Phosphitylating Reagent Research Articles

Microwave-assisted phosphitylation of DNA and RNA nucleosides and their analogs.

Microwave-assisted chemical phosphitylation of novel nucleoside analogs containing a ribulose sugar unit was successful with yields ranging from 50% to 79% using 2-cyanoethyl-N,N-diisopropyl chlorophosphoramidite as the phosphitylating reagent. The resultant phosphoramidite products remained intact, with no signs of degradation over extended reaction times (up to 60 min) at an elevated temperature (65°C). When the same microwave-mediated phosphitylating protocols were applied to canonical DNA and RNA nucleoside monomers as substrates, using either 2-cyanoethyl-N,N,-diisopropyl chlorophosphoramidite or 2-cyanoethyl-N,N,N',N'-tetraisopropyl phosphane with an activator, 40% to 90% yields of DNA and RNA phosphoramidites were obtained within 10 to 15 min. These results demonstrate that microwave-assisted phosphitylation is an efficient alternative to standard phosphitylating conditions that can be expanded and refined to include a variety of substrates.

Use of a novel 5'-regioselective phosphitylating reagent for one-pot synthesis of nucleoside 5'-triphosphates from unprotected nucleosides.

5'-Triphosphates are building blocks for enzymatic synthesis of DNA and RNA. This unit presents a protocol for convenient synthesis of 2'-deoxyribo- and ribonucleoside 5'-triphosphates (dNTPs and NTPs) from any natural or modified base. This one-pot synthesis can also be employed to prepare triphosphate analogs with a sulfur or selenium atom in place of a non-bridging oxygen atom of the α-phosphate. These S- or Se-modified dNTPs and NTPs can be used to prepare diastereomerically pure phosphorothioate or phosphoroselenoate nucleic acids. Even without extensive purification, the dNTPs or NTPs synthesized by this method are of high quality and can be used directly in DNA polymerization or RNA transcription. Synthesis and purification of the 5'-triphosphates, as well as analysis and confirmation of natural and sulfur- or selenium-modified nucleic acids, are described in this protocol unit.

2-Cyanoethyl N,N,N′,N′-Tetraisopropylphosphorodiamidite

2-Cyanoethyl N,N,N′,N′-tetraisopropylphosphorodiamidite is a colorless viscous liquid, which is soluble in most organic solvents. It is a widely used phosphitylating reagent for the preparation of various phosphorylated biomolecules, such as nucleoside carbohydrate conjugates, phospholipids and glycopeptides.[ 1 ] In particular, this reagent is highly effective for automated solid-phase DNA/RNA oligonucleotide synthesis.[ 2 ]

Convenient synthesis of nucleoside 5′-(α-P-thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA)

Modified deoxy- and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S -modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy- and ribo-nucleoside 5′-(α-P-thio)triphosphates (dNTPαS and NTPαS) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5′-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTPαS and NTPαS analogs (mixtures of Sp and Rp diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPαS and NTPαS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the Sp diastereomers of dNTPαS and NTPαS analogs, respectively, while the Rp diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.

Improved Method for the Solid-Phase Synthesis of Oligoribonucleotide 5′-Triphosphates

We have developed an improved solid-phase method for the synthesis of 5'-triphosphates (5'-TPs) of oligoribonucleotides. The method is based on the use of salicyl phosphorochloridite as the phosphitylating reagent and the improvement is characterized by the use of the highly reactive pyrophosphorylating reagent tris(tetra-n-butylammonium) hydrogen pyrophosphate instead of the conventional tri-n-butylammonium salt for the nucleophilic substitution reaction to form the cyclic ester intermediate. The improved method can be used to generate oligoribonucleotide 5'-TPs efficiently and reproducibly.

Convenient synthesis of nucleoside 5′-(α-P-thio)triphosphates and phosphorothioate nucleic acids (DNA and RNA)

Modified deoxy- and ribo-nucleoside triphosphates are chemically synthesized in multiple steps due to the protection and deprotection of the nucleoside functionalities. To conveniently synthesize the S -modified triphosphates for enzymatically preparing phosphorothioate DNAs and RNAs (PS-DNA and PS-RNA) as potential therapeutics, herein we report a one-pot strategy to synthesize the deoxy- and ribo-nucleoside 5′-(α-P-thio)triphosphates (dNTPαS and NTPαS) without protecting any nucleoside functionalities. This facile synthesis is achieved by treating the nucleosides with a mild phosphitylating reagent, reacting selectively with the 5′-hydroxyl group of each unprotected nucleoside, followed by sulfurization and hydrolysis to afford the crude dNTPαS and NTPαS analogs (mixtures of S p and R p diastereomers). We also demonstrated that after just simple precipitation (without HPLC and ion-exchange purification), the quality of the synthesized dNTPαS and NTPαS analogs is excellent for direct DNA polymerization and RNA transcription, respectively. Since Klenow DNA polymerase and T7 RNA polymerase accept the S p diastereomers of dNTPαS and NTPαS analogs, respectively, while the R p diastereomers are neither substrates nor inhibitors, the diastereomerically-pure PS-DNAs and PS-RNAs can be conveniently synthesized enzymatically.

Protection-Free One-Pot Synthesis of 2′-Deoxynucleoside 5′-Triphosphates and DNA Polymerization

By differentiating the functional groups on nucleosides, we have designed and developed a one-pot synthesis of deoxyribonucleoside 5'-triphosphates without any protection on the nucleosides. A facile synthesis is achieved by generating an in situ phosphitylating reagent that reacts selectively with the 5'-hydroxyl groups of the unprotected nucleosides. The synthesized triphosphates are of high quality and can be effectively incorporated into DNAs by DNA polymerase. This novel approach is straightforward and cost-effective for triphosphate synthesis.

Convenient synthesis of nucleoside 5′-triphosphates for RNA transcription

By generating a selective phosphitylating reagent in situ, nucleoside 5'-triphosphates can be conveniently synthesized in one pot. This novel strategy without nucleoside protection has been developed to largely simplify synthesis of the nucleoside triphosphates. This demonstrated principle can be applied to the 5'-triphosphate synthesis of both native and modified nucleosides.

NMR measurement of free and esterified sterols

AbstractThis short account describes a novel analytical technique for the determination of total, free and esterified sterols in olive oil developed in our laboratory. This methodology is based on 1H and 31P NMR spectroscopy. The latter spectroscopic analysis requires first the derivatization of the sterolic hydroxyl groups with a phosphitylating reagent. This NMR method shows a number of advantages over conventional methods for sterols determination, amongst which speed and simplicity are the most beneficial ones. The possibility of applying the NMR spectroscopy to other food matrices is discussed.

ChemInform Abstract: New Phosphitylating Reagent in the Nucleotide Chemistry Containing Two 4-Nitrophenoxy Leaving Groups. Remarkably Fast and Clean Phosphitylations Activated by DBU Leading to Thio- and Seleno- Oligonucleotides.

ChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

Synthesis of Nucleoside Mono-, Di-, and Triphosphoramidates from Solid-Phase cycloSaligenyl Phosphitylating Reagents

Chloromethyl polystyrene resin was reacted with 5-hydroxysalicylaldehyde in the presence of potassium carbonate to afford polymer-bound 2-hydroxybenzaldehyde. Subsequent reduction with borane solution produced polymer-bound 2-hydroxybenzyl alcohol. The reaction of immobilized 2-hydroxybenzyl alcohol with appropriate phosphitylating reagents yielded solid-phase cycloSaligenyl mono-, di-, and triphosphitylating reagents, which were reacted with unprotected nucleosides, followed by iodine oxidation, deprotection of cyanoethoxy groups, and the basic cleavage, respectively, to afford 5'-O-nucleoside mono-, di-, and triphosphoramidates in 52-73% overall yield.

Solid‐Supported Reagents for Synthesis of Nucleoside Monothiophosphates, Dithiodiphosphates, and Trithiotriphosphates

This unit describes procedures for the selective synthesis of nucleoside monothiophosphates, dithiodiphosphates, and trithiotriphosphates from solid-supported phosphitylating reagents. Rigid and sterically hindered polymer-bound 1,3,2-oxathiaphospholane is reacted selectively with the 5'-hydroxyl group of nucleosides in the presence of 1H-tetrazole. Sulfurization in the presence of Beaucage's reagent (3H-1,2-benzodithiole-3-one 1,1-dioxide) followed by ring-opening with 3-hydroxypropionitrile and basic cleavage in the presence of 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) through the elimination of polymer-bound ethylene episulfide afford nucleoside monothiophosphates. Furthermore, reaction of polymer-bound diphosphitylating and triphosphitylating reagents, prepared from polymer-bound benzyl alcohol, with unprotected nucleosides, sulfurization with Beaucage's reagent, and acidic cleavage using trifluoroacetic acid cocktail produce nucleoside dithiodiphosphates and trithiotriphosphates in moderate yields.

A Versatile One-Pot Procedure to Phosphate Monoesters and Pyrophosphates Using Di(p-methoxybenzyl)-N,N-diisopropylphosphoramidite

A one-pot procedure for the preparation of phosphoramidates, phosphorothioates, pyrophosphates, phosphodiesters, and phosphofluoridates has been devised using di(p-methoxybenzyl)-N,N-diisopropylphosphoramidite as the common phosphitylating reagent.

Synthesis of poly(1‐hexene)s end‐functionalized with phenols

AbstractElectrophilic alkylations of phenol/2,6‐dimethylphenol were performed with vinylidene‐terminated poly(1‐hexene)s using BF3·OEt2 catalyst. Vinylidene‐terminated poly(1‐hexene)s with Mn varying from 400 to 10000 were prepared by bulk polymerization of 1‐hexene at 50 to −20 °C using Cp2ZrCl2/MAO catalysts. The phenol/2,6‐dimethylphenol‐terminated poly(1‐hexene)s was characterized by NMR (1H, 13C), UV, IR and vapor phase osmometer (VPO). The isomer distribution (ortho, para and ortho/para) was determined by 13P NMR using a phosphitylating reagent, namely 2‐chloro‐1,3,2‐dioxaphospholane. The number‐average degree of functionality (Fn) >0.9 with >95% para selectivity could be achieved using low‐molecular‐weight oligomers of poly(1‐hexene)s. Copyright © 2005 Society of Chemical Industry

Solid-Phase Reagents for Selective Monophosphorylation of Carbohydrates and Nucleosides

Two classes of aminomethyl polystyrene resin-bound linkers of p-acetoxybenzyl alcohol were subjected to reactions with 2-cyanoethyl N,N-diisopropylchlorophosphoramidite to produce the corresponding polymer-bound phosphitylating reagents. These were reacted with a number of unprotected nucleosides and carbohydrates in the presence of 1H-tetrazole. Oxidation with tert-butyl hydroperoxide followed by removal of the cyanoethoxy group with 1,8-diazabicyclo[5.4.0]undec-7-ene afforded the corresponding polymer-bound phosphate diesters. Acidic cleavage of the p-acetoxybenzyl alcohol linker yielded monophosphorylated products with high regioselectivity and trapped linkers on the resins that can be reused.

Synthesis of Phosphorothioate Oligonucleotides with Stereodefined Phosphorothioate Linkages

A method for solid-phase synthesis of stereodefined PS-oligos via an oxathiaphospholane approach using pure P-diastereomers of nucleoside oxathiaphospholane monomers is described. The oxathiaphospholane monomers are synthesized by phosphitylation of 5'-O-DMTr-N-protected deoxyribonucleosides with 2-chloro-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane followed by sulfurization. The procedure is general and may be applied to other analogs, depending on the aldehyde (or mercaptoalcohol) used. Starting from an 18O-labeled mercaptoalcohol, the corresponding 18O-labeled phosphitylating reagent and nucleoside monomers can be obtained and used for synthesis of labeled stereodefined PS-oligos, which are useful for studying mechanisms of enzymatic reactions. Details are provided for chromatographic separation of the 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s into their P-diastereomers, and for manual solid-phase synthesis of PS-oligos. Oxidation of 5'-O-DMTr-N-protected-deoxyribonucleoside-3'-O-(2-thio-spiro-4,4-pentamethylene-1,3,2-oxathiaphospholane)s with selenium dioxide yields their 2-oxo-analogs, which are suitable either for elongation of stereodefined PS-oligos with segments consisting of unmodified nucleotide units possessing phosphate internucleotide linkages, or for generating isotopomeric 18O-labeled PO-oligos of predetermined P-chirality.

Caged phospho-amino acid building blocks for solid-phase peptide synthesis.

Three 1-(2-nitrophenyl)ethyl-caged phospho-amino acids have been synthesized for use in standard N(alpha)-fluorenylmethoxycarbonyl-based solid-phase peptide synthesis (SPPS). The most common naturally occurring phospho-amino acids, serine, threonine, and tyrosine, were prepared as protected caged building blocks by modification with a unique phosphitylating reagent. In previous work, caged phospho-peptides were made using an interassembly approach (Rothman, D. M.; Vazquez, M. E.; Vogel, E. M.; Imperiali, B. Org. Lett. 2002, 4, 2865-2868). However, this technique is limited to creating peptides without oxidation sensitive residues C-terminal to the amino acid to be modified and the methodology involves synthetic manipulations on the solid phase that may limit the utilization of the methodology. Herein we report the facile synthesis of N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-serine 1, N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-threonine 2, and N-alpha-Fmoc-phospho(1-nitrophenylethyl-2-cyanoethyl)-L-tyrosine 3. These building blocks allow the synthesis of any caged phospho-peptide sequence using standard Fmoc-based SPPS procedures.

Polymer-Supported reagents for methylphosphorylation and phosphorylation of carbohydrates

Two polymer-supported reagents for methylphosphorylation and phosphorylation of carbohydrates have been developed. p-Hydroxybenzyl alcohol and β-mercaptoethanol were immobilized on cross-linked divinylbenzene-polystyrene copolymer and conjugated with methyl N, N-diisopropylchlorophosphoramidite. Carbohydrates were reacted with polymer-bound phosphitylating reagents. Further oxidation, with or without the methoxy group deprotection, and cleavage yielded methylphosphorylated or phosphorylated carbohydrates, respectively.

ChemInform Abstract: Novel Phosphitylating Reagents Containing a Phosphorus—Fluorine Bond and Their Application in Efficient Synthesis of Phosphorofluoridates and Phosphorofluoridothionates.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.

ChemInform Abstract: Di‐tert‐butyl Diethylphosphoramidite as the Phosphitylating Reagent in the Preparation of 3‐Deoxy‐3‐C‐methylene‐D‐ribo‐hexose‐6‐phosphate and 3‐Deoxy‐3‐C‐methylene‐D‐erythro‐pentose‐5‐phosphate.

AbstractChemInform is a weekly Abstracting Service, delivering concise information at a glance that was extracted from about 100 leading journals. To access a ChemInform Abstract of an article which was published elsewhere, please select a “Full Text” option. The original article is trackable via the “References” option.