for oligomers containing 6-thioguanosine. The results were validated by various methods (UV, HPLC, enzymatic digestion). Pioneer work in utilization of the versatile sulphur group for post-synthetic modification was also tested. Post-synthetic modification was also carried out on DNA with 6- deoxythioguanosine. Electrophilic reagents with various functional groups (alphatic, aromatic, fluorescent) and bi-functional groups have been attached with the oligomers.

A copper-hydrogen peroxide redox system induces dityrosine cross-links and chemokine oligomerisation

The activity of the chemoattractant cytokines, the chemokines, in vivo is enhanced by oligomerisation and aggregation on glycosaminoglycan (GAG), particularly heparan sulphate, side chains of proteoglycans. The chemokine RANTES (CCL5) is a T-lymphocyte and monocyte chemoattractant, which has a minimum tetrameric structure for in vivo activity and a propensity to form higher order oligomers. RANTES is unusual among the chemokines in having five tyrosine residues, an amino acid susceptible to oxidative cross-linking. Using fluorescence emission spectroscopy, Western blot analysis and LCMS-MS, we show that a copper/H2O2 redox system induces the formation of covalent dityrosine cross-links and RANTES oligomerisation with the formation of tetramers, as well as higher order oligomers. Amongst the transition metals tested, namely copper, nickel, mercury, iron and zinc, copper appeared unique in this respect. At high (400 µM) concentrations of H2O2, RANTES monomers, dimers and oligomers are destroyed, but heparan sulphate protects the chemokine from oxidative damage, promoting dityrosine cross-links and multimer formation under oxidative conditions. Low levels of dityrosine cross-links were detected in copper/H2O2-treated IL-8 (CXCL8), which has one tyrosine residue, and none were detected in ENA-78 (CXCL5), which has none. Redox-treated RANTES was fully functional in Boyden chamber assays of T-cell migration and receptor usage on activated T-cells following RANTES oligomerisation was not altered. Our results point to a protective, anti-oxidant, role for heparan sulphate and a previously unrecognised role for copper in chemokine oligomerisation that may offer an explanation for the known anti-inflammatory effect of copper-chelators such as penicillamine and tobramycin.

Introduction of structural diversity into oligonucleotides containing 6-thioguanine via on-column conjugation

A method is described for the introduction of structural diversity into the thiocarbonyl group of 6-thioguanine within support-bound, fully protected oligonucleotides via 'on-column′ conjugation. 2′-Deoxy-6-thioguanosine with a chemically-labile trigger at its 6-thio function was incorporated at defined sites into chemically synthesized oligonucleotides. Following selective removal of the thio-protection group the support-immobilized oligonucleotides were conjugated with various groups on-column, and then deprotected and purified to produce a number of oligomers each containing a different modified base. Since the modification is accomplished on-column without affecting other functional and protecting groups in the oligomers this method is compatible with introducing structural diversity at multiple sites in DNA. © 2003 Elsevier Science Ltd. All rights reserved.

Chapter 1 : nucleobases with designed patterns of hydrogen bonding

The chemistry used in key bond-forming steps to prepare nucleobases with designed patterns of hydrogen bonding is surveyed. Incorporation of the nucleobases into DNA and RNA oligomers is achieved either chemically using building blocks such as nucleoside phosphoramidites or enzymatically using nucleotide triphosphates. By varying the hydrogen bonding pattern within nucleobases, and by incorporating additional substituents, new structures have been designed that "reach over" so that contacts with both strands in targeted duplex DNA can be made in antigene strategies to control gene expression. Various new base-pairing systems have been evaluated that expand the genetic alphabet beyond Watson-Crick base pairs A.T and G.C. For example, benzo-homologated analogs of the natural DNA bases represent a new genetic set of orthogonal, size-expanded derivatives that have been shown to encode amino acids of a protein in a living organism.

Production, purification and characterization of recombinant, full-length human claudin-1

The transmembrane domain proteins of the claudin superfamily are the major structural components of cellular tight junctions. One family member, claudin-1, also associates with tetraspanin CD81 as part of a receptor complex that is essential for hepatitis C virus (HCV) infection of the liver. To understand the molecular basis of claudin-1/CD81 association we previously produced and purified milligram quantities of functional, full-length CD81, which binds a soluble form of HCV E2 glycoprotein (sE2). Here we report the production, purification and characterization of claudin-1. Both yeast membrane-bound and detergent-extracted, purified claudin-1 were antigenic and recognized by specific antibodies. Analytical ultracentrifugation demonstrated that extraction with n-octyl-ß-d-glucopyranoside yielded monodispersed, dimeric pools of claudin-1 while extraction with profoldin-8 or n-decylphosphocholine yielded a dynamic mixture of claudin-1 oligomers. Neither form bound sE2 in line with literature expectations, while further functional analysis was hampered by the finding that incorporation of claudin-1 into proteoliposomes rendered them intractable to study. Dynamic light scattering demonstrated that claudin-1 oligomers associate with CD81 in vitro in a defined molar ratio of 1:2 and that complex formation was enhanced by the presence of cholesteryl hemisuccinate. Attempts to assay the complex biologically were limited by our finding that claudin-1 affects the properties of proteoliposomes. We conclude that recombinant, correctly-folded, full-length claudin-1 can be produced in yeast membranes, that it can be extracted in different oligomeric forms that do not bind sE2 and that a dynamic preparation can form a specific complex with CD81 in vitro in the absence of any other cellular components. These findings pave the way for the structural characterization of claudin-1 alone and in complex with CD81.

Nonaqueous capillary zone electrophoresis of synthetic organic polypeptides

Poly(Nε-trifluoroacetyl-l-lysine) was used as a model solute to investigate the potential of nonaqueous capillary electrophoresis (NACE) for the characterization of synthetic organic polymers. The information obtained by NACE was compared to that derived from size exclusion chromatography (SEC) experiments, and the two techniques were found to be complimentary for polymer characterization. On one hand, NACE permitted (i) the separation of oligomers according to their molar mass and (ii) the separation of the polymers according to the nature of the end groups. On the other hand, SEC experiments were used for the characterization of the molar mass distribution for higher molar masses. Due to the tendency of the solutes (polypeptides) to adsorb onto the fused-silica capillary wall, careful attention was paid to the rinsing procedure of the capillary between runs in order to keep the capillary surface clean. For that purpose, the use of electrophoretic desorption under denaturating conditions was very effective. Optimization of the separation was performed by studying (i) the influence of the proportion of methanol in a methanol/acetonitrile mixture and (ii) the influence of acetic acid concentration in the background electrolyte. Highly resolved separation of the oligomers (up to a degree of polymerization n of ∼50) was obtained by adding trifluoroacetic acid to the electrolyte. Important information concerning the polymer conformations could be obtained from the mobility data. Two different plots relating the effective mobility data to the degree of polymerization were proposed for monitoring the changes in polymer conformations as a function of the number of monomers.

Pseudoperiodic "Living" and/or controlled cationic ring-opening copolymerization of oxetane with tetrahydropyran:microstructure of polymers vs kinetics of chain growth

The "living" and/or controlled cationic ring-opening bulk copolymerization of oxetane (Ox) with tetrahydropyran (THP) (cyclic ether with no homopolymerizability) at 35°C was examined using ethoxymethyl-1 -oxoniacyclohexane hexafluoroantimonate (EMOA) and (BF3 · CH3OH)THP as fast and slow initiator, respectively, yielding living and nonliving polymers with pseudoperiodic sequences (i.e., each pentamethylene oxide fragment inserted into the polymer is flanked by two trimethylene oxide fragments). Good control over number-average molecular weight (Mn up to 150000 g mol-1) with molecular weight distribution (MWD ∼ 1.4-1, 5) broader than predicted by the Poison distribution (MWDs > 1 +1/DPn) was attained using EMOA as initiating system, i.e., C 2H5OCH2Cl with 1.1 equiv of AgSbF6 as a stable catalyst and 1.1 equiv of 2,6-di-tert-butylpyridine used as a non-nucleophilic proton trap. With (BF3 · CH 3OH)THP, a drift of the linear dependence M n(GPC) vs Mn(theory) to lower molecular weight was observed together with the production of cyclic oligomers, ∼3-5% of the Ox consumed in THP against ∼30% in dichloromethane. Structural and kinetics studies highlighted a mechanism of chains growth where the rate of mutual conversion between "strain ACE species" (chain terminated by a tertiary 1-oxoniacyclobutane ion, Al) and "strain-free ACE species" (chain terminated by a tertiary 1-oxoniacyclohexane ion, Tl) depends on the rate at which Ox converts the stable species T1 (kind of "dormant" species) into a living "propagating" center A1 (i.e., k aapp[Ox]). The role of the THP solvent associated with the suspension of irreversible and reversible transfer reactions to polymer, when the polymerization is initiated with EMOA, was predicted by our kinetic considerations. The activation -deactivation pseudoequilibrium coefficient (Qt) was then calculated in a pure theoretical basis. From the measured apparent rate constant of Ox (kOxapp) and THP (kTHPapp = ka(endo)app) consumption, Qt and reactivity ratio (kp/kd, k a(endo)/ka(exo), and ks/ka(endo) were calculated, which then allow the determination of the transition rate constant of elementary step reactions that governs the increase of Mu with conversion. © 2009 American Chemical Society.

Amyloid β 1-42 induces hypometabolism in human stem cell-derived neuron and astrocyte networks

Alzheimer's disease (AD) is the most common form of dementia, affecting more than 35 million people worldwide. Brain hypometabolism is a major feature of AD, appearing decades before cognitive decline and pathologic lesions. To date, the majority of studies on hypometabolism in AD have used transgenic animal models or imaging studies of the human brain. As it is almost impossible to validate these findings using human tissue, alternative models are required. In this study, we show that human stem cell-derived neuron and astrocyte cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose, pyruvate, lactate, and glutamate. In addition, a significant increase in the glycogen content of cells was also observed. These changes were accompanied by changes in NAD+ /NADH, ATP, and glutathione levels, suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. Further research using this model may elucidate the mechanisms associated with Aβ-induced hypometabolism.

Oxidised LDL-lipids alter redox ratio, lipid raft formation and increase amyloid beta production by SHSY-5Y cells

Elevated cholesterol in mid-life has been associated with increased risk of dementia in later life. We have previously shown that low density lipoprotein (LDL) is more oxidised in the plasma of dementia patients although total cholesterol levels remained unchanged [1]. We have investigated the hypothesis that amyloid beta production and neurodegeneration can be driven by oxidised lipids derived from LDL following the loss of blood brain barrier integrity with ageing. Therefore, we have investigated amyloid beta formation in SHSY5Y cells treated with LDL, minimally modified (ox) LDL, and lipids extracted from both forms of LDL. LDL-treated SHSY-5Y cell viability was not significantly decreased with up to 8 μg LDL/2 × 104 cells compared to untreated cells. However, 8 μg oxLDL protein/2 × 104 cells decreased the cell viability significantly by 33.7% (P < 0.05). A more significant decrease in cell viability was observed when treating cells with extracted lipids from 8 μg of LDL (by 32.7%; P < 0.01) and oxLDL (by 41%; P < 0.01). In parallel, the ratio of reduced to oxidised GSH was decreased; GSH concentrations were significantly decreased following treatment with 0.8 μg/ml oxLD-L (7.35 ± 0.58;P < 0.01), 1.6 μg/ml (5.27 ± 0.23; P < 0.001) and 4 μg/ml (5.31 ± 0.31; P < 0.001). This decrease in redox potential was associated with an increase acid sphingomyelinase activity and lipid raft formation which could be inhibited by desipramine; SHSY5Y cells treated with oxLDL, and lipids from LDL and oxLDL for 16 h showed significantly increased acid sphingomyelinase activity (5.32 ± 0.35; P < 0.05, 5.21 ± 0.6; P < 0.05, and 5.58 ± 0.44; P < 0.01, respectively) compared to control cells (2.96 ± 0.34). As amyloid beta production is driven by the activity of beta secretase and its association with lipid rafts, we investigated whether lipids from ox-LDL can influence amyloid beta by SHSY-5Y cells in the presence of oxLDL. Using ELISA and Western blot, we confirmed that secretion of amyloid beta oligomers is increased by SHSY-5Y cells in the presence of oxLDL lipids. These data suggest a mechanism whereby LDL, and more significantly oxLDL lipids, can drive amyloid beta production and cytotoxicity in neuronal cells. [1] Li L, Willets RS, Polidori MC, Stahl W, Nelles G, Sies H, Griffiths HR. Oxidative LDL modification is increased in vascular dementia and is inversely associated with cognitive performance. Free Radic Res. 2010 Mar; 44(3): 241–8.

The development of functional astrocyte-neuron networks derived from stem cells

There is currently great scientific and medical interest in the potential of tissue grown from stem cells. These cells present opportunities for generating model systems for drug screening and toxicological testing which would be expected to be more relevant to human outcomes than animal based tissue preparations. Newly realised astrocytic roles in the brain have fundamental implications within the context of stem cell derived neuronal networks. If the aim of stem cell neuroscience is to generate functional neuronal networks that behave as networks do in the brain, then it becomes clear that we must include and understand all the cellular components that comprise that network, and which are important to support synaptic integrity and cell to cell signalling. We have shown that stem cell derived neurons exhibit spontaneous and coordinated calcium elevations in clusters and in extended processes, indicating local and long distance signalling (1). Tetrodotoxin sensitive network activity could also be evoked by electrical stimulation. Similarly, astrocytes exhibit morphology and functional properties consistent with this glial cell type. Astrocytes also respond to neuronal activity and to exogenously applied neurotransmitters with calcium elevations, and in contrast to neurons, also exhibited spontaneous rhythmic calcium oscillations. Astroctyes also generate propagating calcium waves that are gap junction and purinergic signalling dependent. Our results show that stem cell derived astrocytes exhibit appropriate functionality and that stem cell neuronal networks interact with astrocytic networks in co-culture. Using mixed cultures of stem cell derived neurons and astrocytes, we have also shown both cell types also modulate their glucose uptake, glycogen turnover and lactate production in response to glutamate as well as increased neuronal activity (2). This finding is consistent with their neuron-astrocyte metabolic coupling thus demonstrating a tractable human model, which will facilitate the study of the metabolic coupling between neurons and astrocytes and its relationship with CNS functional issues ranging from plasticity to neurodegeneration. Indeed, cultures treated with oligomers of amyloid beta 1-42 (Aβ1-42) also display a clear hypometabolism, particularly with regard to utilization of substrates such as glucose (3). Both co-cultures of neurons and astrocytes and purified cultures of astrocytes showed a significant decrease in glucose uptake after treatment with 2 and 0.2 μmol/L Aβ at all time points investigated (p <0.01). In addition, a significant increase in the glycogen content of cells was also measured. Mixed neuron and astrocyte co-cultures as well as pure astrocyte cultures showed an initial decrease in glycogen levels at 6 hours compared with control at 0.2 μmol/L and 2 μmol/L P <0.01. These changes were accompanied by changes in NAD+/NADH (P<0.05), ATP (P<0.05), and glutathione levels (P<0.05), suggesting a disruption in the energy-redox axis within these cultures. The high energy demands associated with neuronal functions such as memory formation and protection from oxidative stress put these cells at particular risk from Aβ-induced hypometabolism. As numerous cell types interact in the brain it is important that any in vitro model developed reflects this arrangement. Our findings indicate that stem cell derived neuron and astrocyte networks can communicate, and so have the potential to interact in a tripartite manner as is seen in vivo. This study therefore lays the foundation for further development of stem cell derived neurons and astrocytes into therapeutic cell replacement and human toxicology/disease models. More recently our data provides evidence for a detrimental effect of Aβ on carbohydrate metabolism in both neurons and astrocytes. As a purely in vitro system, human stem cell models can be readily manipulated and maintained in culture for a period of months without the use of animals. In our laboratory cultures can be maintained in culture for up to 12 months months thus providing the opportunity to study the consequences of these changes over extended periods of time relevant to aspects of the disease progression time frame in vivo. In addition, their human origin provides a more realistic in vitro model as well as informing other human in vitro models such as patient-derived iPSC.