Marc L. Rothstein and Dianne M. Rothstein
Prime Synthesis, Inc.
2 New Road, Suite 126
Aston, PA 19014
Controlled Pore Glass (CPG) has been widely used as a support for the solid phase synthesis of oligonucleotides. It provides a unique combination of uniform pore size, dimensional stability in synthesis solvents and a pore network structure that facilitates rapid mass transfer during a synthesis. Pore diameters of 500 to 2000 Angstroms are commonly used for this application.
To avoid steric hindrances, the larger pore sizes are used for synthesizing longer oligos. However, since the surface area of CPG is inversely related to the pore size and the density of silanol attachment groups are a function of surface area, large pore CPG has less ligand loading capacity. Additionally, not all silanol attachment points are evenly distributed, with some being too crowded together to be useful for full-length oligo growth. For this reason, maximum loading densities are not used for high purity synthesis. Thus, due principally to steric considerations, oligo length and purity is limited by pore size and loading density. A further limitation of CPG is that some reagents are corrosive to the glass substrate and can cause inconvenient silica precipitates during the oligo work-up or even complete failure in the case of reagents such as acidic fluorinated deblocking solutions.
Porous polystyrene beads have also been popular for oligo synthesis. Highly crosslinked polystyrene is used for micro-scale synthesis. This type of polystyrene is preferred for use in pre-packed synthesis columns, since it does not swell upon exposure to synthesis solvents. The combination of high crosslinking with a relatively broad pore size distribution limits useful ligand loading densities to about 30 µmoles/g. For large scale oligosynthesis, polystyrene of lower crosslinking levels can be used with ligand loadings up to 200-400 µmoles/g. Although these supports can swell up to 6 times their dry volume in synthesis solvents, they can be used in large scale synthesizers by allowing room in the reactor column for this expansion. They have become popular for the synthesis of oligos up to about a 24-mer. However, at these loadings, synthesis of longer oligos is not feasible due to backpressure increases in the reactor bed caused by additional expansion of the polystyrene to accommodate the growing oligo product volume.
HybridCPG™ consists of CPG particles conformally coated with a very thin crosslinked polymer film based on polystyrene. The molecular structure of the polymer coating is designed to have a very high density of evenly distributed attachment points for optimum oligo synthesis. In this way, the pore size - loading density trade-off is minimized and the chemical resistance to glass-aggressive reagents is greatly improved. Although the nano-scale coating is subject to swelling, the rigid pore structure of the CPG substrate accommodates this and, at the same time, maintains a uniform pore space for the oligo synthesis. Thus, HybridCPG exhibits no bulk swelling in the synthesis solvents and allows much higher ligand loadings for a given pore size compared to conventional CPG. As illustrated in Figure 1, the combination of HybridCPG’s high ligand loading capacity and freedom from bulk swelling results in much larger column capacities than either conventional CPG or swellable polystyrenes.
|Synthesis & Support Properties||Uncoated CPG*||Hybrid CPG*||Uncoated CPG||Hybrid CPG||Hybrid CPG||Hybrid CPG|
|Pore Size (Å)||1000||1500||1000||1000||1360||1780|
|Support Loading (µmol/g)||35||90||34||141||137||122|
|Synthesis Scale (µmol)||65||144||45||238||211||223|
|Total FLP (OD's,A260)||5,276||19,285||5,250||27,354||28,027||27,826|
|*Data courtesy of NOXXON Pharma, AG
Sequence A: 5' AGU GAA GC GUG GCU CUG CG-DdT-3' (L-RNA)
Sequence B: 5' ATA CCG ATT AAG CGA AGT TT-3' (DNA)
Table 1 demonstrates the high performance of a large pore HybridCPG support for some example syntheses made on a large-scale synthesizer (GE Healthcare AKTA 100). Data for two sequences are shown. Note the relative independence of Hybrid CPG pore size to ligand loading. Thus, while it is important to establish a minimum pore size in optimizing the synthesis conditions, the tradeoff of loading capacity is not seen as the pore size is further increased.
Prime Synthesis has partnered with Glen Research to begin offering HybridCPG™ to the research market. We are initially offering two popular universal supports, Glen UnySupport™ and US III. Both supports have a loading of around 75 µmoles/g and support oligo synthesis in the 30-40mer length range. The structures of these supports are shown in Figure 2, Back Page.
The high synthesis performance on these supports is demonstrated by the RP HPLC of a 40mer prepared on US III HybridCPG, as illustrated in Figure 3, Back Page.
HybridCPG™ is a trademark of Prime Synthesis, Inc. Glen UnySupport™ is a trademark of Glen Research Corporation.
Universal Support III HybridCPGTM (28-5010-xx) has been discontinued.