Time‐Dependent Thermocontrol of the Hydrophilic and Lipophilic Properties of DNA Oligonucleotide Prodrugs
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- Table of Contents
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Abstract
This unit describes the preparation of alkylthioalkylated and formamidoalkylated alcohols, an amidoalkylated alcohol, a hydroxylalkylated phosphoramidate, and their phosphoramidothioate derivatives, all of which have been identified as heat?sensitive thiophosphate?protecting groups in the development of thermolytic immunostimulatory DNA prodrugs. The alcohols are converted to their deoxyribonucleoside phosphoramidite derivatives, which are then used in the preparation of thermosensitive dinucleoside phosphorothioates. The thiophosphate?protecting groups of these dinucleoside phosphorothioates presumably undergo thermolytic cyclodeesterification at elevated temperature under essentially neutral conditions to release the desired phosphorothioate diester function. On the basis of their thermolytic deprotection kinetics, one can identify those thiophosphate?protecting groups that (i) may be useful for thiophosphate protection of CpG motifs of immunostimulatory DNA oligonucleotides (CpG ODNs); (ii) are suitable for protection of phosphodiester functions flanking the CpG motifs; and (iii) offer adequate protection of terminal phosphodiester functions against ubiquitous extracellular and intracellular exonucleases that may be found in biological environments. Curr. Protoc. Nucleic Acid Chem. 43:4.42.1?4.42.31. © 2010 by John Wiley & Sons, Inc.
Keywords: oligonucleotide prodrugs; thiophosphate protecting groups; thermolytic deprotection; thermolytic conditions; solid?phase oligonucleotide synthesis; CpG ODNs; lipophilicity; hydrophilicity
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PDF or HTML at Wiley Online LibraryTable of Contents
- Introduction
- Basic Protocol 1: Synthesis, Purification, and Characterization of Alkylthioalkylated Alcohols
- Basic Protocol 2: Synthesis, Purification, and Characterization of Formamidoalkylated Alcohols
- Basic Protocol 3: Synthesis, Purification, and Characterization of Hydroxyalkylated Phosphoramidate and Phosphoramidothioate Derivatives
- Basic Protocol 4: Synthesis, Purification, and Characterization of Alkylthioalkylated, Formamidoalkylated, Phosphoramidated, and Phosphoramidothioated Deoxyribonucleoside Phosphoramidite Derivatives
- Basic Protocol 5: Solid‐Phase Synthesis of Thermosensitive Dinucleoside Phosphorothioate Triesters and Their Conversion to Phosphorothioate Diesters Under Thermolytic Conditions
- Basic Protocol 6: Automated Solid‐Phase Synthesis of a Heat‐Sensitive DNA Prodrug Model and Its Selective Thermolytic Deprotection
- Commentary
- Literature Cited
- Figures
- Tables
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PDF or HTML at Wiley Online LibraryMaterials
Basic Protocol 1: Synthesis, Purification, and Characterization of Alkylthioalkylated Alcohols
Materials
Basic Protocol 2: Synthesis, Purification, and Characterization of Formamidoalkylated Alcohols
Materials
Basic Protocol 3: Synthesis, Purification, and Characterization of Hydroxyalkylated Phosphoramidate and Phosphoramidothioate Derivatives
Materials
Basic Protocol 4: Synthesis, Purification, and Characterization of Alkylthioalkylated, Formamidoalkylated, Phosphoramidated, and Phosphoramidothioated Deoxyribonucleoside Phosphoramidite Derivatives
Materials
Basic Protocol 5: Solid‐Phase Synthesis of Thermosensitive Dinucleoside Phosphorothioate Triesters and Their Conversion to Phosphorothioate Diesters Under Thermolytic Conditions
Materials
Basic Protocol 6: Automated Solid‐Phase Synthesis of a Heat‐Sensitive DNA Prodrug Model and Its Selective Thermolytic Deprotection
Materials
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Figure 4.42.1 Synthesis of 5‐(methylthio)pentan‐1‐ol (S.5 ) and 5‐(isopropylthio)pentan‐1‐ol (S.6 ) from methyl 5‐chlorovalerate. See Brown et al. () for the preparation of (CH3 )2 CHSNa. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.2 Synthesis of 4‐(trifluoromethylthio)butan‐1‐ol (S.7 ) from the reaction of 4‐mercaptobutan‐1‐ol with 1‐trifluoromethyl‐1,3‐dihydro‐3,3‐dimethyl‐1,2‐benziodoxole. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.3 Preparation of 4‐(methoxymethylthio)butan‐1‐ol (S.8 ) from 4‐mercaptobutan‐1‐ol. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.4 Preparation of 2‐(2‐methylthioethoxy)ethanol (S.9 ) from 2‐(methylthio)ethanol (S.2 ). Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.5 Synthesis of 3‐(methylthiomethoxy)propan‐1‐ol (S.10 ) and 2‐(methylthiomethoxy)ethanol (S.11 ) from methythiomethyl acetate. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.6 Preparation of 3‐( N ‐formyl‐ N ‐methylamino)propan‐1‐ol (S.13 ) from 3‐aminopropan‐1‐ol. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.7 General synthesis of the hydroxyalkylated phosphoramidate (S.19 ) and phosphoramidothioate derivatives (S.20 ‐S.25 ). Abbreviations: DMTr, 4,4′‐dimethoxytrityl; Et, ethyl; i ‐Pr, isopropyl; N(CH2 CH2 )2 O, morpholinyl. Adapted from Grajkowski et al. (). Reproduced by permission of the Royal Society of Chemistry (RSC) for the Centre National de la Recherche Scientifique (CNRS) and the RSC. View Image -
Figure 4.42.8 Preparation of the deoxyribonucleoside phosphoramidites S.27 ‐S.48 . Abbreviations: CE, 2‐cyanoethyl; DMTr, 4,4′‐dimethoxytrityl; Et, ethyl; i ‐Pr, isopropyl; Thy, thymin‐1‐yl; N(CH2 CH2 )2 O, morpholinyl. Adapted from Ausín et al. () with permission from Elsevier, and from Grajkowski et al. () with permission from the Royal Society of Chemistry. View Image -
Figure 4.42.9 Solid‐phase synthesis of dinucleoside phosphorothioate triesters S.50 ‐S.71 and removal of the thiophosphate protecting groups under thermolytic conditions. Abbreviations: CPG, long‐chain alkylamine controlled‐pore glass; DMTr, 4,4′‐dimethoxytrityl; R, thermosensitive thiophosphate‐protecting groups derived from S.1 ‐S.15 and S.19 ‐S.25 ; Thy, thymin‐1‐yl. Adapted from Ausín et al. () with permission from Elsevier, and from Grajkowski et al. () with permission from the Royal Society of Chemistry. View Image -
Figure 4.42.10 PAGE analysis of the thermolytic deprotection of DNA oligonucleotide S.72 under denaturing conditions. Left lane: RP‐HPLC‐purified control DNA S.73 . Right lane: RP‐HPLC‐purified DNA S.72 heated in PBS (pH 7.4) for 3 hr at 90°C. Oligonucleotides were visualized as blue bands after staining with Stains‐All. Bromphenol blue was used as a marker and shows as a large band at the bottom of the gel in each lane. Reprinted from Ausín et al. () with permission from Elsevier. View Image -
Figure 4.42.11 Relative solubility of DNA oligonucleotide S.72 upon thermal deprotection of the fma thiophosphate‐protecting groups. 100% relative solubility is defined as the maximum absorbance ( A max ) of the DNA sequence at 268 nm and 90°C. Thus, % relative solubility = ( A / A max ) × 100 for each time point. Reprinted from Ausìn et al. () with permission from Elsevier. View Image
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