Deprotection of methylphosphonate oligonucleotides using a novel one-pot procedure.
Deprotection of methylphosphonate oligonucleotides with ethylenediamine was evaluated in a model system. Methylphosphonate sequences of the form 5'-TTTNNTTT, where N was either N4-bz-dC, N4-ibu-dC, N2-ibu-O6-DPC-dG, N2-ibu-dG, N6-bz-dA, or T, were used to determine the extent of modifications that occur during deprotection. Up to 15% of N4-bz-dC was found to transaminate at the C4 position when treated with ethylenediamine. A similar displacement reaction with ethylenediamine was observed at the O6 position of N2-ibu-O6-DPC-dG, and to a much lesser extent of N2-ibu-dG. Side reactions were not observed when oligonucleotides containing N4-ibu-dC, N6-bz-dA, or T were treated with ethylenediamine. A novel method of deprotecting methylphosphonate oligonucleotides was developed from these studies. The method incorporates a brief treatment with dilute ammonia for 30 minutes followed by addition of ethylenediamine for 6 hours at room temperature to complete deprotection in a one-pot format. The solution is then diluted and neutralized to stop the reaction and prepare the crude product for chromatographic purification. This method was used to successfully deprotect a series of oligonucleotides at the 1, 100, and 150 mumole scales. These deprotection results were compared to a commonly used two-step method and found to be superior in yield of product by as much as 250%.
Genta, Inc., San Diego, CA 92121.
R I
RI
Hogrefe
M M
MM
Vaghefi
M A
MA
Reynolds
K M
KM
Young
L J
LJ
Arnold
D000641:Ammonia; D001483:Base Sequence; D002851:Chromatography, High Pressure Liquid; D003841:Deoxycytidine / Q000737:chemistry; D003849:Deoxyguanosine / Q000737:chemistry; D005029:Ethylenediamines / Q000737:chemistry*; D005821:Genetic Techniques; D007201:Indicator Dilution Techniques; D008969:Molecular Sequence Data; D009500:Neutralization Tests; D009838:Oligodeoxyribonucleotides / Q000737:chemistry*; D009943:Organophosphorus Compounds / Q000737:chemistry*
D005029:Ethylenediamines; D009838:Oligodeoxyribonucleotides; D009943:Organophosphorus Compounds; D003841:Deoxycytidine; C032627:methylphosphonic acid; C031234:ethylenediamine; D000641:Ammonia; D003849:Deoxyguanosine