H-phosphonate method

Stereodefined oligo(ribonucleoside phosphorothioate)s were available only by the enzymatic method [51 -54] or by partially stereocontrolled H-phosphonate procedure [55,56]. However, both procedures could provide only products with RP configuration. Alternatively, compounds containing the single phosphorothioate modification were synthesized by nonstereospecific phosphoramidite or H-phosphonate methods and then separated into diastereomers by tedious HPLC procedure with various degree of success [57-59]. Such constructs became important tools for investigation of the mechanism of action of ri-bozymes. 

The synthesis of the cyclic 2 -deoxyribodinucleotides 82 by a solution-phase H-phosphonate method has been described in which a 5 -protected nucleoside-3 -H-phosphonate (83) is first coupled to a nucleoside 3 -H-phosphonate diester (84). Removal of the cyanoethyl group from the 3 -phosphonate group reveals an H-phosphonate monoester function which remains intact during oxidation of the internucleoside H-phosphonate diester linkage. After further deprotection, this 3 -H-phosphonate is reacted to produce the cyclic derivative 82 in 15% overall yield. 

An additional reason for the inferior performance of the H-phosphonate method compared to the phosphoramidite lies in its mechanism, which makes the synthesis very sensitive to pre-activation of the H-phosphonate monoesters with the conventional coupling agents, which must be avoided [138], However, the main appeal of the H-phosphonate approach lies in the ease it offers for the synthesis of backbone analogues modified at the phosphorus atom phosphorothioates [139], phos-photriesters [126] and especially phosphoramidates [126, 140, 141]. Now it is rarely used for routine oligonucleotide synthesis but very often for the preparation of variously modified analogues. Further developments in the H-phosphonate method have been made in the last decade by Polish [142] and Japanese researchers [143]. 

Reports on the preparation of 2-a-mannopyranosyl-1-phosphatidyl-D-myo-inositol were published in 1977 (Ref.117) and 1989.118 After these reports, recently the 2,6-dimannoside (PIM2, 363) has been synthesized. Van Boom s groupie introduced the mannosyl moieties to the 2 position and subsequently to the 6 position in myo- inositol derivative 359 to yield 361. Dimannoside 361 was phosphorylated after removal of the allyl group by the H-phosphonate method and finally deprotected giving the final product PIM2 (Scheme 6-6). ... 

Notice that in the phosphoramidite method an oxidation is done each time a monomer is added, whereas in the H-phosphonate method a single oxidation is carried out after the entire strand has been synthesized. 

The metabolite 205, produced during t-RNA splicing, has been synthesized by making the pyrophosphate bond through interaction of a-D-ribofuranose-1,2-cyclic phosphate and a mixed anhydride of AMP. Cyclic dinucleotides of type 206 have been prepared by an H-phosphonate method in solution, ... 

Deoxynucleoside 3 -C-phosphonates 188 have been prepared by reaction of 3 -ketonucleosides with tris(Tms) phosphite. Some of the chemistry of these geminal hydroxyphosphonates was also explored. 3 -Methylene-H-phosphon-ates 189 [X = H, OMe, F, 0(CH2)20Me], and also 5 -0-Dmtr species of use for oligonucleotide synthesis by the H-phosphonate method, have been prepared by reaction of iodomethylene compounds with bis(trimethylsilyoxy)phosphine. ... 

We reported earlier that the presence in a molecule of phosphodiester units does not interfere with the next 0-phosphoryIation step by means of the H-phosphonate method (i.e., P-protection for phosphodiesters is not required). Indeed, compound 40 (containing a phosphodiester at 0-6" ) was successfully phospholipidated (Scheme 6) by pivaloyl chloride assisted reaction with the acylalkylglyceiyl hydrogenphosphonate 7 followed by oxidation (in situ) with iodine to provide the fully protected oleic ester GPI [Sp = -1.4 and -1.2 (0.5 P each),... 

All ribooligonucleotides described in the following were chemically synthesized using the H phosphonate method [34, 35]. It was demonstrated that this method allows the preparation of the relatively large amounts (> 10 mg) of RNA being necessary for 2D NMR studies at reasonable costs and with the required purity. In addition, as compared to phosphora-midite chemistry, the H phosphonate chemistry offers the advantage of recovery and reusing of synthons. This is particularly important for expensive modified or isotopically labelled nucleotides. 

The oxazoline method and two modifications of the H-phosphonate method have been used to obtain glycosyl phosphatidylinositol derivatives. ... 

Vasser, M., Ng, P, Jhurani, P., and Bischofberger, N. (1990) Error rates in oligonucleotides synthesized by the H-phosphonate method. Nucl. Acids Res 18,3089. 

Gaffney, B L, and Jones, R. A (1988) Large-scale oligonucleotide synthesis by the H-phosphonate method. Tetrahedron Lett. 29, 2619-2622. 

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