Phosphodiester method

This so-called phosphodiester method [92] was later also performed on cross-linked polystyrene [93], macroporous 25% cross-linked polystyrene [94], silica [95], poly(-vinyl alcohol) [96,97], proteins [98], PEG [99], and polyacrylamide [100]. This extensive evaluation of different supports stemmed from the observation that cross-linked polystyrene or other gel-type supports were not ideal for the preparation of phospho-... 

From the late 1950s oligonucleotide synthesis has become dominated for almost two decades by the method introduced by Khorana and coworkers [16] and dubbed the phosphodiester method (Scheme 19.2, method I) [9]. Its central dogma requires the phosphate groups to be left completely unprotected, which renders them sus-... 

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. 

While Merrifield experimented with the polystyrene cross-linked with 2% and later 1% DVB [55], Letsinger adopted the popcorn polystyrene [54] which relies on less cross-linking agent (0.05-0.2%) to achieve complete insolubility in common organic solvents. Ah of his early ohgodeoxyribonucleohde syntheses by the phos-photriester method were performed on this type of support [53, 58, 59], Other researchers have used popcorn PS as well in the syntheses by the phosphodiester method [144] and, later, more advanced phosphotriester method [145], However, with the widespread use of low cross-linked PS and the advent of polyacrylamide and especially silica gel supports (Section 19.2.3), use of popcorn polystyrene in oligonucleotide synthesis has practically ceased. 

Low cross-linked polystyrene resins were first adopted for oligodeoxyribonucleo-tide synthesis within the framework of the phosphodiester method [146-150],... 

Interest in highly cross-linked (20-30% DVB) macroporous polystyrene arose in the early days of oligonucleotide synthesis [158]. It has been argued that this type of support allows for faster reaction kinetics than the low cross-linked gel-type polystyrene. Modified with a trityl-type tinker, it has been tested by Roster and Cramer [159] in the phosphodiester method. The limitations of the chemistry hampered its use for oligomers longer than a few nucleotides. 

The most common trityl anchor was monomethoxytrityl (Figure 19.5), employed in the phosphodiester method on PS-1% DVB by Melby and Strobach [146], on macroporous PS by Roster and Cramer [159] and on PTFE grafted polystyrene by Potapov d. al. [167] A dimethoxytrityl linker was used by Roster and Cramer for the phosphodiester chemistry on popcorn PS [144] and by Belagaje and Brush [155] for their original adaptation of the phosphotriester method for the synthesis in 5 -to-3 direction (Figure 19.5). Roster has also described a trityl anchor linked to a silica gel support [185]. Similar linkers have been exploited for liquid-phase oligonucleotide synthesis (Section 19.4). 

The answer to the former problem came once again from peptide synthesis, when Bayer and coworkers [309] reported the first use of polyethylene glycol) (PEG) as a soluble support for peptide synthesis. The idea was immediately picked up by Koster, who prepared a PEG-20000 terminating in a dimethoxytrityl linker (Figure 19.16) and demonstrated the attachment of thymidine [310]. Later, Bayer and coworkers [311] assembled up to a pentanucleotide by the phosphodiester method on a PEG-NH2 support with a phosphoramidate linker cleavable by isoamyl... 

The phosphotriester method overcomes some of the disadvantages of the phosphodiester method by blocking each intermediate phosphodiester function during the synthesis of the required sequence. This method was used to synthesize 67 different oligonucleotides of chain length 10-20 which were then spliced to gen-... 

Three types of method are used for the chemical synthesis of oligodeoxyribo-nucleotides. The phosphodiester method, in which a nucleotide with a protected 5 -hydroxy group is condensed with a nucleoside-5 -monophosphate with a protected 3 -OH, has been extended by use of the polymer-support method analogous to that used for peptide synthesis. The phosphotriester method has been modified by Narang et al., to use a two-step coupling procedure starting with a nucleotide with a fully masked 3 -phosphate. The most recent type of... 

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