Solid Support Synthesis of Oligonucleotides

Gene (sense/antisense) Overlap Species Sense gene properties 

Proto-oncogene, possible transcription factor DNA-binding protein DNA precursor synthesis, mRNA binding protein Proto-oncogene DNA repair DNA repair 

Vitamin D3 and cholesterol-27 hydroxylation, mitochondrial Structural protein Structural protein Peptide hormone Growth factor Ribosomal protein 

Antisense is by no means an artificial concept. Apart from basic coding/non-coding strand interactions, a series of cases has been found in nature, in which gene regulation is effected by antisense-type nucleic acid/nucleic acid interactions. Frequently, control of translation and accelerated depletion of target mRNA is effected by antisense ODNs in eucaryotic organisms like yeast, insects or mammals. In Table 2 a series of reported natural sense/antisense interaction is listed. In case given, sequences involved in such interactions are also potential elements of therapeutic antisense action. 

Unmodified ODNs are by far more stable when compared with unmodified RNA, which is cleaved easily by almost ubiquitous enzymes. In addition, RNA synthesis requires an additional protective group at the 2 -hydroxy group. Thus, it is no wonder that automated RNA synthesis was first published only about 10 years ago by Ogilvie [33], who modified the synthetic phosphoramidite protocol previously established for ODN synthesis. 

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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]. 

For the automated solid-phase synthesis of oligonucleotides, however, silica was found to be the support of choice [192-197]. Silica with large pore size (25-300 nm), so-called controlled pore glass (CPG), is generally used for this purpose. The main advantages of CPG, as compared with silica gel, are its more regular particle size and shape, and greater mechanical stability. 

Attachment of suitable linkers to the surface of silica can be achieved by transesterification with (3-aminopropyl)triethoxysilane, which leads to the support 2 (Figure 2.8) [198-200]. Alternatively, silica can be functionalized by reaction with alkyltri-chlorosilanes [201]. For the solid-phase synthesis of oligonucleotides, supports with a longer spacer, such as that in 3, have proven more convenient than 2 [202-206]. Supports 3, so-called LCAA-CPG (long chain alkylamine CPG [194,195]), are commercially available (typical loading 0.1 mmol/g) and are currently the most commonly used supports for the synthesis of oligonucleotides. For this purpose, protected nucleosides are converted into succinic acid monoesters, and then coupled to LCAA-CPG. CPG functionalized with a 3-mercaptopropyl linker has been used for the solid-phase synthesis of oligosaccharides [207]. 

Bis(hydroxymethyl)phosphonic acid esters that incorporated thymine were employed as a backbone to prepare short oligonucleotide chains. This chain was prepared by condensation of the bis(4,4 -dimethoxytrityl) protected phosphonic acid and iV or N -(2-hydroxyethyl)thymine in the presence of l-(2-mesitylenesul-fonyl)-3-nitro-l,2,4-triazole or by an Appel reaction with or N -(2-aminoethyl)thymine (89a-h). Selective removal of one DMT-group and phos-phitylation yielded the building blocks for solid supported synthesis of the short oligomers by the phosphoramidite approach. Holy has reported the synthesis of 8-amino and 8-substituted amino derivatives of acyclic purine nucleotide analogues. The 8-amino, 8-methylamino- and 8-dimethylamino-adenine and -guanine analogues of iV-(2-phosphonomethoxyethyl) and (S)-iV-(3-hydroxy-2-phosphono-methoxy-propyl) derivatives of purines (90a-i), were prepared by... 

The solid support synthesis of oligopeptides and oligonucleotides is routinely carried out by reacting a support-bound structure (acceptor) with an excess of a solution-... 

Figure 7 illustrates the different steps in solid support synthesis of an oligonucleotide. After detritylation, tetrazole-catalyzed coupling - the most crucial reaction step - is carried out. Figure 8 indicates the mechanism of tetrazole action. 

The solid-phase synthesis of oligonucleotides by the phosphoramidite approach requires the use of a solid support that is functionalized with an appropriate nucleoside. Solid supports are typically macroporous structures... 

While a large number of improvements in procedures for solid phase synthesis of oligonucleotides have been described, the techniques have not altered fundamentally from those described in previous reports. Solid-phase synthesis using a continuous-flow phosphotriester method on a kieselguhr-polyamide support has been described. Other solid phases used as supports for synthesis include derivatized h.p.l.c.-grade silica gel, . derivatized cross-linked poly-... 

Compared to solid-phase poljq)eptide synthesis, there has been far less activity in polymer-supported oligosaccharide synthesis. Unlike the solid-phase synthesis of oligonucleotides (Letsinger and Mahadevan, 1965), which closely followed the first reported solid-phase polypeptide... 

Wafers and Capsules. Another solution of compartmentalization of resin beads into permeable containers was reported by Beattie and Frost, who invented porous wafers that housed insoluble supports for the multiple solid-phase synthesis of oligonucleotides and peptides. The porous wafer was made from a Teflon ring covered on both sides by a porous Teflon membrane to form a cylindrical permeable container. The use of wafers was reduced to practice in a specialized column-based oligonucleotide synthesizer. ... 

The solid phase synthesis of oligonucleotides has not reached the level of sophistication as that for peptides. The design of an appropriate solid support has been difficult with such problems as undesirable polymer characteristics and irreversible adsorption of reagents onto the support. Only recently have appropriate polymer matrices (e.g., HPLC silica) been described that appear to have potential for the synthesis of nucleotide polymers. In fact, a major impetus for the development of solid phase methodology for the synthesis of polydeoxyribonucleotide (DNA) sequences has been the industrial application of the so-called biotechnology or recombinant DNA technology. 

William Fraser was born in Hamilton. He studied at the other of the two local universities, Strathclyde, where he obtained a first class B.Sc. honors degree in 1986 and Ph.D. in 1989 under the direction of Professor Colin J. Suckling and Professor Hamish C. S. Wood. He was awarded a Royal Society European Exchange Postdoctoral Fellowship and worked in the laboratories of Professor Albert Eschenmoser at the ETH, Zurich. In 1991, he took up his present position as lecturer in medicinal chemistry at Aston University, Birmingham. His scientific interests include nucleoside and nucleic acid chemistry, solid-supported, synthesis, and study of base-modified antigene oligonucleotides targeted to DNA. 

Synthetic oligonucleotides are very important tools in the study and manipulation of DNA, including such techniques as site-directed mutagenesis and DNA amplification by the polymerase chain reaction (PCR). The techniques for chemical synthesis of oligonucleotides have been highly developed. Very efficient and automated methodologies based on synthesis on a solid support are used widely in fields that depend on the availability of defined DNA sequences.152... 

The subsequent explosion of array technologies has been sparked by two key inno-vations. The first is the use of non-porous solid support, such as glass, which has facilitated the miniaturization of the array and the development of fluorescence-hybridization detection (16, 17, 18). The second critical iimovation has been the development of methods for high-density spatial synthesis of oligonucleotides, which allows the analysis of thousands of genes at the same time. Because DNA cannot bind directly to the glass, the surface is first treated with silane to covalently attach reactive amine, aldehyde, or epoxies groups that allow stable attachment of DNA, proteins, and other molecules. 

The synthetic protocols used for the preparation of oligonucleotides on supports can also be used to prepare oligomers from diols other than nucleosides. Symmetric or unsymmetric diols, such as N-acylated 4-hydroxyprolinol [268] or cyclopentane-derived diols (carbocyclic deoxyribose analogs [269]), can be selectively mono-trity-lated and then converted into a phosphoramidite that is suitable for the solid-phase synthesis of oligophosphates. An illustrative synthesis of protected //-phosphonates from diols, as well as their conversion on CPG into oligomeric phosphoramidates, are outlined in Figure 16.28. 

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