Precise redox-sensitive cleavage sites for improved bioactivity of siRNA lipopolyplexes

Lipo-oligomers have been proven as potent siRNA carriers based on stable electrostatic and hydrophobic complex formation and endosomal membrane destabilization. Although high stability of siRNA polyplexes is desirable in the extracellular space and cellular uptake, intracellular disassembly is important for the cytosolic release of siRNA and RNA-induced silencing complex formation. To improve the release, bioreducible sequence-defined lipo-oligomers were synthesized by solid-phase assisted synthesis using the disulfide building block Fmoc-succinoyl-cystamine for precise positioning of a disulfide unit between a lipophilic diacyl (bis-myristyl, bis-stearyl or bis-cholestanyl) domain and an ionizable oligocationic siRNA binding unit. Reducible siRNA polyplexes show higher gene silencing efficacy and lower cytotoxicity than their stable analogs, consistent with glutathione-triggered siRNA release and reduced lytic activity

Photoswitchable precision glycooligomers and their lectin binding

The synthesis of photoswitchable glycooligomers is presented by applying solid-phase polymer synthesis and functional building blocks. The obtained glycoligands are monodisperse and present azobenzene moieties as well as sugar ligands at defined positions within the oligomeric backbone and side chains, respectively. We show that the combination of molecular precision together with the photoswitchable properties of the azobenzene unit allows for the photosensitive control of glycoligand binding to protein receptors. These stimuli-sensitive glycoligands promote the understanding of multivalent binding and will be further developed as novel biosensors

Synthese von sequenz-definierten Glykooligomeren zur Studie von multivalenten Effekten

This thesis presents a novel synthetic approach towards sequence defined, monodisperse glycooligomers employing solid phase polymer synthesis and their use as precision glycomimetics. The synthetic principle is based on the stepwise assembly of functional building blocks on a solid support thereby allowing for the control of the overall chain length as well as the positioning of building blocks within the chain. In a first step, two sets of functional building blocks were introduced: First, a functional building block carrying an alkyne side chain allowing for the conjugation with sugar ligands via CuAAC reaction was designed. Secondly, spacer building blocks were synthesized enabling the incorporation of a desired distance between the sugar moieties and modulate polymer backbone properties such as hydrophobicity and flexibility. The building blocks developed in this thesis were then applied for solid phase synthesis of a) homomultivalent glycooligomers b) heteromultivalent glycooligomers, and c) photoswitchable glycooligomers with tunable backbone properties. Homomultivalent glycooligomers were synthesized by simultaneous conjugation of the same type of sugar ligand after backbone assembly. Heteromultivalent glycooligomers were generated by a sequential coupling-conjugation protocol during backbone assembly. Photoswitchable glycooligomers were synthesized by using the synthetic approach of homomultivalent structures but using a different spacer building block. A library of glycooligomers was synthesized varying specific parameters known to influence multivalent binding: Number and spacing of sugar ligands was varied for ten homomultivalent structures. Five heteromultivalent glycooligomers presenting combinations of mannose together with galactose or glucose ligands displaying heterogeneity of sugar ligands were obtained. A change in backbone properties and spacing of sugar ligands was achieved by four photoswitchable structures incorporating a hydrophobic, stiff, azobenzene-moiety containing spacer in contrast to an ethyleneglycol based flexible, hydrophilic spacer used for the homo- and heteromultivalent glycooligomers. With this novel set of precision glycomimetics, fundamental investigations on multivalent ligand- receptor interactions were performed. Different binding assays were employed to study specific effects of multivalent binding towards sugar-recognizing lectin receptors Con A and PA-IL.Diese Arbeit beschäftigte sich mit einem neuen Ansatz zur Synthese von sequenzdefinierten, monodispersen Glykooligomeren mit Hilfe der Festphasen- Polymersynthese sowie der Untersuchung der erhaltenen Makromoleküle als neuartige Glykomimetika. Das Prinzip basiert auf der schrittweisen Kupplung geeigneter Bausteine an einer Festphase. Durch die Kontrolle der einzelnen Additionsschritte werden so monodisperse Ketten erhalten und durch die Wahl der Bausteine die Positionierung funktioneller Gruppen in der Kette möglich. Im ersten Schritt wurden daher zunächst geeignete funktionelle Bausteine hergestellt: Zum einen wurde ein Baustein mit Alkinseitenkette entwickelt, der die Anbindung von Zuckerliganden mithilfe der CuAAC Reaktion ermöglicht. Zum anderen wurden Spacer Bausteine hergestellt, die sowohl den Abstand der Zuckerliganden entlang der Kette kontrollieren als auch die Eigenschaften des Oligomerrückgrats, etwa Hydrophobizität und Flexibilität, beeinflussen. Diese neu entwickelten Bausteine wurden dann in der Festphasensynthese eingesetzt zur Herstellung von a) homomultivalenten Glykooligomeren, b) heteromultivalenten Glykooligomeren und c) fotoschaltbaren Glykooligomeren mit veränderbaren Eigenschaften des Oligomerrückgrats. Homomultivalente Glykooligomere wurden mithilfe einer simultanen Anbringung des gleichen Zuckerliganden im Anschluss an den Aufbau der Oligomerkette hergestellt. Heteromultivalente Glykooligomere wurden durch einen sequentiellen Kupplungs- Konjugations-Ansatz während der Festphasensynthese erzeugt. Fotoschaltbare Glykooligomere wurden durch den gleichen synthetischen Ansatz wie die homomultivalenten Glykooligomere hergestellt, aber unter Benutzung eines anderen Spacer-Bausteins. Mit Hilfe dieser Syntheseplattform wurde dann eine Bibliothek von Glykooligomeren erzeugt und spezifische strukturelle Parameter variiert, von denen bekannt ist, dass sie multivalente Bindungen beeinflussen: Anzahl und Abstand der Zuckerliganden wurden bei zehn homomultivalenten Strukturen verändert. Fünf verschiedene heteromultivalente Glykooligomere präsentieren Kombinationen aus bindenden (Mannose) und nicht- bzw. schwächer bindenden Liganden (Galaktose- oder Glukoseliganden). Eine Veränderung der strukturellen Eigenschaften des Oligomerrückgrats und Abstand der Zuckerliganden wurde bei vier verschiedenen fotoschaltbaren Strukturen erzeugt durch den Einbau von hydrophoben, steifen AZO-Bausteinen anstelle der zuvor exklusiv verwendeten flexiblen, hydrophilen Ethylenglykol-Spacern. Mit dieser ersten Bibliothek hoch-definierter Glykomimetika wurden dann Studien zur multivalenten Ligand-Rezeptor-Wechselwirkung durchgeführt. Hierzu wurden verschiedene Bindungsassays benutzt, um so spezifische Effekte multivalenter Bindung an Con A und PA IL Lektinen zu erforschen

La Lanterne : journal politique quotidien

04 juin 19251925/06/04 (N17473,A51)

Sequence-Defined Glycopolymer Segments Presenting Mannose: Synthesis and Lectin Binding Affinity

We present for the first time the synthesis of sequence-defined monodisperse glycopolymer segments via solid-phase polymer synthesis. Functional building blocks displaying alkyne moieties and hydrophilic ethylenedioxy units were assembled stepwise on solid phase. The resulting polymer segments were conjugated with mannose sugars via 1,3-dipolar cycloaddition. The obtained mono-, di-, and trivalent mannose structures were then subject to Con A lectin binding. Surface plasmon resonance studies showed a nonlinear increase in binding regarding the number and spacing of sugar ligands. The results of Con A lectin binding assays indicate that the chemical composition of the polymeric scaffold strongly contributes to the binding activities as well as the spacing between the ligands and the number of presented mannose units. Our approach now allows for the synthesis of highly defined glycooligomers and glycopolymers with a diversity of properties to investigate systematically multivalent effects of polymeric ligands

Neutral Gold Complexes with Tridentate SNS Thiosemicarbazide Ligands

Na[AuCl4].2H(2)O reacts with tridentate thiosemicarbazide ligands, H(2)L1, derived from N-[N',N'-dialkylamino(thiocarbonyl)]benzimidoyl chloride and thiosemicarbazides under formation of air-stable, green [AuCl(L1)] complexes. The organic ligands coordinate in a planar SNS coordination mode. Small amounts of gold(I) complexes of the composition [AuCl(L3)] are formed as side-products, where L3 is an S-bonded 5-diethylamino-3-phenyl-1-thiocarbamoyl-1,2,4-triazole. The formation of the triazole L3 can be explained by the oxidation of H(2)L1 to an intermediate thiatriazine L2 by Au3+, followed by a desulfurization reaction with ring contraction. The chloro ligands in the [AuCl(L1)] complexes can readily be replaced by other monoanionic ligands such as SCN- or CN- giving [Au(SCN)(L1)] or [Au(CN)(L1)] complexes. The complexes described in this paper represent the first examples of fully characterized neutral Gold(III) thiosemicarbazone complexes. All the [AuCl(L1)] compounds present a remarkable cell growth inhibition against human MCF-7 breast cancer cells. However, systematic variation of the alkyl groups in the N(4)-position of the thiosemicarbazone building blocks as well as the replacement of the chloride by thiocyanate ligands do not considerably influence the biological activity. On the other hand, the reduction of Au-III to Au-I leads to a considerable decrease of the cytotoxicity.DAADDAADCAPESCAPESCNPqCNPqFAPESPFAPES

Specific Adhesion of Carbohydrate Hydrogel Particles in Competition with Multivalent Inhibitors Evaluated by AFM

Synthetic glycooligomers have emerged as valuable analogues for multivalent glycan structures in nature. These multivalent carbohydrates bind to specific receptors and play a key role in biological processes. In this work, we investigate the specific interaction between mannose ligand presenting soft colloidal probes (SCPs) attached to an atomic force microscope (AFM) cantilever and a Concanavalin A (ConA) receptor surface in the presence of competing glycooligomer ligands. We studied the SCP–ConA adhesion energy via the JKR approach and AFM pull-off experiments in combination with optical microscopy allowing for simultaneous determination of the contact area between SCP and ConA surface. We varied the contact time, loading rate and loading force and measured the resulting mannose/ConA interaction. The average adhesion energy per mannose ligand on the probe was 5 kJ/mol, suggesting that a fraction of mannose ligands presented on the SCP bound to the receptor surface. Adhesion measurements via competitive binding of the SCP in the presence of multivalent glycooligomer ligands did not indicate an influence of their multivalency on the glycooligomer displacement from the ConA surface. The absence of this “multivalency effect” indicates that glycooligomers and ConA do not associate via chelate complexes and shows that steric shielding by the glycooligomers does not slow their displacement upon competitive binding of a ligand presenting surface. These results highlight the high reversibility of carbohydrate–surface interactions, which could be an essential feature of recognition processes on the cell surface

Exploiting Oligo(amido amine) Backbones for the Multivalent Presentation of Coiled-Coil Peptides

The investigation of coiled coil formation for one mono- and two divalent peptide–polymer conjugates is presented. Through the assembly of the full conjugates on solid support, monodisperse sequence-defined conjugates are obtained with defined positions and distances between the peptide side chains along the polymeric backbone. A heteromeric peptide design was chosen, where peptide K is attached to the polymer backbone, and coiled-coil formation is only expected through complexation with the complementary peptide E. Indeed, the monovalent peptide K-polymer conjugate displays rapid coiled-coil formation when mixed with the complementary peptide E sequence. The divalent systems show intramolecular homomeric coiled-coil formation on the polymer backbone despite the peptide design. Interestingly, this intramolecular assembly undergoes a conformational rearrangement by the addition of the complementary peptide E leading to the formation of heteromeric coiled coil–polymer aggregates. The polymer backbone acts as a template bringing the covalently bound peptide strands in close proximity to each other, increasing the local concentration and inducing the otherwise nonfavorable formation of intramolecular helical assemblies

Neutral Gold Complexes with Tridentate <i>SNS</i> Thiosemicarbazide Ligands

Na­[AuCl₄]·2H₂O reacts with tridentate thiosemicarbazide ligands, H₂L1, derived from <i>N</i>-[<i>N</i>′,<i>N</i>′-dialkylamino­(thiocarbonyl)]­benzimidoyl chloride and thiosemicarbazides under formation of air-stable, green [AuCl­(L1)] complexes. The organic ligands coordinate in a planar <i>SNS</i> coordination mode. Small amounts of gold­(I) complexes of the composition [AuCl­(L3)] are formed as side-products, where L3 is an S-bonded 5-diethylamino-3-phenyl-1-thiocarbamoyl-1,2,4-triazole. The formation of the triazole L3 can be explained by the oxidation of H₂L1 to an intermediate thiatriazine L2 by Au³⁺, followed by a desulfurization reaction with ring contraction. The chloro ligands in the [AuCl­(L1)] complexes can readily be replaced by other monoanionic ligands such as SCN^– or CN^– giving [Au­(SCN)­(L1)] or [Au­(CN)­(L1)] complexes. The complexes described in this paper represent the first examples of fully characterized neutral Gold­(III) thiosemicarbazone complexes. All the [AuCl­(L1)] compounds present a remarkable cell growth inhibition against human MCF-7 breast cancer cells. However, systematic variation of the alkyl groups in the N(4)-position of the thiosemicarbazone building blocks as well as the replacement of the chloride by thiocyanate ligands do not considerably influence the biological activity. On the other hand, the reduction of Au^III to Au^I leads to a considerable decrease of the cytotoxicity