4.17. Chemical Synthesis of DNA

Definition: Oligonucleotide synthesis is the chemical synthesis of relatively short fragments of nucleic acids with defined chemical structure/sequence. The technique is extremely useful in current laboratory practice because it provides a rapid and inexpensive access to custom-made oligonucleotides of the desired sequence. Whereas enzymes synthesize DNA and RNA in a 5' to 3' direction, chemical oligonucleotide synthesis is carried out in the opposite, 3' to 5' direction. Currently, the process is implemented as solid-phase synthesis using phosphoramidite method and phosphoramidite building blocks derived from protected 2'-deoxynucleosides (dA, dC, dG, and T), ribonucleosides (A, C, G, and U), or chemically modified nucleosides, e.g. LNA. To obtain the desired oligonucleotide, the building blocks are sequentially coupled to the growing oligonucleotide chain in the order required by the sequence of the product. Upon the completion of the chain assembly, the product is released from the solid phase to solution, deprotected, and collected. The occurrence of side reactions sets practical limits for the length of synthetic oligonucleotides (up to about 200 nucleotide residues) because the number of errors accumulates with the length of the oligonucleotide being synthesized. Products are often isolated by HPLC to obtain the desired oligonucleotides in high purity. Typically, synthetic oligonucleotides are single-stranded DNA or RNA molecules around 15–25 bases in length. They are most commonly used as antisense oligonucleotides, small interfering RNA, primers for DNA sequencing and amplification, probes for detecting complementary DNA or RNA via molecular hybridization, tools for the targeted introduction of mutations and restriction sites, and for the synthesis of artificial genes.

4.17.1. Types of Chemical Synthesis

The evolution of oligonucleotide synthesis comprised of four major methods of the formation of inter-nucleosidic linkages.

4.17.1.1. Early work and contemporary H-phosphonate synthesis

1. Todd’s synthesis: ‎ In the early 1950s, Alexander Todd’s group pioneered H-phosphonate and phosphate triester methods of oligonucleotide synthesis. The reaction of compounds 1 and 2 to form H-phosphonate diester 3 is an H-phosphonate coupling in solution while that of compounds 4 and 5 to give 6 is a phosphotriester coupling.



Scheme 4.1: Todd’s DNA synthesis.

2. H-Phosphonate Synthesis: Thirty years later, this work inspired, independently, two research groups to adopt the H-phosphonate chemistry to the solid-phase synthesis using nucleoside H-phosphonate monoesters 7 as building blocks and pivaloyl chloride, 2,4,6-triisopropylbenzenesulfonyl chloride (TPS-Cl), and other compounds as activators. The practical implementation of H-phosphonate method resulted in a very short and simple synthetic cycle consisting of only two steps, detritylation and coupling (Scheme 4.2). Oxidation of inter-nucleosidic H-phosphonate diester linkages in 8 to phosphodiester linkages in 9 (X = O) with a solution of iodine in aqueous pyridine is carried out at the end of the chain assembly rather than as a step in the synthetic cycle. Alternatively, 8 can be converted to phosphorothioate 9 (X = S).



Scheme 4.2: Synthetic cycle in H-phosphonate method of oligonucleotide synthesis.

4.17.1.2. Phosphodiester synthesis

In the 1950s, Khorana and co-workers developed a phosphodiester method where 3’-O-acetylnucleoside-5’-O-phosphate 2 (Scheme 4.3) was activated with N,N’-dicyclohexylcarbodiimide (DCC) or 4-toluenesulfonylchloride (Ts-Cl) and a 5’-O-protected nucleoside 1 was reacted with the activated species to give a protected dinucleoside monophosphate 3. Upon the removal of 3’-O-acetyl group using base-catalyzed hydrolysis, further chain elongation was carried out. Following this methodology, sets of tri- and tetradeoxyribonucleotides were synthesized and enzymatically converted to longer oligonucleotides, which allowed elucidation of the genetic code. The major limitation of the phosphodiester method consisted in the formation of pyrophosphate oligomers and oligonucleotides branched at the internucleosidic phosphate. The method seems to be a step back from the more selective chemistry described earlier; however, at that time, most phosphate-protecting groups available now had not yet been introduced. The lack of the convenient protection strategy necessitated taking a retreat to a slower and less selective chemistry to achieve the ultimate goal of the study.

Scheme 4.3: Oligonucleotide coupling by phosphodiester method.