Solution Stability and Degradation Pathway of Deoxyribonucleoside Phosphoramidites in Acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O‐cyanoethyl‐N,N‐diisopropyl‐phosphoramidites of 5′‐O‐dimethoxytrityl (DMT) protected deoxyribonucleosides (dG^ib, dA^bz, dC^bz, T) were analyzed by HPLC‐MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile‐induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid‐catalyzed hydrolysis could also be reduced by addition of small amounts of base.     

Solution stability and degradation pathway of deoxyribonucleoside phosphoramidites in acetonitrile

The impuritiy profiles of acetonitrile solutions of the four standard O-cyanoethyl-N,N-diisopropyl-phosphoramidites of 5'-O-dimethoxytrityl (DMT) protected deoxyribonucleosides (dG(ib), dA(bz), dC(bz), T) were analyzed by HPLC-MS. The solution stability of the phosphoramidites decreases in the order T, dC>dA>dG. After five weeks storage under inert gas atmosphere the amidite purity was reduced by 2% (T, dC), 6% (dA), and 39% (dG), respectively. The main degradation pathways involve hydrolysis, elimination of acrylonitrile and autocatalytic acrylonitrile-induced formation of cyanoethyl phosphonoamidates. Consequently, the rate of degradation is reduced by reducing the water concentration in solution with molecular sieves and by lowering the amidite concentration. Acid-catalyzed hydrolysis could also be reduced by addition of small amounts of base.     

Diastereomeric Process Control in the Synthesis of 2′-O-(2-Methoxyethyl) Oligoribonucleotide Phosphorothioates as Antisense Drugs

Abstract

Coupling of 2′-O-methoxyethylsubstituted nucleoside phosphoramidites to 5′-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

Diastereomeric process control in the synthesis of 2'-O-(2-methoxyethyl) oligoribonucleotide phosphorothioates as antisense drugs

Coupling of 2'-O-methoxyethylsubstituted nucleoside phosphoramidites to 5'-hydroxyl group of a nucleoside or nucleotide on solid support is under stereochemical process control and is independent of scale, concentration, synthesizer, ratio of amidite diastereomers, solid support etc. However, activators and phosphate protecting groups do play a role in influencing the ratio of phosphorothioate diesters obtained by sulfurization of phosphite triesters.     

A Phosphoramidite-Based Synthesis of Phosphoramidate Amino Acid Diesters of Antiviral Nucleosides

Abstract

A general synthetic procedure is presented for the preparation of 5′-amino acid phosphoramidates of zidovudine (AZT), 3′-deoxy-2′,3′-didehydrothymidine (D4T), and 3′-fluoro-3′-deoxythymidine (FLT) from their corresponding phosphoramidites. These water soluble amino acid phosphoramidates are more non-polar than the parent nucleoside and exhibit high stability in aqueous media.     

Synthesis of Novel Nucleoside 5′-Triphosphates and Phosphoramidites Containing Alkyne or Amino Groups for the Postsynthetic Functionalization of Nucleic Acids

A series of novel nucleoside 5′-triphosphates and phosphoramidites containing alkyne or amino groups for the postsynthetic functionalization of nucleic acids were designed and synthesized. For this purpose, the new 3-aminopropoxypropynyl linker group was used. It contains two alternative functional capabilities: an amino group for the reaction of amino–alkynyl-modified oligonucleotides with corresponding activated esters and an alkyne group for the copper(I)-catalyzed azide–alkyne cycloaddition (CuAAC) reaction. It was shown that a variety of methods of the attachment of the new linker can be used to synthesize a diversity of modified pyrimidine nucleosides.     

Modified base compositions at degenerate positions of a mutagenic oligonucleotide enhance randomness in site-saturation mutagenesis.

Site-saturation mutagenesis, using degenerate oligonucleotide primers, is a frequently used method in introducing various mutations in a selected target codon. Oligonucleotides that are synthesized using equimolar concentrations of nucleoside phosphoramidites (dA, dC, dG, dT) in the positions to be saturated, result in a mutant population that is biased towards the original nucleotides. We found that this bias could be eliminated by modifying the concentrations of nucleoside phosphoramidites during the oligonucleotide synthesis. We synthesized eight degenerate oligonucleotides to saturate eight different codons, and sequenced a total of 344 mutagenized codons. In six of these eight oligonucleotides, we reduced to varying extents the concentrations of those nucleotides in the target positions that would form base pairs with the template. From the data, we analyzed the effects of different base compositions in the oligonucleotides when mutagenizing different codons, the influence of the positions of mismatches, and the significance of different non-Watson-Crick base pairs. Based on these results, we suggest levels to which different phosphoramidites should be reduced when synthesizing oligonucleotides for site-saturation mutagenesis.     

Hydroxyl-Functionalized DNAs with Different Linkers and Their Complmentary Duplex Stability

Tert-butyldiphenylsilyl (TBDPS) was testified to be an appropriate orthogonal protecting group for novel 7-hydroxyl-functionalized 8-aza-7-deaza-2′-deoxyadenosine analogues. It was stable in partial and complete hydrogenation reactions used for the different linker preparation. The corresponding phosphoramidites and hydroxyl-functionalized oligodeoxynucleotides were synthesized and identified. The thermal effect of the hydroxyl group with different linkers on DNA duplexes was evaluated. It provided a feasible strategy for the preparation of hydroxyl-functionalized DNAs for the nucleic acid research.     

Organometallic Intermediates in the Synthesis of Nucleoside Analogs

Abstract

The common nucleosides, modified or derivatized in some way at the heterocyclic ring carbons, include examples of structures which a r e useful as biological probes and chemotherapeutic agents. Like previous authors, we will use the term “nucleoside analog” for structures related to one of the common naturally occurring nucleosides. Nucleosicle analogs can be derivatives which differ by such minor modification as replacement of hydrogen by a single atom or derivatives which are grossly modified at both the carbohydrate and the base. Examples of the former include 5-fluoro-2′-deoxyuridine, an inhibitor of thymidylate synthetase as its 5′-phosphate, and 5′-iodo-2′-deoxyuridine, a clinically useful antiviral agent. Larger groups have frequently been linked to nucleoside as probes for enzymatic processes. Side chains in “nonrestricted positions” may be used to carry spectroscopic or chemically reactive probes, or provide the means to attach a molecule to an affinity column. Ultimately with positions of bulk tolerance defined, it may be possible to design “active site directed irreversible enzyme inhibitors” as defined by B.R. Baker. Nucleoside structures in which a side chain is attached at a pyrimidine or purine carbon will undoubtedly, in some instances be the most appropriate structure. Yet, these have typically been more difficult to synthesize than analogs with side chains attached to heteroatoms.     

Polymer Support Oligonucleotide Synthesis XVI: Synthesis of Oligonucleotides Using Suitably Protected Deoxynucleoside-N-morpholinophosphoramidites on Porous Glass Beads

Abstract

Several oligodeoxynucleotides had been synthesized on controlled pore glass beads using pure and stable 3′-O(5′-O, N-protected) nucleoside-methyl-N-morpholino phosphoramidites. The average coupling yield at each condensation step was about 94%.     

2,6-Diaminopurine nucleoside derivative of 9-ethyloxy-2-oxo-1,3-diazaphenoxazine (2-amino-Adap) for recognition of 8-oxo-dG in DNA

8-Oxo-2′-deoxyguanosine (8-oxo-dG) is a nucleoside resulting from oxidative damage and is known to be mutagenic. 8-Oxo-dG has been related to aging and diseases, including neurological disorders and cancer. Recently, we reported that a fluorescent nucleoside derivative, adenosine-1,3-diazaphenoxazine (Adap), forms a stable base pair with 8-oxo-dG in DNA with accompanying efficient quenching. In this study, a new Adap derivative having an additional 2-amino group on the adenosine moiety (2-amino-Adap) was designed with the anticipation of additional hydrogen bonding with the 8-oxo group of 8-oxo-dG. The properties of the ODN containing 2-amino-Adap were evaluated by measuring thermal stability and fluorescence quenching. In contrast to the previously designed Adap, the base-pairing and fluorescence quenching properties of 2-amino-Adap varied depending on the ODN sequence, and there was no clear indication of an additional hydrogen bond with 8-oxo-dG. Instead, the base pairing of 2-amino-Adap with dG was significantly destabilized compared with that of Adap with dG, resulting in improved selectivity for 8-oxo-dG in the human telomere DNA sequence. Thus, the telomere-targeting ODN probe containing 2-amino-Adap displayed selective, sensitive and quantitative detection of 8-oxo-dG in the human telomere DNA sequence in a light-up detection system using SYBR Green.     

4,5-BIS(ETHOXYCARBONYL)-[1,3]DIOXOLAN-2-YL AS A NEW ORTHOESTER-TYPE PROTECTING GROUP FOR THE 2′-HYDROXYL FUNCTION IN THE CHEMICAL SYNTHESIS OF RNA

We wish to report 4,5-bis(ethoxycarbonyl)-[1,3]dioxolan-2-yl as a new protecting for the 2′-hydroxyl function. Our cyclic orthoester-type group is compatible with the DMTr strategy for oligonucleotide synthesis. This group was introduced to the 2′-hydroxyl group of appropriately protected nucleoside derivatives in good yields under mild acidic conditions. Post-synthetic conversion of the moiety of this protecting group with an amine resulted in formation of a new amide moiety that is more stable to acid deprotection in aqueous solution, but it can still be easily removed by treatment with acids in organic solvents. In this article, we also describe the stability of not only the original and modified protecting groups but also internucleotidic phosphate linkages of protected RNA intermediates under deprotection conditions.     

DNA-Triesters - The Synthesis and Absolute Configuration Assignments at P-Stereogenic Centres

Abstract

Decadeoxyribonucleotide GGGAATTCCC and nine diastereomeric pairs of its mono-O-ethyl ester analogues were synthesized via phosphoramidite approach using the combination of 5′-DMT-base protected (except T) nucleoside 3′-(2-cyanoethyl N,N-diisopropyl phosphoramidites) and 3′-(0-ethyl N,N-diisopropyl phosphoramidites). Under conditions of release from solid support and removal of base-protecting groups (25% NH₄OH, 25°C, 48 h) 2-cyanoethyl groups were removed while O-ethyl phosphate triester functions were practically intact. Isolation of products and separation of diastereomers were performed by means of RP-HPLC. Absolute configuration at P-stereogenic centres was established via degradation of decamers into corresponding dinucleoside O-ethyl phosphates and stereochemical correlation with dinucleoside phosphorothioates of known configuration at phosphorus. Decadeoxyribonucleotide mono-O-ethyl esters were used for mapping the contact points between DNA and Eco RI endonuclease - the restriction enzyme which recognizes canonical sequence. GAATTC and cleaves unmodified DNA strands giving G and p AATTC.     

An Efficient Protection-Free One-Pot Chemical Synthesis of Modified Nucleoside-5′-Triphosphates

A simple, reliable, and an efficient “one-pot, three step” chemical method for the synthesis of modified nucleoside triphosphates such as 5-methylcytidine-5′-triphosphate (5-MeCTP), pseudouridine-5′-triphosphate (pseudoUTP) and N¹-methylpseudouridine-5′-triphosphate (N¹-methylpseudoUTP) starting from the corresponding nucleoside is described. The overall reaction involves the monophosphorylation of nucleoside, followed by the reaction with pyrophosphate and subsequent hydrolysis of the cyclic intermediate to furnish the corresponding NTP in moderate yields with high purity (>99.5%).     

Synthesis of oligodeoxynucleoside methylphosphonates utilizing the tert-butylphenoxyacetyl group for exocyclic amine protection.

Oligodeoxynucleoside methylphosphonates were synthesized using methylphosphonamidite monomers incorporating the base labile tert-butylphenoxyacetyl (t-BPA) protecting group on the exocyclic amines of dA, dC, and dG. Synthesis of the oligodeoxynucleoside methylphosphonates required only a small change in oxidation solution from standard DNA synthesis. The increased lability of the t-BPA group over the standard benzoyl and isobutyryl protection permitted the use of milder basic deprotection conditions. Deprotection of the nucleoside bases and release from support was best accomplished by a short treatment with ammonia saturated methanol. This procedure resulted in minimal backgone degradation with no base modifications. Analysis of the resultant oligodeoxynucleoside methylphosphonates by reverse phase HPLC and MALDI-TOF mass spectroscopy are described.     

Structural requirements for hybridization at the 5'-position are different in α-l-LNA as compared to β-D-LNA

The synthesis and biophysical evaluation of R and S-5'-Me-α-l-LNA nucleoside phosphoramidites and modified oligo-2'-deoxyribonucleotides is reported. Synthesis of the nucleoside phosphoramidites was accomplished in multi-gram quantities starting from diacetone glucose. The 5'-methyl group in the S configuration was introduced by reacting the sugar 5'-aldehyde with MeMgBr. Synthesis of the R-5'-Me isomer was accomplished from the S-5'-Me nucleoside by a late stage inversion using Mitsunobu conditions. Evaluation of the modified oligonucleotides in thermal denaturation experiments revealed that R-5'-Me-α-l-LNA showed similar RNA affinity as α-l-LNA while the S-5'-Me analog was less stabilizing. This result is in contrast to the β-d-series where the S-5'-Me isomer showed LNA-like affinity for RNA while the R-5'-Me group completely reversed the stabilization effect on duplex thermostability.     

HYDROLYSIS OF 2′-DEOXY AND 2′-FLUORONUCLEOSIDE-3′-PHOSPHODlESTERS

Abstract

Two nucleoside analogs were synthesized to test the ribose conformational and electronic effects on phosphate hydrolysis at the 3′ position. It was found that under alkaline conditions, a 2′-fluoro-nucleoside (C3′-endo) resulted in a phosphate degradation that was ten times faster than the 2′-deoxynucleoside analog (C2′-endo). In addition to kinetic differences, product distributions will be presented.     

Synthesis of 3'-thioribonucleosides and their incorporation into oligoribonucleotides via phosphoramidite chemistry.

Oligoribonucleotides containing 3'-S-phosphorothiolate linkages are valuable probes in nucleic acid biochemistry, but their accessibility has been limited because 3'-thioribonucleoside phosphoramidites have not been available. We synthesized 3'-thioribonucleoside derivatives (C, G, and U) via glycosylations of nucleoside bases with 3-S-thiobenzoyl-5-O-toluoyl-1,2-O-diacetylfuranose 5, which was obtained from 1 ,2-O-isopropylidene-5-O-toluoyl-3-trifluoromethane-sulfonyl-alpha-D-x ylofuranose 2 by SN2 displacement with sodium thiobenzoate. Additionally, a 3'-thioinosine derivative was prepared from inosine via direct modification of the ribose, analogous to the previously reported synthesis of 3'-thioadenosine, except that the intermediate 2',3'-epoxide 9 was first protected as the 5'-O-tert-butyldiphenylsilyl ether prior to subsequent synthetic steps. This hydrophobic silyl group facilitated extraction and isolation of synthetic intermediates. After removal of the protecting groups, the 3'-thionucleosides (C, G, U, and I) were treated with 2,2'-dipyridyl disulfide to protect the free thiol group as a disulfide. The 3'-thionucleosides were converted to the corresponding phosphorothioamidites using procedures analogous to those for standard phosphoramidites. The amino groups of 3'-thiocytidine and 3'-thioguanosine were protected as benzoyl and isobutyryl amides, respectively, and the 5'- and 2'-hydroxyl groups of each nucleoside were protected as dimethoxytrityl and tert-butyldimethylsilyl ethers, respectively. The 3'-thiol group was deprotected by reduction with DTT and phosphitylated to afford analytically pure 3'-S-phosphorothioamidites 15, which were incorporated into oligoribonucleotides by solid-phase synthesis. Chemical assays and mass spectrometry of the synthetic RNA showed that ribose-3'-S-phosphorothiolate linkages were installed correctly and efficiently into RNA oligonucleotides using phosphoramidite chemistry.     

Selective O-phosphitilation with nucleoside phosphoramidite reagents.

In contrast to tetrazole, pyridine hydrochloride/imidazole converts nucleoside phosphoramidites to intermediates that show a high preference for phosphitilating hydroxyl groups relative to nucleoside amino groups. Use of this activating agent and incorporation of a pyridine hydrochloride/aniline wash step in the synthetic cycles permit synthesis of mixed base twenty-mer oligonucleotides from nucleoside reagents containing unprotected amino groups. This approach should be useful for the synthesis of oligonucleotide analogues containing substituents sensitive to reagents used in conventional deblocking steps. Pyridine hydrochloride itself is an effective reagent for activating nucleoside methylphosphonoamidites and ribonucleoside phosphoramidites, as well as deoxyribonucleoside phosphoramidites, when high O/N selectivety is not needed.     

TrimerDimer: an oligonucleotide-based saturation mutagenesis approach that removes redundant and stop codons

9-fluorenylmethoxycarbonyl (Fmoc) and 4,4′-dimethoxytrityl (DMTr) are orthogonal hydroxyl protecting groups that have been used in conjunction to assemble oligonucleotide libraries whose variants contain wild-type and mutant codons randomly interspersed throughout a focused DNA region. Fmoc is labile to organic bases and stable to weak acids, whereas DMTr behaves oppositely. Based on these chemical characteristics, we have now devised TrimerDimer, a novel codon-based saturation mutagenesis approach that removes redundant and stop codons during the assembly of degenerate oligonucleotides. In this approach, five DMTr-protected trinucleotide phosphoramidites (dTGG, dATG, dTTT, dTAT and dTGC) and five Fmoc-protected dinucleotide phosphoramidites (dAA, dTT, dAT, dGC and dCG) react simultaneously with a starting oligonucleotide growing on a solid support. The Fmoc group is then removed and the incorporated dimers react with a mixture of three DMTr-protected monomer phosphoramidites (dC, dA and dG) to produce 15 trinucleotides: dCAA, dAAA, dGAA, dCTT, dATT, dGTT, dCAT, dAAT, dGAT, dCGC, dAGC, dGGC, dCCG, dACG and dGCG. After one mutagenic cycle, 20 codons are generated encoding the 20 natural amino acids. TrimerDimer was tested by randomizing the four contiguous codons that encode amino acids L64–G67 of an engineered, nonfluorescent GFP protein. Sequencing of 89 nonfluorescent mutant clones and isolation of two fluorescent mutants confirmed the principle.