Holm Frauendorf

Template control over dimerization and guest selectivity of interpenetrated coordination cages.

We have previously shown that the self-assembly of dibenzosuberone-based bis-monodentate pyridyl ligands L(1) with Pd(II) cations leads to the quantitative formation of interpenetrated coordination cages [BF4@Pd4L(1)8]. The BF4(-) anion inside the central cavity serves as a template, causing the outer two pockets to show a tremendous affinity for allosteric binding of two small chloride anions. Here we show that derivatization of the ligand backbone with a bulky aryl substituent allows us to control the dimerization and hence the guest-binding ability of the cage by the choice of the templating anion. Steric constraints imposed by L(2) prevent the large BF4(-) anion from serving as a template for the formation of interpenetrated double cages. Instead, a single isomer of the monomeric cage [Pd2L(2)4] is formed. Addition of the small anionic template Cl(-) permits dimerization, yielding the interpenetrated double cage [Cl@Pd4L(2)8], whose enlarged outer pockets show a preference for the binding of large anions such as ReO4(-).

Assembly and Stepwise Oxidation of Interpenetrated Coordination Cages Based on Phenothiazine

A breath of fresh air is sufficient for the eightfold S-monooxygenation of an interpenetrated double cage based on eight phenothiazine ligands and four square-planar-coordinated Pd(II) cations. Besides these two cages, which were both characterized by X-ray crystallography, an eightfold S-dioxygenated double-cage was obtained under harsher oxidation conditions.

Towards click bioconjugations on cube-octameric silsesquioxane scaffolds

Cube-octameric silsesquioxane (POSS) based conjugation scaffolds for copper catalysed azide-alkyne [3+2] cycloaddition are reported. The synthetic route to octaazido and octaalkyno functionalised POSS templates without cage rearrangements is described. A set of click couplings is conducted including the first effective conjugation with a fully unprotected functional peptide towards a POSS assembled peptide octamer.

Head-to-Tail Cyclized Cystine-Knot Peptides by a Combined Recombinant and Chemical Route of Synthesis

Cyclic peptides form an important class of naturally occurring or synthetic compounds with a large variety of biological activities as, for example, hormones, ion carriers, cancerostatics, antibiotics, antimycotics, or toxins. Biological studies with cyclopeptides have often indicate increased metabolic stability, improved receptor selectivity, and improved activity profiles in comparison with their linear counterparts. Among the group of natural circular peptides and proteins isolated in the last few years from microorganisms, plants, and even from humans, cyclotides provide an especially interesting topology. This family of circular plant proteins displays a head-totail cyclized peptide backbone together with a cystine knot (CK) motif based on disulfide bonds formed by six conserved Cys residues (Figure 1). Two disulfide bonds and their connecting backbone segments form a ring that is penetrated by the third disulfide bond to give a pseudo-knot structure that is ACHTUNGTRENNUNGinvariably associated with the nearby b sheet structure. The cystine knot in combination with the cyclic backbone appears to be a highly efficient motif for structure stabilization, resulting in exceptional conformational rigidity, together with stability against denaturing conditions, as well as against proteolytic degradation. CK-containing peptides are found in almost 20 different protein families with activities such as ion channel blocking (conotoxins and spider toxins), protease inhibition (squash inhibitors), and antiinsecticidal activity (plant cyclotides). Head-to-tail macrocyclic cystine knot peptides have been isolated from plants in the Rubiaceae, Violaceae, and Cucurbitaceae families. Several members of these family have been introduced as versatile scaffolds in drug design and biomolecular engineering. Because of their sizes, in the range of 30–40 amino acids, ACHTUNGTRENNUNGcyclotides are amenable both to recombinant production through bacterial expression and to chemical synthesis. In both routes, two steps of post-synthetic processing—oxidation of six cysteines to form three disulfide bonds and head-to-tail cyclization—are required to obtain the final cyclic product. Although the processes by which cyclotide backbone cyclization occurs naturally are largely unknown, two major strategies have been applied to generate synthetic macrocyclic CK peptides. The first approach relies on recombinant synthesis and makes use of modified protein splicing elements known as inteins to form a C-terminal thioester that reacts with the N terminus to result in macrocyclization. The second strategy is based on a solid-phase synthesis of the target peptide, followed by oxidation and cyclization. Fully deprotected peptides have successfully been “zipped” into macrocycles, followed by oxidation and cystine knot formation. Here we present a strategy for the backbone cyclization of already folded miniproteins based on the formation of a stable hydrazone. This method takes advantage of the combination of cheap and high-yielding recombinant production of linear peptide precursors that are already folded and oxidized. Chemical synthesis efficiently provides the artificial linkage of the termini, not interfering with the fold of the knotted motif stabilized by disulfide bonds. In a comparison of linear and cyclized derivatives, an increased efficiency in tryptase inhibition is reported for a representative iminocyclotide; this also indicates Figure 1. Structure of the cystine knot peptide McoEeTI. b Strand secondary structure elements are indicated as arrows. The disulfide bonds forming the cystine knot architecture are indicated as sticks; cysteine residues are numbered from I to VI beginning from the N terminus. The macrocycleforming loop was tentatively added and is shown as a dashed line.

Determination of the Biological Activity and Structure Activity Relationships of Drugs Based on the Highly Cytotoxic Duocarmycins and CC-1065

The natural antibiotics CC‑1065 and the duocarmycins are highly cytotoxic compounds which however are not suitable for cancer therapy due to their general toxicity. We have developed glycosidic prodrugs of seco-analogues of these antibiotics for a selective cancer therapy using conjugates of glycohydrolases and tumour-selective monoclonal antibodies for the liberation of the drugs from the prodrugs predominantly at the tumour site. For the determination of structure activity relationships of the different seco-drugs, experiments addressing their interaction with synthetic DNA were performed. Using electrospray mass spectrometry and high performance liquid chromatography, the experiments revealed a correlation of the stability of these drugs with their cytotoxicity in cell culture investigations. Furthermore, it was shown that the drugs bind to AT-rich regions of double-stranded DNA and the more cytotoxic drugs induce DNA fragmentation at room temperature in several of the selected DNA double-strands. Finally, an explanation for the very high cytotoxicity of CC-1065, the duocarmycins and analogous drugs is given.

Locked by Design: A Conformationally Constrained Transglutaminase Tag Enables Efficient Site-Specific Conjugation.

Based on the crystal structure of a natural protein substrate for microbial transglutaminase, an enzyme that catalyzes protein crosslinking, a recognition motif for site-specific conjugation was rationally designed. Conformationally locked by an intramolecular disulfide bond, this structural mimic of a native conjugation site ensured efficient conjugation of a reporter cargo to the therapeutic monoclonal antibody cetuximab without erosion of its binding properties.

Genetically encoding lysine modifications on histone H4.

Post-translational modifications of proteins are important modulators of protein function. In order to identify the specific consequences of individual modifications, general methods are required for homogeneous production of modified proteins. The direct installation of modified amino acids by genetic code expansion facilitates the production of such proteins independent of the knowledge and availability of the enzymes naturally responsible for the modification. The production of recombinant histone H4 with genetically encoded modifications has proven notoriously difficult in the past. Here, we present a general strategy to produce histone H4 with acetylation, propionylation, butyrylation, and crotonylation on lysine residues. We produce homogeneous histone H4 containing up to four simultaneous acetylations to analyze the impact of the modifications on chromatin array compaction. Furthermore, we explore the ability of antibodies to discriminate between alternative lysine acylations by incorporating these modifications in recombinant histone H4.

A Practical One-Pot Synthesis of Positron Emission Tomography (PET) Tracers via Nickel-Mediated Radiofluorination.

Recently a novel method for the preparation of 18F-labeled arenes via oxidative [18F]fluorination of easily accessible and sufficiently stable nickel complexes with [18F]fluoride under exceptionally mild reaction conditions was published. The suitability of this procedure for the routine preparation of clinically relevant positron emission tomography (PET) tracers, 6-[18F]fluorodopamine (6-[18F]FDA), 6-[18F]fluoro-l-DOPA (6-[18F]FDOPA) and 6-[18F]fluoro-m-tyrosine (6-[18F]FMT), was evaluated. The originally published base-free method was inoperative. However, a “low base” protocol afforded protected radiolabeled intermediates in radiochemical conversions (RCCs) of 5–18 %. The subsequent deprotection step proceeded almost quantitatively (>95 %). The simple one-pot two-step procedure allowed the preparation of clinical doses of 6-[18F]FDA and 6-[18F]FDOPA within 50 min (12 and 7 % radiochemical yield, respectively). In an unilateral rat model of Parkinsons disease, 6-[18F]FDOPA with high specific activity (175 GBq μmol−1) prepared using the described nickel-mediated radiofluorination was compared to 6-[18F]FDOPA with low specific activity (30 MBq μmol−1) produced via conventional electrophilic radiofluorination. Unexpectedly both tracer variants displayed very similar in vivo properties with respect to signal-to-noise ratio and brain distribution, and consequently, the quality of the obtained PET images was almost identical.

Synthesis of 18F-labelled β-lactams by using the Kinugasa reaction

Owing to their broad spectrum of biological activities and low toxicity, β-lactams are attractive lead structures for the design of novel molecular probes. However, the synthesis of positron emission tomography (PET)-isotope-labelled β-lactams has not yet been reported. Herein, we describe the simple preparation of radiofluorinated β-lactams by using the fast Kinugasa reaction between (18)F-labelled nitrone [(18)F]-1 and alkynes of different reactivity. Additionally, (18)F-labelled fused β-lactams were obtained through the reaction of a cyclic nitrone 7 with radiofluorinated alkynes [(18)F]-6 a,b. Radiochemical yields of the Kinugasa reaction products could be significantly increased by the use of different Cu(I) ligands, which additionally allowed a reduction in the amount of precursor and/or reaction time. Model radiofluorinated β-lactam-peptide and protein conjugates ([(18)F]-10 and (18)F-labelled BSA conjugate) were efficiently obtained in high yield under mild conditions (aq. MeCN, ambient temperature) within a short reaction time, demonstrating the suitability of the developed method for radiolabelling of sensitive molecules such as biopolymers.

Efficient Oxidative Degradation of Azo Dyes by a Water‐Soluble Manganese Porphyrin Catalyst

Dyes in wastewater severely affect the nature and quality of water by inhibiting the sunlight penetration into the stream, which thereby reduces the photosynthesis reaction. This poses a serious environmental threat in many developing countries. Recently, new methods for the treatment of colored dye effluent streams have attracted much attention. The [MnIII(tmpyp)]/H2O2 system (tmpyp=meso‐tetrakis(1‐methylpyridinium‐4‐yl)porphyrinato) was now found to degrade various azo dyes with remarkably high efficiency under ambient conditions in aqueous solution at certain pH values. Main products of the catalytic degradation of the dye amaranth by [MnIII(tmpyp)]/H2O2 were analyzed. The reaction mechanism was studied in more detail by using rapid‐scan stopped‐flow spectrophotometry as a function of pH, [catalyst], [H2O2], [dye], and [surfactants]. Spectral analyses and kinetic data suggested rapid formation of an intermediate [MnIII(tmpyp)(OOH)] (a compound 0‐type intermediate), followed by the formation of a relatively stable trans‐dioxomanganese(V) porphyrin complex, [MnV(O)2(tmpyp)] (a compound I analog). The one‐electron reduction of [MnV(O)2(tmpyp)] to [MnIV(O)(tmpyp)] (a compound II analog) was accelerated greatly by amaranth. On the basis of the kinetic and spectroscopic data, a reaction mechanism of the formation of reactive intermediates [MnIII(tmpyp)(OOH)], [MnV(O)2(tmpyp)], and [MnIV(O)(tmpyp)] was proposed.