Microwave assisted rapid deprotection of oligodeoxyribonucleotides
Microwave assisted rapid deprotection of oligodeoxyribonucleotidesP. Kumar and K. C. Gupta*
Nucleic Acids Research Laboratory, Centre for Biochemical Technology, Mall Road, University Campus, Delhi 110 007, India
Received July 10, 1997;Revised and Accepted October 8, 1997
ABSTRACT
A novel method for the deprotection of oligodeoxyribonucleotides under microwave irradiation has been developed. The oligodeoxynucleotides having base labile, phenoxyacetyl (pac), protection for exocyclic amino functions were fully deprotected in 0.2 M sodium hydroxide (methanol:water : : 1:1, v/v) = A and 1 M sodium hydroxide (methanol:water : : 1:1, v/v) = B using microwaves in 4 and 2 min, respectively. The deprotection of oligodeoxyribonucleotides carrying conventional protecting groups, dAbz, dCbz and dGpac, for exocyclic amino functions was achieved in 4 min in B without any side product formation. The deprotected oligonucleotides were compared with the oligomers deprotected using standard deprotection conditions (29% aq. ammonia, 16 h, 55°C) with respect to their retention time on HPLC and biological activity.
The commercial availability of synthetic oligonucleotides has revolutionized the modern biological sciences. The recent applications of these molecules as antisense oligonucleotides as future pharmaceuticals have thrown challenges before nucleic acid chemists to devise convenient and economical methods for their synthesis (1 -4 ). The solid phase assembling of these molecules following phosphoramidite chemistry is now well established and is being used commercially to produce them in large numbers as well as in reasonable quantities. However, the post-synthesis work-up has still been the most time consuming in oligonucleotide synthesis and has attracted the attention of oligonucleotide chemists. Recently, this problem has been addressed in two ways, i.e., by introducing base labile protecting groups (pac, dmf, t-bpac etc.) (5 -7 ) for nucleic bases and employing rapid deprotection conditions, i.e., (i) aq. ammonia-methyl amine (8 ), and (ii) gaseous amimes (9 ). However, first condition requires special protection for cytosine and the second one needs a specially deviced stainless steel pressure container for deprotection. We have tried to address this problem using commonly available reagents and equipments under microwaves. Koster et al. (10 ) in 1981 recommended the use of 0.2 N sodium hydroxide (water-methanol) as safe and clean deprotecting reagent for N-acylnucleosides. However, the t1/2 (deprotection) reported for various N-acyl nucleoside derivatives was considerably too high to be adapted in DNA synthesis.
Recently, we have reported (11 ) rapid synthesis of 5'-S-trityl (acyl)-2',5'-dideoxynucleoside and their deprotection under microwaves without any side product formation. Encouraged by this finding and the fast reaction kinetics under microwaves (12 ), we decided to perform the deprotection of oligonucleotides in A and B under microwaves. Since two types of N-acylating groups, conventional and base labile, are being used for routine synthesis of oligomers, it was, therefore, considered necessary to study deprotection kinetics of oligonucleotides synthesized using phosphoramidite synthons carrying conventional and base labile protecting groups for nucleic bases. In case of conventional protecting groups Gibu was replaced with Gpac, owing to its considerably longer deprotection t1/2 in aq. ammonia.
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