Matthias Mag

Synthesis and selective cleavage of an oligodeoxynucleotide containing a bridged non-chiral internucleotide 3′-phosphoramidate linkage

Abstract A self complementary oligodeoxynucleotide dodecamer containing an achiral bridged 3′-phosphoramidate linkage 3′-NH-P-O-5′ has been prepared using the solid phase phosphoramidite procedure. Cleavage of the PN bond can be accomplished selectively by mild acidolysis.

Nuclease stability as dominant factor in the antiviral activity of oligonucleotides directed against HSV-1 IE110

The anti-herpes simplex virus type 1 (anti-HSV-1) efficacy of a series of oligonucleotides was determined as a function of their chemical structure. All oligonucleotides consisted of the same sequence directed against the translation initiation codon of the HSV-1 immediate early gene. The oligonucleotides were modified with phosphorothioate linkage patterns according to various protection strategies against nucleolytic degradation. We show that nuclease resistance is the dominant factor that determines the antiviral efficacy in this series. A minimal protection strategy, consisting of end-capping and pyrimidine protection, has proven to be particularly useful, because it not only yields nuclease-resistant oligonucleotides but also minimizes non-sequence-specific effects.

Enhanced Cellular Uptake of G-Rich Oligonucleotides

Abstract Sequence-dependency of cellular uptake of oligonucleotides into Vero cells has been studied. Cellular uptake of 5′-[35S]-labelled homopolymers decreased in the order (dG)16 >> (dT)16> (dA)16 > (dC)16. The change of two base-pairs (dG → dA) in a dG-rich antisense oligonucleotide with good antiviral activity dramatically decreased cellular uptake and abolished antiviral activity.

Synthesis of dinucleotides containing a bridged non-chiral internucleotide 5′- or 3′-phosphoramidate linkage

Abstract The synthesis of several dinucleoside phosphate derivatives which are linked by phosphoramidate bonds [ 3′- O - P ( O )( O − )- NH -5′ or 3′- NH - P ( O )( O − )- O -5′ ] has been accomplished. The internucleoside phosphoramidate linkage was performed using the Staudinger reaction which is directly followed by a Michaelis-Arbuzov type transformation. Due to the exclusive use of base labile blocking groups deprotection could be carried out very easily in a single step by treatment with concentrated ammonia for 24 hours at 55° C.

Double Protection of the Heterocyclic Base of Xanthosine and 2′-Deoxyxanthosine

Abstract Reaction of O-protected xanthosines with p-nitrophenyl ethanol under Mitsunobu conditions yields the doubly alkylated O2,O6- and N1-,O2-derivatives. Deoxyxanthosine protected on both oxygens with a 2-(4-nitrophenyl)-ethyl group was synthesized starting from deoxyguanosine. Both protecting groups can be eliminated with DBU in pyridine.

Phosphoramidate Analogs of Dinucleotides: Synthesis and 1H Assignment by Two Dimensional NMR Spectroscopy (1H,1H-COSY)

Abstract The synthesis of several dinucleoside phosphate derivatives which are linked by phosphoramidate bonds 3′-OP(O)NH-5′ are described. One of these dimer units can be used in automated solid phase DNA synthesis by the phosphoramidite procedure. In order to study the conformational change which is induced on substituting O-P-0 against O-P-N we have also p-repared the fully deprotected dimer analog. The constitution of the dimer units were confirmed by means of 2D-300MHZ homonuclear chemical shift correlation spectroscopy (1H,1H-COSY).

Synthesis and duplex stability of oligodeoxynucleotides containing stereoregular or stereorandom octylphosphonate linkages

Abstract The synthesis of oligodeoxynucleotide pentadecamers containing two octylphosphonate linkages [3′-O-P(=O)(n-C8H17)-O-5′] with stereoregular or stereorandom chirality is described. The introduction of random octylphosphonate linkages was performed using a monomeric nucleoside octylphosphonamidite as synthon whereas the introduction of stereoregular linkages could be accomplished by the use of stereoregular dimers containing a preformed octylphosphonate linkage. The novel oligodeoxynucleotides were characterized by electrospray ionization mass spectrometry and the influence of chirality of the modified linkages on the duplex stability was studied. Furthermore, end-capped oligodeoxynucleotides having two octylphosphonate linkages at either end which were directed against HSV-1 mRNA have been synthesized for investigation as antisense drugs.

HIV Inhibition by Antisense Oligodeoxynucleotides

Abstract A series of oligodeoxynucleotides containing phosphorothioates and normal internucleotide linkages were synthesized and tested for their antiviral activity against HIV 1.

Amide Protection in Oligodeoxynucleotide Synthesis

Abstract Several O6 -protected deoxyguanosine- as well as O4 -protected thymidine-phosphoramidites were prepared according to the Mitsunobu reaction and Michael addition and were tested in a solid-phase automated DNA synthesizer.

Modified antisense oligodeoxynucleotides against the splice acceptor site of tat do not inhibit in vitro hematopoietic colony growth in HIV-positive patients

The hematopoietic failure in the majority of patients with progressive HIV infection is further aggravated by virustatic agents like azidothymidine. As an alternative therapeutic attempt, three derivatives of an antisense oligodeoxynucleotide (ODN) against the splice acceptor site of the tat gene have been shown to inhibit HIV replication in vitro. This study was aimed at examining whether these agents are toxic to the hematopoietic progenitor cells. To this end, bone marrow cells from HIV-positive and healthy persons were depleted from adherent cells to eliminate fibroblasts. In further experiments, the cells were additionally enriched for CD34-positive hematopoietic progenitor cells or were depleted fromδTCS-1-positive T lymphocytes. At concentrations of 1.25–10 ΜM, the three antisense ODN did not inhibit any erythrocyte or granulocyte-monocyte colony growth from CD34-positive cells, either from the HIV-positive or from the HIV-negative cohort. In contrast to azidothymidine, which served as inhibitory control, a significant increase of colony growth was seen after depletion of fibroblasts, ofδTCS-1-positive cells, or without cell separation. In conclusion, the three oligodeoxynucleotides do not exert any hematotoxic effect but do increase colony formation from low-density bone marrow cells in vitro and could therefore be useful in future clinical studies.