Coupling oligonucleotide synthesis

Oligonucleotide synthesis involves specialized blocking and coupling reactions the chemistry of which is beyond the scope of a typical introductory course The in terested reader is referred to http //WWW bi umist ac uk/ users/dberrisford/1 MBL/ nucleicacidB html... 

Although use of automated oligonucleotide synthesis is widespread, work continues on the optimization of protecting groups, coupling conditions, and deprotection methods, as well as on the automated devices.56... 

The microspheres—synthesised via a two-step process (acid-catalysed hydrolysis and condensation of 3-mercaptopropyltrimethoxysilane (MPS) in aqueous solution, followed by condensation catalysed by triethanolamine)—have a narrow size distribution (Figure 5.16) and are considerably more stable than polystyrene divinylbenzene microspheres as shown in phosphoramidite oligonucleotide synthesis by the excellent retention of fluorescence intensity in each of the reagent steps involved in phosphoramidite DNA synthesis (Figure 5.17, in which the organo-silica microsphere free thiol groups are derivatized with ATTO 550 maleimide coupled to the entrapped dye). 

Palladium-mediated (Sonogashira) coupling [332] of 5-iodouracil with alkynes can be performed under mild reaction conditions, and is compatible with the supports, linkers, and protective groups used in solid-phase oligonucleotide synthesis (Entries 12 and 13, Table 15.28). This coupling reaction has been used to prepare modified oligonucleotides on polystyrene and on CPG. The reaction of halopyrimidines with amines to yield aminopyrimidines is discussed in Section 10.1.2. 

This group was developed for 5 -hydroxyl protection in oligonucleotide synthesis. It is stable to the conditions for nucleotide coupling using the phosphoramidite approach. It is not stable to acid or to 12/pyridine/THF, conditions used for phosphite oxidation. It has been used to prepare a 20-mer. ... 

However, even the pre-synthesized tetrazolides were still too reactive to be routinely used in the automated solid phase synthesis. The major advance that solved the problem was made in 1981, when Beaucage and Caruthers, who were experimenting with the nucleoside 3 -phosphoramidite derivatives [83] following some previous Russian work on the phosphorus(III)-amino compounds [84], discovered that these otherwise pretty stable compounds can be rapidly and very efficiently coupled to a solid-supported nucleoside in the presence of a mildly acidic nucleophilic catalyst, tetrazole [85]. This discovery combined with the already existing solid-phase assembly layout [68, 69, 82] paved the way for the very rapid expansion of the polymer-supported oligonucleotide synthesis, which has been summarized in the timely book edited by Gait [12a]. 

The only exception was an attempt at the 5 -to-3 solid-phase oligonucleotide synthesis using nucleoside 3 -H-phosphonate monoesters, polymer-bound through a trityl-type linker, and oxidative coupling conditions (HgCl2) [123], The H-phosphonates were produced by phosphitylation of the polymer-bound N-unpro-tected nucleoside with phosphorus trichloride followed by hydrolysis by aqueous pyridine [61c]. However, despite some advanced features [124] the stepwise yields were comparable with those in the contemporary phosphodiester method-that is they were too low for the method to be truly competitive. 

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]. 

---