Phosphite triester Approach,Gentle Acid Hydrolysis,Di-Isopropylammonium Tetrazolide,Alkaline Hydrolysis,Microprocessor

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	Phosphite triester Approach - Nucleosides having protected bases and 5' -OH groups are the basic materials. The 3'-OH of the terminal nucleotide is fixed to a solid support and, then, its 5'-OH is freed by gentle acid hydrolysis. The subsequent nucleotides are used as 3'phosphoramidities, which are readily produced by coupling the nucleotides with di-isopropylammonium tetrazolide. The phosphoramidites are stable and efficient coupling agents, and are readily synthesized. The desired nucleotide 3' phosphoramidite is now added and is activated for coupling by the addition of tetrazole; the immediate product is a phosphite, which is oxidized to phosphate by iodine (12), This phosphate remains as a triester.
The 5' -OH of the second nucleotide is now freed, and the third desired nucleotide 3'-phosphoramidite is added and joined to the growing chain. In this manner, the oligo nucleotide chain is elongated. Finally, the various protective groups are removed and the chain is freed from the solid support by alkaline hydrolysis. This approach using silica based or controlled pore glass beads solid support is used for automated synthesis of oligonucleotides. It takes less than 15 minutes for adding one nucleotide to the chain, and chains as long as 50 nucleotides can be prepared in good yields. The automated DNA synthesizers are popularly called gene machines; they are microprocessor controlled and carry out all the operations automatically.