The oligoadenylate synthetase-ribonuclease L pathway is a major player in the interferon-induced antiviral defense mechanism of cells. Upon sensing viral dsRNA, 5'-phosphorylated 2',5'-oligoadenylates are synthesized, and subsequently activate latent RNase L. To determine the influence of 5'-phosphate end on the activation of human RNase L, four sets of 5'-phosphonate modified oligoadenylates were prepared on solid-phase. The ability of these 5'-modified oligoadenylates bearing shortened, isosteric and prolonged phosphonate linkages to activate RNase L was explored. We found that isosteric linkages and linkages prolonged by one atom were in general well tolerated by the enzyme with the EC50 values comparable to that of the natural activator. In contrast, linkages shortened by one atom or prolonged by two atoms exhibited decrease in the activity.
- MeSH
- adeninnukleotidy chemická syntéza chemie farmakologie MeSH
- endoribonukleasy metabolismus MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- oligoribonukleotidy chemická syntéza chemie farmakologie MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
A series of conformationally constrained uridine-based nucleoside phosphonic acids containing annealed 1,3-dioxolane and 1,4-dioxane rings and their "open-structure" isosteres were synthesized and evaluated as potential multisubstrate-like inhibitors of the human recombinant thymidine phosphorylase (TP, EC 2.4.2.4) and TP obtained from peripheral blood mononuclear cells (PBMC). From a large set of tested nucleoside phosphonic acids, several potent compounds were identified that exhibited Ki values in the range of 0.048-1 μM. The inhibition potency of the studied compounds strongly depended on the degree of conformational flexibility of the phosphonate moiety, the stereochemical arrangement of the sugar-phosphonate component, and the substituent at position 5 of the pyrimidine nucleobase.
- MeSH
- inhibitory enzymů farmakologie MeSH
- kyseliny fosforité chemie farmakologie MeSH
- lidé MeSH
- molekulární konformace MeSH
- pyrimidinové nukleosidy farmakologie MeSH
- thymidinfosforylasa antagonisté a inhibitory MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Solid phase synthesis of phosphonate-modified oligoribonucleotides using 2'-O-benzoyloxymethoxymethyl protected monomers is presented in both 3'→5' and 5'→3' directions. Hybridisation properties and enzymatic stability of oligoribonucleotides modified by regioisomeric 3'- and 5'-phosphonate linkages are evaluated. The introduction of the 5'-phosphonate units resulted in moderate destabilisation of the RNA/RNA duplexes (ΔT(m)-1.8 °C/mod.), whereas the introduction of the 3'-phosphonate units resulted in considerable destabilisation of the duplexes (ΔT(m)-5.7 °C/mod.). Molecular dynamics simulations have been used to explain this behaviour. Both types of phosphonate linkages exhibited remarkable resistance in the presence of ribonuclease A, phosphodiesterase I and phosphodiesterase II.
- MeSH
- exonukleasy metabolismus MeSH
- fosfodiesterasa I metabolismus MeSH
- kyseliny fosforité chemická syntéza chemie metabolismus MeSH
- oligoribonukleotidy chemická syntéza chemie metabolismus MeSH
- pankreatická ribonukleasa metabolismus MeSH
- simulace molekulární dynamiky MeSH
- techniky syntézy na pevné fázi MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The synthesis of the novel nucleotide analogues 5'-deoxynucleoside-5'-S-methylphosphonates, starting from 5'-deoxy-5'-haloribonucleosides, 5'-O-tosylribonucleosides, and 2'-O-triflylnucleosides, is described. The phosphonothiolation of these compounds was achieved using S-(diisopropylphosphonomethyl)isothiouronium tosylate, a new, odourless, and efficient equivalent of mercaptomethylphosphonate. The thiolate anion of mercaptomethylphosphonate was generated in situ from the isothiouronium salt in both protic and aprotic solvents using two equivalents of sodium iso-propoxide. The prepared nucleoside 5'-S-methylphosphonates were deprotected, and the free phosphonic acids were transformed into diphosphoryl derivatives (the NTP analogues). Both mononucleotides and NTP analogues were studied as substrates/inhibitors of several enzymes that are involved in the nucleoside/nucleotide metabolism.
In an attempt to prepare a library of short oligoadenylate analogues featuring both the enzyme-stable internucleotide linkage and the 5'-O-methylphosphonate moiety and thus obtain a pool of potential RNase L agonists/antagonists, we studied the spontaneous polycondensation of the adenosin-5'-O-ylmethylphosphonic acid (p(c)A), an isopolar AMP analogue, and its imidazolide derivatives employing N,N'-dicyclohexylcarbodiimide under nonaqueous conditions and uranyl ions under aqueous conditions, respectively. The RP LC-MS analyses of the reaction mixtures per se, and those obtained after the periodate treatment, along with analyses and separations by capillary zone electrophoresis, allowed us to characterize major linear and cyclic oligoadenylates obtained. The structure of selected compounds was supported, after their isolation, by NMR spectroscopy. Ab initio calculation of the model structures simulating the AMP-imidazolide and p(c)A-imidazolide offered the explanation why the latter compound exerted, in contrast to AMP-imidazolide, a very low stability in aqueous solutions.
- MeSH
- adeninnukleotidy chemie metabolismus MeSH
- adenosinmonofosfát analogy a deriváty MeSH
- molekulární modely MeSH
- molekulární struktura MeSH
- nukleární magnetická rezonance biomolekulární MeSH
- oligoribonukleotidy chemie metabolismus MeSH
- oxidace-redukce MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
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.
- MeSH
- CHO buňky MeSH
- Cricetulus MeSH
- inhibitory enzymů chemie farmakologie MeSH
- křečci praví MeSH
- kyseliny fosforité chemie MeSH
- lidé MeSH
- nukleosidy chemie farmakologie MeSH
- thymidinfosforylasa antagonisté a inhibitory chemie MeSH
- trombocyty enzymologie MeSH
- vztahy mezi strukturou a aktivitou MeSH
- zvířata MeSH
- Check Tag
- křečci praví MeSH
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Phosphonate analogs of mono- and oligonucleotides with the P-C-O-C5' moiety have interesting biochemical and biophysical properties. A series of novel compounds, S-methylphosphonate-based nucleotides, with the P-C-S-C5' linkage was prepared as monomers for solid phase synthesis of modified oligonucleotides. Replacement of the 5'-oxygen atom with more nucleophilic, bulky, and lipophilic sulfur atom may influence the physicochemical and biological properties of nucleoside 5'-S-methylphosphonates and chimeric oligonucleotides as well.
We report on synthesis of new fluorescent probes suitable for site-specific incorporation into oligonucleotides. Coumarin derivatives were used as sensitive fluorescent labels and were attached to glycerol unit by two types of linkers as potential building blocks for oligonucleotide synthesis. Spectral characteristics of the functionalized coumarin building blocks were measured.
