Removal of t-butyldimethylsilyl protection in RNA-synthesis. Triethylamine trihydrofluoride (TEA, 3HF) is a more reliable alternative to tetrabutylammonium fluoride (TBAF) min) of crude mixtures from treatment of all-2'-O-TBDMS-(Up)20U with; (a) 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF) for 2 h; (b) 1 M TBAF in THF containing an additional 3 M water for 24 h; (c) neat triethylamine trihydrofluoride (TEA, 3HF) for 1 h; (d) neat TEA, 3HF containing an additional 3 M water for

Abstract

The t-butyldimethylsilyl (TBDMS) group is the most widely used 2'-OH protection in synthesis of oligoribonucleotides. The TBDMS-group became much used particularly after it was combined with phosphoroamidite methodology (1) and was also the choice when H-phosphonate based RNA-synthesis was introduced (2). Removal of TBDMS-protection is most commonly done with 1 M tetrabutylammonium fluoride (TBAF) in tetrahydrofuran (THF). These conditions has proven efficient, but it has been reported that deprotection may be incomplete for longer RNA's such as a t-RNA (3). It has also been noted by a number of workers in the field (own experience and personal communication) that this can be the case even for shorter oligomers and that the results are not always reproducible. Moisture as a likely cause of such problems has recently been addressed by us (4) and others (5). Lyophilising the TBAF from dioxane (-1 mmol in 5 ml) overnight (which leaves 2-2.5 eq. water 4-5 % w/w by KF titration) before preparing the reagent solutions has given us consistent results for oligomers around the size of 20-mers (standard conditions being 1 M TBAF in THF overnight). With all-2'-O-TBDMS-(Up)20U a small quantity of TBDMS-containing oligomer remains after 2 h but most of it is deprotected (Fig. la) and seems intact after 24 h (chromatogram not shown but is distinguishable from that in Fig. la only by the absence of the last eluting peak). With 3 additional equivalents of water present (i. e. about 5.5 eq. in total 11 % w/w) the deprotection is only about half-complete even after 24 h (Fig. lb). In a recent report on desilylation of different dimers and an octamer containing 2'-O-TBDMS groups it was concluded that for efficient desilylation the TBAF solution should not contain more than 5% water (5). Drying of the TBAF solution with molecular sieves was recommended (our way of making sure that the reagent does not contain too much water is simply another alternative). The qualitative results of our study on deprotection of 2 1-mers with TBAF are in good agreement with their findings. Thus, after finding out about the work by Hogrefe et al. we thought that together with their results the picture is complete enough not to pursue this part of the study further, particularly having our results with TEA, 3HF in mind (see below). Neat triethylamine trihydrofluoride was reported to be a more efficient desilylating agent than TBAF and successful in deprotecting a synthetic t-RNA (3). When we used TEA, 3HF directly from the bottle, our all-2'-O-TBDMS-(UP)20U was deprotected faster than with the standard TBAF solution. TEA, 3HF removes the silyl groups within lh (Fig. lc). Importantly the rate of desilylation with this reagent is also quite insensitive to the presence of moisture. A reagent with 1 M higher water concentration (from added extra water, 1.8%-total water content 2.2% w/w) still caused complete deprotection after 1 h (not shown) and this was true even with 3 M higher water content (total water content-5.8% w/w, Fig. Id). The TEA, 3HF reagent also does not seem to take up moisture particularly fast since bottles that were opened and reopened over more than a month only contained about 0.3-0.35 M water (-0.6-0.7% w/w, typical water content when newly opened was around 0.4% w/w). Deprotection when using TEA, 3HF is clearly not particularly sensitive to moisture and this should give a high (c) Figure 1. RP-HPLC analysis (Supelcosil LC-18, 3 am, 4.6 x 150 mm, 1 ml/min and a linear gradient of 0-25% MeCN in 0.1 M triethylammonium acetate buffer (TEAA, pH-6.5) during 60