Rolf Breinbauer

Suzuki and Heck reactions catalyzed by preformed palladium clusters and palladiumnickel bimetallic clusters

Abstract Soluble palladium clusters and palladium nickel bimetallic clusters stabilized by tetraalkylammonium salts or poly(vinylpyrrolidone) are effective catalysts in Suzuki and Heck reactions involving iodo-, bromo- or activated chloroaromatics, whereas chlorobenzene is not a suitable reaction partner.

Photochemical Surface Patterning by the Thiol-Ene Reaction†

The immobilization of proteins on solid substrates while controlling the size and dimensions of the generated patterns is increasingly relevant in biotechnology. Site-specific immobilization and thus control over the orientation of proteins is particularly important because, as opposed to nonspecific adsorption, it generates homogeneous surface coverage and accessibility to the active site of the protein. Consequently, different types of bioorthogonal reactions have been developed to attach proteins site-specifically to surfaces and to control protein patterning. Herein, we report the photochemical coupling of olefins to thiols to generate a stable thioether bond for the covalent surface patterning of proteins and small molecules. This reaction has been applied previously in solution for carbohydrate and peptide coupling. The thiol-ene photoreaction proceeds at close to visible wavelengths (l = 365–405 nm) and in buffered aqueous solutions. As a result of its specificity for olefins, this photoreaction can be considered to be bioorthogonal, unlike other photochemical methods used previously for protein immobilization. To adopt the thiol-ene reaction for the immobilization of biomolecules, surfaces functionalized with thiols and biomolecules derivatized with olefins were prepared (Figure 1). Polyamidoamine (PAMAM) dendrimers were attached covalently to silicon oxide surfaces. An aminocaproic acid spacer was attached to the dendrimers to create distance from the surface. Cystamine was coupled to the spacer, and subsequent reduction of the disulfide yielded the desired thiolterminated surfaces. A liquid layer of terminal-olefinfunctionalized molecules dissolved in ethylene glycol was spread onto these wafers, which were then covered immediately with a photomask. Subsequent irradiation of the surfaces through the photomask led to patterning with adducts of covalently attached thioethers. To establish the method, we photochemically attached the biotin derivative 1 to a thiol-functionalized surface as described above (Figure 1). After the removal of unreacted biotin molecules, the surface was incubated with Cy5-labeled streptavidin (SAv) to produce a SAv-patterned surface. Fluorescence images of the resulting surface (Figure 1) demonstrated that lateral gradients and patterns with micrometer-sized features (5–100 mm) over areas of centimeters in width (Figure 1A) were readily accessible. Figure 1B,C and the fluorescence-intensity profile in Figure 1D show that the patterns have a well-defined shape and are homogeneous over large distances. When prolonged sonication (4 h) and stringent washing were carried our after irradiation, SAv patterns with similar fluorescence intensities were observed, whereas control experiments with biotin that lacked the olefin linker showed no distinctive SAv patterns. These results indicate that the covalent attachment of biotin to the surface occurs specifically through the proposed thiol-ene reaction and that the nonspecific adsorption of biotin is insignificant. Figure 1E shows that the amount of material immobilized can be modified by changing the irradiation time. The procedure reproducibly requires a short irradiation time of 60 s to yield sufficient surface coverage for fabricating dense SAv patterns. To obtain homogeneous fluorescence signals of the patterns, the starting concentration of the solution that is drop cast onto the surface is also important. When the solution of 1 was diluted (to 1 mm), the Cy5-fluorescence intensity decreased considerably. Further dilution (below 500 mm) resulted eventually in disrupted SAv patterns. The application of more concentrated solutions of 1 (> 20 mm) resulted in the saturation of the fluorescence intensity of the SAv patterns. This behavior corresponds well with the effects observed upon varying the irradiation time. Longer irradi[*] Dr. D. N sse, Dr. H. Schroeder, Dr. R. Wacker, Prof. Dr. C. M. Niemeyer Faculty of Chemistry Biological-Chemical Microstructuring Technical University of Dortmund Otto-Hahn-Strasse 6, 44227 Dortmund (Germany) Fax: (+49)231-755-7082 E-mail: christof.niemeyer@tu-dortmund.de

Azide–Alkyne Coupling: A Powerful Reaction for Bioconjugate Chemistry

In their studies of biological systems, molecular biologists or chemists often encounter the challenge of covalently linking two molecular entities, for example, fusing two proteins together, linking a complex carbohydrate with a peptide, or attaching a small molecular probe (fluorescent dye, radical probe, affinity tag, etc.) onto a biopolymer. As biological systems are rich in structural complexity and diverse in functional reactivity, one has to find chemoselective ligation reactions that allow the coupling of two mutually and uniquely reactive functional groups, in most cases in an aqueous environment under physiological conditions. These uniquely reactive functional groups should be selective for each other and also tolerate a diverse array of other functionality, which renders the use of protecting groups unneccesary and, in the ideal case, allows application of the molecules in the complex environment of a living cell. While several bioconjugation techniques are available for the in vitro preparation of bioconjugates exhibiting a limited set of functional groups, truly chemoselective ligation reactions are rather limited. One reason for this problem is that most ligation reactions rely on the reaction of an electrophile with a nucleophile. As biological systems are rich in diverse electrophilic and nucleophilic sites, only a few functional groups are available that exhibit reactivity orthogonal to other functional groups existing within the system. Recently, two reactions have been introduced that use the azide moiety as a functional group and offer two advantages: 1) the azide moiety is absent in almost all natural exisiting compounds and 2) despite a high intrinsic reactivity, azides allow selective ligation with a very limited set of reaction partners. Bertozzi et al. have redesigned the long-known Staudinger reaction between azides and phosphines so that the highly reactive aza-phosphor-ylide intermediate can undergo a follow-up reaction with cleverly positioned substituents on the phosphine to form a thermodynamically stable amide bond. The usefulness and the generality of this approach has been highlighted by the metabolic engineering of cell surfaces. Sharpless et al. realized that even higher functional group compatibility could be achieved if one could surpass the limitations imposed by electrophile ± nucleophile reactions and turn instead to electrocylic reactions. The Huisgen 1,3dipolar cycloaddition of azides and acetylenes to give 1,2,3-triazoles was identified as an interesting candidate for such an approach. This water-tolerant reaction is thermodynamically favorable by approximately 30 ± 35 kcalmol . The groups of Sharpless and Finn applied the azide ± alkyne coupling in the parallel synthesis of a highly active Azide ±Alkyne Coupling: A Powerful Reaction for Bioconjugate Chemistry Rolf Breinbauer* b] and Maja Kˆhn b]

Size‐Selective Electrochemical Preparation of Surfactant‐Stabilized Pd‐, Ni‐ and Pt/Pd Colloids

A detailed study concerning the size-selective electrochemical preparation of R4N+Br- -stabilized palladium colloids is presented. Such colloids are readily accessible using a simple electrolysis cell in which the sacrificial anode is a commercially available Pd sheet, the surfactant serving as the electrolyte and stabilizer. It is shown that such parameters as solvent polarity, current density, charge flow, distance between electrodes and temperature can be used to control the size of the Pd nanoparticles in the range 1.2-5 nm. Characterization of the Pd colloids has been performed using transmission electron microscopy (TEM), small angle X-ray scattering (SAXS) and X-ray powder diffractometry (XRD) evaluated by Debye-function-analysis (DFA). Possible mechanisms of particle growth are discussed. Experiments directed towards the size-selective electrochemical fabrication of (n-C6H13)4N+Br- -stabilized nickel colloids are likewise described. Finally, a new strategy for preparing bimetallic colloids (e.g., Pt/Pd nanoparticles) electrochemically is presented, based on the use of a preformed colloid (e.g., (n-C8H17)4N+Br- -stabilized Pt particles) and a sacrificial anode (e.g., Pd sheet).

Natural Product Guided Compound Library Development

Natural products are biologically validated starting points for the design of combinatorial libraries, as they have a proven record of biological relevance. This special role of natural products in medicinal chemistry and chemical biology can be interpreted in the light of new insights about the domain architecture of proteins gained by structural biology and bioinformatics. In order to fulfil the specific requirements of the individual binding pocket within a domain family it is necessary to optimise the natural product structure by chemical variation. Solid-phase chemistry is becoming an efficient tool for this optimisation process, and recent advances in this field are highlighted in this review article.

Of Two Make One: The Biosynthesis of Phenazines

Physicians of the 19th century were familiar with the conspicuous occurrence of “blue pus”, which they sometimes observed in patients with severe purulent wounds. Even older are reports of and folk remedies against “blue milk”, a coloration that sometimes developed in fresh milk after some days. Key insight into these phenomena was provided in 1859—exactly 150 years ago—when Mathurin-Joseph Fordos, at a session of the Societ d’ mulation pour les Sciences Pharmaceutiques, reported the isolation of the blue pigment “pyocyanine” (from the Greek words for “pus” and “blue”; pyocyanine is nowadays more commonly spelled as pyocyanin) from purulent wound dressings by chloroform extraction. Pyocyanin (5-N-methyl-1hydroxophenazinium betaine) was the first example of a phenazine natural product, a compound class that has grown to well over 100 members since this first report. Due to the improved understanding of their importance to the phenazinegenerating and also to commensal species, the phenazines have been reviewed several times in recent years. Here, we provide a historical perspective of the more than 100 years of research that led us to our current picture of the interesting biosynthesis of phenazine natural products. The details of Fordos’ pyocyanin isolation method, chloroform extraction followed by acidification and partition into an aqueous phase, were published one year later and are still in use today, but it took until 1882 for the French pharmacist Carle Gessard to show that the blue coloration in pus was due to the presence of a microorganism that he then termed Bacillus pyocyaneus. B. pyocyaneus is nowadays known as Pseudomonas aeruginosa, and the Latin term still reflects this strain’s capacity to secrete colored compounds in the modern name: “aerugo” is the Latin word for verdigris, the blue–green coating that develops on copper after long exposure to air. P. aeruginosa is an important human opportunistic pathogen responsible for a large number of nosocomial infections, and it is also the main course of low life expectancy in patients with cystic fibrosis due to chronic infections of the lungs. The production of pyocyanin is used both for identification in the clinic and as a reporter signal in P. aeruginosa research until today. The occurrence of blue milk, on the other hand, is probably due to an environmental strain of P. fluorescens, and it is not yet clear if this coloration also is a consequence of phenazine production. Gessard’s discovery of P. aeruginosa was resonated in many publications from the medical field, but it required more than 50 years before the correct chemical structure of pyocyanin was established. The chemical composition was first studied by Ledderhose, who derived a formula that was later corrected by McCombie and Scarborough and by Wrede and Strack. Wrede and Strack were also the first to discover a phenazine moiety in a breakdown product of pyocyanin, but their studies were hampered by the fact that they could only obtain a defined molecular weight when working in glacial acetic acid, under which circumstances they obtained a pyocyanin dimer. This dimer was questioned by the results of electrochemical studies by Elema and by Friedheim and Michaelis, before Hillemann finally derived the correct structure in 1938. In retrospect, it seems possible that the conditions employed by Wrede and Strack induced a 1:1 charge-transfer complex of reduced and oxidized pyocyanin, similar to the phenazine derivative chlororaphin, which is also produced by P. aeruginosa (Figure 1). Jensen and Holten later measured the dipole moment of pyocyanin and found that its zwitterionic mesomer is present in considerable amounts. In the course of these studies, it became clear that pyocyanin is a redox-active compound that changes its color depending on pH and oxidation state. This also explained the “chameleon phenomenon”, which describes a temporary color change of P. aeruginosa cultures on solid media after exposure to air by disturbance with a platinum needle. Since the first isolation by Fordos, more than 100 phenazine derivatives modified at all positions of the ring system and colored in all shades of

Development of small-molecule inhibitors targeting adipose triglyceride lipase

Adipose triglyceride lipase (ATGL) is rate-limiting in the mobilization of fatty acids from cellular triglyceride stores. This central role in lipolysis marks ATGL as interesting pharmacological target since deregulated fatty acid metabolism is closely linked to dyslipidemic and metabolic disorders. Here we report on the development and characterization of a small-molecule inhibitor of ATGL. Atglistatin is selective for ATGL and reduces fatty acid mobilization in vitro and in vivo.

PhzA/B catalyzes the formation of the tricycle in phenazine biosynthesis.

Phenazines are redox-active bacterial secondary metabolites that participate in important biological processes such as the generation of toxic reactive oxygen species and the reduction of environmental iron. Their biosynthesis from chorismic acid depends on enzymes encoded by the phz operon, but many details of the pathway remain unclear. It previously was shown that phenazine biosynthesis involves the symmetrical head-to-tail double condensation of two identical amino-cyclohexenone molecules to a tricyclic phenazine precursor. While this key step can proceed spontaneously in vitro, we show here that it is catalyzed by PhzA/B, a small dimeric protein of the Delta(5)-3-ketosteroid isomerase/nuclear transport factor 2 family, and we reason that this catalysis is required in vivo. Crystal structures in complex with analogues of the substrate and product suggest that PhzA/B accelerates double imine formation by orienting two substrate molecules and by neutralizing the negative charge of tetrahedral intermediates through protonation. HPLC-coupled NMR reveals that the condensation product rearranges further, which is probably important to prevent back-hydrolysis, and may also be catalyzed within the active site of PhzA/B. The rearranged tricyclic product subsequently undergoes oxidative decarboxylation in a metal-independent reaction involving molecular oxygen. This conversion does not seem to require enzymatic catalysis, explaining why phenazine-1-carboxylic acid is a major product even in strains that use phenazine-1,6-dicarboxylic acid as a precursor of strain-specific phenazine derivatives.

Preparation of Biomolecule Microstructures and Microarrays by Thiol–ene Photoimmobilization

A mild, fast and flexible method for photoimmobilization of biomolecules based on the light‐initiated thiol–ene reaction has been developed. After investigation and optimization of various surface materials, surface chemistries and reaction parameters, microstructures and microarrays of biotin, oligonucleotides, peptides, and MUC1 tandem repeat glycopeptides were prepared with this photoimmobilization method. Furthermore, MUC1 tandem repeat glycopeptide microarrays were successfully used to probe antibodies in mouse serum obtained from vaccinated mice. Dimensions of biomolecule microstructures were shown to be freely controllable through photolithographic techniques, and features down to 5 μm in size covering an area of up to 75×25 mm were created. Use of a confocal laser microscope with a UV laser as UV‐light source enabled further reduction of biotin feature size opening access to nanostructured biochips.

Activity-Based Probes for Studying the Activity of Flavin-Dependent Oxidases and for the Protein Target Profiling of Monoamine Oxidase Inhibitors**

High profile: new activity-based protein profiling (ABPP) probes have been designed that target exclusively monoamine oxidases A and B within living cells (see picture; FAD=flavin adenine dinucleotide, FMN=flavin monodinucleotide). With these probes it could be shown that the MAO inhibitor deprenyl, which is in clinical use against Parkinsons disease, shows unique protein specificity despite its covalent mechanism of action.