Protecting nucleosides

The protected nucleoside-3-phosphoramidite monomer units such as 671 are used in the solid-phase oligonucleotide synthesis. In the 60mer synthesis, 104 allylic protective groups are removed in almost 100% overall yield by the single Pd-catalyze reaction with formic acid and BuNH2[432], N,(9-protection of uridine derivatives was carried out under pha.se-transfer conditions[433]. 

The reaction has been extended (106) to the nucleoside field and provides a means for the direct iodination of suitably protected nucleosides. Thus treatment of 2,3-O-isopropylidene uridine (68) with tri-phenylphosphite methiodide in N,2V-dimethylformamide at room temperature afforded the corresponding crystalline 5-deoxy-5-iodo analog 69 in 77% yield. 

By using a phosphoric diester monotetrazolide, made in situ from the corresponding phosphoric chloride and tetrazole in the presence of triethylamine, die 3 -OH of a suitably protected nucleoside could be phosphorylated to give a fully protected nucleotide [241... 

Reaction of a 5 -0-protected nucleoside with moipholinophosphorous ditetrazolide (phosphitylation),... 

The catalysis by a protected nucleoside of the aminolysis by butylamine of / -nitrophenyl acetate in benzene (Scheme 3) has been reported. Interestingly, only 2, y, 5 -<9-tris(t-butyldimethylsilyl)cytidine showed any marked catalytic effect, the adenosine, guanosine and uridine analogues behaving merely as weak general base... 

An interesting approach149 to the synthesis of partially protected nucleosides involved fusion for 12 hours at 130° of 5 -0-acetyladenosine and 2, 3, 5 -tri-0-acetyladenosine, followed by separation, by countercurrent distribution, of the so-formed mixture of 3, 5 -di-0-acetyl-, 5 -0-acetyl-, and 2, 3, 5 -tri-0-acetyladenosine. It is noteworthy that no 2, 5 -diester was isolated. A variation154 of this procedure was the fusion, at —200°, of a mixture of 2, 3, 5 -tri-0-acetyladenosine and adenosine, which yielded the 2, 3, 5 -triacetate, the 2, 3 -diacetate, and the 3, 5 -diacetate, with the last preponderating. 

Polynucleotides. These compounds are now generally prepared by use of a bifunctional phosphitylating agent such as o-chlorophenylphosphorodichloridite (6, 114-115) or methoxydichlorophosphine, CHjOPCl,. The intermediate nucleoside phosphites from these reagents tend to be unstable. This difficulty can be alleviated by use of I, which reacts with suitably protected nucleosides to form stable phosphoramidales 2 in good yield. These products can be activated for formation of a dinucleotide phosphite (4) by treatment with a weak acid such as N,N-dimethylaniline hydrochloride or l/Z-tetrazole (5). 

The synthetic procedure currently used consists in the direct oxidation of an isolated hydroxyl group in the sugar moiety of suitably protected nucleosides. In the meantime, the synthesis of some keto derivatives of aldopentose nucleosides by selective-elimination processes has been reported.4 8... 

Attachment of suitable linkers to the surface of silica can be achieved by transesterification with (3-aminopropyl)triethoxysilane, which leads to the support 2 (Figure 2.8) [198-200]. Alternatively, silica can be functionalized by reaction with alkyltri-chlorosilanes [201]. For the solid-phase synthesis of oligonucleotides, supports with a longer spacer, such as that in 3, have proven more convenient than 2 [202-206]. Supports 3, so-called LCAA-CPG (long chain alkylamine CPG [194,195]), are commercially available (typical loading 0.1 mmol/g) and are currently the most commonly used supports for the synthesis of oligonucleotides. For this purpose, protected nucleosides are converted into succinic acid monoesters, and then coupled to LCAA-CPG. CPG functionalized with a 3-mercaptopropyl linker has been used for the solid-phase synthesis of oligosaccharides [207]. 

In the 1,3,2-dioxaphosphole method a bis(2-butene-2,3-diyl) pyrophosphate is used as the condensing agent. It allows two successive esterifications of one phosphate group to be performed without additional activation. First a 5 -O-protected nucleoside is added in methylene chloride in the second reaction an unprotected nucleoside can be used, since only the 3 OH group is able to attack the cyclic enediol 3 -nucleosidyl phosphotriester. Protected dinucleoside triesters are obtained in 80% yield. Removals of protective groups, methoxytrityl by means of trifluoroacetic acid in methylene chloride and 1-methylacetonyl by aqueous triethyl-amine, also give about 80% yield (F. Ramirez, 1975, 1977). 

Deacetylation of the Protected Nucleosides 8 and 9 General Procedure. A 1 M solution of NaOMe (200 ylL, 0.2 mmol) was added to the solution of the protected nucleoside 8 or 9 (0.5—0.8 mmol) in MeOH (20 mL) and the mixture was stirred at ambient temperature overnight. The crystals (if formed) were filtered off. Then the solution was neutralized by addition of Dowex 50x8 (H+) (ca. 100 mg) and filtered. The ion-exchanger was washed with saturated methanolic ammonia (5 mL) followed by methanol (20 mL) and the combined filtrates were evaporated to dryness. The collected crystals and residue was recrystallized from EtOH/toluene to give the nucleosides 12 or 13. 

Although, the photolysis requires quite short wavelengths (254 nm), the reaction is clean and high-yielding (up to 84% of liberated alcohol) in acetonitrile (a different and nonproductive reaction occurs in benzene) with a variety of primary, secondary, and allylic alcohols, as well with 3 - and 5 -protected nucleosides. The chemical stability is very high, approaching that of triisopropylsilyl (TIPS) in some cases (R2 = Me). 

Activated aromatic compounds are chlorinated readily by (dichloroiodo)benzene. Generally, negligible amounts of ortAo-chloro isomers are produced from diverse aromatic compounds, e.g. salicylic acid gave its 5-chloroderivative (89%) [16]. Exceptions were noted in the chlorination of the triphenyl phosphinimine of aniline which afforded o- and p-products in the ratio 7 3 [17], and of 2-naphthol which gave its 1-chloroderivative (84%) [16]. (Dichloroiodo)benzene was superior for the chlorination of uracil bases and protected nucleosides, e.g. [18] ... 

Alkylation on ring nitrogen remains the most frequently cited reaction of a heteroatom. Due to the considerable interest in nucleosides for their biological importance, this will be dealt with further. Standard treatment of the dioxo compound (28) with hexamethyldisilazane, followed by 1-0-acetyl-2,3,5-tri-0-benzoyl-/f-D-ribose gives the protected nucleoside (29) (Equation (6)), which can be converted into the free nucleoside with methanolic ammonia (92JHC343). 

The common use of amino and cyano groups ortho to each other to create a pyrimidine ring has also been employed. For example, the nucleoside (242), which has been synthesized from simple carbohydrate precursors, can be treated with formamidine acetate to give the protected nucleoside (243) in 80% yield (Equation (82)). Deprotection of this compound using standard techniques leads to the free nucleoside <90MI 707-01). 

A number of protected nucleosides (336) have been prepared by cyclization of pyrimidine nucleosides <84H(21)289, 84MI 716-02). 

---