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The Chemical Synthesis of Oligonucleotides

10/01/2018 · Chemical Synthesis and Purification of Oligonucleotides

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[Chemical synthesis of oligonucleotides].

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:

The Chemical Synthesis of Oligonucleotides By Andrei Laikhter and Klaus D

Oligonucleotides are chemically synthesized using nucleosidephosphoramidites. A nucleoside phosphoramidite is a derivative ofnatural or synthetic nucleosides with protection groups added toits reactive exocyclic amine and hydroxy groups. As mentionedearlier (see Phosphodiester synthesis above), the naturallyoccurring nucleotides (nucleoside-3'- or 5'-phosphates) areinsufficiently reactive to afford the synthetic preparation ofoligonucleotides. A dramatically more reactive N,N-diisopropylphosphoramidite group is therefore attached to the 3'-hydroxy groupof a nucleoside to form nucleoside phosphoramidite. To preventundesired side reactions, all other functional groups ofnucleosides have to be rendered unreactive (protected) by attaching. Upon the completion ofthe oligonucleotide chain assembly, all the protecting groups areremoved. Below, the protecting groups currently used incommercially available and most common nucleoside phosphoramiditebuilding blocks arebriefly reviewed:

Chemical synthesis of oligonucleotides. 3: Synthesis …

In the 1970s, substantially more reactive P(III) derivatives ofnucleosides, 3'-O-chlorophosphites, were successfully usedfor the formation of internucleosidic linkages. Thisled to the discovery of the triester methodology. The group ledby M. Caruthers took the advantage of less aggressive and moreselective 1H-terazolidophosphites and implemented themethod on solid phase. Veryshortly after, the workers from the same group further improved themethod by using more stable nucleoside phosphoramidites as buildingblocks. Theuse of 2-cyanoethyl phosphite-protecting group in place of a lessuser-friendly group led to the nucleosidephosphoramidites currently used in oligonucleotide synthesis (seePhosphoramidite building blocks below).Many later improvements to the manufacturing of building blocks,instrumentation, and synthetic protocols made the phosphoramiditechemistry a very reliable and expedite method of choice for thepreparation of synthetic oligonucleotides.

One may visualize an oligonucleotide microarray as a miniaturemulti-well plate where physical dividers between the wells (plasticwalls) are intentionally removed. With respect to the chemistry,synthesis of oligonucleotide microarrays is different from theconventional oligonucleotide synthesis in two respects:

The Chemical Synthesis of DNA and RNA Oligonucleotides …

In the 1950s, and co-workers developed a method where3’-O-acetylnucleoside-5’-O-phosphate2 was activated withN,N’-dicyclohehylcarbodiimide (DCC) or4-toluenesulfonylchloride (Ts-Cl) and a 5’-O-protectednucleoside 1 was reacted with the activatedspecies to give a protected dinucleoside monophosphate3. Uponthe removal of 3’-O-acetyl group using base-catalyzedhydrolysis, further chain elongation was carried out. Followingthis methodology, sets of tri- and tetradeoxyribonucleotides weresynthesized and enzymatically converted to longer oligonucleotides,which allowed elucidation of the . The major limitation of thephosphodiester method consisted in the formation of pyrophosphateoligomers and oligonucleotides branched at the internucleosidicphosphate. The method seems to be a step back from the moreselective chemistry described earlier; however, at that time, mostphosphate-protecting groups available now had not yet beenintroduced. The lack of the convenient protection strategynecessitated taking a retreat to a slower and less selectivechemistry to achieve the ultimate goal of the study.

The use of synthetic oligonucleotides and their mimics to inhibit gene expression by hybridizing with their target sequences has been hindered by their poor cellular uptake and inability to reach the nucleus. Covalent postsynthesis or solid-phase conjugation of peptides to oligonucleotides offers a possible solution to these problems. As feasible chemistry is a prerequisite for biological studies, development of efficient and reproducible approaches for convenient preparation of peptide−oligonucleotide conjugates has become a subject of considerable importance. The present review gives an account of the main synthetic methods available to prepare covalent conjugation of peptides to oligonucleotides.

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    (1997) Chemical Synthesis of Oligonucleotides, in Route Maps in Gene Technology, Blackwell Publishing Ltd., Oxford, UK

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Oligonucleotide synthesis - YouTube

Modern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.

DNA Oligonucleotide Synthesis | Sigma-Aldrich

Modern nucleic acid synthesizers utilize phosphite triester chemistries that employ stable phosphoramidite monomers to build a growing polymer. These robust reactions allow easy generation of specific oligodeoxyribo‐ and oligoribonucleotides with a variety of labels, modified linkages, and nonstandard bases. Strategies are given for the maximization of synthetic yield, the generation of sequences containing site‐specific modifications, and the isolation of synthetic oligonucleotides. Protocols describe monitoring the progress of synthesis via the trityl assay and methods for deprotection.

Consiga Chemical synthesis aqui

Glen Research is delighted to introduce a GalNAc modification strategy using a monomeric GalNAc support and the equivalent GalNAc phosphoramidite. Our experimental work has shown that these products are fully compatible with regular oligonucleotide synthesis and deprotection. Oligonucleotides containing GalNAc can be deprotected using standard procedures during which the acetyl protecting groups on the GalNAc group are removed. Glen Research offers these GalNAc C3 products under an agreement with AM Chemicals LLC.

Oligonucleotide synthesis - Revolvy

The use of a sulfurizing reagent during the regular synthesis cycle using phosphoramidite chemistry has revolutionized the production of phosphorothioate oligonucleotide analogues. Undoubtedly, this ease of preparation of phosphorothioates has made this oligonucleotide modification by far the most common in research. Glen Research was one of the first sources of the sulfurizing reagent, 3H-1,2-benzodithiol-3-one 1,1-dioxide, popularly known as Beaucage Reagent (1).1 This sulfurizing reagent has found common use in the face of a plethora of rival reagents over the years because of its high efficiency, fast reaction time, and widespread availability. The one mild flaw we have found with Beaucage Reagent is that, although it is quite stable in acetonitrile solution in a silanized amber bottle, it is has relatively poor stability in solution once installed on the DNA synthesizer. Consequently, we have not been able to supply a sulfurizing solution, preferring to supply the powdered reagent along with an appropriate silanized bottle. The customer then weighs an appropriate amount of reagent into the silanized bottle and adds acetonitrile at a concentration of 1g/100mL. Over the years, we have considered other sulfurizing reagents but we were not able to find another reagent that exhibits the same fast sulfurization kinetics along with improved stability on the synthesizer. RNA Sulfurization

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