Leo Lh Koole

Conformational transmission in four- and five-coordinated phosphorus compounds. Solvent effects on the conformation in 5'-phosphorylated model nucleosides

The C4,-C5, conformation of the 5’-PIV model compounds 1-3 and the 5’-Pv TBP model compounds 4-6 has been determined in various solvents with 300- and 500-MHz ‘H NMR. It is found that lowering the solvent polarity results in a substantial increase of the g- populations for the model compounds 1, 2, 4, and 5 (e.g., for 1 in D20, x(g) = 0.00; CCI4, x(g-) = 0.23). This effect can be attributed to an enhanced electrostatic repulsion between the charge densities on Os and the endocyclic oxygen(s) at lower solvent polarities. This conclusion is supported by the experimental finding that the 5’-PIV system 3 and the 5’-Pv TBP system 6, in which the endocyclic oxygen(s) are replaced by C(H,), do not show a C4,-C5, conformational change when the medium polarity is changed. Furthermore, it is found that the 5’-PIV nucleotides 7 and 8, which are made soluble in apolar solvents by triesterification of the phosphorus, show also increased g- populations upon lowering the solvent polarity. The present results confirm our earlier proposal that enhanced charge repulsion between 05a,n d 01, in mononucleotides drives a rotation around the C 4 4 , bond toward g-.

Characterization of the African HIV-1 isolate CBL-4 (RUT) by partial sequence analysis and virus neutralization with peptide antibody and antisense phosphate-methylated DNA.

The HIV-1 isolate CBL-4 (RUT), originating from Tanzania, was characterized using a comprehensive virus-typing system. This system included sequence analysis of the region coding for the neutralization domain in the third variable region (V3) of the external envelope and of the tat responsive (TAR) region after polymerase chain reaction (PCR) amplification of these sequences from cellular DNA in the CBL-4 (RUT) producer line. Based on independent cluster analysis of TAR and V3 sequences the CBL-4 (RUT) virus was positioned closest to the Z6 (and ELI) African virus family. The V3 amino acid sequence on the surface of the virus particle was confirmed by the inhibition of neutralization of CBL-4 (RUT) by a synthetic peptide derived from the nucleic acid sequence. Using antisense phosphate-methylated DNA covering the TAR loop region of LAV-1/HTLV-IIIB, inhibition of HTLV-IIIB and HTLV-IIIRF infection was seen, whereas no inhibition was observed for CBL-4 (RUT), indicating two or more mismatches in the TAR loop region, a characteristic shared with Z6 virus, but not with ELI. We propose a virus-typing system based on sequence analysis confirmed by virus neutralization with a peptide binding antibody and inhibition by antisense phosphate-methylated DNA to group viruses for laboratory use and vaccine design.

Optimization of the separation of the Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides

The separation by reversed-phase high-performance liquid chromatography of Rp and Sp diastereomers of phosphate-methylated DNA and RNA dinucleotides was studied with respect to pH, organic modifier type and concentration and reversed-phase packing material. Drylab G was used to deduce optimum conditions. On the basis of the observed discrepancies between the computer predictions and experimental results, the gradient operation procedure with volatile buffers was improved. By repetitive chromatography on a 250 x 22 mm I.D. reversed-phase column, fourteen diastereomeric pairs were obtained in at least 97% purity and 60% yield, in amounts of 10-100 mg.

Conformational transmission in 5'-P(IV) and 5'-P(V) TBP phosphorylated nucleosides : implications for DNA structure and dynamics

Abstract Specific conformational changes in DNA and RNA can be induced via a transient pentacoordination of the phosphorus atoms in the helix backbone. The details of this conformational transmission mechanism are discussed briefly, using experimental data that were obtained with a set of 5′-pIV and 5′-pV phosphorylated tetrahydrofurfuryl systems. A conformational study on the more realistic model system, the dinucleotide 2 in which a stabilized pentacoordinated phosphorus forms the internucleoside linkage, is presented. Furthermore, it has been found that methylation of the phosphate groups in d(TpTpTpTpTpT) results in the formation of a non-Watson & Crick type parallel duplex DNA structure, in which the two strands are joined via hydrogen bonding between the thymidine bases. Various physico-chemical techniques (e.g. NMR methods and UV hyperchromicity) were used to elucidate the structural details of the parallel duplex. Characteristic properties (parallellity, slimness, symmetry) are presented.

Pseudorotation and conformational transmission in pentacoordinated phosphorous compounds

Abstract This poster reviews our recent work on conformational transmission and pseudorotation in the pentacoordinated phosphorus (Pv) compounds 1a-d and 2a-d (1,2).

Conformational Transmission in DNA

Abstract This poster presents our recent results on DNA dimers in which a stable trigonal bipyramidal pentacoordinated phosphorus (Pv) structure forms the internucleoside linkage. Conformational analysis of the systems 1-4 with 300 and 500 1H NMR has shown that the Pv structure results in a distorted conformation of the backbone structuE (1).

Phosphate-methylated DNA fragments involved in parallel and antiparallel duplex formation : novel aspects of structure : stability and biological activity

Abstract Parallel duplexes are encountered for the phosphate-methylated DNA dinucleotides d(CpC) and d(TpC; for the Sp configuration exclusively. since inward location of the methyl group (Rp,) encounters severe steric interactions in the groove. A parallel duplex with T-T base pairs is found for the natural system d(T10) only after complexation of the cationic peptides polylysine and polyornithine with the phosphate groups, which diminishes phosphate-phosphate repulsions. For natural d(C10), parallel duplex formation is seen exclusively after complexation with polylysine, which only uses pro-S phosphate oxygens in the complexation. Polyornithine complexates with both pro-S and pro-R phosphate oxygens, leading to steric hindrance in the groove of the C-C duplex. For antiparallel duplexes of phosphate-methylated DNA (‘antisense’) with natural polynucleotides (‘sense’) it is found that the hybridization has a cooperative character. Furthermore, it is mentioned that long phosphate-methylated DNA fragments ca...

A comparative high-field n.m.r. study on tri-, tetra-, penta-, and hepta-mer ribonucleotides modelling the branch site of Group II and nuclear messenger RNA spliced lariat introns

Conformational analysis of the branched RNA systems (1)–(4) using variable-temperature 600 MHz proton and 223 MHz phosphorus-31 n.m.r. spectroscopy revealed that (i), generation of a lariat structure, typical for Group II and nuclear messenger RNA splicing, can occur with preservation of the A-geometry of the RNA strand; (ii), mimicry of the lariat branch site requires the presence of at least one nucleotide linked to the 5′-site of the branch point [as in tetramer (2) and heptamer (4)], since the absence of this nucleotide [as in trimer (1) and pentamer (3)] results in an unnatural molecular conformation which is largely dominated by adenine 2′–5′ guanine base stacking.