Small synthetic fluorophores are in many ways superior to fluorescent proteins as labels for imaging. A major challenge is to use them for a protein-specific labeling in living cells. Here, we report on our use of noncanonical amino acids that are genetically encoded via the pyrrolysyl-tRNA/pyrrolysyl-RNA synthetase pair at artificially introduced TAG codons in a recoded E. coli strain. The strain is lacking endogenous TAG codons and the TAG-specific release factor RF1. The amino acids contain bioorthogonal groups that can be clicked to externally supplied dyes, thus enabling protein-specific labeling in live cells. We find that the noncanonical amino acid incorporation into the target protein is robust for diverse amino acids and that the usefulness of the recoded E. coli strain mainly derives from the absence of release factor RF1. However, the membrane permeable dyes display high nonspecific binding in intracellular environment and the electroporation of hydrophilic nonmembrane permeable dyes severely impairs growth of the recoded strain. In contrast, proteins exposed on the outer membrane of E. coli can be labeled with hydrophilic dyes with a high specificity as demonstrated by labeling of the osmoporin OmpC. Here, labeling can be made sufficiently specific to enable single molecule studies as exemplified by OmpC single particle tracking.

Solid phase peptide synthesis (SPPS) is the method of choice to produce peptides. Several protecting groups enable specific modifications. However, complex peptide conjugates usually require a rather demanding conjugation strategy, which is mostly performed in solution. Herein, an efficient strategy is described using an on-resin Diels-Alder reaction with inverse electron demand (DARinv). This method is compatible with the standard Fmoc/tBu strategy and is easy to monitor. As a proof of concept a titanium binding peptide was modified with a cyclic cell binding peptide (RGD) by DARinv on a solid support applying different tetrazines and alkenes. The generated bulky DARinv linkers were employed to act as the required spacer for RGD mediated cell adhesion on titanium. In vitro studies demonstrated improved cell spreading on DARinv-conjugated peptides and revealed, in combination with molecular dynamics-simulation, new insights into the design of spacers between the RGD peptide and the surface. Performing the DARinv on resin expands the toolbox of SPPS to produce complex peptide conjugates under mild, catalyst free conditions with reduced purification steps. The resulting conjugate can be effectively exploited to promote cell adhesion on biomaterials.

Personalized anti-cancer medicine is boosted by the recent development of molecular diagnostics and molecularly targeted drugs requiring rapid and efficient ligation routes. Here, we present a novel approach to synthetize a conjugate able to act simultaneously as an imaging and as a chemotherapeutic agent by coupling functional peptides employing solid phase peptide synthesis technologies. Development and the first synthesis of a fluorescent dye with similarity in the polymethine part of the Cy7 molecule whose indolenine-N residues were substituted with a propylene linker are described. Methylating agent temozolomide is functionalized with a tetrazine as a diene component whereas Cy7-cell penetrating peptide conjugate acts as a dienophilic reaction partner for the inverse Diels-Alder click chemistry-mediated ligation route yielding a theranostic conjugate, 3-mercapto-propionic-cyclohexenyl-Cy7-bis-temozolomide-bromide-cell penetrating peptide. Synthesis route described here may facilitate targeted delivery of the therapeutic compound to achieve sufficient local concentrations at the target site or tissue. Its versatility allows a choice of adequate imaging tags applicable in e.g. PET, SPECT, CT, near-infrared imaging, and therapeutic substances including cytotoxic agents. Imaging tags and therapeutics may be simultaneously bound to the conjugate applying click chemistry. Theranostic compound presented here offers a solid basis for a further improvement of cancer management in a precise, patient-specific manner.

AbstractDie Verbesserung der Zelladhäsion auf einer Biomaterialoberfläche ist entscheidend für den Langzeiterfolg eines Titanimplantats. Ein neues Konzept ist die Kombination aus sehr affiner und stabiler Adhäsion an Titanoberflächen mit spezifisch zellbindenden Motiven in einem Molekül. Ein l‐3,4‐Dihydroxyphenylalanin‐haltiges Peptid wurde synthetisiert und auf seine Affinität für Titan untersucht. Die Modifizierung mit einem cyclischen RGD‐Peptid und einem Heparin‐Bindepeptid (HBP) gelang mittels einer effizienten Festphasenkombination aus einer Diels‐Alder‐Reaktion mit inversem Elektronenbedarf und einer CuI‐katalysierten Azid‐Alkin‐Cycloaddition. Darüber hinaus wurde das Peptid durch Thiol‐Michael‐Addition fluoreszenzmarkiert. Die Konjugation von RGD und HBP in einem Molekül führte zu verbesserter Ausbreitung, Proliferation, Viabilität und Entstehung eines gut ausgebildeten Aktinzytoskeletts sowie fokaler Adhäsionskomplexe von Osteoblast‐ähnlichen Zellen.

Promotion of cell adhesion on biomaterials is crucial for the long-term success of a titanium implant. Herein a novel concept is highlighted combining very stable and affine titanium surface adhesive properties with specific cell binding moieties in one molecule. A peptide containing L-3,4-dihydroxyphenylalanine was synthesized and affinity to titanium was investigated. Modification with a cyclic RGD peptide and a heparin binding peptide (HBP) was realized by an efficient on-resin combination of Diels-Alder reaction with inverse electron demand and Cu(I) catalyzed azide-alkyne cycloaddition. The peptide was fluorescently labeled by thiol Michael addition. Conjugating the cyclic RGD and HBP in one peptide gave improved spreading, proliferation, viability, and the formation of well-developed actin cytoskeleton and focal contacts of osteoblast-like cells.

A modular approach combining inverse electron-demand Diels-Alder coupling (DAR(inv)) and oxime ligation expands the toolbox of bioorthogonal peptide chemistry. Applicability of versatile site-specific bifunctional building blocks is demonstrated by generation of defined conjugates comprising linear, cystine-bridged and multi-disulfide functional peptides as well as their conjugation with hybrid silsesquioxane nanoparticles.

Disastrous effects of tobacco consumption on health remain in the focus of preclinical and clinical research. Exposures to tobacco smoke are closely connected to acute and chronic smoke lung dysfunctions and the effects are not restricted to airway inflammation processes. The facts that smoke exposure result in aberrant gene expression and changes in cellular phenotype resulting in clinical patterns. Here we pyrolyzed constituents of tobacco smoke, induced a stress response in human embryonic lung (HEL) cells, which respond with an altered expression of a broad spectrum of genes. We systematically analyzed the genetic expression, using the microarray-technology. After exposure of HEL cells to alkaline or acidic extracts of pyrolyzed smoke, already 2h after exposition the most affected genes (HMOX1, CYP1B1, ID3, and ID2) were rapidly up-regulated, whereas after 24 hours the genes were almost down-regulated. Using DAVID bioinformatics we detected annotation clusters with significant enrichment scores allowing insight into pharmacological processes and molecular functions. In the alkaline and acidic probes in a ratio 24 h versus 2h we identified annotation clusters with enrichment scores (between 5.77 and 2.89) representing upregulated genes. A negative transcription control leads to the conclusion that a "loss of function" can be possible. STRING tools give insight into the functional network of the gene products of affected genes. Noticeable is the fact that the detected genes with late response encode predicted proteins with unknown function (LOC100134504, LOC645157, LOC653156). With Blast2GO we generated and analysis and graphs of reliable functions.

Advances in imaging diagnostics using magnetic resonance tomography (MRT), positron emission tomography (PET) and fluorescence imaging including near infrared (NIR) imaging methods are facilitated by constant improvement of the concepts of peptide synthesis. Feasible patient-specific theranostic platforms in the personalized medicine are particularly dependent on efficient and clinically applicable peptide constructs. The role of peptides in the interrelations between the structure and function of proteins is widely investigated, especially by using computer-assisted methods. Nowadays the solid phase synthesis (SPPS) chemistry emerges as a key technology and is considered as a promising methodology to design peptides for the investigation of molecular pharmacological processes at the transcriptional level. SPPS syntheses could be carried out in core facilities producing peptides for large-scale scientific implementations as presented here.

The highly organized DNA architecture inside of the nuclei of cells is accepted in the scientific world. In the human genome about 3 billion nucleotides are organized as chromatin in the cell nucleus. In general, they are involved in gene regulation and transcription by histone modification. Small chromosomes are localized in a central nuclear position whereas the large chromosomes are peripherally positioned. In our experiments we inserted fusion proteins consisting of a component of the nuclear lamina (lamin B1) and also histone H2A, both combined with the light inducible fluorescence protein KillerRed (KRED). After activation, KRED generates reactive oxygen species (ROS) producing toxic effects and may cause cell death. We analyzed the spatial damage distribution in the chromatin after illumination of the cells with visible light. The extent of DNA damage was strongly dependent on its localization inside of nuclei. The ROS activity allowed to gain information about the location of genes and their functions via sequencing and data base analysis of the double strand breaks of the isolated DNA. A connection between the damaged gene sequences and some diseases was found.

Here, we review the development of prospective image processing systems in molecular diagnostics and of pharmacologically active ingredients for patient-specific therapeutic approaches. These projects require not only high demands on quality, safety, and specificity but also, rapid, efficient and irreversible ligation routes during the synthesis of future pharmaceuticals. The Diels-Alder ligation reaction with inverse electron demand (DAR(inv)) is an eligible technology not restricted to medical applications, but valuable in selective modification by functionalization of polymers, organic and inorganic surfaces and micro arrays. Additionally, the DARinv technology is considered as an attractive strategy-platform for efficient syntheses of promising pharmacologically active components and derivatives of natural molecules with an optimized therapeutic index. We like to encourage scientists working with the brilliant concept of Sharpless. s "Click chemistry", to intensify their research with this valuable DARinv technology able to open the door for regioselective, stereospecific, and bioorthogonal exigent syntheses of substances of highest quality inconceivable so far.

In contrast to the problematic health and economic effects of acute and chronic smoke exposure on lung function and airway inflammation, there are still few data dealing with the effects of smoking. Smoke exposure can result in aberrant cell growth. In our experiments, pyrolyzed components of cigarettes have been shown to induce a strong stress response in cultured cells. We used human embryonic lung (HEL) cells, which respond with an altered expression of a broad spectrum of genes. Therefore we performed a systematic analysis of the genetic expression behaviour, using the established whole genome microarray-technology which should be able to reveal the cellular effects. With these data we aim to generate a qualitative spectrum of cellular stress response activity. It is noticeable that after cells. exposure to pyrolyzed tobacco smoke components the products of the most affected genes, e.g. ID1, inhibitor of DNA binding, are up-regulated as a rapid response after 2 h with a factor 3.8 and RPS2, ribosomal protein S2, is down regulated to nearly 50 % after 24 hours. In databases they are documented as still uncharacterized and hypothetical proteins. The DDIT4 gene, encoding the DNAdamage-inducible transcript 4, associated with regulation and development of DNA processes after damage by ionizing radiation and in p53 mediated apoptotic processes, is up-regulated. The exposure leads to a rapid cellular stress response of genes like induction of the ID1, ID2, and ID3 genes, located on different chromosomes, already after two hours. They interact normally with DNA binding proteins under heterodimer formation and are considered as negative regulators of transcription. After 24 hours, a return back to normal was not observed and the genes remained stably down-regulated. The suppression of the GADD45B gene which is involved in the cell cycle regulation and after DNA damage a cell cycle arrest is mediated by the gene product. The C14orf4 gene (IRF2BPL) suggests a bifunctional role in transcription control as a promotor. s activator as well as a repressor. Recent data indicate a prominent role of this gene transcript in the control of female reproductive function. On balance, the role of the predominant amount of affected genes is focused on cellular stress response and DNA metabolism.

The personalized medicine, also documented as “individualized medicine”, is an effective and therapeutic approach. It is designed to treat the disease of the individual patient whose precise differential gene expression profile is well known. The trend in the biomedical and biophysical research shows important consequences for the pharmaceutical drug and diagnostics research. It requires a high variability in the design and safety of target-specific pharmacologically active molecules and diagnostic components for imaging of metabolic processes. A key technology which may fulfill the highest demands during synthesis of these individual drugs and diagnostics is the solid phase synthesis which is congenial to automated manufacturing. Additionally the choice of tools like resins and reagents is pivotal to synthesize drugs and diagnostics in high quality and yields. Here we demonstrate the solid phase synthesis effects dependent on the choice of resin and of the deprotection agent.