CPG solid support

Load an empty synthesis column with 8 mg of the DNA CPG solid support (—25 /xmol/g) corresponding to the first nucleotide at the 3 -end of the oligonucleotide. 

CPG = Controlled Pore Glass (solid support) 1 and 2 (Bj, B2, B3, B4), commercially available... 

Even controlled-pore glass (CPG) could be successfully employed as solid support with (9-glycosyl trichloroacetimidates as glycosyl donors. Thus, limitations of solvents and reaction temperatures in the glycosylation step, as experienced with the Merrifield resin, are restricted to those observed in solution-phase synthesis. Therefore, regio- and stereocontrol of the glycosylation reactions should be available from well-established solution-phase methodologies. 

A method for the synthesis of cyclic oligoribonucleotides has been described. A TOM-protected RNA phosphoramidite is attached to a CPG linker via 3-chloro-4-hydroxyphenylacetic acid, attached to the phosphoramidite, which is then oxidised to yield the phosphotriester attached to the solid support. RNA synthesis is carried out in the usual manner, and cydisation of the terminal 5 -hydroxyl group to the 3 -phosphodiester occurs using MSNT. A variety of cyclic ODNs were prepared, though cydisation yields were only of the order of 15%. 

The biocatalyzed phenylacetyl removal can be carried out using both solubilized or immobilized substrates1771. The latter methodology has been developed using controlled pore glass (CPG) as a solid support (Fig. 18-7). 

Silyl-based linkers were investigated to effect a nonbasic fluoride-mediated cleavage of the oligonucleotide from the solid support. The siloxyl, 42 [112,113], or disiloxyl, 43 [114], backbones were grafted on CPG or polystyrene supports. Release of the oligonucleotide was achieved by use of 0.5 M tetrabutylammonium fluoride in THF or DMF. Such conditions render the use of this linker structure less compatible with conventional 2 -0-TBDMS RNA chemistry. 

Another important feature of the synthesis of oligonucleotide-peptide conjugates is the choice of a solid support. Polystyrene supports are used in peptide synthesis and CPG supports are incorporated for oligonucleotide synthesis. Most reports describe the use of CPG for the synthesis of oligonucleotide-peptide conjugates [13,14,26,27,36,63,74]. However, in some cases low coupling yields have been reported with CPG and alternative supports have been described, such as Teflon [34], polystyrene (PS)... 

Oligodeoxynudeotide synthesis today is virtually always carried out on a solid support. Initially, the polymeric solid supports were polystyrene cross-linked with divinylbenzene, but controlled-pore glass beads (CPG beads) with defined porosities are now used in preference. The 3 -hydroxyl of the first protected deoxynucleotide is precoupled to the solid support and then solid phase, multistage oligodeoxynudeotide synthesis may commence in the 3 5 direction, in direct analogy to SPPS (Figure 2.5). 

Figure 2.5 Solid Phase DNA Synthesis Cycle (Contd.). Most frequently used base protecting groups are shown Bz Af-6 benzoyl (adenine), W-4 benzoyl (cytosine) N-2 isobutyroyl (guanine). All are base sensitive. DNA chain is built up from 3 to 5 on controlled-pore glass (CPG) bead solid support. Post global deprotection and resin removal, the desired product oligo-/polydeoxynucleotide is then separated initially by precipitation by means of an agent such as ethanol and purified finally by reversed phase liquid chromatography, or ion exchange chromatography as appropriate (see later in Chapter 2).

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