ABSTRACT
A complete series of the 2 '-5 ' and 3 '-5 ' regioisomeric types of r(ApA) and 2 '-d(ApA) analogues with the α-hydroxy-phosphonate C3 '-O-P-CH(OH)-C4 â³ internucleotide linkage, isopolar but non-isosteric with the phosphodiester one, were synthesized and their hybridization properties with polyU studied. Due to the chirality on the 5 '-carbon atom of the modified internucleotide linkage bearing phosphorus and hydroxy moieties, each regioisomeric type of ApA dimer is split into epimeric pairs. To examine the role of the 5 '-hydroxyl of the α-hydroxy-phosphonate moiety during hybridization, the appropriate r(ApA) analogues with 3 '(2 ')-O-P-CH(2)-C4 â³ linkage lacking the 5 '-hydroxyl were synthesized. Nuclear magnetic resonance (NMR) spectroscopy study on the conformation of the modified sugar-phosphate backbone, along with the hybridization measurements, revealed remarkable differences in the stability of complexes with polyU, depending on the 5 '-carbon atom configuration. Potential usefulness of the α-hydroxy-phosphonate linkage in modified oligoribonucleotides is discussed.
Subject(s)
Organophosphonates/chemistry , Poly U/chemistry , Chemistry Techniques, Synthetic , Magnetic Resonance Spectroscopy , Models, Molecular , Molecular Conformation , Organophosphonates/chemical synthesisABSTRACT
Structurally diverse, sugar-modified, thymine-containing nucleoside phosphonic acids were evaluated for their ability to inhibit thymidine phosphorylase (TP, EC 2.4.2.4) purified from spontaneous T-cell lymphomas of an inbred Sprague-Dawley rat strain. From a large set of tested compounds, among them a number of pyrrolidine-based derivatives, 10 nucleotide analogues with IC(50) values below 1 microM were selected. Out of them, four compounds strongly inhibited the enzyme with IC(50) values lying in a range of 11-45 nM. These most potent compounds might be bi-substrate analogues.
Subject(s)
Lymphoma, T-Cell/enzymology , Nucleosides/chemistry , Organophosphonates/chemistry , Thymidine Phosphorylase/antagonists & inhibitors , Animals , Dose-Response Relationship, Drug , Humans , Inhibitory Concentration 50 , Nucleosides/pharmacology , Organophosphonates/pharmacology , Rats , Rats, Sprague-Dawley , Structure-Activity Relationship , Thymidine Phosphorylase/metabolismABSTRACT
The synthetic approach towards selected monomers of ribonucleosidyl-5'-C-phosphonates I compatible with the phosphotriester condensation method is presented, together with the surprising and unexpected effect of TEA.3HF on the phosphonate moiety observed upon removal of the O-silyl protecting groups.
Subject(s)
Oligonucleotides/chemical synthesis , Organophosphonates/chemistry , Ribonucleotides/chemical synthesis , Aldehydes/chemistry , Oligonucleotides/chemistry , Ribonucleotides/chemistryABSTRACT
A number of structurally diverse nucleoside phosphonic acids have been tested against human recombinant thymidine phosphorylase and human platelets supernatant using 2'-deoxy-5-nitrouridine as the substrate. We have selected several inhibitors working at micromolar level as lead structures for further evaluation.
Subject(s)
Enzyme Inhibitors/chemistry , Nucleosides/chemistry , Nucleosides/pharmacology , Organophosphonates/chemistry , Thymidine Phosphorylase/antagonists & inhibitors , Animals , Blood Platelets/enzymology , CHO Cells , Cricetinae , Cricetulus , Enzyme Inhibitors/pharmacology , Humans , Structure-Activity Relationship , Thymidine Phosphorylase/chemistryABSTRACT
We have optimized surface plasmon resonance (SPR) biosensor technology for a rapid, direct, and low-consumption label-free multianalyte screening of synthetic oligonucleotides (ONs) with modified internucleotide linkages potentially applicable in antisense therapy. Monitoring of the ONs hybridization is based on the formation of complex between the natural oligonucleotide probe immobilized on the sensor surface and the ON in solution in contact with the sensor surface. An immobilization chemistry utilizing the streptavidin-biotin interaction was employed to obtain desired ligand density and high hybridization efficiency. It was demonstrated that the sensor is capable of detecting complementary 23-mer ONs in concentrations as low as 0.1 nM with high specificity and reproducibility.
Subject(s)
Biosensing Techniques/methods , Nucleic Acid Hybridization/methods , Surface Plasmon Resonance/methods , Biosensing Techniques/instrumentation , Nanotechnology/instrumentation , Nanotechnology/methods , Oligonucleotide Array Sequence Analysis/instrumentation , Oligonucleotide Array Sequence Analysis/methods , Oligonucleotide Probes/chemistry , Oligonucleotide Probes/geneticsABSTRACT
This work deals with isopolar, phosphonate-based nucleotide analogues containing a bridging P-C bond instead of the ester P-O linkage. Specifically, starting from activated derivatives 1, 2, and 3, a simple process for preparation of mixtures of short oligomers and their analyses were elaborated.
Subject(s)
Adenine Nucleotides/chemistry , Adenine Nucleotides/chemical synthesis , Oligoribonucleotides/chemistry , Oligoribonucleotides/chemical synthesis , Indicators and Reagents , Molecular Structure , Organophosphonates , ZalcitabineABSTRACT
The synthesis of monomers (S)-1, (R)-1 and 2 derived from (5'S)-, (5'R)-2'-deoxythymidine-5'-C-phosphonic acids and 2',5'-dideoxythymidine-5'-C-phosphonic acids was elaborated. The protection of the 5'-hydroxyl by the methoxycarbonyl group was a key step of the synthesis. Prepared monomers were used for the solid-phase assembly of several types oligothymidylate 15-mers (S)-3, (S)-4, (S)-5, (R)-4 and (R)-5 containing the chiral 3'-O-P-CH(OH)-5'' internucleotide linkage. Their hybridization properties with dA15 and rA15 were studied as well as their resistance against nuclease cleavage.
Subject(s)
DNA/chemistry , Oligodeoxyribonucleotides/chemistry , Oligodeoxyribonucleotides/chemical synthesis , Organophosphonates , Thymidine/analogs & derivatives , Indicators and Reagents , Molecular Structure , Nucleic Acid ConformationABSTRACT
The recently described epimeric nucleosidyl-5'-C-phosphonates (alpha-hydroxyphosphonates) represent novel nucleotide analogues that can be incorporated into chimeric oligonucleotides by the phosphotriester condensation method. In order to prepare suitable protected monomer(s) we have studied condensation reaction between protected 2'-deoxythymidine and 2'-deoxythymidinyl-5'-C-phosphonate, both as model compounds, in dependence on the nature of the 5'-hydroxyl protecting group. We have found that the O-acetyl group is unstable in the presence of TPSCl or MSNT used as condensing agents for activation of the phosphorus moiety. This instability negatively influences the scope of the condensation process. On the other hand, introduction of the O-methoxycarbonyl group gave excellent results. The O-methoxycarbonyl group does not participate in the condensation process, and its quantitative introduction into the nucleotide analo gues is accomplished using a novel acylating agent, methoxycarbonyl tetrazole.