Michael Gottschaldt

Matrix-free UV-laser desorption/ionization (LDI) mass spectrometric imaging at the single-cell level: Distribution of secondary metabolites of Arabidopsis thaliana and Hypericum species

The present paper describes matrix-free laser desorption/ionisation mass spectrometric imaging (LDI-MSI) of highly localized UV-absorbing secondary metabolites in plant tissues at single-cell resolution. The scope and limitations of the method are discussed with regard to plants of the genus Hypericum. Naphthodianthrones such as hypericin and pseudohypericin are traceable in dark glands on Hypericum leaves, placenta, stamens and styli; biflavonoids are also traceable in the pollen of this important phytomedical plant. The highest spatial resolution achieved, 10 microm, was much higher than that achieved by commonly used matrix-assisted laser desorption/ionization (MALDI) imaging protocols. The data from imaging experiments were supported by independent LDI-TOF/MS analysis of cryo-sectioned, laser-microdissected and freshly cut plant material. The results confirmed the suitability of combining laser microdissection (LMD) and LDI-TOF/MS or LDI-MSI to analyse localized plant secondary metabolites. Furthermore, Arabidopsis thaliana was analysed to demonstrate the feasibility of LDI-MSI for other commonly occurring compounds such as flavonoids. The organ-specific distribution of kaempferol, quercetin and isorhamnetin, and their glycosides, was imaged at the cellular level.

Simplified NaCl Based 68Ga Concentration and Labeling Procedure for Rapid Synthesis of 68Ga Radiopharmaceuticals in High Radiochemical Purity

A simple sodium chloride (NaCl) based (68)Ga eluate concentration and labeling method that enables rapid, high-efficiency labeling of DOTA conjugated peptides in high radiochemical purity is described. The method utilizes relatively few reagents and comprises minimal procedural steps. It is particularly well-suited for routine automated synthesis of clinical radiopharmaceuticals. For the (68)Ga generator eluate concentration step, commercially available cation-exchange cartridges and (68)Ga generators were used. The (68)Ga generator eluate was collected by use of a strong cation exchange cartridge. 98% of the total activity of (68)Ga was then eluted from the cation exchange cartridge with 0.5 mL of 5 M NaCl solution containing a small amount of 5.5 M HCl. After buffering with ammonium acetate, the eluate was used directly for radiolabeling of DOTATOC and DOTATATE. The (68)Ga-labeled peptides were obtained in higher radiochemical purity compared to other commonly used procedures, with radiochemical yields greater than 80%. The presence of (68)Ge could not be detected in the final product. The new method obviates the need for organic solvents, which eliminates the required quality control of the final product by gas chromatography, thereby reducing postsynthesis analytical effort significantly. The (68)Ga-labeled products were used directly, with no subsequent purification steps, such as solid-phase extraction. The NaCl method was further evaluated using an automated fluid handling system and it routinely facilitates radiochemical yields in excess of 65% in less than 15 min, with radiochemical purity consistently greater than 99% for the preparation of (68)Ga-DOTATOC.

Prospects of metal complexes peripherally substituted with sugars in biomedicinal applications.

Metal complexes possess unique tunable properties, such as radioactivity, cytotoxicity or photophysical features, enabling them to act as diagnostic tracers or therapeutic agents. In applying them in biological systems, it is often necessary to enhance their solubility and biocompatibility. To achieve such goals, like the targeting of binding domains, transport systems and enzyme activities, the attachment of carbohydrate moieties appears to be suitable. Sugar-substitution in the periphery of metal complexes has therefore become a strongly growing field of research. Outlined herein is a selection of recent examples.

Poly(2-oxazoline) functionalized surfaces: from modification to application.

Poly(2-oxazoline)s (POxs) are a versatile class of biocompatible polymers, which have been investigated as poly(ethylene glycol) (PEG) alternatives. In recent years, POxs have drawn significant attention as coatings for antifouling applications. In this tutorial review different approaches to immobilize POxs on surfaces as well as properties and applications of POx coated surfaces will be presented.

Poly(2-oxazoline) glycopolymers with tunable LCST behavior

A series of thermo-responsive glyco-poly(2-oxazoline)s based on 2-ethyl-2-oxazoline and 2-(dec-9-enyl)-2-oxazoline were prepared. To study the effect of the sugar content on the solution behavior in water, two sets of copolymers with constant monomer-to-initiator ratios of 20 and 50 and varying amounts of the hydrophobic alkene functionalized monomer were synthesized. The glycopolymers were obtained by the photoaddition of 2,3,4,6-tetra-O-acetyl-1-thio-β-D-glycopyranose onto the double bonds followed by deacetylation of the saccharide residues. Turbidimetry measurements of the respective glycopolymers revealed a decreasing cloud point temperature with increasing amount of sugar moieties, proposed to be caused by hydrogen bonding between the sugars and the polymer amide groups, which is enabled by the flexibility of the long decyl spacer. Due to the linear relationship between cloud point temperatures and the sugar content, the cloud points can be easily tailored for specific applications.

New Building Blocks for the Design of Oligonuclear Copper Complexes Based on Amino Carbohydrates

Remarkably high magnetic coupling and O(2)-activation ability analogous to that of catechol oxidase are characteristics of the first structurally defined, low-symmetry oligonuclear copper complexes of tridentate beta-oxoenamine ligands based on amino carbohydrates (the structure of a bis(acetylbutenonylaminoglucosidato)bis(µ-acetato)tricopper(II) complex is shown).

Sugar-Selective Enrichment of a D-Glucose-Substituted Ruthenium Bipyridyl Complex Inside HepG2 Cancer Cells

Ruthenium bipyridyl (bpy) complexes display luminescent properties and a rich photochemistry associated with the visible-light-induced formation of a long-lived MLCT state (t up to 1 ms), which can be efficiently quenched by oxygen under the formation of singlet oxygen (F=0.86). These properties render the complexes interesting candidates for fluorescent sensors and photodynamic-therapy applications. However, the in vivo potential of these complexes depends heavily upon both the molecular (sensor applications) and the cellular (photodynamic therapy applications) recognition of the luminescent probes. In addition, Ru(bpy)3 -type complexes are positively charged ions and therefore possess only limited cellmembrane permeability for intact cells ; this significantly limits their potential applications. It has been shown recently that the latter limitation can be overcome by using more lipophilic derivatives such as Ru(DIP)2dppz 2+ (DIP=4,7-diphenyl-1,10phenanthroline, dppz=dipyridophenazine). Cellular uptake into HeLa cells by nonspecific passive diffusion could be observed by using confocal laser scanning microscopy (CLSM) and flow cytometry. 6] A dinuclear Ru complex was observed to bind to DNA in cellulo. Modification of bipyridyl ruthenium complexes in the 4,4’-position can influence their allocation in cellular matrices as shown for a recently reported 4,4’-di(N,Ndiethyl-amino)-substituted homoleptic compound that selectively binds to the cell membrane. One further example towards the design of biosensors is the modification of bipyridyl ligands with biotin, which acts as a binding site for avidin. Carbohydrate-modified complexes present a highly interesting alternative, as it has been shown that their presence enhances the specificity of the interaction with biological probes. These luminescent sensors for the recognition of lectins were tested in vitro as probes for the influenza virus and show different lectin affinities and amplified luminescence depending on the spacer used. Other advantages of the introduction of these groups include increased biocompatibility, a reduction of toxicity and utilization of the targeting properties of sugar-specific receptors or metabolic pathways. Sugar-containing metal complexes are therefore increasingly examined. Recently, mannosylated bipyridyl based ligands with branched spacers possessing a dendrimeric structure were used to obtain Ru complexes. Under irradiation these complexes were able to produce singlet oxygen and to act as lectin biosensors, in particular if an appropriately attached quencher was displaced by the binding lectin. However, while molecular selectivity can be introduced, cellular selectively has so far not been demonstrated. In this contribution we present the synthesis of new ruthenium polypyridyl complexes with peripherally attached sugar substituents. These complexes show, for the first time, a distinct uptake into cancer cells but still retain their luminescent properties. In all known examples the linkages between saccharides and metal-chelating units was realized by using a biodegradable amide or O-glycosidic bonds with flexible spacers; these compounds focused on lectin binding and did not address cellular uptake. To avoid lectin binding in serum or on cell surfaces, the carbohydrates in the compounds reported in this work were bound close to the bipyridine. For biodistribution studies of the intact glycoconjugates we synthesized S-glycosylated disubstituted bipyridines (L1–L3) because it is known that Sglycosidic bonds resist endogenous hydrolysis catalysed by ubiquitous glycosidases. The symmetric ligands were complexed to Ru by using Ru(DMSO)2Cl2 under reflux in water. This results in the formation of compact “ball-shaped” ruthenium complexes of the general formula Ru(L)3Cl2 (Scheme 1). After purification utilizing a Sephadex LH-20 column, the complexes were obtained as red powders. For comparison, the complex Ru(dmbpy)3Cl2 from 4,4’-dimethyl-2,2’-bipyridine (dmbpy) was synthesized by using the same reaction conditions. [a] Dr. M. Gottschaldt, Prof. U. S. Schubert Laboratory for Organic and Macromolecular Chemistry, Friedrich-Schiller-University Jena Humboldtstrasse 10, 07743 Jena (Germany) Fax: (+49)3641-948202 E-mail : michael.gottschaldt@uni-jena.de [b] Prof. U. S. Schubert Laboratory of Macromolecular Chemistry and Nanoscience, Eindhoven University of Technology Den Dolech 2, 5600MB Eindhoven (The Netherlands) [c] Prof. S. Rau Friedrich Alexander Universit t Erlangen–N rnberg Department for Chemistry and Pharmacy Egerlandstrasse 1, 91058 Erlangen (Germany) [d] Prof. S. Yano Innovative Collaboration Center, Kyoto University Nishikyo-ku, Kyoto-daigaku Katsura, Kyoto 615-8520 (Japan) [e] Prof. J. G. Vos Solar Energy Conversion SRC, School of Chemical Sciences Dublin City University Dublin 9 (Ireland) [f] Dr. T. Kroll, Dr. J. Clement Clinics of Internal Medicine, University Hospital Jena Erlanger Allee 101, 07747 Jena (Germany) [g] Prof. I. Hilger Institute of Diagnostic and Interventional Radiology University Hospital Jena Erlanger Allee 101, 07747 Jena (Germany) E-mail : ingrid.hilger@med.uni-jena.de Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.200900769.

Cationic poly(2-oxazoline) hydrogels for reversible DNA binding

A new 2-oxazoline monomer with a Boc-protected amino group in the side chain (BocOx) was synthesized. Homopolymerization as well as copolymerization with 2-ethyl-2-oxazoline (EtOx) revealed a pseudo first order kinetic. A series of homopolymers was synthesized, deprotected and characterized regarding their structure and thermal properties. The copolymerization with EtOx yielded a series of water soluble polymers with varying amino contents. After deprotection it was shown by the ethidium bromide assay that these polymers were able to form complexes with DNA. Treatment with epichlorohydrin leads to the formation of hydrogels. The swelling properties of the gels were investigated and it could be demonstrated that also the polymeric scaffolds were able to immobilize DNA from aqueous solution. Furthermore, the release of the DNA was accomplished using heparin.

Cell type-specific delivery of short interfering RNAs by dye-functionalised theranostic nanoparticles

Efficient delivery of short interfering RNAs reflects a prerequisite for the development of RNA interference therapeutics. Here, we describe highly specific nanoparticles, based on near infrared fluorescent polymethine dye-derived targeting moieties coupled to biodegradable polymers. The fluorescent dye, even when coupled to a nanoparticle, mimics a ligand for hepatic parenchymal uptake transporters resulting in hepatobiliary clearance of approximately 95% of the dye within 45 min. Body distribution, hepatocyte uptake and excretion into bile of the dye itself, or dye-coupled nanoparticles can be tracked by intravital microscopy or even non-invasively by multispectral optoacoustic tomography. Efficacy of delivery is demonstrated in vivo using 3-hydroxy-3-methyl-glutaryl-CoA reductase siRNA as an active payload resulting in a reduction of plasma cholesterol levels if siRNA was formulated into dye-functionalised nanoparticles. This suggests that organ-selective uptake of a near infrared dye can be efficiently transferred to theranostic nanoparticles allowing novel possibilities for personalised silencing of disease-associated genes.

Linear Poly(ethylene imine)-Based Hydrogels for Effective Binding and Release of DNA

A series of copolymers containing both amine groups of linear poly(ethylene imine) (LPEI) and double bonds of poly(2-(3-butenyl)-2-oxazoline) (PButEnOx) was prepared. To this end, a poly(2-ethyl-2-oxazoline) (PEtOx) precursor was hydrolyzed to the respective LPEI and functionalized in an amidation reaction with butenyl groups resulting in the double bond containing poly(2-(3-butenyl-2-oxazoline)-co-ethylene imine) (P(ButEnOx-co-EI)). Hydrogels were obtained by cross-linking with dithiols under UV-irradiation resulting in networks with different properties in dependence of the content of double bonds. The developed method allows the exact control of the amount of ethylene imine units within the copolymer and, thus, within the resulting hydrogels. The gel structures were characterized by solid state NMR and infrared spectroscopy. In addition the water uptake behavior from the liquid and the gas phase was investigated. It was shown by an ethidium bromide assay (EBA) that the copolymers and the respective hydrogels were able to bind and release DNA. Furthermore, the influence of the ethylene imine content on this interaction was investigated.