Deprotection of oligonucleotides

Scheme 7.30 Deprotection of oligonucleotides on controlled pore glass (cpg).

Aqueous ammonia, dioxane. - The addition of nitromethane in the cleavage reaction will scavenge the released acrylonitrile and prevent it from reacting with the nucleohase during deprotection of oligonucleotides/ ... 

A comparative evaluation of methylamine, methylamine/anunonium hydroxide and ammonium hydroxide/ethanol for the deprotection of oligonucleotides has also been undertaken. In order to avoid the possible transamination of cytosine when benzoyl protection is employed for the amino group an acetyl protected monomer was used instead. Methylamine or methylamine/ammonium hydroxide mixtures were found to liberate the oligoribonucleotide at room temperature in 90 minutes and improve the yield of the desired product. 

The most serious problems of CPG are the presence, in the crude product, of silica and polymeric siloxanes released during cleavage and deprotection of oligonucleotides. Furthermore, the silane coupling chemistry used to functionalize the inorganic surface of CPG beads is complex, leading to variation in substitution levels from preparation to preparation. 

Strongly basic nonnucleophilic conditions such as 0.5 M DBU in di-oxane or pyridine were used by Eritja et al. [115] to effect the -elimination cleavage of a 4-ethyl-3-nitrobenzoyl carbonate linker, 44, allowing complete deprotection of oligonucleotides synthesized with p-nitrophenylethyl-based protecting groups under totally ammonia-free conditions [116]. 

Reddy MP, Hanna NB, Farooqui F. Fast cleavage and deprotection of oligonucleotides. Tetrahedron Lett 35 4311-4314, 1994. 

This amide, readily formed from an amine and the anhydride or enzymatically using penicillin amidase, is readily cleaved by penicillin acylase (pH 8.1, A -methylpyrrolidone, 65-95% yield). This deprotection procedure works on peptides, phosphorylated peptides, and oligonucleotides, as well as on nonpeptide substrates. The deprotection of racemic phenylacetamides with penicillin acylase can result in enantiomer enrichment of the cleaved amine and the remaining amide. An immobilized form of penicillin G acylase has been developed. ... 

The steps involved in automated oligonucleotide synthesis illustrate the current use of protective groups in phosphate chemistry (Scheme 1). Oligonucleotide synthesis involves the protection and deprotection of the 5 -OH, the amino groups on adenine, guanine, and cytosine, and -OH groups on phosphorus. 

In the early solution phase syntheses of oligonucleotides, coupling of phosphate diesters was used. A mixed 3 -ester with one aryl substituent, usually o-chlorophenyl, was coupled with a deprotected 5 -OH nucleotide. The coupling reagents were sulfonyl halides, particularly 2,4,6-tri-i-propylbenzenesulfonyl chloride,53 and the reactions proceeded by formation of reactive sulfonate esters. Coupling conditions... 

Critical to the success of this procedure was the development of a suitable temporary ligand that is sufficiently inert to conditions of oligonucleotide deprotection (typically aqueous ammonia, 6h at 50° C) and can subsequently be replaced by chloride. The nucleobase derivative 1-cyclohexamethyl-thymine 41 was found to satisfy these criteria and can be converted to the trans-Pt-Cl species by treatment with dilute HC1 (pH 2.3, 40°C, 48 h). This last step restricts the range of possible oligonucleotide sequences to those that are not susceptible to depurination, but clearly establishes a prototype system for future development. 

Several different methods of sidewall functionalisation, such as fluorination, radical addition, nucleophilic addition, electrophilic addition and cycloaddition, have been developed (Tasis et al., 2006). The sidewalls of vertically aligned CNTs have been functionalised with DNA using azide units as photoactive components. The azi-dothymidine reacted photochemically with sidewalls of CNTs utilising [2+1] cycloaddition. The oligonucleotides were grown in situ on the sidewalls of CNTs and the DNA-modified CNTs were obtained after the deprotection of the nucleic acid (Moghaddam et al., 2004). 

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