Nucleic Acids Research: VOLUME 37 ISSUE 9 2009

The modified triester approach has been further improved and refined to the synthesis of defined sequences of deoxyribooligonucleotides. Improvements include arylsulfonyltetrazoles as faster and milder condensing agents, benzenesulfonic acid to avoid depurination during deblocking of trityl protecting groups and improved chromatographic procedures for purification of triester intermediates and purification of the final product containing 3'-5' phosphodiester linkages. The efficiency of these new modifications in terms of yields and time has been illustrated by the synthesis of various defined sequences related to the lactose-operator region of E. coli, its analogues and two restriction enzyme recognition regions. INTRODUCTION The synthetic availability of various deoxyribooligonucleotides containing the sequence of various control regions of a gene could be of great importance in the study of the mechanism of sequence specific DNA-protein interaction. The triester chemical method3 seems to offer the first opportunity for large scale synthesis of oligonucleotides. Previously, we reported on the use of modified triester method developed in our laboratory for the chemical synthesis of a 21-mer duplex with demonstrated biological activity of the lactose operator gene of E. coli4'1. During these studies we developed arylsulfonyltriazoles5 as new coupling reagents which were found to give much higher yields of condensation products especially in the synthesis of oligonucleotides containing guanine base. However, these reagents were rather slow in completing the condensation reactions 1 (3-5 days) . In this paper we wish to report on: 353 C) Information Retrieval Limited 1 Falconberg Court London Wl V 5FG England Nucleic Acids Research (i) arylsulfonyltetrazoles as new and more reactive coupling reagents; (ii) further improvements in the triester approach to remove unreacted starting material; and (iii) purification of the 6 final diester products by PEI-TLC plate The usefulness of these modifications has been demonstrated by the efficient synthesis of various deoxyribooligonucleotides constituting the duplex sequence of the lac-operator region7 containing Eco RI restriction endonuclease recognition sequence8 (A), symmetrical lac-operator (B), its bromouracil analogues9 (C), decanucleotide containing the Bam I restriction enzyme sequence8,10 (D), and second decanucleotide containing the Hind III restriction enzyme recognition sequence8'1l (E) as outlined in Figure 1. The sequence of these oligonucleotides was confirmed by electro12 phoresis homochromatography mobility-shift analysis A. LACTOSE-OPERATOR CONTAINING Eco RI RECOGNITION SEQUENCE I II 5 '-A-A-T-T-C-A-A-T-T-G-T-G-A-G-C-G-G-A-T-A-A-C-A-A-T-T-31 3'-T-T-A-A-C-A-C-T-C-G-C-C-T-A-T-T-G-T-T-A-A-C-T-T-A-A-5' III I B. SYMMETRICAL LACTOSE OPERATOR IV 5 '-A-A-T-T-G-T-G-A-G-C-G-G-C-T-C-A-C-A-A-TT-31' 3'-T-T-A-A-C-A-C-T-C-G-C-C-G-A-G-T-G-T-T-A-A-5' V C. LACTOSE-OPERATOR (BROMO-URACIL ANALOGS)