Jean Louis Imbach

Intracellular delivery of nucleoside monophosphates through a reductase-mediated activation process

On the basis of three different models (namely: ddU, AZT and PMEA), mononucleotide phosphotriester derivatives were designed to be able to liberate the corresponding monophosphate (or phosphonate) inside the cell through a reductase-mediated activation process. It was demonstrated that the use of bis[S-(2-hydroxyethylsulfidyl)-2-thioethyl] esters of ddUMP (11), AZTMP (12) and PMEA (17) resulted in intracellular delivery of the parent monophosphate (or phosphonate). This point was corroborated by observation of an anti-HIV effect of, 11 in various cell lines, for 12 in CEM TK- cells and by the enhanced activity observed for 17. Furthermore, the reported decomposition data in cell extracts fully confirm the validity of this approach and show unambiguously the potential for intracellular reductase-mediated activation of the starting drug.

Equal inhibition of the replication of human immunodeficiency virus in human T-cell culture by ddA BIS(SATE)phosphotriester and 3′-azido-2′,3′-dideoxythymidine

It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway.

Template. Phosphorothioate oligonucleotides duplexes as inhibitors of HIV-1 reverse transcriptase

We have investigated the interaction between a number of 14 mers phosphorothioate oligonucleotides and HIV-1 reverse transcriptase. Two methods were used to measure the affinity of the analogs for the enzyme. In the first, the oligonucleotide or its duplex with Poly(rl) were used as inhibitors of the enzyme using Poly(rA).(dT)14 as template primer. In the second, the oligonucleotides or their duplexes were used to displace a fluorescent template primer complex of known affinity from its binding site on reverse transcriptase. The two methods gave the same relative order of affinity. Phosphorothioate oligodeoxyribonucleotides had a much higher affinity than oligo(dC)14 and it was increased on hybridization. Quantitatively similar results were obtained for S(dC)14 or its analog with bases in the alpha-configuration. Of the analogs tested, only S(dC)14 showed priming activity.

Synthesis and biological activity of some unsaturated 6-azauracil acyclonucleosides.

A useful route is described for obtaining Z and E unsaturated alkylating agents 3 and 4. Coupling 6-azauracils 5 and 6 with unsaturated alkylating agent followed by the deprotection with H+ resin gave acyclonucleosides 11–14 in good overall yields. Unsaturated acyclonucleosides phosphonates 19 and 20 were prepared using potassium carbonate as base and 4-bromobut-2-enyl diethyl phosphonate 16 as the alkylating agent. The introduction of a propargyl group at the N-3 position of acyclonucleosides 7, 8, 17, 18, 19, and 20 was achieved using potassium carbonate in DMF. In honor and celebration of the 70th birthday of Professor Leroy B. Townsend. Financial support by the scientific program CNRST (Morocco)/DFG (Germany) and CNRST (Morocco)/CNRS (France) are gratefully acknowledged. We thank Professor De Clercq (Rega Institute for Medical Research Catholic University, Leuven, Belgium) For providing biological tests. We thank Dr. C. Kwong (Southern Research Institute, Birmingham, Alabama) for reviewing this manuscript, and Dr. O. Moukha-Chafiq (Southern Research Institute, Birmingham, Alabama) for his help. The authors thank the referee for his comments and for the very interesting discussion.

NAC/MEA conjugate: a new potent antioxidant which increases the GSH level in various cell lines.

I-152 is a prodrug of NAC and MEA with potent pro-GSH effects in human macrophages, astrocytes and lymphocytes. This molecule could be of interest in HIV infection in respect to its antioxidant and anti-HIV activities, but also in other diseases to counteract oxidative stress, that is, inflammation, cardiovascular diseases, and neurodegenerative diseases.

A Reinvestigation of Sulfenyl Groups as Amino Protecting Groups for the Synthesis of Oligonucleotides on Solid Support by Phosphoramidite Chemistry

Abstract Although sulfenyl groups as protectors of heterocyclic amines of nucleosides appeared satisfactory during the synthesis of DNA and RNA via the phosphotriester approach, their usefulness in automated synthesis of oligonucleotides using phosphoramidite chemistry has not been investigated. Herein, we examined the stability and efficiency of 2-nitrophenylsulfenyl- and tritylsulfenyl-nucleosides upon the conditions applied in oligonucleotide synthesis by the phosphoramidite approach. This article is dedicated to the memory of Professor Tsujiaki Hata

Synthesis and HIV-1 reverse transcriptase inhibition properties of two new methylenephosphonate analogues of 3′-azido-3′-deoxythymidine-5′-triphosphate

Abstract Two new 5′-phosphorylated derivatives of 3′-azido-3′-deoxythymidine (AZT), namely α,β;β,γ-bis (methylene) AZT-5′-triphosphate 1 and α,β-propylene AZT 5′-diphosphate 2 , were synthesized. When evaluated for their inhibitory effects on human immunodeficiency virus (HIV) reverse transcriptase, these compounds were about 1000-fold less active than AZT-5′-triphosphate (AZTTP) as competitive inhibitors of this enzyme.

Rapid determination of the affinity of 28- and 14-mer phosphorothioate oligonucleotides for HIV-1 reverse transcriptase by fluorescence spectroscopy

Intrinsic fluorescence of human immunodeficiency virus type 1 reverse transcriptase (E.C. 2.7.7.49) and displacement experiments of a fluorescent template.primer probe were used to study the interaction of the enzyme with several types of 28- and 14-mer normal or phosphorothioate oligodeoxycytidinylates and their duplexes with poly(rI). The two methods gave convergent results and allowed in each case fast determinations of ligand affinities for the enzyme. The dissociation constants (Kd) obtained from intrinsic fluorescence changes were slightly lower than those determined from the less direct competitive displacement experiments. In all cases, the enzyme displayed better recognition of the hybrid than of the unannealed oligonucleotide. The Kd values of phosphorothioate oligomers and their hybrids were lower than those of the corresponding normal oligomers and hybrids, but the difference was not as significant as in the case of the Ki constants for (dC)28 and S(dC)28 (Majumdar et al. (1989) Biochemistry 28, 1340). The affinities of the annealed phosphorothioate oligodeoxycytidinylates for the enzyme were found to be larger than for any other compounds in this series (Kd of poly(rI).S(dC)28: 0.28 nM at 25 degrees C). Changing the beta stereochemistry of the oligomer bases to alpha did not alter the affinity of the oligodeoxycytidinylate and its hybrids for the enzyme.

Prooligonucleotide metabolism in a crude cell extract followed by matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry.

Using matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOFMS) we studied the decomposition pathway of a prooligonucleotide during incubation in a crude cell extract. This study, involving no sample processing, except drop dailysis to remove salts, led us to identify more than thirty metabolites from several metabolic pathways.

Synthesis of 2',3',5'-trideoxyuridine-5'-methylphosphonic acid and its diphosphate derivative: study of the interaction with HIV-1 reverse transcriptase

2′,3′,5′-Trideoxyuridine-5′-methylphosphonate, [8], was synthesized from ddU. The 5′,6′ carbon-carbon bond was formed by condensing the 5′-aldehyde of ddU and a Wittig reagent. The binding interaction of the diphosphate derivative [10] of the phosphonate [8] with HIV-1 reverse transcriptase (RT) was studied using methods based primarily on fluorescence spectroscopy. From the quenching of intrinsic tryptophan fluorescence of RT during its titration against [10], a dissociation constant of 24 μm was calculated at 25°C. In the presence of a DNA/DNA template/primer of defined sequence and RT, [10] and a fluorescent derivative of ddTTP competed for binding to RT without incorporation of ddU-5′-methylphosphonate. In the presence of a 0.5 mm concentration of [10], the RT activity measured with Poly(rA)/(dT)15 and [3H] dTTP was lowered to 65%. All our observations are consistent with suppression of the catalysis of bond formation between the OH at the primer 3′-end and α-P of [10] after formation of the complex between RT, template/primer and [10].