Diiron Complexes with a Bridging Functionalized Allylidene Ligand: Synthesis, Structural Aspects, and Cytotoxicity

Regio- and stereoselective nucleophilic attack of cyanide (from NBu4CN) to cationic diiron vinyliminium compounds [Fe2Cp2(CO)(mu-CO){mu-eta(1):eta(3)-C-(R')C(R '')-CNMe2}]CF3SO3 ([1a-f]CF3SO3) affords the nitrile-aminoallylidene derivatives 2a-f in good to excellent yield. The analogous reaction of [1g]CF3SO3, comprising two different N substituents, gives 4 (63%) as a mixture of two stereoisomers. The new products [lg]CF3SO3, 2a-f, and 4 were characterized by IR and NMR spectroscopy and in a number of cases by IR-spectroelectrochemistry and single-crystal X-ray diffraction. The allylidene complexes are air-stable and robust in aqueous solution; however, in general they undergo oxidation within a biologically relevant range of potentials. DFT calculations were carried out to rationalize the observed stereoselectivity of the synthesis reaction and other structural and thermodynamic aspects. The cytotoxicity of 2a-f was assessed on cisplatin-sensitive and -resistant human ovarian carcinoma (A2780 and A2780cisR) cell lines and human embryonic kidney (HEK-293) cells. Experiments reveal that treatment with the compounds leads to ROS production, with an absence of direct interactions with double-stranded DNA (calf thymus) and bovine serum albumin.

Synthesis, structural characterization and applications of hyperbranched polymers based on L-lysine

Markus Scholl

L-lysine is an essential a-amino acid and a necessary building block for all proteins in the body. As an essential amino acid, L-lysine is not synthesized by humans or animals. Industrially produced L-lysine has therefore a big market as food additive, e.g. in pig food. In the body, L-lysine plays a major role in calcium absorption, building muscle proteins, recovering from surgery or sports injuries, and the body's production of hormones, enzymes, and antibodies.1 From a chemical point of view, L-lysine is an AB2 building block that contains two amino groups and one carboxylic acid group, which makes it an interesting monomer for the synthesis of dendrimers and hyperbranched polymers. L-lysine indeed has been extensively used to synthesize dendrimers2-4 or conjugates of dendrimers and linear polymers.5,6 These dendrimers and linear-dendritic hybrid architectures have attracted interest for various medical applications, including gene delivery,3,6-8 as drug carriers,9 for the development of multiple antigen peptide systems,10,11 and as magnetic resonance imaging agents.12 The drawback of these dendrimers and linear-dendritic hybrid architectures is their time consuming synthesis and their expensive large scale production. This thesis evaluates the feasibility of L-lysine as a monomer to synthesize hyperbranched polymers and investigates the potential of these materials as encapsulation agents and for various biomedical applications. As the hyperbranched polylysines are prepared in a single step and can be produced conveniently at a large scale, they may offer an interesting alternative to their widely used dendritic analogues. Including this introduction, this thesis consists of 8 chapters, which are briefly described below. Chapter 2 gives a general overview of dendritic and hyperbranched polymers build up of amide bonds. Their synthesis and applications in medicine and catalysis as well as their self-assembling and encapsulation properties will be discussed. Chapter 3 reports on the synthesis of hyperbranched polylysines via the thermal polymerization of L-lysine hydrochloride.13 Different catalysts were investigated to improve the reaction kinetics. The structure of the polymers was determined by 1H-NMR spectroscopy, and the degree of branching and the average number of branches were calculated. Chapter 4 will discuss the feasibility of different approaches to control polymer architecture during the thermal hyperbranched polymerization of L-lysine hydrochloride.14 The reactivity of the more reactive ε-NH2 group was modulated by introducing temporary protective groups. The distribution of structural units was analyzed by 1H-NMR spectroscopy and the degree of branching and the average number of branches were calculated. Chapter 5 will describe the dilute solution and solid state structure of hyperbranched polylysines and polyelectrolyte complexes generated from hyperbranched polylysine and various anionic, sodium alkyl sulfate surfactants.15 Structural models for the obtained liquid crystalline phases will be proposed. Chapter 6 will show some preliminary results of the end group modification of hyperbranched polylysine with different hydrophilic and hydrophobic agents. The feasibility of these hydrophobically modified hyperbranched polylysines to encapsulate hydrophilic dyes and transfer them from aqueous to organic media has been investigated. Chapter 7 provides a first insight into the biological properties of hyperbranched polylysine. In this chapter the results from preliminary cytotoxicity and cellular uptake experiments will be discussed. Chapter 8 will give a short summary of the work done in this thesis and an outlook. References Nelson, D. L.; Cox, M. M. "Lehninger, Principles of Biochemistry" 3rd Ed. Worth Publishing: New York, 2000. ISBN 1-57259-153-6. Denkewalter, R. G.; Kolc, J. F.; Lukasavage, W. J. In U.S. Pat. 4289872; Allied Corporation, 1981; Chem. Abstr. 1981, 102, 79324. Ohsaki, M.; Okuda, T.; Wada, A.; Hirayama, T.; Niidome, T.; Aoyagi, H. Bioconjugate Chem. 2002, 13, 510-517. Driffield, M.; Goodall, D. M.; Smith, D. K. Org. Biomol. Chem. 2003, 1, 2612-2620. Lübbert, A.; Nguyen, T. Q.; Sun, F.; Sheiko, S. S.; Klok, H.-A. Macromolecules 2005, 38, 2064-2071. Choi, J. S.; Joo, D. K.; Kim, C. H.; Kim, K.; Park, J. S. J. Am. Chem. Soc. 2000, 122, 474-480. Vlasov, G. P.; Korol'kov, V. I.; Pankova, G. A.; Tarasenko, I. I.; Baranov, A. N.; Glazkov, P. B.; Kiselev, A. V.; Ostapenko, O. V.; Lesina, E. A.; Baranov, V. S. Russ. J. Bioorg. Chem. 2004, 30, 12-20. Choi, J. S.; Lee, E. J.; Choi, Y. H.; Jeong, Y. J.; Park, J. S. Bioconjugate Chem. 1999, 10, 62-65. Sakthivel, T.; Toth, I.; Florence, A. T. Pharm. Res. 1998, 15, 776-782. Tam, J. P. Proc. Natl. Acad. Sci. U. S. A. 1988, 85, 5409-5413. Pessi, A.; Bianchi, E.; Bonelli, F.; Chiappinelli, L. J. Chem. Soc., Chem. Commun. 1990, 8-9. Nicolle, G. M.; Tóth, E.; Schmitt-Willich, H.; Radüchel, B.; Merbach, A. E. Chem. Eur. J. 2002, 8, 1040-1048. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. J. Polym. Sci. Part A Polym. Chem. 2007, 45, 5494-5508. Scholl, M.; Nguyen T. Q.; Bruchmann, B.; Klok, H.-A. Macromolecules 2007, 40, 5726-5734. Canilho, N.; Scholl, M.; Mezzenga, R.; Klok, H.-A. Macromolecules 2007, 40, 8374-8383.

EPFL2007

Solution behavior of peptide and peptide-hybrid materials

Harald Nuhn

Peptide based and peptide hybrid materials represent two interesting and challenging classes of biomaterials. Both classes consist of and contain, respectively, one or more polypeptide blocks, endowing the resulting polymers with enhanced self-assembly properties, biocompatibility or novel characteristics. Peptide based materials, solely comprised of amino acids, benefit from their advanced self-assembly properties. In the case of peptide hybrid materials, being conjugates of peptides and synthetic polymers, either the polypeptide or the synthetic polymer block can drive the self-assembly process. Furthermore, the latter class benefits from combining the advantages of peptides and synthetic polymers, thus overcoming limitations related to the use of the individual components. The application of these materials is still challenging because it is not possible to predict their final self-assembly behavior based on their design. In order to facilitate a target-oriented design of novel materials for application such as drug delivery, tissue engineering, or self-healing materials, a detailed understanding of the complex interactions within and between these molecules is essential. The work presented in this thesis addresses these fundamental questions. Over the last decades, a plethora of peptide and peptide hybrid materials has been reported. Chapter 1 gives an overview of selected articles describing different synthesis routes for peptide as well as peptide hybrid materials, showing examples of molecules and their self-assembly into superstructures, and highlighting recent applications. Chapter 2 describes the synthesis and characterization of different peptide hybrid diblock oligomers composed of two classes of peptide sequences covalently attached to a poly(ethylene glycol) (PEG) block. The first class of peptide sequences contains the intrinsic propensity to form coiled coils, whereas the second class is composed of "switch" peptides and is able to adopt both amphiphilic α-helical and β-strand conformations. Upon dissolution, the first class yielded superstructures comprised of dimeric and tetrameric coiled coil aggregates, whereas the second class formed fibrillar assemblies. In both cases, the self-assembly properties were driven by the peptide block, and the final superstructure consisted of a peptidic core wrapped by the synthetic polymer block. Chapter 3 presents a detailed study on the aqueous solution self-assembly of two poly(styrene)n-b-poly(L-lysine)m peptide hybrid oligomers. The aim was to clarify whether the observed rod-like micelles, obtained by direct dissolution of the diblock oligomers in aqueous solution, reflect intrinsic self-assembly properties of the hybrid diblock oligomers or whether they present structures that are energetically trapped due to the glassy nature of the polystyrene block. To address this question, the influence of a variety of sample preparation techniques, including the use of organic solvents, dialysis from an organic solvent and several thermal treatments, was investigated. All treatments resulted in solution aggregates that differed in size and shape from samples prepared by direct dissolution in aqueous solution. Thus, it seems likely that direct dissolution of polystyrene-b-poly(L-lysine) diblock oligomers in aqueous solution results in kinetically trapped, rod-like aggregates, which can be transformed into non-spherical micelles by the appropriate treatment. Chapter 4 contains a comprehensive study on peptide-based materials, investigating four different series of short elastin-like peptides (ELPs) based on the GVGVP motif. The objective was to test whether and how defined changes in the primary structure of short ELPs affect the secondary structure and the lower critical solution temperature (LCST) behavior. Therefore, the number of repeats was altered, and within one peptide of constant length, all valines were successively substituted by the more hydrophobic amino acids isoleucine, leucine and phenylalanine, respectively. In parallel, a theoretical approach based on the partition coefficient log P was used to predict the shift of the LCST as function of chain length and composition. For short ELPs, the LCST is strongly affected by changes in the primary structure. This has also been predicted by calculating log P. An increase in the hydrophobicity resulted in a shift of the LCST towards lower temperatures. Furthermore, it was shown for the first time that the sensitivity of short ELPs towards external factors, such as salt concentration, is determined by the intrinsic propensity of the incorporated amino acids to form either α-helices or β-strands. In summary, the obtained results on peptide based and peptide-hybrid materials provides deeper insights into their self-assembly characteristics and highlights the potential of these materials to be used for technical and medical applications.

EPFL2008

Biologically active secondary metabolites from cyanobacteria

Cyril Portmann

For millennia, nature was the only source of medicine for mankind. Even today natural products represent a large proportion of the drugs in use. In some areas, like cancer or infectious diseases, about 50 % of the compounds used are of natural origin. Cyanobacteria, also known as blue green algae are prokaryotic photoautotrophs. They have been on earth for ~3.5 billion years and were able to adapt to almost every conceivable environment. They have evolved to produce a large diversity of secondary metabolites including alkaloids, terpenes, polyketides and peptides. These compounds display a large array of biological activities and are therefore an interesting source for new drugs or biological tools. However they can also represent a threat to humans because of their potent toxic activities. In this thesis we report the isolation, characterization and the biological evaluation of different cyanobacterial secondary metabolites. The first compounds are the heterocyclic ribosomal peptides aerucyclamides A-D isolated from Microcystis aeruguinosa PCC 7806. Their structure was established by NMR spectroscopy and chemical transformation and degradation. We also proposed a structure revision for the microcyclamide 7806A isolated from the same strain. Antiplasmodial evaluation established submicromolar IC50 values for aerucyclamide B against Plasmodium falciparum; low micromolar values for aerucyclamide C were found against Trypanosoma brucei rhodesiense. The compounds were selective for the parasites over a cell line of L6 rat myoblasts and are thus being considered for further study as antimalarial agents. The ecological role of these compounds was also investigated, aerucyclamides A-C were found to be toxic to the freshwater crustacean Thamnocephalus platyurus, exhibiting LC50 values in the range of 30 µM. The second compound, cyanopeptolin 1020 is an aminohydroxypiperidone (Ahp)-containing depsipeptide, which was isolated from Microcystis aeruguinosa UV-006. Its structure was established by NMR and chemical degradation. This compound is a very potent picomolar trypsin inhibitior (IC50 = 670 pM) and also an inhibitor of human kallikrein (4.5 nM) and factor XIa (3.9 nM). Cyanopeptolin 1020 was found to be toxic against the freshwater crustacean Thamnocephalus platyrus (LC50 = 8.8 µM), which is in the same range as some of the well-known microcystins. These data support the hypothesis that cyanopeptolins can be considered as a second class of toxins in Microcystis and may suggest a re-evaluation of drinking water guidelines solely focussing on microcystins at present. In the third part we describe the new microcystin variants [D-Asp3,(E)-Dhb7]-MC-HtyY, [D-Asp3,(E)-Dhb7]-MC-HtyHty isolated from from Planktothrix rubescens strain No80 and MC-RY, [MeAsp3]-MC-RY and [NMeSer7]-MC-YR isolated from a fresh water lake in Uganda. NMR spectroscopy or MS/MS experiments were used to characterize the structure of these potent protein phosphatase inhibitors. The next chapter describes the biosynthesis of nostocarboline, a chlorinated and N-methylated carbolinium alkaloid produced by Nostoc 78-12A. This compound displays potent and selective inhibition of photoautotrophic organisms. New derivatives of nostocarboline incorporating Br, F or methyl substituents have been obtained through precursor-directed biosynthesis. The mode of action of nostocarboline was investigated using chlorophyll-a fluorescence imaging of and it was demonstrated that a decrease in photosynthesis precedes cell death, thus establishing the phytotoxic properties of this alkaloid. In chapters 6 and 7 we describe the evaluation of 27 crude cyanobacterial extracts for anticancer and antiplasmodial activities respectively. For anticancer activity we used the Telospot method to find inhibitors of telomerase, an attractive target for new chemotherapeutic agents. All the extracts displayed telomerase inhibitor activity. By bioassay guided fractionation we were able to identify four sulfoquinovosyl diacylglycerols from Microcystis aeruguinosa PCC 7806 that inhibit telomerase in the micromolar range (IC50 16 µM). For antiplasmodial activity, extracts of Scytonema cf. crustaceum EAWAG 179a, Tolypothrix distorata var. symplocoides EAWAG 224a, Phormidium foveolarum EAWAG 132, Scytonema sp. CCALA 177 and Anabaena flos-aquae EAWAG 116 displayed promising activity against Plasmodium falciparum. In the last chapter we used the cyanobacterium Lyngbya sp. as a model organism for the evaluation of modified surfaces for inhibition of aquatic biofouling. We demonstrated that a PLL-g-(DHPAA; PEG) copolymer layer on a titanium oxide-coated microscopy glass slide successfully prevent attachment of this aquatic phototroph in culture for up to 100 days.

EPFL2010