Deoxynucleoside Phosphoramidites Research Articles

Synthesis of 4'-Methoxy 2'-Deoxynucleoside Phosphoramidites for Incorporation into Oligonucleotides.

This unit contains detailed synthetic protocols for the preparation of 4'-methoxy 2'-deoxynucleoside phosphoramidite monomers for A, G, C, T, and U. First, 3'-silyl-protected 2'-deoxynucleosides (dNs) are converted in two steps to 4',5'-enol acetates as the key starting compounds. Next, 4'-methoxy dNs are prepared by a one-pot procedure comprising N-iodosuccinimide-promoted methoxylation, hydrolysis, and reduction of the formed intermediates. Finally, 3'-phosphoramidites of 4'-methoxy dNs are obtained by a routine three-step procedure. Title phosphoramidite monomers are suitable for the synthesis of oligonucleotides on solid phase according to conventional amidite chemistry. 4'-Methoxy substitution represents a simple modification of the sugar part of dNs, where β-D-erythro epimers preferentially adopt N-type (C3'-endo, RNA-like) conformation. Moreover, it imparts superior chemical stability, nuclease resistance, and excellent hybridization properties to modified 4'-methoxyoligodeoxynucleotides. The strong tendency toward RNA-selective hybridization suggests its potential utilization in antisense and/or RNAi technologies. © 2016 by John Wiley & Sons, Inc.

The Degradation of dG Phosphoramidites in Solution

The reaction of 2′-deoxynucleoside phosphoramidites with water is an important degradation reaction that limits the lifetimes of reagents used for chemical deoxyoligonucleotide synthesis. The hydrolysis of nucleoside phosphoramidites in solution has therefore been investigated. The degree of degradation depends not only on the presence of water but also on the specific nucleoside, 2′-deoxyguanosine (dG) being especially susceptible. Additionally, the nature of the group protecting the exocyclic amine on the nucleoside base strongly influences the rate of hydrolysis. For dG, the degradation is second order in phosphoramidite concentration, indicating autocatalysis of the hydrolysis reaction. Comparison of the degradation rates of dG phosphoramidites with different protecting groups as well as with phosphoramidites containing bases that are structurally similar to dG affords clues to the nature of how dG catalyzes its own destruction and indicates a direct correlation between ease of protecting group removal and propensity to undergo autocatalytic degradation.

2-Benzamidoethyl group--a novel type of phosphate protecting group for oligonucleotide synthesis.

A number of 5'-O-(4,4'-dimethoxytrityl)thymidine N,N-diisopropylamino phosphoramidites protected at P(III) with derivatives of 2-benzamidoethanol were synthesized and incorporated into synthetic oligonucleotides. Depending on substitution patterns at the alkyl chain, amido group, and phenyl ring, the time required for removal of these protecting groups using concentrated ammonium hydroxide varied from 48 h at 55 degrees C to 25 min at 25 degrees C. Of the 11 groups studied, 2-[N-isopropyl-N- (4-methoxybenzoyl)amino]ethyl- (H) and omega-(thionobenzoylamino)alkyl protections (I and K) were most easily removed. Derivatives of the 2-[N-methyl-N-benzoylamino]ethyl group (E-G) demonstrated moderate stability, but those of the 2-(N-benzoylamino)ethyl group (A-C) were the most stable. For the most reactive group, H, a phosphitylating reagent, bisamidite 60, was synthesized and used in the preparation of four deoxynucleoside phosphoramidites 28 and 65-67, plus the 2'-O-(2-methoxyethyl)-5-methyluridine phosphoramidite 68. All of these novel building blocks were successfully tested in the preparation of natural, 20-mer oligonucleotides and their phosphorothioate analogues. With the model phosphotriester 37, the mechanism of deprotection was studied and revealed, in the case of group H, a pH-independent formation of the 2-oxazolinium cation 47. Under aqueous conditions, 47 gave 54, which in turn was converted in the presence of ammonia to a number of identified products. It is important to note that none of the products formed was reactive toward the oligonucleotide backbone or nucleic bases. Thus, a general strategy for protection of internucleosidic phosphodiester groups is described, which may also find application in synthetic organic chemistry of phosphorus(III) and (V).

Production by quantitative photolithographic synthesis of individually quality checked DNA microarrays.

For DNA chip analyses, oligonucleotide quality has immense consequences for accuracy, sensitivity and dynamic range. The quality of chips produced by photolithographic in situ synthesis depends critically on the efficiency of photo-deprotection. By means of base-assisted enhancement of this process using 5'-¿2-(2-nitrophenyl)-propyloxycarbonyl-2'-deoxynucleoside phosphoramidites, synthesis yields improved by at least 12% per condensation compared to current chemistries. Thus, the eventual total yield of full-length oligonucleotide is increased more than 10-fold in the case of 20mers. Furthermore, the quality of every individual array position was checked quantitatively after synthesis. Subsequently, the quality tested chips were used in successive hybridisation experiments.

Proofing of Photolithographic DNA Synthesis with 3‘,5‘-Dimethoxybenzoinyloxycarbonyl-Protected Deoxynucleoside Phosphoramidites

We have evaluated in a microchip format the photochemical solid-phase phosphoramidite DNA synthesis method we previously developed. A set of nucleoside building blocks with “easy-off” base protecting groups was prepared bearing photolabile 5‘-O-dimethoxybenzoincarbonate (DMBOC) groups. Photolysis rates and cycle yields for these DMBOC-protected nucleotides covalently attached to planar, derivatized glass surfaces were determined by fluorescence imaging-based methods earlier developed by McGall et al. and described in detail elsewhere. Data were obtained for both 280/310 and 365/400 nm irradiation in a range of solvents. Deprotection of the DMBOC occurs fastest in a nonpolar medium or without solvent. The coupling efficiency of these amidites in the synthesis of homopolymers was determined to be in the range 80−97%, with purines generally showing lower efficiency than pyrimidines. These DMBOC-protected monomers were used to prepare a 4 × 4 array of 16 decanucleotides of the sequence 5‘-AAXTAXCTAC−chip, whe...

The Efficiency of Light-Directed Synthesis of DNA Arrays on Glass Substrates

New methods based on photolithography and surface fluorescence were used to determine photodeprotection rates and stepwise yields for light-directed oligonucleotide synthesis using photolabile 5‘-(((α-methyl-2-nitropiperonyl)oxy)carbonyl)(MeNPOC)-2‘-deoxynucleoside phosphoramidites on planar glass substrates. Under near-UV illumination (primarily 365 nm) from a mercury light source, the rate of photoremoval of the MeNPOC protecting group was found to be independent of both the nucleotide and length of the growing oligomer (t1/2 = 12 s at 27.5 mW/cm2). A moderate dependence on solvent polarity was observed, with photolysis proceeding most rapidly in the presence of nonpolar solvents or in the absence of solvent (e.g., t1/2 = 10−13 s at 27.5 mW/cm2). In solution, the photolysis rate was linearly dependent on light intensity over the range 5−50 mW/cm2. Average stepwise yields for the synthesis of dodecamer oligonucleotides were in the range of 92−94%, using monomers based on N6-(phenoxyacetyl)-2‘-deoxyadenos...

Insertion of the fragile 2′-deoxyribosylurea residue into oligodeoxynucleotides

Deoxyribosylurea is an alkali sensitive DNA defect induced by the action of ionizing radiation. Oligonucleotides bearing this residue were synthesized using 5′-O-(4-methoxytrilyl)-2′-deoxyribosylurea-3′-O-(2-cyanoethyl-N,N'-diisopropyl) phosphoramidite and PAC protected deoxynucleoside phosphoramidites.

Chemical synthesis of DNA

Overview of DNA synthesis methodologies and polymer-supported synthesis of DNA using deoxynucleoside phosphoramidites.

ChemInform Abstract: Modern Methods of Nucleic Acid Synthesis on Polymeric Supports

Abstract Deoxyoligonucleotides can be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid phase synthesis of silica-based support and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and genes. Oligoribonucleotides have also been prepared. The most successful approach so far developed involves using 2′-protected nucleosides as synthons and bis(diisopropylamino)methoxyphosphine as a phosphitylating reagent. A regioselective condensation route leading to the exclusive synthesis of 3′–5′ internucleotide linkages is possible.

Modern methods of nucleic acid synthesis on polymeric supports

Abstract Deoxyoligonucleotides can be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid phase synthesis of silica-based support and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and genes. Oligoribonucleotides have also been prepared. The most successful approach so far developed involves using 2′-protected nucleosides as synthons and bis(diisopropylamino)methoxyphosphine as a phosphitylating reagent. A regioselective condensation route leading to the exclusive synthesis of 3′–5′ internucleotide linkages is possible.

Gene synthesis machines: DNA chemistry and its uses.

Deoxyoligonucleotides can now be synthesized rapidly and in high yield because of recent advances in nucleic acid chemistry. Key innovations include solid-phase synthesis on silica-based supports and the development of stable deoxynucleoside phosphoramidites as synthons. When incorporated into manual, semiautomatic, or automatic instruments, these new procedures can be used to prepare probes, mixed probes, deoxyoligonucleotides for priming DNA synthesis, analogues of deoxyoligonucleotides, and DNA segments containing more than 100 deoxynucleotides.

Conceptual basis of the selective activation of bis(dialkylamino)methoxyphosphines by weak acids and its application toward the preparation of deoxynucleoside phosphoramidites in situ

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTConceptual basis of the selective activation of bis(dialkylamino)methoxyphosphines by weak acids and its application toward the preparation of deoxynucleoside phosphoramidites in situMichael F. Moore and Serge L. BeaucageCite this: J. Org. Chem. 1985, 50, 12, 2019–2025Publication Date (Print):June 1, 1985Publication History Published online1 May 2002Published inissue 1 June 1985https://doi.org/10.1021/jo00212a003RIGHTS & PERMISSIONSArticle Views354Altmetric-Citations23LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (2 MB) Get e-Alerts Get e-Alerts

In situ activation of bis-dialkylaminophosphines--a new method for synthesizing deoxyoligonucleotides on polymer supports.

Deoxynucleoside phosphoramidites can be prepared in good yield from deoxynucleosides, bis- dialkylaminophosphines , and the corresponding dialkylamine hydrotetrazolide or tetrazole as catalysts. These phosphoramidites generated in situ lead to the direct synthesis of deoxyoligonucleotides on polymer supports.

Improved procedure for the preparation of deoxynucleoside phosphoramidites: Arylphosphoramidites as new convenient intermediates for oligodeoxynucleotide synthesis

A simplified procedure for the preparation of deoxynucleoside methyl- and arylphosphoramidites is described. Both types of phosphoramidites can be conveniently activated by N-methylaniline trifluoracetate for their use in oligodeoxynucleotide synthesis.

A simple and efficient preparation of deoxynucleoside phosphoramidites in situ

A highly selective activation of bis-(pyrrolidino) methoxyphosphine by 4,5-dichloroimidazole has been developed and successfully used in the preparation of deoxynucleoside phosphoramidites.

ChemInform Abstract: NEW METHODS FOR SYNTHESIZING DEOXYOLIGONUCLEOTIDES

Deoxynucleoside phosphoramidites are prepared in good yield from doexynucleosides, bis-dialkylamino-phosphines, with the corresponding dialkylamine hydrotetrazolide or tetrazole as catalyst for the reaction. These phosphoramidites generated in situ lead to the direct synthesis of deoxyoligonucleotides on polymer supports.

An investigation of several deoxynucleoside phosphoramidites useful for synthesizing deoxyoligonucleotides

Various deoxynucleoside N,N-dialkylaminomethoxyphosphines were examined for stability and reactivity in deoxyoligonucleotide synthesis. Of those examined, the N-morpholino and N,N-diisopropylamino derivatives most completely satisfied all criteria.

Substituted 5-phenyltetrazoles: improved activators of deoxynucleoside phosphoramidites in deoxyoligonucleotide synthesis.

5-(p-nitrophenyl)tetrazole, a tetrazole of enhanced acidity relative to 5-H-tetrazole, is a superior activator of the N-morpholinophosphoramidite in the synthesis of deoxyoligonucleotides.

A new and general procedure for the preparation of deoxynucleoside phosphoramidites

Nucleoside phosphoramidites can be obtained in a one pot reaction by reacting successively an alkyloxy (or aryloxy) bis -triazolylphosphine with a 3'-OH free deoxynucleoside and a trimethylsilylated secondary amine.

Deoxynucleoside phosphoramidites—A new class of key intermediates for deoxypolynucleotide synthesis

The development of a new class of nucleoside phosphites is described. These compounds are stable to normal laboratory conditions, are activated by mild acid treatment, and are observed to react essentially quantitatively with protected nucleosides.