Oligonucleotides deprotection

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. 

Post-synthesis modification may be used to introduce a number of O - alkylated guanosine derivatives into oligonucleotides. Using a 0 -methoxycar-bonylmethyl-dG phosphoramidite and different oligonucleotide deprotection conditions leads to a number of different alkylated guanosine derivatives. 

McBride LJ, Kierzek R, Beaucage SL, Caruthers, MH. Amidine protecting groups in oligonucleotide synthesis. J Am Chem Soc 108 2040-2048, 1986. Vinayak R, Anderson P, McColum C, Hampel A. Chemical synthesis of RNA using fast oligonucleotide deprotection chemistry. Nucleic Acids Res 20 1265-1269, 1992. 

Vu H, McColum C, Jacobson K, Theisen P, Vinayak R, Spiess E, Andras A. Fast oligonucleotide deprotection phosphoramidite chemistry for DNA synthesis. Tetrahedron Lett 31 7269-7272, 1990. 

A phosphoramidite derivative of A -isobutryT2 -0-methylcytidine has been synthesised in order to decrease the risk of transamination reactions which occur at the C4-position of cytidine when alternatives to ammonia are used for oligonucleotide deprotection. The monomer was prepared from cytidine protected with 1,1,3,3-tetraisopropyldisiloxane. Transient protection of the 2 -hydroxyl as the trimethylsilyl derivative followed by reaction with isobutyryl chloride gave the protected nucleoside which could be reacted with methyl iodide and silver oxide after removal of the transient protection. Suitable protection and derivatisation for synthesis yield the required synthon (131). 

One synthesis cycle is now closed. A new cycle can start with 5 -detritylation (step 1). When the last cycle is closed, the terminal 5-0-(4,4 -dimethoxytrityl) group of the desired oligonucleotide is either removed or left attached before the deprotection, liberation and puriHcation of the product. 

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. 

Step 4 With the coupling accomplished, the phosphite product is oxidized to a phosphate by treatment with iodine in aqueous tetrahydrofuran in the presence of 2,6-dimethylpyridine. The cycle (1) deprotection, (2) coupling, and (3) oxidation is then repeated until an oligonucleotide chain of the desired sequence has been built. 

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... 

Although use of automated oligonucleotide synthesis is widespread, work continues on the optimization of protecting groups, coupling conditions, and deprotection methods, as well as on the automated devices.56... 

Figure 27.8 SATA may be used to modify a 5 -amine derivative of an oligonucleotide, forming a protected sulf-hydryl. Deprotection with hydroxylamine results in generation of a free thiol.

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

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). 

The ability to synthesize chemically short sequences of single-stranded DNA (oligonucleotides) is an essential part of many aspects of genetic engineering. The method most frequently employed is that of solid-phase synthesis, where the basic philosophy is the same as that in solid-phase peptide synthesis (see Section 13.6.3). In other words, the growing nucleic acid is attached to a suitable solid support, protected nucleotides are supplied in the appropriate sequence, and each addition is followed by repeated coupling and deprotection cycles. 

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