Synthesis of chemically modified DNA
To make the solid support material suitable for oligonucleotidesynthesis, non-nucleosidic linkers or nucleoside succinates arecovalently attached to the reactive amino groups in AminopropylCPG, LCAA CPG, or Aminomethyl MPPS. The remaining unreacted aminogroups are capped with . Typically, threeconceptually different groups of solid supports are used.
Oligonucleotide synthesis - Wikipedia
In contrast to organic solid-phase synthesis and , the synthesis of oligonucleotides proceeds best onnon-swellable solid supports. The two most often used solid-phasematerials are Controlled Pore (CPG) and macroporous (MPPS).
Oligonucleotide synthesis is carried out by a stepwise additionof nucleotide residues to the 5'-terminus of the growing chainuntil the desired sequence is assembled. Each addition is referredto as a synthetic cycle (Scheme 6) and consists of four chemicalreactions:
Oligonucleotide synthesis - an overview | ScienceDirect …
Thrity years later, this work inspired, independently, tworesearch groups to adopt the H-phosphonate chemistry to thesolid-phase synthesis using nucleoside H-phosphonate monoesters7 as building blocks and pivaloyl chloride,2,4,6-triisopropylsulfonyl chloride (TPS-Cl), and other compoundsas activators. Thepractical implementation of H-phosphonate method resulted in a veryshort and simple synthetic cycle consisting of only two steps,detritylation and coupling (Scheme 2). ofinternucleosidic H-phosphonate diester 8 tophosphodiester 9 with a solution of in aqueous is carried out atthe end of the chain assembly rather than as a step in thesynthetic cycle. Alternatively, 8 can be convertedto phosphorothioate 9 (X =S).
9.08.2.1 Chemical Synthesis of Oligonucleotides
Oligonucleotide phosphorothioates (OPS) are modifiedoligonucleotides where one of the oxygen atoms in the phosphatemoiety is replaced by sulfur. Only the phosphorothioates havingsulfur at a non-bridging position as shown in Figure are widelyused and are available commercially. The replacement of thenon-bridging oxygen with sulfur creates a new center of at . In a simplecase of a dinucleotide, this results in the formation of a pair of Sp- andRp-dinucleoside monophosphorothioates whose structuresare shown in Figure. In a n-mer oligonucleotide where all(n - 1) internucleosidic linkages are phosphorothioatelinkages, the number of diastereomers m is calculated asm = 2(n - 1). Being non-naturalanalogs of nucleic acids, OPS are substantially more stable towards by , the class of that destoy nucleicacids by breaking the bridging P-O bond of the phosphodiestermoiety. This property determines the use of OPS as antisenseoligonucleotides in and applications where the extensive exposure tonucleases is inevitable. Similarly, to improve the stability of , at leastone phosphorothioate linkage is often introduced at the 3'-terminusof both and antisensestrands. In chirally pure OPS, all-Sp diastereomers are more stableto enzymatic degradation than their all-Rp analogs.However, the preparation of chirally pure OPS remains a syntheticchallenge. In laboratory practice, mixtures of diastereomers of OPSare commonly used. Synthesis of OPS is very similar to that ofnatural oligonucleotides. The difference is that the oxidation stepis replaced by sulfur transfer (sulfurization) and that the cappingstep is performed after the sulfurization. Of many reportedreagents capable of the efficient sulfur transfer, only three arecommercially available:
[Chemical synthesis of oligonucleotides].
In the early 1950’s, ’s group pioneeredH-phosphonate and triester methods of oligonucleotidesynthesis. Thereaction of compounds 1 and 2 toform H-phosphonate diester 3 is an H-phosphonatecoupling in solution while that of compounds 4 and5 to give 6 is a phosphotriestercoupling (see Phosphotriester synthesis below).