[89992-70] · C9H18ClN2OP · (MW 236.68)
(reagent widely used for the phosphinylation of the 3´-hydroxy function of modified and unmodified nucleosides, and of various reporter groups for incorporation into oligonucleotides)
Alternate Name: 2-cyanoethyl N,N-diisopropylphosphonamidic chloride; chloro-(2-cyanoethoxy)-N,N-diisopropylaminophosphine; chloro(diisopropylamino)-b-cyanoethoxyphosphine; 2-cyanoethyl N,N-diisopropylphosphoramidochloridite.
Physical Data: bp 103-104 °C (0.08 mmHg; d 1.061 g cm-3).
Solubility: soluble in most organic solvents.
Form Supplied in: colorless liquid.
Analysis of Reagent Purity: 31P NMR, 1H NMR, EI-MS.
Preparative Methods: the reagent is prepared from the reaction of N,N-diisopropylamine or its N-trimethylsilyl derivative with (2-cyanoethoxy)dichlorophosphine in anhydrous diethyl ether and isolated by vacuum distillation.
Purification: vacuum distillation.
Handling, Storage, and Precautions: the reagent is moisture-sensitive and corrosive. It should therefore be stored in a dry environment, in a tightly closed container at -20 °C. Caution should be exercised when vacuum-distilling the reagent as it may explode if heated at a temperature (internal) exceeding 140 °C. Distillation should be carried out only if a vacuum of less than 0.1 mmHg can be generated.
Although 2-cyanoethyl diisopropylchlorophosphoramidite has been used extensively for the phosphinylation of unmodified nucleosides toward solid-phase oligonucleotide synthesis,1 its application to the preparation of modified nucleoside phosphoramidites for incorporation into oligonucleotides has recently become the focus of intense scrutiny. For example, 2-cyanoethyl diisopropylchlorophosphoramidite is used to phosphinylate 1,N6-ethano-2´-deoxyadenosine.2 Insertion of the modified nucleoside phosphoramidite (1) at a specific location in an oligodeoxyribonucleotide is reported as a means to gather information about the repair, mutagenesis, and structural properties of the lesion. Studies such as these should lead to a better understanding of the cytotoxic and carcinogenic mechanisms of 1,3-bis(2-chloroethyl)nitrosourea, which has been frequently used in cancer treatment; 1,N6-ethano-2´-deoxyadenosine is one of the many adducts generated from the reaction of the nitrosourea with DNA.
It should be noted that 3,N4-etheno-2´-deoxycytidine and 3,N4-ethano-2´-deoxycytidine have also been converted to the corresponding phosphoramidites upon reaction with 2-cyanoethyl diisopropylchlorophosphoramidite.3 The incorporation of these modified nucleoside phosphoramidites into oligonucleotides at predetermined sites provides additional models for studying the cytotoxic mechanisms of 1,3-bis(2-chloroethyl)nitrosourea, vinyl chloride, and other a,b-unsaturated compounds. In this context, polycyclic aromatic hydrocarbons are metabolized to bay-region diol epoxides, which are thought to exert their carcinogenic and other genotoxic effects predominantly through alkylation of DNA.4 In order to investigate the biochemical events associated with cancer induction subsequent to DNA-adduct formation, site-specific integration of a bay-region diol epoxide adduct into an oligonucleotide may indicate whether a preference for specific mismatches opposite the adduct is achieved during replication of the adducted DNA. Thus, reaction of 2-cyanoethyl diisopropylchlorophosphoramidite with a tetrahydrophenanthrene-2´-deoxyadenosine derivative affords the modified deoxyribonucleoside phosphoramidite derivative (2) for incorporation into a nonanucleotide at predetermined sites.4
Similar syntheses of oligonucleotides containing both cis5 and trans6 ring-opened adducts of 3,4-epoxy-1,2,3,4-tetrahydrophenanthrene with 2´-deoxyadenosine, and that of an analogous trans adduct with 2´-deoxyguanosine7 have been reported.
Oligonucleotides modified with a specific chemical functionality, such as a transition or lanthanide metal complex, are of widespread interest for analytical and therapeutic applications, and for mechanistic studies. The use of solid-phase DNA synthesis methodologies for site-specific labeling oligonucleotides with a transition metal complex has led to the preparation of a novel ruthenium-nucleoside phosphoramidite. Specifically, phosphinylation of a Ru(bpy)32+-labeled 2´-deoxyuridine derivative with 2-cyanoethyl diisopropylchlorophosphoramidite produces the ruthenium-labeled phosphoramidite (3), which was inserted at selected sites in hexadecanucleotides.8 These ruthenium-modified oligonucleotides form stable luminescent DNA duplexes at ambient temperature and are amenable to photophysical studies.8
Oligonucleotides composed of carbohydrate-modified nucleosides have been extensively investigated in an attempt to identify those oligonucleotides that would demonstrate an increased affinity for complementary DNA/RNA oligomers while maintaining sequence specificity in hybridization experiments. In this regard, the incorporation of two anthraquinonylmethyl groups into oligonucleotides at specific carbohydrate residues has been accomplished by solid-phase synthesis.9 Thus, condensation of 2-cyanoethyl diisopropylchlorophosphoramidite with 5´-o-(4,4´-dimethoxytrityl)-2´-O-(2-anthraquinonylmethyl) uridine gives the modified nucleoside phosphoramidite (4) required for oligonucleotide synthesis. An oligonucleotide having two 2´-o-anthraquinonylmethyl groups at sites separated by four nucleotides exhibits the highest affinity for both unmodified complementary DNA (dTm = + 5.7 °C per modification) and RNA (dTm = + 3.3 °C per modification) oligonucleotides without altering binding specificity.9
‘Locked nucleic acids’ (LNA) composed of 2´-O,4´-C-methylene bicyclonucleosides are efficiently prepared from the phosphoramidite derivative of these bicyclonucleosides.10 2-Cyanoethyl diisopropylchlorophosphoramidite has been efficient, as a phosphinylating agent, in the synthesis of the modified phosphoramidites (5).
LNA display unprecedented binding affinity for unmodified complementary DNA and RNA oligonucleotides; dTm values of + 3 to + 5 °C per modification and + 4 to + 8 °C per modification were determined for LNA-DNA and LNA-RNA hybrids, respectively. LNA also demonstrate excellent stability against 3´-exonucleases.11
Replacement of the five-membered furanose ring of natural DNA by a cyclohexene ring leads to cyclohexene nucleic acids (CeNA) as a novel class of structurally modified nucleic acids. Cyclohexenyl nucleosides12 are converted to their corresponding phosphoramidites such as 6 upon reaction with 2-cyanoethyl diisopropylchlorophosphoramidite, and then incorporated into oligonucleotides according to standard solid-phase synthesis.13
The integration of cyclohexenyladenosine into oligonucleotides results only in a small decrease of the stability of DNA-DNA hybrids; a dTm/mod of -0.5 °C is observed. However, when cyclohexenyladenosine-modified DNA oligonucleotides are mixed with complementary unmodified RNA oligomers, the stability of these DNA-RNA hybrids increases; the dTm/mod ranges from + 0.8 to + 1.7 °C depending on the incorporation site of cyclohexenyladenosine. CeNA are stable against fetal calf serum 3´-exonuclease relative to unmodified DNA and, remarkably, CeNA/RNA hybrids are able to activate Escherichia coli RNase H, thereby resulting in cleavage of the RNA strand of each hybrid.13 Thus, CeNA represent a promising class of oligonucleotide analogs for evaluation in antisense therapeutic applications.
2-Cyanoethyl diisopropylchlorophosphoramidite has also been applied to the synthesis of cyclohexanyl nucleoside phosphoramidites14 and hexitol nucleoside phosphoramidites15 toward the preparation of cyclohexanyl nucleic acids (CAN) and hexitol nucleic acids (HNA), respectively. HNA are completely stable to 3´-exonucleases and form stable duplexes with complementary DNA and RNA oligonucleotides.15,16 It is worth mentioning that, almost a decade ago, hexopyranosyl nucleoside phosphoramidites were prepared using 2-cyanoethyl diisopropylchlorophosphoramidite, and inserted in oligonucleotides to evaluate base-pairing properties and enzymatic stability of this still maturing class of carbohydrate-modified oligonucleotides.17,18
Acyclic nucleoside phosphoramidites such as 7 and 8 are prepared by phosphinylation of the parent acyclic nucleosides with 2-cyanoethyl diisopropylchlorophosphoramidite.19
Incorporation of these phosphoramidites into oligonucleotides at the 3´-terminus results in significant stabilization of the modified oligonucleotides to 3´-exonucleases like snake venom phosphodiesterase.19 2-Cyanoethyl diisopropylchlorophosphoramidite has similarly been employed in the synthesis of glyceronucleoside phosphoramidites20 and silicon-containing acyclic nucleoside phosphoramidites21 for integration into oligonucleotides. The stability of DNA-DNA hybrids incorporating acyclic nucleosides decreases by 9-15 °C for each flexible nucleoside incorporated into a strand,20 and thus casts doubt about the usefulness of flexible oligonucleotide analogs in antisense experiments or as diagnostic probes.
An efficient approach to the synthesis of oligonucleotides containing methylene acetal linkages serves as a model to the preparation of therapeutic oligonucleotides containing modified internucleoside linkages.22 Oligonucleotides such as these are not only resistant to serum nucleases but are designed to facilitate cellular uptake by decreasing the highly charged nature of native phosphodiester groups. The incorporation of nucleoside dimers containing (3´-5´)-methylene linkages into oligonucleotides is achieved by converting the dimers to phosphoramidite derivatives comparable to 9 by treatment with 2-cyanoethyl diisopropylchlorophosphoramidite followed by conventional solid-phase synthesis.22
2-Cyanoethyl diisopropylchlorophosphoramidite has found an important application in the synthesis of photolabile 5´-o-(a-methyl-6-nitropiperonyloxycarbonyl)-N-acyl-2´-deoxyribonucleoside phosphoramidites (10).23 These phosphoramidites are used in the preparation of densely packed oligonucleotides on microarrays. The precise removal of selected 5´-O-photolabile protecting groups can be achieved through properly designed photolithographic masks.23
For similar applications, the synthesis of photolabile 5´-o-(3´´, 5´´-dimethoxybenzoin)carbonyl-N-acyl-2´-deoxyribonucleoside phosphoramidites24,25 and 5´/3´-O-[2-(2-nitrophenyl) ethoxycarbonyl]-N-protected-2´-deoxyribonucleoside phosphoramidites26,27 has been accomplished using 2-cyanoethyl diisopropylchlorophosphoramidite.
Chemical methods have been developed for the 5´-phosphitylation of fully protected oligodeoxyribonucleotides. Phosphinylation of 2-(4,4´-dimethoxytrityloxyethylsulfonyl)ethanol with 2-cyanoethyl diisopropylchlorophosphoramidite affords the phosphoramidite derivative 11.28
The reagent is used on automated DNA synthesizers to functionalize synthetic oligonucleotides with either a 5´-phosphate or a 5´-phosphorothioate group. The coupling efficiency of the reagent can be monitored upon release of the 4,4´-dimethoxytrityl cation under acidic conditions.28 Other functional groups have been added to the 5´-terminus of oligonucleotides using a similar strategy. For example, the reaction of 2-cyanoethyl diisopropylchlorophosphoramidite with 2-(N-trifluoroacetyl) aminoethanol,29 2-[N-(9-fluorenylmethoxycarbonyl)amino]ethanol,30 6-(N-monomethoxytrityl)aminohexanol,31 3-(tritylthio)propanol,31 6-(tritylthio)hexanol31,32a and 2-[2-(2-acetylthioethoxy)ethoxy] ethanol,32b gives the parental phosphoramidite derivatives required for subsequent addition of a 5´-amino or a 5´-mercapto function to oligonucleotides. The conjugation of 5´-aminoalkylated or 5´-mercaptoalkylated oligonucleotides with a variety of ligands has been extensively reviewed in the literature.33,34 In addition, phosphinylation of 3-(4,4´-dimethoxytrityloxy)propan-1-ol,35 18-(4,4´-dimethoxytrityloxy)-3,6,9,12,15,18-hexaoxaoctadecan-1-ol,36 and N-[6-(4,4´-dimethoxytrityloxy)hexyl]-N´-(6-hydroxyhexyl)terephthalamide,37 6-(3-hydroxypropyl)-N1,N5, N10, N14-tetraphenoxyacetylspermine,38 1,3-O-bis(N-[3-o-(4,4´-dimethoxytrityloxy)propyl] carbamoyl-2,2-bis[N-(3-hydroxypropyl) carbamoyl]oxymethyl)-1,3-propanediol,39 and N-Fmoc-tyramine40 with 2-cyanoethyl diisopropylchlorophosphoramidite has enabled the integration of the resulting phosphoramidites into oligonucleotides. These modified oligonucleotides are useful in assessing DNA triplex formation,37-39 the mechanism of ligation effected by the FLP recombinase and mammalian topoisomerase I,40 the quality of oligonucleotides on glass supports for biosensor development,36 and the folding and binding of oligonucleotides in solution.37
Condensation of 1,6-bis(4,4´-dimethoxytrityloxy)hexan-2-ol,41 2,2-bis(4,4´-dimethoxytrityloxymethyl)propan-1-ol,42 and tris-2, 2,2-[3-(4,4´-dimethoxytrityloxy) propyloxymethyl]ethanol43 with 2-cyanoethyl diisopropylchlorophosphoramidite gives the phosphoramidites (12-14), which can be incorporated into oligonucleotides to allow attachment of multiple ligands41,42 or generate oligonucleotide dendrimers.43
Many ligands functionalized with linkers have been converted to phosphoramidites upon reaction with 2-cyanoethyl diisopropylchlorophosphoramidite, and then added singly or repeatedly to the 5´-terminus of oligonucleotides to generate conjugates with specific properties. These ligands include acridine (15),44a psoralen (16),44 2,4-dinitrophenyl (17),45 biotin (18),46-49 phosphotyrosine (19),48 mannopyranosyl (20),42 phenanthrodihydrodioxin (21),50 and vitamin E (22)51 derivatives.
Several carboranyl alcohol derivatives, such as 23,52 have been converted to the corresponding phosphoramidite derivatives by treatment with 2-cyanoethyl diisopropylchlorophosphoramidite toward the preparation of boron-rich oligophosphates for the treatment of cancer by boron neutron capture therapy.
An analogous approach to the synthesis of boron-rich oligophosphates uses S-alkyl derivatives of mercaptoundecahydrododecaborate as starting materials for phosphoramidite monomers.53
2-Cyanoethyl diisopropylchlorophosphoramidite has been particularly useful in the preparation of phosphoramidites toward the synthesis of cyclic sphingosine 1,3-phosphate,54 bis-phosphatidylcholine lipids,55 lysophosphatidylnucleosides,56 glycosyl phospholipids of 5-fluorouridine and arabinofuranosylcytidine,57 lactosyl thiophosphate diester derivatives of arabinofuranosylcytidine and arabinofuranosyladenosine,58 D- and L-myo-inositol 1-phosphorothioate,59 and myo-inositol 1,4,5-trisphosphorothioate.60
Food and Drug Administration, Bethesda, Maryland, USA