The Experts below are selected from a list of 261 Experts worldwide ranked by ideXlab platform
Wojciech J. Stec - One of the best experts on this subject based on the ideXlab platform.
-
mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10 23
FEBS Journal, 2007Co-Authors: Barbara Nawrot, Kinga Widera, Marzena Wojcik, Beata Rebowska, Genowefa Nowak, Wojciech J. StecAbstract:The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10–23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3 mm Mg2+ or 3 mm Mn2+. A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proRP or proSP) located at seven positions of the core (P2, P4 and P9–13) involved in direct coordination with a divalent metal ion(s). By contrast, Phosphorothioates at positions P3, P6, P7 and P14–16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg2+, and this was reversed in the presence of Mn2+. Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proRP oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G6 contributes to metal ion binding and that this interaction is essential for 10–23 deoxyribozyme catalytic activity.
-
Mapping of the functional phosphate groups in the catalytic core of deoxyribozyme 10–23
The FEBS Journal, 2007Co-Authors: Barbara Nawrot, Kinga Widera, Marzena Wojcik, Beata Rebowska, Genowefa Nowak, Wojciech J. StecAbstract:The RNA phosphodiester bond cleavage activity of a series of 16 thio-deoxyribozymes 10–23, containing a P-stereorandom single phosphorothioate linkage in predetermined positions of the catalytic core from P1 to P16, was evaluated under single-turnover conditions in the presence of either 3 mm Mg2+ or 3 mm Mn2+. A metal-specificity switch approach permitted the identification of nonbridging phosphate oxygens (proRP or proSP) located at seven positions of the core (P2, P4 and P9–13) involved in direct coordination with a divalent metal ion(s). By contrast, Phosphorothioates at positions P3, P6, P7 and P14–16 displayed no functional relevance in the deoxyribozyme-mediated catalysis. Interestingly, phosphorothioate modifications at positions P1 or P8 enhanced the catalytic efficiency of the enzyme. Among the tested deoxyribozymes, thio-substitution at position P5 had the largest deleterious effect on the catalytic rate in the presence of Mg2+, and this was reversed in the presence of Mn2+. Further experiments with thio-deoxyribozymes of stereodefined P-chirality suggested direct involvement of both oxygens of the P5 phosphate and the proRP oxygen at P9 in the metal ion coordination. In addition, it was found that the oxygen atom at C6 of G6 contributes to metal ion binding and that this interaction is essential for 10–23 deoxyribozyme catalytic activity.
-
new approach to the synthesis of oligodeoxyribonucleotides modified with Phosphorothioates of predetermined sense of p chirality
Tetrahedron Letters, 2005Co-Authors: Barbara Nawrot, Beata Rebowska, Katarzyna Cieślinska, Wojciech J. StecAbstract:Abstract Appropriately protected, diastereomerically pure dinucleoside Phosphorothioates, obtained by the stereocontrolled oxathiaphospholane method, were S -alkylated with 2-nitrobenzyl bromide and then converted into their 3′- O -phosphoramidites. The corresponding S -protected dinucleotide building blocks were successfully used for the synthesis of oligonucleotides containing P -stereodefined phosphorothioate bonds at preselected positions.
-
the p stereocontrolled synthesis of po ps chimeric oligonucleotides by incorporation of dinucleoside Phosphorothioates bearing an o 4 nitrophenyl phosphorothioate protecting group
European Journal of Organic Chemistry, 2005Co-Authors: Lucyna A. Wozniak, Marcin Góra, Sophie Mourgues, Geneviève Pratviel, Bernard Meunier, Malgorzata Bukowieckamatusiak, Wojciech J. StecAbstract:The synthesis of protected model dinucleoside (3',5')-O-aryl Phosphorothioates, their separation into pure diastereomers, and their successful incorporation into oligonucleotides followed by stereospecific deprotection of the O-aryl phosphorothioate function with oximate ion (inversion) enables the preparation of chimeric PO/PS-oligonucleotides with a predetermined sense of P-chirality at each internucleotide phosphorothioate position. The absolute configuration at the phosphorus of the internucleotide O-aryl phosphorothioate in “dimeric building blocks” has been assigned. The 3'-terminal SP-phosphorothioate linkages effectively protect such chimeric constructs from degradation by human plasma exonuclease (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
-
The P‐Stereocontrolled Synthesis of PO/PS‐Chimeric Oligonucleotides by Incorporation of Dinucleoside Phosphorothioates Bearing an O‐4‐Nitrophenyl Phosphorothioate Protecting Group
European Journal of Organic Chemistry, 2005Co-Authors: Lucyna A. Wozniak, Marcin Góra, Malgorzata Bukowiecka-matusiak, Sophie Mourgues, Geneviève Pratviel, Bernard Meunier, Wojciech J. StecAbstract:The synthesis of protected model dinucleoside (3',5')-O-aryl Phosphorothioates, their separation into pure diastereomers, and their successful incorporation into oligonucleotides followed by stereospecific deprotection of the O-aryl phosphorothioate function with oximate ion (inversion) enables the preparation of chimeric PO/PS-oligonucleotides with a predetermined sense of P-chirality at each internucleotide phosphorothioate position. The absolute configuration at the phosphorus of the internucleotide O-aryl phosphorothioate in “dimeric building blocks” has been assigned. The 3'-terminal SP-phosphorothioate linkages effectively protect such chimeric constructs from degradation by human plasma exonuclease (© Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005)
Sang Soo Hah - One of the best experts on this subject based on the ideXlab platform.
-
two step synthesis of 5 deoxy 5 thioguanosine 5 monophosphorothioate and its incorporation efficiency into 5 terminus of rna for preparation of thiol functionalized rna
Tetrahedron Letters, 2010Co-Authors: Il-hyun Kim, Seonmi Shin, Yong-joo Jeong, Sang Soo HahAbstract:Abstract Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal Phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to Phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.
-
Two-step synthesis of 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate and its incorporation efficiency into 5′-terminus of RNA for preparation of thiol-functionalized RNA
Tetrahedron Letters, 2010Co-Authors: Il-hyun Kim, Seonmi Shin, Yong-joo Jeong, Sang Soo HahAbstract:Abstract Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal Phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to Phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.
Il-hyun Kim - One of the best experts on this subject based on the ideXlab platform.
-
two step synthesis of 5 deoxy 5 thioguanosine 5 monophosphorothioate and its incorporation efficiency into 5 terminus of rna for preparation of thiol functionalized rna
Tetrahedron Letters, 2010Co-Authors: Il-hyun Kim, Seonmi Shin, Yong-joo Jeong, Sang Soo HahAbstract:Abstract Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal Phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to Phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.
-
Two-step synthesis of 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate and its incorporation efficiency into 5′-terminus of RNA for preparation of thiol-functionalized RNA
Tetrahedron Letters, 2010Co-Authors: Il-hyun Kim, Seonmi Shin, Yong-joo Jeong, Sang Soo HahAbstract:Abstract Several 5′-modifications of RNA molecules have been shown to have broad applications in studying RNA structures, mapping RNA–protein interactions, and in vitro selection of catalytic RNAs. While phosphorothioate modification is one of the most popular methods for functionalizing the 5′-terminus of RNA by a transcription or kinase reaction, conjugation of terminal Phosphorothioates with fluorophores has been achieved only with a low efficiency. To overcome this limitation, we have developed a two-step synthetic method for 5′-deoxy-5′-thioguanosine-5′-monophosphorothioate by combining two known reactions and measured its incorporation efficiency into the 5′-terminus of RNA by in vitro transcription using T7 RNA polymerase that requires guanosine to efficiently initiate transcription, followed by treatment of alkaline phosphatase, yielding a terminal sulfhydryl group at the 5′-termini of RNA molecules. Since the sulfhydryl group can be used as an alternative to Phosphorothioates, our method may provide a useful route to efficiently introduce reporters, such as fluorophores, into the 5′-terminus of RNA via a stable thio-linker, or to tether the oligomer to a solid support.
Alex Azhayev - One of the best experts on this subject based on the ideXlab platform.
-
Synthesis of peptide-oligonucleotide phosphorothioate conjugates by convergent or stepwise solid-phase strategies.
Nucleosides Nucleotides and Nucleic Acids, 2001Co-Authors: Maxim Antopolsky, Alex AzhayevAbstract:A convergent strategy was employed to link eight 10-27-mer peptides to oligonucleotide Phosphorothioates, resulting in twenty-six various conjugates. A stepwise synthesis strategy for the preparation of peptide-oligonucleotide phosphorothioate conjugates, employing Fmoc peptide chemistry, was developed and applied to the synthesis of four conjugates. Three of these conjugates contained either a 10 or 16-mer peptide, incorporating either 2 or 3 arginine residues, respectively.
-
stepwise solid phase synthesis of peptide oligonucleotide phosphorothioate conjugates employing fmoc peptide chemistry
Tetrahedron Letters, 2000Co-Authors: Maxim Antopolsky, Alex AzhayevAbstract:Abstract A straightforward stepwise method for the preparation of peptide–oligonucleotide phosphorothioate conjugates, was developed, based on the highly efficient Fmoc peptide solid phase synthesis, followed by oligonucleotide phosphorothiate chain assembly. The three conjugates synthesized contained 15- or 17-mer oligonucleotide Phosphorothioates and 10- or 16-mer peptides, incorporating two or three arginine residues.
-
Stepwise solid-phase synthesis of peptide–oligonucleotide phosphorothioate conjugates employing Fmoc peptide chemistry
Tetrahedron Letters, 2000Co-Authors: Maxim Antopolsky, Alex AzhayevAbstract:Abstract A straightforward stepwise method for the preparation of peptide–oligonucleotide phosphorothioate conjugates, was developed, based on the highly efficient Fmoc peptide solid phase synthesis, followed by oligonucleotide phosphorothiate chain assembly. The three conjugates synthesized contained 15- or 17-mer oligonucleotide Phosphorothioates and 10- or 16-mer peptides, incorporating two or three arginine residues.
Radhakrishnan P. Iyer - One of the best experts on this subject based on the ideXlab platform.
-
in vivo stability disposition and metabolism of a hybrid oligonucleotide phosphorothioate in rats
Biochemical Pharmacology, 1995Co-Authors: Ruiwen Zhang, Zhiwei Jiang, Hui Zhao, Xueshu Zhang, Robert B. Diasio, Ivan Habus, L U Zhihong, Radhakrishnan P. IyerAbstract:Oligodeoxynucleotide Phosphorothioates containing segments of 2′-O-methyloligoribo-nucleotide Phosphorothioates at both 3′- and 5′-ends (hybrid oligonucleotide) have been shown to be potent antisense agents. In the present study, in vivo biostability, disposition, and excretion of a 25-mer hybrid oligonucleotide were determined in rats after i.v. bolus administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. The plasma disappearance curve for the hybrid oligonucleotide could be described by a two-compartmental model, with half-lives of 0.34 and 52.02 hr, respectively. The majority of the radioactivity in plasma was associated with the intact hybrid oligonucleotide. Urinary excretion represented the major pathway of elimination, with 21.98 ± 3.21% (mean ± SD) of the administered dose excreted within 24 hr and 38.13 ± 2.99% over 240 hr post-dosing. The majority of the radioactivity in urine was associated with the degradative products with lower molecular weights, but the intact form was also detected by HPLC analysis. Fecal excretion was a minor pathway of elimination with 2.34 ± 0.13% of the administered dose excreted over 24 hr and 6.74 ± 0.40% over 240 hr post-dosing. A wide tissue distribution of hybrid oligonucleotide was observed based on radioactivity levels, and analysis by HPLC showed that the majority of the radioactivity in tissues was associated with the intact hybrid oligonucleotide. Further analyses of the experimental data provided a comprehensive pharmacokinetic analysis of hybrid oligonucleotide in each tissue. Compared with a previously examined oligodeoxynucleotide phosphorothioate (GEM 91) that has a similar nucleotide sequence, the hybrid oligonucleotide had a shorter distribution half-life and a longer elimination half-life, based on the quantitation of radioactivity in plasma. Although it had a similar tissue distribution pattern compared with other oligonucleotide Phosphorothioates such as GEM 91, the hybrid oligonucleotide was more stable in vivo, which may be important in the development of antisense oligonucleotides as therapeutic agents.
-
prodrugs of oligonucleotides the acyloxyalkyl esters of oligodeoxyribonucleoside Phosphorothioates
Bioorganic Chemistry, 1995Co-Authors: Radhakrishnan P. Iyer, Sudhir AgrawalAbstract:Abstract In model studies toward development of prodrugs of oligonucleoside Phosphorothioates, R p and S p , S -alkyl phosphorothiolates 3a-d were prepared by chemoselective S-alkylation of R p acid S p dinucleoside phosphorothioate 2 with iodoalkyl acylates 9a-d. When incubated with human serum or porcine liver esterase (PLE), stereospecific conversion of 3a-d to R p and S p 2 was observed. Stereodifferentiation was also noted in the hydrolysis- S p faster than R p 3a-c, with serum, and R p faster than S p 3a-c, with PLE. In the case of hindered analogs 3c, desulfurized product 4 was also formed along with 2. Mechanisms to account for this product profile obtained during the hydrolysis are proposed. Similarly, bioreversion of acyloxyalkyl oligonucleoside Phosphorothioates 15, 16, and 17 to 18, 19, and 20, respectively, was demonstrated by 31 P NMR and PAGE.
-
In vivo stability, disposition and metabolism of a “hybrid” oligonucleotide phosphorothioate in rats
Biochemical Pharmacology, 1995Co-Authors: Ruiwen Zhang, Zhiwei Jiang, Hui Zhao, Xueshu Zhang, Robert B. Diasio, Ivan Habus, Radhakrishnan P. IyerAbstract:Oligodeoxynucleotide Phosphorothioates containing segments of 2′-O-methyloligoribo-nucleotide Phosphorothioates at both 3′- and 5′-ends (hybrid oligonucleotide) have been shown to be potent antisense agents. In the present study, in vivo biostability, disposition, and excretion of a 25-mer hybrid oligonucleotide were determined in rats after i.v. bolus administration of the 35S-labeled oligonucleotide at a dose of 30 mg/kg. The plasma disappearance curve for the hybrid oligonucleotide could be described by a two-compartmental model, with half-lives of 0.34 and 52.02 hr, respectively. The majority of the radioactivity in plasma was associated with the intact hybrid oligonucleotide. Urinary excretion represented the major pathway of elimination, with 21.98 ± 3.21% (mean ± SD) of the administered dose excreted within 24 hr and 38.13 ± 2.99% over 240 hr post-dosing. The majority of the radioactivity in urine was associated with the degradative products with lower molecular weights, but the intact form was also detected by HPLC analysis. Fecal excretion was a minor pathway of elimination with 2.34 ± 0.13% of the administered dose excreted over 24 hr and 6.74 ± 0.40% over 240 hr post-dosing. A wide tissue distribution of hybrid oligonucleotide was observed based on radioactivity levels, and analysis by HPLC showed that the majority of the radioactivity in tissues was associated with the intact hybrid oligonucleotide. Further analyses of the experimental data provided a comprehensive pharmacokinetic analysis of hybrid oligonucleotide in each tissue. Compared with a previously examined oligodeoxynucleotide phosphorothioate (GEM 91) that has a similar nucleotide sequence, the hybrid oligonucleotide had a shorter distribution half-life and a longer elimination half-life, based on the quantitation of radioactivity in plasma. Although it had a similar tissue distribution pattern compared with other oligonucleotide Phosphorothioates such as GEM 91, the hybrid oligonucleotide was more stable in vivo, which may be important in the development of antisense oligonucleotides as therapeutic agents.
-
stereospecific bio reversibility of dinucleoside s alkyl phosphorothiolates to dinucleoside Phosphorothioates
Bioorganic & Medicinal Chemistry Letters, 1994Co-Authors: Radhakrishnan P. Iyer, Sudhir AgrawalAbstract:In model studies towards prodrugs of oligonucleoside phosphorothiolates eg., 1, the dinucleoside S-alkyl phosphorothiolates 3a-c were prepared by chemoselective alkylation of d[TpsT] with the iodoalkyl acylates 8a-c. Upon incubation with serum or porcine liver esterase, stereospecific conversion of Rp and Sp 3c to 2 was observed along with the desulfurized product 4.