One focus of our research is to synthesize molecules with the ability to selectively interact with biomolecular targets. This includes studies on the reactivity of functional biomolecules. It also includes studies on compounds whose properties are improved over those of unmodified biomolecules and to employ this capability to develop new functional entities. The molecular recognition phenomena of interest also include the recognition of transition states, i.e. the generation of new biomimetic catalysts. The following is a brief list of selected projects from recent years.
Many key techniques of modern molecular biology and medicine, including genomics, rely on sequence selective hybridization between oligonucleotide strands. It has long been known that base pairing fidelity at the termini is poor, as evidenced, e.g. by the degeneracy of the genetic code (Crick, F. H. C. J. Mol. Biol.1966, 19, 548). These laboratories have declared the development of oligonucleotide derivatives with high base pairing fidelity throughout their length one of their goals. The bile acid-modified terminus shown on the left is one recent example where improved base discrimination has been achieved at the 5'-terminus. Some of the caps have been commercialized.
L. Zhang, H. Zhu, M.C. Sajimon, J.A. Rojas Stütz, K. Siegmund, C. Richert, V. Shafirovich, F.D. Lewis, Stabilization of DNA hairpins by stilbene capping of the terminal base pair. J. Chin. Chem. Soc.2006, 53, 1501-1507.
J. Tuma, W.H. Connors, D.H. Stitelman, C. Richert, On the Effect of Covalently Appended Quinolones on Termini of DNA-Duplexes. J. Am. Chem. Soc. 2002, 124, 4236-4246.
O.P. Kryatova, W.H. Connors, C.F. Bleczinski, A.A. Mokhir, C. Richert, A 2'-acylamido cap that increases the stability of oligonucleotide duplexes. Org. Lett.2001, 3, 987-990.
Bleczinski, C. F.; Richert, C. Steroid-DNA interactions increasing stability, sequence-selectivity, DNA/RNA discrimination, and hypochromicity of oligonucleotide duplexes. J. Am. Chem. Soc.1999, 121,10889-10894.
The template-directed extension of primers is the pivotal reaction of DNA replication and transcription. It is also the basis of key biotechnological and diagnostic applications, including, but not limited to, the polymerase chain reaction (PCR), DNA sequencing by the dideoxy method, and most SNP-genotyping techniques. Traditionally, these reactions can only be performed with polymerases as catalysts. Elegant work from L. Orgel's lab and several other research groups has shown that replication can also be induced without enzymes. However, these non-enzymatic reactions are very slow and stop, if several weak base pairs are encountered. We have an interest in performing non-enzymatic primer extension reactions where the rate and specificity of the reactions are enhanced through affordable, non-biological catalysts, as well as biomedical applications for the chemically catalyzed primer extension.
R. Eisenhuth, C. Richert, Convenient syntheses of 3'-amino-2',3'-dideoxynucleosides, their 5'-monophosphates, and 3'-aminoterminal oligodeoxynucleotide primers. J. Org. Chem.2009, 74, 26-37.
M. Röthlingshöfer, E. Kervio, T. Lommel, U. Plutowski, A. Hochgesand, C. Richert, Chemical primer extension in seconds. Angew. Chem.2008, 120, 6154-6157; Angew. Chem. Int. Ed.2008, 47, 6065-6068.
We study DNA-Nanoparticle, DNA-Nanotube, and DNA-DNA-interactions with the longterm goal of creating molecular electronics circuits based on sequence-selective recognition events. This work is supported by the Center Of Functional Nanostructures (CFN) at the University of Karlsruhe.
Publications:
N. L. Wenz, S. Piasecka, M. Kalinowski, A. Schneider, C. Richert, C. Wege, Building expanded structures from tetrahedral DNA branching elements, RNA and TMV protein. Nanoscale2018, 10, 6496-6510.
Singh, A.; Tolev, M.; Schilling, C. Bräse, S.; Griesser, H.; Richert, C. Solution-phase synthesis of branched DNA hybrids via H-phosphonate dimers.
J. Org. Chem., published online February 27, 2012, DOI: 10.1021/jo202508n.
Griesser, H.; Tolev, M.; Singh, A.; Sabirov, T.; Gerlach, C.; Richert, C. Solution-phase synthesis of branched DNA hybrids based
on dimer phosphoramidites and phenolic or nucleosidic cores. J. Org. Chem., published online February 27, 2012, DOI: 10.1021/jo202505h.
A. Singh, M. Tolev, M. Meng, K. Klenin, O. Plietzsch, C. I. Schilling, T. Muller, M. Nieger, S. Bräse, W. Wenzel, C. Richert, Branched DNA that forms a solid at 95 °C.Angew. Chem. 2011, 123, 3285-3289; Angew. Chem. Int. Ed.2011, 50, 3227-3231.
M. Meng, C. Ahlborn, M. Bauer, O. Plietzsch, S. A. Soomro, A. Singh, T. Muller, W. Wenzel, S. Bräse, C. Richert, Two base pair duplexes suffice to build a novel material. ChemBioChem2009, 10 , 1335-1339.
C. Richert, M. Meng, K. Müller, K. Heimann, The third dimension: DNA-driven formation of nanoparticle crystals (Highlight). Small2008, 4, 1040-1042.
S. R. Vogel, K. Müller, U. Plutowski, M. M. Kappes, C. Richert, DNA-carbon nanotube interactions and nanostructuring based on DNA. Phys. Stat. Sol. B, 2007, in press (DOI 10.1002/pssb.200776108).
U. Plutowski, S.S. Jester, S. Lenhert, M.M. Kappes, C. Richert, DNA-based self-sorting of nanoparticles on gold surfaces. Adv. Mat.2007, 19, 1951-1956. Highlighted in: M. Berger,The long road to molecular electronics could be paved with DNA.Nanowerk Spotlight, 2007, August 6.
Taking full advantage of the base pairing selectivity of arrayed oligonucleotide probes (DNA chips) requires duplex stabilities that are independent of G/C-content. Developing sequence independent high affinity/selectivity probes is one of the challenges in today's nucleic acid chemistry. Click here for a recent short essay (in German) and here for the pdf version of an older essay (in English) on this topic.
Publications:
M. Printz, C. Richert, Pyrenylmethyldeoxyadenosine: A 3'-cap for universal DNA hybridization probes. Chem. Eur. J.2009, 15, 3390-3402.
C. Ahlborn, K. Siegmund, C. Richert, Isostable DNA. J. Am. Chem. Soc. 2007, 129, 15218-15232.
K. Siegmund, C. Ahlborn, C. Richert, ChipCheckII - Predicting binding curves for multiple analyte strands on small DNA microarrays, Nucleosides Nucleotides Nucl. Acids, 2008, 27, 376-388.
Oligonucleotides (both DNA and RNA) stimulate the innate immune system, presumably through signalling by Toll-like receptors (TLRs). We study the structural basis of this phenomenon through chemical synthesis. Biological evaluation (in collaboration with the groups of profs. Dalpke and Heeg at the University of Heidelberg). This work is supported by the Landesstiftung Baden-Württemberg.
Structure Elucidation of non-natural RNA Analogs and Peptide-Nucleic Acid Hybrids
Our NMR-based structural work has several motivations. First, we want to gain structural insight into new modes of interaction between potential therapeutic agents and their targets. Particularly, we are interested in the duplexes formed between antisense oligonucleotides and their genetic targets. Second, we want to better understand ligand-nucleic acid and particularly protein-nucleic acid interactions. To this end, we use peptide-nucleic acid hybrid molecule. Linking entropically favors complex formation and provides structural preorientation. Structure elucidation starts with the acquisition of two- and, if necessary, three-dimensional NMR spectra, followed by peak assignments and generation of distance constraints. Restrained molecular dynamics are performed with both simulated annealing and the distance geometry algorithms. We employ the GIFA and XPLOR program packages, the most recent version of CNS (by A. Bruenger, Yale University), along with some software developed in-house. Spectroscopic work is performed both at U. Constance and at the Francis Bitter Magnet Labs at the MIT.
Key Techniques: Multidimensional NMR, Restrained molecular dynamics
W.C. Ho, C. Steinbeck, C. Richert, Solution Structure of the Aminoacyl-Capped Oligodeoxyribonucleotide Duplex (W-TGCGCAC)2, Biochemistry, 1999, 38, 12597-12606.
C. Steinbeck, C. Richert, The Role of Ionic Backbones in RNA Structure: An Unusually Stable non-Watson-Crick Duplex of a Non-Ionic Analog in an Apolar Medium. J. Am. Chem. Soc., 1998, 120, 11576-11580.
C. Steinbeck, C, Richert, Solution structure of a backbone-modified UC-dimer: An unusual recognition motif. Poster to be presented at the Conference "Multidimensional NMR in Structural Biology", Lucca (Italy), August 17-21, 1997.
Combinatorial Synthesis and in Vitro Selection: Searching Structure Space Efficiently
These experiments are being employed to search structure space for oligonucleotide derivatives with high affinity for single- and double-stranded targets, and have been shown to allow rapid genotyping of DNA from patient samples.
J. Störker, C.N. Tetzlaff, J. Mayo, D.A. Sarracino, I. Schwope, C. Richert,
Rapid genotyping via MALDI-monitored nuclease selection from probe libraries.
Nature Biotechn. 2000, 18, 1213 - 1216.
Jay Störker, Jason Mayo, Charles N. Tetzlaff, David A. Sarracino, and
Clemens Richert, Rapid Genotyping Using Spectrometrically Monitored
Nuclease Selections, presented at the Conference Mass Spectrometry for
Pharmaceutical Research, Orlando, FL, December 9-10, 1999.
K. Berlin, D.A. Sarracino, R.K. Jain, C. Steinbeck, C. Richert,
MALDI-TOF analysis of combinatorial libraries of porphyrins.
Poster presented at ASMS, Orlando, May 31-June 4, 1998.
K. Berlin, R.K. Jain, C. Tetzlaff, C. Steinbeck, C. Richert,
Spectrometrically monitored selection experiments: quantitative laser
desorption mass spectrometry of small chemical libraries. Chemistry &
Biology1997, 4, 63-77; 1997, 4, 237-
238.
In order to search structure space efficiently for new functional molecules, our lab has developed monitored selection assays. These involve small chemical libraries and a spectrosc opic technique for detecting the library components simultaneously. We find MALDI-TOF mass spectrometry to be particularly useful in this context, as it produces essentially fragmentation-free spectra or, in the case of isotopically resolved spectra, one peak cluster of minimal spectral width. The size of libraries that can be employed in such experiments is limited, among other things, by the spectral overlap, i.e. by the number of isobaric library components. Optimization of mass spectrometrically mo nitored selection experiments therefore called for a computational tool that would facilitate the choice of library components. MASP, a computer program that scans meta-library space, is such a tool.
Publications:
C. Steinbeck, K. Berlin, C. Richert, MASP - a program predicting mass spectra of combinatorial libraries. J. Chem. Inf. Comput. Sci.,1997, 37, 449-457.
Natural products are important building blocks for our combinatorial work and interesting synthetic targets in themselves. We synthesize and modify natural products.
Publications:
D.L. Daniels, C. Richert, One-pot synthesis of L-felinine. Tetrahedron Lett.1999, 40, 4463-4465.
Peptide-DNA Hybrids: Synthesis and Selection of Acyl-DNA Hybrids
The long term goal of this research project is to use the combined DNA and peptide structure space to search for new therapeutic agents that bind to DNA or RNA targets. Presently, we focus on hybrids between single stranded DNA and short peptides with the underlying theme to tune the bioavailability and recognition properties of oligonucleotides by complexing them with ligands rather than by replacing atoms in their backbone. Peptide-DNA hybrids are "selectable" entities. This work uses combinatorial synthesis and in vitro selection to identify hybrids with increased stability against enzymatic degradation and with increased affinity for target strands. Critical positions in the hybrids are randomized to produce small libraries. The survival of the individual members of these libraries under selection conditions is monitored directly, using quantitative laser desorption mass spectrometry in a new form of assay established in these laboratories (Berlin, et al. & Richert Chemistry & Biology 4 (1997) 63).
Covalently linking a peptide and an oligonucleotide entropically favors the formation of a complex. The hybrids proposed here are therefore ideal entities for studying interactions that may otherwise be too weak or too unspecific to be studied in solution. Our work will involve elucidation of the three-dimensional structure of selected peptide-DNA hybrids by multidimensional NMR and molecular dynamics.
Key Techniques: Semi-automated synthesis of peptide-DNA hybrids, Quantitative MALDI-TOF mass spectrometry, Enzymatic in vitro selection and footprinting.
C.N. Tetzlaff, D.A. Sarracino, I. Schwope, J.A. Steinberg, C.F. Bleczinski, M.T. Vergo, W. Ho, C. Richert, 5'-Peptidyl-DNA:Synthesis and Binding Studies. Presented at the 216th ACS Meeting, Boston, August 23-27, 1998.
Charles N. Tetzlaff, Ina Schwope, Colleen F. Bleczinski, Joshua A. Steinberg, Clemens Richert, A convenient synthesis of 5'-amino-5'-deoxythymidine and preparation of peptide-DNA hybrids. Tetrahedron Lett.,1998, 39, 4215-4218.
K. Berlin, R.K. Jain, C. Tetzlaff, C. Steinbeck, C. Richert, Spectrometrically monitored selection experiments: quantitative laser desorption mass spectrometry of small chemical libraries. Chemistry & Biology1997, 4, 63-77; 1997, 4, 237-238.
C. Tetzlaff, D. Sarracino, I. Schwope, C. Bleczinski & C. Richert, "Synthesis and in Vitro Selection of Peptide-DNA Hybrids: Towards Ligand-Shielded Oligonucleotides." presented at the Nature Biotechnology Conference "Antisense 97", Cambridge, Massachusetts, May 1-2, 1997
Porphyrins are photoactive heterocyclic macro cycles with important biological functions. Our synthetic efforts focus on porphyrins that can be employed as sensitizers for photodynamic therapy and porphyrins that interact with nucleic acids. Among possible nucleic acid targets, single-stranded DNA and RNA are of particular interest to us. We have established combinatorial syntheses for a number of different tetraphenylporphyrin species and have also succeeded in incorporating an alkylporphyrin in the backbone of DNA.
M.D. Simon, C. Richert, Synthesis and Selection of Peptidyl-Tetraphenylporphyrins: Toward Target-Specific Nucleic-Acid Ligands. Poster presented at the 217th American Chemical Society National Meeting, Anaheim, CA, March 1999: Abstr. Pap. Am. Chem. Soc.1999, 217, 558.
K. Berlin, R. K. Jain, C. Richert, Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins. Biotechn. Bioengin.: Combin. Chem.1998, 61, 107-118.
K. Berlin, R.K. Jain, M.D. Simon, C. Richert, A porphyrin embedded in DNA. J. Org. Chem,1998, 63, 1527-1535.
Rishi K. Jain, David A. Sarracino, and Clemens Richert, A tetraphenylporphyrin-peptide hybrid with high affinity for single-stranded DNA. Chem. Comm.,1998, 423-424.
Photodynamic therapy is a binary treatment modality for neoplastic tissue. Photodynamic therapy of tumors has b een approved for treatment of esophageal cancer (USA), lung and esophageal cancer (France and Netherlands), esophageal and bladder cancer (Canada), and early-stage lung cancer, superficial and esophageal cancer, superficial and early stage gastric cancer, early stage cervical cancer, and cervical dysplasia (Japan) with photofrin, a porphyrin mixture as sensitizer. Our work in this area focuses on the chemical and physical principles that underlie tumor selective localization and site-selective action.
Publications:
K. Berlin, R. K. Jain, C. Richert, Are porphyrin mixtures favorable photodynamic anticancer drugs? A model study with combinatorial libraries of tetraphenylporphyrins. Biotechn. Bioengin.: Combin. Chem., 1998, 61, 107-118.
M. Dellian, C. Richert, F. Gamarra, A.E. Götz, Photodynamic eradication of amelanotic melanoma of the hamster with fast acting photosensitizers. Int. J. Cancer, 1996, 65, 246-248.
C. Richert, J.M. Wessels, M. Mller, M. Kisters, T. Benninghaus, A.E. Götz, Photodynamic antitumor agents: b-methoxyethyl groups give access to functionalized porphycenes and enhance cellular uptake and activity. J. Med. Chem., 1994, 37, 2797-2807.
M. Leunig, C. Richert, F. Gamarra, W. Lumper, E. Vogel, D. Jocham, A.E. Götz, Tumour localization kinetics of photofrin and three synthetic porphyrinoids in an amelanotic melanoma of the hamster. Br. J. Cancer, 1993, 68, 225-234.
F. Gamarra, F. Yuan, A.E. Götz, M. Leunig, C. Richert, R.K. Jain, K. Messmer, Monitoring extravascular transport of porphycenes. In P. Spinelli, M. Dal Fante, R. Marchesini (Eds.) "Photodynamic therapy and biomedical lasers", Elsevier, Amsterdam, Excerpta Med., 1992, 1011, 706-709.
Two of the structures frequently employed in our work:
