OLIGONUCLEOTIDE CHEMISTRY

Members Fields of Research Cooperations References

Members

Petra Bongardt (PB)	Ulrike Goetz (UG)	Tatjana Bauer (TB)

Klaus Charissé (KC)	Michael Gerster (MG)	Martin Maier (MM)

Klaus Brischwein (KB)

• Solid phase synthesis of oligonucleotides on PS-PEG-tentacle polymers (MM, MG, UG)

• Synthesis and characterisation of lipid-modified oligonucleotides (MG; MM)

• Synthesis of stereogenic homogenous phosphorothioates by PCR (TB)

• of oligonucleotides (MG; MM)

• Synthesis and characterisation of phosphorodithioates (UG)

• Synthesis and characterisation of oligonucleotide-peptide-hybrides(KC)

• Multiple synthesis and characterisation of very short oligonucleotide-peptide-conjugates by affinity chromatography and ES-MS (PB)

• Development of drug carrier systems for antisense and gene therapy (MM; MG; KC)

• Enzymatic degradation of oligonucleotides investigated by CE and ES-MS (MM; MG; UG)

• Design and synthesis of antisense oligonucleotides (MM; MG; KC; UG)

• Antisense in vitro experiments for suppression of human mdr genes and gene transfection experiments (KB)

Research group of Molecular Oncology, Prof. Dr. H. Kalthoff, Clinic for General Surgery,University of Kiel, Arnold-Heller-Str. 7, D-24105 Kiel:

• Effects of antisense oligonucleotides on mutated p53 protein expression and tumor cell proliferation
• Cellular uptake of fluorescently labeled oligonucleotides and their conjugates (confocal laser scanning microscopy, fluorescence microscopy)
• Investigations of the suitability of drug delivery sytems for gene transfer

Research group of P.D. Dr. Dr. Schrenk, Institute of Toxicology, University of Tübingen, Wilhelmstr. 56, D-72074 Tübingen:

• Suppression of MDR gene expression in rat primary hepatocytes with antisense oligonucleotides
• Investigations of the effects of anti mdr1b oligonucleotides on the P-glycoprotein function

• PS-PEG-tentacle polymers

  Polystyrene-polyethylene glycol (PS-PEG) tentacle polymers with loadings of up to 60 µmol/g were used for standard oligonucleotide synthesis. As these resins are easy to handle and stable under reaction and cleavage conditions they may be used alternatively to controlled pore glass (CPG) as the most commonly used solid support for oligonucleotide synthesis. However, structural and chemical properties of the PS-PEG resins require modified conditions to guarantee syntheses with high coupling efficiencies. Oligonucleotides (ODN) of various sequences and lengths have successfully been synthesized using HPLC and capillary electrophoresis (CE) for purity control. Additionally, electrospray mass spectrometry (ESMS) was used for product identification.

  E. Bayer, K. Bleicher and M. Maier, Z. Naturforsch. 50b 1096-1100 (1995).
  

• lipid modified oligonucleotides:

  Oligonucleotides conjugated with lipophilic residues at their 3'-terminus were synthesized using modified solid supports. Palmityl-, cholesteryl- and tocopheryl-groups were introduced via trifunctional linker systems while PEG-modified oligonucleotides were prepared using resins derivatized with DMT-PEG-succinates. Special phosphoramidites were applied for modification of the 5'-terminus of the oligonucleotides. The products are characterized by capillary electrophoresis and ion spray mass spectrometry. These new methods are very efficient for purity control as it is required for potential antisense drugs.

  E. Bayer, M. Maier, K. Bleicher and H.-J. Gaus, Z. Naturforsch. 50b , 671-676 (1995).
  

• Large scale synthesis:

  Sulphurization is a crucial step during synthesis of phosphorothioate oligonucleotides. Insufficient reaction leads to inhomogeneous products with phosphodiester defects and subsequently to destabilization of the oligomers in biological media. To achieve a maximum extent of sulphur incorporation, various sulphurizing agents have been investigated. Solely, the use of Beaucage reagent provided satisfactory results on PS-PEG supports. Based on our investigations in small scale synthesis (1 µmol) with continuous-flow technique, upscaling to the 0.1-0.25 mmolar range has been achieved using a peptide synthesizer. The syntheses were performed in batch mode with standard phosphoramidite chemistry. Additionally, large scale synthesis of a phosphodiester oligonucleotide has been carried out on PS-PEG with optimized protocols and compared to small scale synthesis on different supports. Products were analysed by 31P-NMR, capillary gel electrophoresis and electrospray mass spectrometry. An extent of sulphurization of 99% and coupling effiencies of more than 99% were obtained and the products proved to have similar purity compared to small scale syntheses on CPG resins.

  M. Gerster, M. Maier, N. Clausen, J. Schewitz and E. Bayer, Z. Naturforsch. 52b , 110-117 (1997).
  
  

• Enzymatic degradation:

  Efficacy and sequence specific behaviour of antisense oligonucleotides in biological systems are attenuated by enzymatic degradation which is predominantly dependent on the oligonucleotide modification. Quantitative data relating to the kinetics and pattern of enzymatic digestion are thus valuable for the interpretation of biological tests with novel antisense oligonucleotides. To study the stability of modified oligonucleotides against nuclease attack, in vitro experiments of enzymatic degradation have been carried out using micellar electrokinetic capillary chromatography (MECC), capillary gel electrophoresis (CGE) for quantitative analysis and electrospray mass spectrometry (ES-MS) for fragment identification.

  Degradation was investigated using several exo- and endonucleases (snake venom phosphordiesterase, Nuclease P1, etc.) and in serum-containing media.

  M. Maier, K. Bleicher, H. Kalthoff and E. Bayer, Biomed. Pept., Proteins Nucleic Acids , 1 , 235-242 (1995).
  

• p53 protein expression:

  Human pancreatic cancer reveals genomic alterations in the p53 tumor suppressor gene frequently associated with mutations in the c-K-ras oncogene. Based on our findings in pancreatic tumor cell-lines (Oncogene, 1993, 8 , 289) we investigated the regulation of p53 expression by antisense oligonucleotides. The phosphorothioate oligonucleotide Ol(1)p53 (P. Iverson, Leukemia and Lymphoma, 1994, 12 , 223) specifically inhibited both, growth and p53 protein expression, in PancTu-1 cells harbouring a mutation at position 176 in the p53 gene. Tumor cells expressing wild type p53 (A818-4), or normal cells (human fibroblasts), or cells without p53 mRNA (K562) were not or only marginally affected by this treatment. The specific effect was dose- and time-dependent and, furthermore, selectively for p53 protein in contrast to CEA as a control protein. In addition 5'- lipid- and 3'-,5'-hexaethylene glycol-modified PTOs were synthesized in order to improve cellular uptake and overall efficiency.
  

• Cellular uptake:

  Biotinylated versions of modified and unmodified phosphorothioate oligonucleotides served as tools to monitor uptake and subcellular distribution by flourescense (confocal laser scanning) microscopy after staining the treated cells with Cy3-conjugated streptavidin. All investigated derivatives could be detected intracellular predominantly located in the nuclei. The uptake appears to be similar in all cell lines used. However, in human fibroblasts (K 562) a spherical distribution around the nucleus was observed while the distribution was more diffuse in the pancreas tumor cell lines (Panc-Tu-1,

  J.E. Lüttges, K. Bleicher, M. Maier, A. Fiedler, H. Kalthoff, Verh. Dtsch. Ges. Path. 79 , 409 (1995).
  

• mdr gene expression / P-glycoprotein function

  One of the most serious problems during the chemotherapy of cancer is the occurence of multidrug resistance (MDR) i. e. a cross resistance to structurally and functionally unrelated mostly lipophilic cytotoxic drugs (MDR type drugs).Overexpression of MDR genes encoding the transmembrane efflux transporter P-glycoprotein (Pgp) is frequently responsible for this type of resistance. The MDR gene family comprises two members in human (MDR1, MDR2), and three members in the rat (mdr1a, mdr1b, mdr2). Only the overexpression of the MDR1/mdr1-subtypes is associated with the emerge of MDR. Several strategies have been developed to overcome MDR, one of them is the inhibition of MDR1/mdr1 gene expression.
  

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