Liquid phase oligonucleotide synthesis

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). 

Finally, the same Italian group studied monocarboxy-functionalized poly-N-acryloylmorpholine (PAcM) (MW 6000) as a new soluble polymer support for liquid-phase oligonucleotide synthesis [322] and a possible alternative to PEG, although its advantages over the latter were not immediately apparent. Preparation and antisense properties of oligonucleotide-polyacryloylmorpholine conjugates were also described [323]. 

Scheme 4. Scheme of liquid-phase oligonucleotide synthesis... 

Bonora GM, Scremin CL, Colonna FP, Garbesi A, HELP (high efficiency liquid phase) new oligonucleotide synthesis on soluble polymeric support, Nucleic Acid Res., 18 3155-3159, 1990. 

While PEG-based supports are widely used for liquid-phase combinatorial chemistry, other non-PEG-based soluble polymers have also been reported for combinatorial applications. A recent review (276) contains an exhaustive list of homo- and copolym-eric soluble supports used in peptide, oligonucleotide, and oligosaccharide synthesis, including combinatorial chemistry. Two of these supports have also been used for small organic molecule synthesis. Homopolymeric polyvinyl alcohol was used in conjunction with PEG for a protection/derivatization strategy in solution (284), and the copolymer between isopropylacrylamide and acrylic acid was used in the catalytic hydrogenation of a Cbz group (285). 

The repetitive sequential-type synthesis on soluble polymeric supports has also been applied to the synthesis of oligonucleotides. As the coupling kinetics in liquid-phase... 

Scremin CL, Bonora GM. Liquid phase synthesis of phosphorothioate oligonucleotides on polyethylene glycol support. Tetrahedron Lett 34 4663-4666, 1993. 

Bonora GM, Baldan A, Schiavon O, Ferrati P, Veronese FM. Poly(V-acryo-lylmorpholine) as a new soluble support for the liquid phase synthesis of oligonucleotides. Tetrahedron Lett 37 4761-4764, 1996. 

Comparable to peptide synthesis, the liquid-phase synthesis of oligonucleotides is based on the sequential repetition of reaction steps, thus adding one monomeric nucleotide to the other (Scheme 4). 

The selection of the polymer support is the most important step in liquid-phase synthesis of oligonucleotides. The number of soluble polymers, which could serve here as carriers, is very limited due to many restrictions. In most cases synthetic macromolecules are preferred because they fulfill the requirements better than many biopolymers. 

Table 14. Derivatives of polyfvinyl alcohol) investigated in liquid-phase synthesis cd oligonucleotides [152]. -(CH-CHd ...

The preparation and availability of suitable soluble polymer supports are basic requirements for the successful application of liquid-phase synthesis of peptides and nucleotides. Many parameters have to be considered when designing appropriate supports and the route of synthesis and the target product of synthesis must also be considered. Liquid-phase synthesis requires optimum solubility properties of the polymer supports and therefore the adaption of the functional capacity to the solubilizing power of the polymer backbone and side-chains is the determining step before synthesis. The main factors of evaluation are the solubility characteristics of the polypeptide or oligonucleotide to be synthesized. 

Brandstetter, F., Schott, H., and Bayer, E. (1975) Polymeric phosphate groups as protective groups for the liquid phase synthesis of oligonucleotides. Makromol Chem, 176,2163-2175. 

Bonora, G M, Scremin, C L, Colonna, F. P, and Garbesi, A. (1990) HELP (High Efficiency Liquid Phase) new oligonucleotide synthesis on soluble polymeric support Nucl, Acids Res 18,3155-3159... 

Figure 4. Scheme of the HELP (High Efficiency Liquid Phase) synthesis of the MPEG-conjugated oligonucleotides. 

Tilted-plate synthesis technology (26,101,104,105) is applicable not only to solid phase synthesis, but also to fluorous synthesis (106), solid and liquid-liquid extraction, and in-plate washing. A production facility (http //www.illumina. com/prod oligos.asp) with a capacity of more than 30 million oligonucleotides (20 mers) per year was built on the basis of tilted centrifugation (101,107). 

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