Dendrimers have several unique properties that make them attractive scaffolds for use in biomedical applications. To date, multivalent and multimodal dendritic structures have been synthesized predominantly by statistical modification of peripheral groups. However, the potential application of such probes in patients demands well-defined and monodisperse materials that have unique structures. Current progress in the field of chemical biology, in particular chemoselective ligation methods, renders this challenge possible. In this Minireview, we outline the different available synthetic strategies, some applications that already make use of this new generation of multivalent and multimodal architectures, and the challenges for future developments.

Dendrimere haben eine Reihe einzigartiger Eigenschaften, die sie zu interessanten Architekturen fur biomedizinische Anwendungen machen. Bisher wurden multivalente und multimodale dendritische Strukturen uberwiegend durch die statistische Modifikation der Endgruppen hergestellt. Ihre potenzielle Anwendung am Patienten verlangt dagegen nach wohldefinierten, monodispersen Materialien mit einer einheitlichen Struktur. Aktuelle Fortschritte in der chemischen Biologie und bei chemoselektiven Ligationsmethoden ermoglichen es nun, sich dieser Herausforderung zu stellen. In diesem Kurzaufsatz diskutieren wir die unterschiedlichen Synthesestrategien, einige Anwendungen, die diese neue Generation multivalenter und multimodaler Architekturen bereits nutzen, und die Herausforderungen fur zukunftige Weiterentwicklungen.

The mutual recognition of biomacromolecules often is mediated by dedicated interaction modules. We take two main approaches in order to recognize and control nucleic acid-nucleic acid, protein-protein, and protein-nucleic acid interactions. In one, the rules that govern the formation of nucleic acid structures are used to design molecules that respond to the presence of nucleic acid or protein targets by showing changes of conformation or reactivity. For example, hybrid molecules can transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. The other approach takes advantage of protein domains that once may form the basis of sensor materials and control elements. However, the current chemical synthesis methods have still not reached the level of maturity required to provide routine access to folded protein domains. In this article, we also describe recent progress that may facilitate the chemical synthesis of protein interaction domains.

Protein switches use the binding energy gained upon recognition of ligands to modulate the conformation and binding properties of protein segments. We explored whether the programmable nucleic acid mediated recognition might be used to design or mimic constraints that limit the conformational freedom of peptide segments. The aim was to design nucleic acid-peptide conjugates in which the peptide portion of the conjugate would change the affinity for a protein target upon hybridization. This approach was used to control the affinity of a PNA-phosphopeptide conjugate for the signal transduction protein Src kinase, which binds the cognate phosphopeptides in a linear conformation. Peptide-nucleic acid arms were attached to known peptide binders. The chimeric molecules were studied in three modes: 1) as single strands, 2) constrained by intermolecular hybridization (duplex formation) and 3) constrained by intramolecular hybridization (hairpin formation). Of note, duplexes that were designed to accommodate bulged peptide structures (for example, in hairpins or bulges) had lower binding affinities than duplexes in which the peptide was allowed to adopt a more relaxed conformation. Greater than 90-fold differences in binding affinities were observed. It was, thus, feasible to make use of DNA hybridization to reversibly switch from no to almost complete inhibition of Src-SH2-peptide binding, and vice versa. A series of DNA and PNA-based hybridization experiments revealed the importance of charges and conformational effects. Nucleic acid mediated switching was extended to the use of RNA; this enabled a regulation of the enzymatic activity of the Src kinase. The proof-of-principle results demonstrate for the first time that PNA-peptide chimeras can transduce changes of the concentration of a given RNA molecule to changes of the activity of a signal transduction enzyme.

Ein starkes Signal: Der RNA-katalysierte Transfer eines Biotin-Reporters wurde genutzt, um das Signal eines Enzym-basierten Nucleinsäurenachweises zu verstärken und so dessen Empfindlichkeit zu erhöhen. Die Effizienz der Kombination aus Vorverstärkung und kovalentem Anbinden des Biotin-Reporters belegt der Nachweis von 500 attomol einer HIV-RNA-Sequenz. Auch die Abhängigkeit der Transferreaktion von der Flexibilität am Reaktionszentrum wurde untersucht. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2008/z801355_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The rules that govern the formation of DNA-based structures are relatively well understood. This process can not only be used in the design of probe molecules that report the presence of target nucleic acids by responding to changes of structure and reactivity but also in the development of molecules that transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. Applications in the fields of bioanalytical chemistry and synthetic biology are discussed.

Wirkstoffe aus dem Meer: Drei neue Netropsin-artige Antibiotika (1–3) mit einer bisher unbekannten Furangrundstruktur wurden aus marinen Actinomyceten isoliert und ihre Struktur mithilfe von Massenspektrometrie und 2D-NMR-Spektroskopie aufgeklärt. Sie haben Antitumoraktivität und induzieren im Unterschied zu Netropsin eine Hochregulation von p53 und des Cyclinkinase-Inhibitors p21.

Drugs from the sea: Three new netropsin-type antibiotics (1–3) with a hitherto unknown furan core structure have been isolated from marine actinomycete strains and their structures elucidated by means of mass spectrometry and 2D NMR spectroscopy. The compounds show antitumor activity and, in contrast to netropsin, they induce upregulation of p53 and the cyclin kinase inhibitor p21. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z705295_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Oligonucleotide-peptide conjugates have frequently been used to control the localisation of the conjugate molecule. For example, the oligonucleotide segment has allowed spatially addressed immobilization of peptides and proteins on DNA-arrays via hybridisation while the peptide part has most frequently been used to confer transfer of oligonucleotide cargo into live cells. The regulation of functional properties such as the affinity of these bioconjugates for protein targets has rarely been addressed. This review article describes the current developments in the application of smart oligonucleotide-peptide hybrids. The mutual recognition between nucleic acid segments is used to constrain the structure or control the distance between peptide and protein segments. Application of these new type of oligonucleotide-peptide hybrids allowed not only the regulation of binding affinity of peptide ligands but also control of enzymatic and optical activity of proteins.

Strong signals: The RNA-catalyzed transfer of a biotin reporter was used to preamplify a signal that was detected in an enzyme-based readout, thereby increasing its sensitivity. The advantages of combining the covalent attachment of biotin and preamplification were demonstrated by the detection of 500 attomol HIV RNA. The flexibility requirements of DNA- and RNA-catalyzed transfer reactions were tested. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2008/z801355_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

The rules that govern the formation of DNA duplex structures are well known. On one hand, this process is used in the design of probe molecules that report the presence of target nucleic acids by responding to changes of structure and reactivity. On the other hand, molecules may be developed that transduce changes of nucleic acid structure to changes of peptide structure, and vice versa. Applications in the fields of bioanalytical chemistry and synthetic biology are discussed.

The ability to accurately quantify specific nucleic acid molecules in complex biomolecule solutions in real time is important in diagnostic and basic research. Here we describe a DNA-PNA (peptide nucleic acid) hybridization assay that allows sensitive quantification of specific nucleic acids in solution and concomitant detection of select single base mutations in resulting DNA-PNA duplexes. The technique employs so-called FIT (forced intercalation) probes in which one base is replaced by a thiazole orange (TO) dye molecule. If a DNA molecule that is complementary to the FIT-PNA molecule (except at the site of the dye) hybridizes to the probe, the TO dye exhibits intense fluorescence because stacking in the duplexes enforces a coplanar arrangement even in the excited state. However, a base mismatch at either position immediately adjacent to the TO dye dramatically decreases fluorescence, presumably because the TO dye has room to undergo torsional motions that lead to rapid depletion of the excited state. Of note, we found that the use of d-ornithine rather than aminoethylglycine as the PNA backbone increases the intensity of fluorescence emitted by matched probe-target duplexes while specificity of fluorescence signaling under nonstringent conditions is also increased. The usefulness of the ornithine-containing FIT probes was demonstrated in the real-time PCR analysis providing a linear measurement range over at least seven orders of magnitude. The analysis of two important single nucleotide polymorphisms (SNPs) in the CFTR gene confirmed the ability of FIT probes to facilitate unambiguous SNP calls for genomic DNA by quantitative PCR.

Einstellbare „Signalfeuer“: Eine Triplexbildung wurde zur Konstruktion einer Haarnadelsonde genutzt, die sich bei Bindung der Ziel-DNA (rot) öffnet. Der Triplex-basierte „molecular beacon“ besteht aus einem einzelnen DNA-Strang (blau), einem Fluorophor/Fluoreszenzlöscher-Paar (rote, blaue Kreise) und einem stammbildenden Oligomer (schwarz). Der modulare Aufbau ermöglicht den Einbau weiterer Funktionalitäten, z. B. von Löschern, zur Konstruktion von „superquenched beacons“.

Ein DNA-Schalter: Die Hybridisierung mit DNA steuert die Konformation eines Peptidnucleinsäure(PNA)-Peptid-Konjugats. Dieser reversible Prozess kann die Bindungsaffinität eines Peptids für ein Protein, das an der Signaltransduktion beteiligt ist, gezielt aktivieren (siehe Schema) oder desaktivieren. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2001/2007/z603889_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

A DNA switch: Hybridization with DNA controls the conformation of a peptide nucleic acid (PNA)–peptide conjugate. This reversible process can activate (see scheme) or deactivate the binding propensity of the peptide for a protein target involved in cellular signal transduction. Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z603889_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.

Angewandte Chemie International EditionVolume 46, Issue 27 p. 5223-5225 Communication Triplex Molecular Beacons as Modular Probes for DNA Detection† Tom N. Grossmann Dipl.-Chem., Tom N. Grossmann Dipl.-Chem. Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266 Both authors contributed equally to this work.Search for more papers by this authorLars Röglin Dipl.-Chem., Lars Röglin Dipl.-Chem. Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266 Both authors contributed equally to this work.Search for more papers by this authorOliver Seitz Prof. Dr., Oliver Seitz Prof. Dr. [email protected] Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266Search for more papers by this author Tom N. Grossmann Dipl.-Chem., Tom N. Grossmann Dipl.-Chem. Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266 Both authors contributed equally to this work.Search for more papers by this authorLars Röglin Dipl.-Chem., Lars Röglin Dipl.-Chem. Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266 Both authors contributed equally to this work.Search for more papers by this authorOliver Seitz Prof. Dr., Oliver Seitz Prof. Dr. [email protected] Institut für Chemie der Humboldt-Universität zu Berlin, Brook-Taylor-Strasse 2, 12489 Berlin, Germany, Fax: (+49) 30-2093-7266Search for more papers by this author First published: 22 June 2007 https://doi.org/10.1002/anie.200700289Citations: 130 † We acknowledge support from Schering AG. T.N.G. is grateful for a fellowship from the Studienstiftung des deutschen Volkes. Read the full textAboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Graphical Abstract Reporting live: Triplex formation is used to construct a stem–loop probe, which is opened upon binding of the DNA target (red). The triplex molecular beacon is composed of a single DNA strand (blue), a stem-forming oligomer (black), and is labeled with a fluorophore and a quencher (red and blue circles). This concept facilitates the introduction of further functionalities such as additional quenchers in the assembly of “superquenched” beacons. References 1 1aC. A. Foy, H. C. Parkes, Clin. Chem. 2001, 47, 990– 1000; 1bR. T. Ranasinghe, T. Brown, Chem. Commun. 2005, 5487– 5502. 2 2aS. Tyagi, F. R. Kramer, Nat. Biotechnol. 1996, 14, 303– 308; 2bselected review: W. Tan, K. Wang, T. J. Drake, Curr. Opin. Chem. Biol. 2004, 8, 547– 553. 3G. Bonnet, S. Tyagi, A. Libchaber, F. R. Kramer, Proc. Natl. Acad. Sci. USA 1999, 96, 6171– 6176. 4aC. Crey-Desbiolles, D. Ahn, C. Leumann, Nucleic Acids Res. 2005, 33, e 77; 4bL. Wang, C. J. Yang, C. D. Medley, S. A. Benner, W. Tan, J. Am. Chem. Soc. 2005, 127, 15664– 15665; 4cA. Bourdoncle, A. Estevez Torres, C. Gosse, L. Lacroix, P. Vekhoff, T. Le Saux, L. Jullien, J.-L. Mergny, J. Am. Chem. Soc. 2006, 128, 11094– 11105; 4dO. Seitz, Angew. Chem. 2000, 112, 3389– 3392; Angew. Chem. Int. Ed. 2000, 39, 3249– 3252; 4eH. Kuhn, V. V. Demidov, B. D. Gildea, J. M. Fiandaca, J. C. Coull, M. D. Frank-Kamenetskii, Antisense Nucleic Acid Drug Dev. 2001, 11, 265– 270; 4fH. Kuhn, V. V. Demidov, J. C. Coull, J. M. Fiandaca, B. D. Gildea, M. D. Frank-Kamenetskii, J. Am. Chem. Soc. 2002, 124, 1097– 1103. 5I. Trkulja, S. M. Biner, S. M. Langenegger, R. Häner, ChemBioChem 2007, 8, 25– 27. 6C. J. Yang, H. Lin, W. Tan, J. Am. Chem. Soc. 2005, 127, 12772– 12774. 7M. D. Frank-Kamenetskii, S. M. Mirkin, Annu. Rev. Biochem. 1995, 64, 65– 95. 8 8aP. Nielsen, M. Egholm, R. Berg, O. Buchardt, Science 1991, 254, 1497– 1500; 8bselected review: P. E. Nielsen, Curr. Opin. Biotechnol. 2001, 12, 16– 20. 9R. A. J. Darby, M. Sollogoub, C. McKeen, L. Brown, A. Risitano, N. Brown, C. Barton, T. Brown, K. R. Fox, Nucleic Acids Res. 2002, 30, 39e. 10S. A. E. Marras, F. R. Kramer, S. Tyagi, Nucleic Acids Res. 2002, 30, 122e. 11 11aD. J. French, C. L. Archard, T. Brown, D. G. McDowell, Mol. Cell. Probes 2001, 15, 363– 374; 11bN. Svanvik, A. Stahlberg, U. Sehlstedt, R. Sjoback, M. Kubista, Anal. Biochem. 2000, 287, 179– 182; 11cO. Köhler, O. Seitz, Chem. Commun. 2003, 2938– 2939; 11dO. Köhler, D. V. Jarikote, O. Seitz, ChemBioChem 2005, 6, 69– 77. Citing Literature Supporting Information Supporting information for this article is available on the WWW under http://www.wiley-vch.de/contents/jc_2002/2007/z700289_s.pdf or from the author. Please note: The publisher is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article. Volume46, Issue27July 2, 2007Pages 5223-5225 ReferencesRelatedInformation

The use of carboxylates in the carbodiimide-mediated coupling to amines was investigated. The addition of pyridinium p-toluenesulfonate (PPTS) and a tertiary amine was found to significantly improve acylation yields by up to 70%.