Phosphite triester synthesis

Figure 19.2 Alternative oxidation reagents for polymer-supported phosphite triester synthesis m-chloroperbenzoic acid (8), diacetoxyiodobenzene (9), 10-camphorsulfonyloxaziridine (10), dimethyldioxirane (11), ethylmethyldioxirane (12).

In the phosphite triester synthesis (phosphoramidite synthesis), an efficient coupling reaction takes place between the 3 -hydroxyl group of the chain and 5 -DMTr-(A -acy-lated)-deoxyribonucleoside 3 -0-(MA -diisopropylamino)phosphite. 

Because coupling is not always quantitative, the non-reacted terminal deoxynucteoside must be excluded from the following synthesis cycles. Otherwise deletion sequences will render the isolation of the pure final product difficult. Therefore a capping step (step 3) follows, e.g., acetylation with acetic anhydride and N,N-dimethyl-4-pyridinamine (DMAP) in dioxane. Capping times should be as short as possible, especially with 2-cyanoethyl phosphite triesters, which are sensitive to bases such as DMAP. 

The disadvantage of this method is that the dichloridites and monochloridites are sensitive to water and thus could not be used readily in automated oligonucleotide synthesis. This problem was overcome by Beaucage and Caruthers, who developed the phosphoramidite approach. In this method, derivatives of the form R 0P(NR2)2 react with one equivalent of an alcohol (catalyzed by species such as l//-tetrazole) to form diesters, R OP(OR")NR2, which usually are stable, easily handled solids. These phosphoroamidites are easily converted to phosphite triesters by reaction with a second alcohol (catalyzed by l//-tetrazole). Here, again, oxidation of the phosphite triester with aqueous iodine affords the phosphate triester. Over the years, numerous protective groups and amines have been examined for use in this approach. Much of the work has been reviewed. ... 

In the phosphite triester approach to the synthesis of nucleotides, morpholinophos-phorous ditetrazolide is used as the phosphitylating agent. The procedure generally consists of three steps ... 

In this context, it is interesting to note that the first synthesis of 2, 3 -0,0-cyclic phosphorothioate 22a was reported by Eckstein in 1968 [25], He also isolated pure Rp diastereomer by fractional crystallization of the triethylammonium salts [26] and used it as reference to determine the absolute configurations of the other phosphorothioate analogues [27], 2, 3 -0,0-Cyclic H-phosphonate 20a was used as a key substrate for the synthesis of uridine 2, 3 -0,0-cyclic boranophosphate 27. Silylation of H-phosphate 20a gave the phosphite triester 25 (two diastereomers). Its boronation, with simultaneous removal of the trimethylsilyl group, was achieved by its reaction with borane-A.A-diisopropylethylamine complex (DIPEA-BH3). 

A common method for DNA synthesis is the phosphite triester method (Fig. 7-6). A single-stranded oligonucleotide is formed by the sequential creation of diester bonds between the 5 -hydroxyl of one residue and the 3 -phosphate of the next. The 3 -phosphate is activated by substitution by dialkyl phosphoamidite (DPA) and reacts readily with the free 5 -OH of the first nucleotide. To prevent the formation of unwanted linkages the first nucleotide is linked, by the 3 -OH, to a solid support (often silica gel) in a column or funnel the 5 -OH of the second nucleotide is prevented from reacting by a dimethoxytrityl group (DMT). 

The 2-(methyldiphenylsilyl)ethyl (DPSME) group was used to protect a phosphate in a synthesis of Lysobisphosphatidic Acid [Scheme 7.40].82 The phosphate protection was introduced by a phosphite triester approach to give the symmetrical phosphate 40 2. The two isopropylidene groups were removed by acid hydrolysis without harm to the phosphate and the resultant diols selectively protected on their less hindered termini with fert-butyldiphenylsilyl groups. Acylation of the remaining secondary hydroxyls with oleic anhydride afforded the diester 403, whereupon the silyl protecting groups were removed with... 

Figure 6.7. Solid-Phase Synthesis of a DNA Chain by the Phosphite Triester Method. The activated monomer added to the growing chain is a deoxyribonucleoside 3 -phosphoramidite containing a DMT protecting group on its 5 oxygen atom, a p -cyanoethyl ( p CE) protecting group on its 3 phosphoryl oxygen, and a protecting group on the base.

Shaw has reviewed the various approaches to the synthesis of organophos-phate-oligonucleosides and has further commented on their chemical and biophysical properties along with their interactions with various enzymes such as DNA-polymerases, and compared them to other members of the family of phosphorus modified nucleic acids. She also reported the synthesis of P-tyrosinyl(P-0)-5 -P-nucleosidyl boranophosphates (23a,b), as antiviral and anticancer prodrug candidates. The P-boranophosphates were prepared by reacting a phosphoramidite intermediate obtained from protected tyrosine and the protected nucleoside in the presence of IH-tetrazole, followed by in situ borona-tion of the phosphite triester intermediate. The two diastereomers were then separated by reverse-phase HPLC. ... 

The phosphoramidite derivative of N-nitrothymidine (44) has been synthesised and found suitable for oligonucleotide synthesis using a standard phosphite triester solid phase approach. The N-nitrothymidine residues could be converted into a range of N -modified thymidines by reaction with primary alkyl amines. Phosphoramidite derivatives of 4-nitroindazole N and N -(2 -deoxy-p-D-ribofuranosides) (45, 46) have been synthesised, their base pairing properties investigated and found to show ambiguous base pairing. Seela has also reported the syntheses of the phosphoramidite derivatives of 8-aza-7-adenine... 

Shaw reported the synthesis of P-tyrosinyl-(P-0)-5-P-nucleosidyl boranophos-phates (190a,b) obtained in a one-pot synthetic procedure via a phosphoramidite and that of the nucleoside 3, 5 -cyclic boranophosphorothioates (191a,b) prepared from a cyclophosphoramidite intermediate. The cyclophosphoramidite, obtained by heating the nucleoside with HMPA was transformed to the phosphite triester by reaction with 4-nitrophenol in the presence of 5-ethylthio-l//-tetrazole. The boranophosphite was oxidized with Li2S after boronation with BH3.SMe2. 

The first synthesis of a dinucleotide analogue combining phosphorothioate and boranophosphate features has been reported. To prepare dithymidine boranophosphorothioate (71), Fmoc-protected thymidine was phosphitylated by bis(diisopropylamino)-chlorophosphine in the presence of DMAP. The resulting phosphoramidite was treated in situ with a 3 -protected thymidine and tetrazole and was then converted to a phosphite triester with nitrophenol. The phosphite triester was subsequently treated with BH3-SMe2 followed by LiS2 in the presence of 18-crown-6 to yield (71). This novel type of dinucleoside... 

Figure 5.6 Solid-phase synthesis of a DNA chain, by the phosphite triester method.

Polynucleoside Phosphate Derivatives. A convenient method for the oxidation of nucleoside phosphite triesters into phosphate triesters under non-basic and non-aqueous conditions using commercially available ethyl(methyl)diox-irane has been reported (Scheme 5). ° This oxidation method could be used with both N-protected and iV-unprotected nucleosides (80a-m), and was efficiently applied to nucleotide synthesis both in solution phase and on solid support. 

Scheme 19.2 Methods for polymer-supported oligonucleotide synthesis. Dinudeoside phosphate formation in the common methods for oligonucleotide synthesis phosphodiester (I), phosphotriester (II), phosphite triester (III) and H-phosphonate (IV).

An improved synthesis of glycinamide ribonucleotide has been achieved by treating (10) with dibenzyl N, N-diisopropylphosphoramidite and tetrazole, oxidizing the phosphite triester obtained using MCPBA, and conventional deblocking. 

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