Phosphodiester approach

The phosphotriester approach solves some of the problems of the phosphodi-ester method. More recently, the phosphite-triester method has been introduced this makes use of the extreme reactivity of the phosphite reagent, i.e., the two building blocks are joined in a few minutes compared with the hours required by the phosphodiester approach. 

A possible solution to some of the problems inherent in the phosphodiester approach (water solubility, ion exchange chromatography, and lower yields) would accrue if the third dissociation of phosphate was masked, thus creating a neutral organic molecule amenable to the more standard manipulations of organic chemistry. Actually, this approach was first introduced by Michelson and Todd in 1955. 

Phosphorothioates. All three synthetic approaches appHcable to unmodified oligonucleotides can be adapted for synthesis of phosphorothioates (11) (33,46). If all of the phosphodiester linkages in an oligonucleotide are to be replaced with phosphorothioates, the ff-phosphonate method for coupling, followed by oxidation with Sg in carbon disulfide and triethylamine in the final step, is the most straightforward method. 

Finally, a similar approach was undertaken to evaluate the origin of factor 3 in Fig. 15.3c whose score image reveals several small-localized pockets in the epidermis. The loading from factor 3 (Fig. 15.3b) depicts one broad feature at 1090cm 1 not present in the other factor loadings. Based on both the position of this particular band and the spatial distribution of the factor score, the band probably arises from a phosphodiester mode of DNA [30, 31] present in the nuclei of keratinocytes or other cells. An additional feature at 780 cm 1 in both spectra and factors (not shown) arises from cytosine in DNA [32] and is consistent with this interpretation. 

The above potential energy approach of building double helical polynucleotides is illustrated below for some hypothetical B-family poly(dA) poly(dT) chains. For simplicity, the helices are generated as a function of the phosphodiester angles < and u> only. 

The apparent usefulness of the modeling approach suggested that possible active site interactions important in understanding the mode of action of the well-characterized enzymes, ribonuclease (16) and staphylococcal nuclease (17). may be revealed. Both have been the subject of extensive crystallographic studies (18,19) with suitable inactive substrates in place. We considered the first step of hydrolytic action of ribonuclease (RNase) on the dinucleotide substrate uridylyl-(3 -5 )-adenosine(UpA). Our results (20) on the enzyme mechanism were consistent with the main features summarized by Roberts et al (21). The first step is a transphosphorylation leading to cleavage "oT the phosphodiester... 

The results of the above experimental approach will provide excellent proof for an sn-1,2-diacyl glycerol backbone attached by a phosphodiester... 

Oligonucleotides containing the isonucleoside (59), that has an extended phosphodiester linkage were prepared by the phosphoramidite approach. Such homo-oligonucleotides adopt an A-form conformation as demonstrated by... 

This approach has recently been applied to the association of a phosphodiester antisense oligonucleotide directed against the 3 non-translated region of the PKCa gene with nanospheres prepared from PIBCA. These nanospheres were able to inhibit PKCot neoexpression in cultured Hep G6 cells. 

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