Polystyrene oligonucleotide synthesis

Synthetic organic polymers, which are used as polymeric supports for chromatography, as catalysts, as solid-phase supports for peptide and oligonucleotide synthesis, and for diagnosis, are based mainly on polystyrene, polystyrene-divinylbenzene, polyacrylamide, polymethacrylates, and polyvinyl alcohols. A conventional suspension of polymerization is usually used to produce these organic polymeric supports, especially in large-scale industrial production. 

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). 

Figure 5.17 Stability of optical encoding towards oligonucleotide reagents. Organosilica microspheres covalently labelled with ATTO 550 dye are stable towards each of the reagents used in phosphoramidite oligonucleotide synthesis. In contrast, optically encoded polystyrene-divinylbenzene (DVB) beads are unstable in most steps, in particular those involving dichloromethane and tetrahydrofuran. (Reproduced from ref. 28, with permission.)...

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. 

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. 

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. 

Uncross-linked polystyrene grafted onto polytetrafluoroethylene (PTFE, Teflon) was adopted for oligonucleotide synthesis according to the phosphodiester scheme by Potapov et al. [167] To produce their support, the authors y-irradiated PTFE... 

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). 

However, the Japanese group has noted partial cleavage of the diisopropylsi-lanediyl linkage under the acidic conditions needed for the DMTr group removal [245, 247]. To circumvent this nuisance, Kobori et al. [247] have prepared a highly cross-linked polystyrene-supported phenyldiisopropylsilyl ether linker that proved to be completely stable to detritylation and used it successfully for oligonucleotide synthesis without N-protection by O-selective phosphoramidite chemistry [246] and pyrophosphate formation on solid phase [248]. The anchor can be cleaved under almost neutral conditions by 1M TBAF-AcOH in THF (90% release after 1 h) or 0.2 M triethylamine trihydrofluoride in the presence of 0.4 M triethylamine for 4h. 

Polystyrene containing 50% divinylbenzene provides a nonswelling, rigid support that possesses the attractive features of rapid reaction kinetics, efficient washing with organic solvents, and mechanical stability during oligonucleotide synthesis. This support has been derivatized to provide a primary amino functionality (Fig. 2) by the same procedure described for aminomethylated polystyrene resin (see Section II.A). 

McCollum C, Andrus A. An optimized polystyrene support for rapid efficient oligonucleotide synthesis. Tetrahedron Lett 32 4069-4072, 1991. 

Bardella F, Giralt E, Pedroso E. Polystyrene supported synthesis by the phosphite triester approach An alternative for the large scale synthesis of small oligonucleotides. Tetrahedron Lett 31 6231-6234, 1990. 

Birch-Hirschfield E, Foldes-Papp Z, Guhrs KH, Seliger H. Oligonucleotide synthesis on polystyrene grafted poly(tetrafluoroethylene) support. Helv Chim Acta 79 137-150, 1996. 

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)... 

Very soon after the introduction of the solid-jdiase synthesis in peptide chemistry, Cramer et al [40] published a paper on oligonucleotide synthesis with soluble polystyrene as support. They used polystyrene (M = 170 OOOgmol ) with 20% of monomethoxytrityl- functionalized phenyl groups of which 40% could be loaded with 3 -0-acetyl thymidine (Scheme 5). 

The sugar moiety is blocked at C5 as a dimethoxytrityl [di(4-methoxyphenyl)phenylmethyl, DMT] ether, readily cleaved by mild acid through an SnI mechanism (Section 22-1 and Problem 40 of Chapter 22), much like 1,1-dimethylethyl (tcrt-butyl) ethers (Section 9-8, Real Life 9-2). To anchor the first protected nucleoside on the solid support, the C3 -OH is attached to an activated linker, a diester. Unlike the Merrifield medium of polystyrene, the solid used in oligonucleotide synthesis is surface-functionalized silica (Si02) bearing an amino substituent as a hook. Coupling to the anchor nucleoside is by amide formation. 

A large variety of different materials have been tried as insoluble supports during the evolution of oligonucleotide synthesis, and the major materials are described in Table 1. The first solid-phase oligonucleotide syntheses were performed using the same nonpolar popcorn polystyrene resin (3,4) used for peptide synthesis (Fig. 5). Later, polar polyamide resins were developed that were more suitable for the polar solvents used in early phosphodiester (5)/triester (d) methods (Fig. 5). 

However, despite the current popularity of CPG supports, the search for improved materials still continues, and recently two promising new supports based on polystyrene have been introduced. Bayer (25) has prepared a tentacle support (Fig. 5) that contains polyethylene glycol (PEG) chains grafted onto the surface of monodisperse polystyrene beads (PEG-PS). The hydrophilic PEG tentacles impart greatly improved swellability in the polar solvents required for oligonucleotide synthesis, and the uniform size and spherical shape of the mono-... 

Gao, H, Gaffney, B L., and Jones, R. A. (1991) H-Phosphonate oligonucleotide synthesis on a polyethylene glycol/polystyrene copolymer. Tetrahedron Lett. 32,5477-5480. 

Likewise, in the preparation of many ion-exchange resins, suitable functional groups are introduced by secondary reactions of macromolecular substances (that are generally crosslinked see Sect. 5.2). In this context the utilization of crosslinked polystyrene resins or poly(acrylamide) gel in the solid-phase synthesis of polypeptides (Merrifield technique) or even oligonucleotides should be mentioned. After complete preparation of the desired products they are cleaved from the crosslinked substrate and can be isolated. 

The fact that macroporous, highly cross-linked polystyrene does not swell makes this support particularly interesting for continuous-flow synthesis in columns. This support has also been successfully used as an alternative to CPG for the solid-phase synthesis of oligonucleotides [90,91]. Furthermore, because reagents do not need to penetrate into the polystyrene network, enzyme-mediated reactions should also proceed smoothly on macroporous polystyrene [85]. 

Linear polystyrene can be generated on insoluble polymers by /-irradiation of the latter in a solution of styrene [110]. Polystyrene grafted onto polytetrafluoroethylene [111-116], polyethylene [2,110,117], or polypropylene [15] can be functionalized in the same way as cross-linked polystyrene, and loadings of up to 1.0 mmol/g can be attained. These supports, which are also available as crown-shaped pins (Multipin, 2-3 mm diameter, 8-10 pmol per crown), have been used for the synthesis of peptides [2,110,111,118], oligonucleotides [112-115,117,119], and small molecules [120-122]. 

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