The Synthesis of Oligonucleotides

The polycondensation of several nucleoside monophosphates gives oligonucleotides (up to 40-50 units). If the chain is even longer, the polymer is referred to as a polynucleotide. Initial experiments on the polycondensation of nucleotides to give longer chains were carried out about ten years after the discovery of the DNA double helix (G. Schramm, Sect. 6.3). 

Naylor and Gilham (1966) took another route they were able to link short DNA fragments to a complementary matrix without using an enzyme. The reactions were carried out in aqueous solution, and the molecules first had to be converted into a reactive state by chemical activation the activation agent used was a water-soluble carbodiimide. 

The experimental result shows that two conditions must be fulfilled in order for a ligation to take place  

One building block must be activated, and A complementary matrix must be present. 

Even if the Naylor-Gilham experiment was successful, the question as to how the hexamers were formed still remains. 

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This group was effective in the synthesis of oligonucleotides using the phospho-triester approach. 

Although the pre-RNA world is now much more the centre of scientific attention in prebiotic chemistry, there have been several attempts in recent years to understand the synthesis of oligonucleotides from the normal nucleotides by using simulation experiments (Ferris, 1998). In condensation reactions in aqueous media, there is always competition between synthesis and hydrolysis synthesis is generally only successful when supported by catalysts. 

Fig. 6.19 Three different units for the synthesis of oligonucleotides (Herdewijn, 2001) ...

Smith, L.M., Fung, S., Hunkapiller, M.W., Hunkapiller, T.J., and Hood, L.E. (1985) The synthesis of oligonucleotides containing an aliphatic amino group at the 5 terminus Synthesis of fluorescent DNA primers for use in DNA sequence analysis. Nucleic Acids Res. 13, 2399-2412. 

Compounds ROP(NR2)2 are standard reagents in the synthesis of oligonucleotides and their thioanalogues. The most popular are diamidites contain-... 

The synthesis of oligonucleotide primers, based upon conserved sequences between the NGF and BDNF genes, allowed researchers to fish for additional members of the neurotrophin family by PCR analysis. In 1991 this led to the discovery of neurotrophin 3 (NT-3), which is expressed early in — and throughout — embryogenesis. It supports the development of various neuronal populations in culture, although its role in vivo is less well understood. 

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]. 

The formation of dialkyl phosphonates by coupling of monoalkyl phosphonates with support-bound alcohols is discussed in Section 16.2.3. Some additional examples, not related to the synthesis of oligonucleotides, are sketched in Figure 11.3. 

Ribonuclease N, has properties similar to those of RNase Ti in regard to base specificity and molecular size (6). However, it has a higher phosphotransferase activity and far less hydrolase activity than RNase Tl As suggested from this finding, RNase N, is more advantageous than RNase T for the synthesis of oligonucleotides with 5 -terminal... 

Recently, Koike et al. (104) have proved that RNase N, is a more useful tool than RNase Ti for the synthesis of oligonucleotides such as guanylyl-(3, 5 )-nucleoside, oligoguanylic acid, and the oligonucleotides of defined sequence containing guanylyl or inosinylyl residue. 

Resistant to acids and bases and without a tendency to migrate, this group is stable for all the reactions employed in the synthesis of oligonucleotides. It is photolytically cleaved under a controlled pH without affecting the pyrimidine or purine bases [107, 108]. It has also been used as a photoremovable protecting group for the phosphate function [109]. 

The primary structure of DNA consists of nucleotides linked by a phosphodiester bond between the 5 -OH of one unit and the 3 -OH of the next unit. To fully describe a DNA molecule, the base sequence must be known. Methods for sequencing have been developed, and, at present, over 150 bases can be sequenced per day. The counterpart of sequencing, the synthesis of oligonucleotides having known base sequences, is also highly developed. 

Beaucage SL, Iyer RP, Advances in the synthesis of oligonucleotides by the phosphoramidite approach, Tetrahedron, 48 2223-2311, 1992. 

In the Werstiuk-Neilson approach to the synthesis of oligonucleotides by the triester method,60 N-benzoyl protection has been ap-... 

The development of the synthesis of peptides on solid supports has also had a strong impact on other areas of organic synthesis. From the very beginning, some research groups tried, with some success, to export the idea from peptide synthesis to, for instance, the monoalkylation and monoacylation of carboxylic acids,the synthesis of aldehydes and ketones, or to the use of resin-bound dienes or dienophiles to trap reactive intermediates.PI This pioneering work has been fundamental to the much more recent explosion of combinatorial chemistry on solid supports. Synthesis on solid supports has also found application in the synthesis of oligonucleotides, PI oligosaccharides,PI peptide-DNA hybrids,P°l and peptide nucleic acids (PNAs).P l... 

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