Triethylammonium bicarbonate, preparation

Nucleotide thiophosphate analogues. The preparation and purification of [ HJATPyS, pHJGTPyS, s ITPyS (6-thioinosine), cl ITPyS (6-chloroinosine) and [ HJATPyS are described and the general purification was achieved by chromatography of the nucleotide thiophosphates in the minimum volume of H2O placed onto a DEAE-Sephadex A25 column and eluting with a linear gradient of triethylammonium bicarbonate (0.1 to 0.6M for G and I nucleotides and 0.2 to 0.5M for A nucleotides). [Biochim Biophys Acta 276 155 7972]. 

The residue then was dissolved in 30 ml of 0.04 M aqueous triethyl-ammonium bicarbonate buffer (pH 7.5), (resulting pH 5.6). It was chromatographed on a DEAE cellulose column (HCOJ form) (3.4 X 15.0 cm), with elution first with 500 ml of water and then with a linear gradient of trimethylammonium bicarbonate at pH 7.2 (0-0.07 M). XXVII was completely resolved from some of XXVIII which was formed under the above reaction conditions. However, it was still contaminated by small amounts of cAMP and ethyl 2-diazo-malonic acid. These impurities were apparently generated from the hydrolysis of some XXVII when the triethylammonium bicarbonate was removed. Pure samples of XXVII were obtained by preparative paper chromatography on either Whatman 40 or 3 MM paper with overnight development with ethanol 0.5 M ammonium acetate, pH 7.0 (5 2, v/v). 

The tubes containing [ P]8-Br-cAMP are pooled and evaporated to dryness at 30° under reduced pressure with a Buchi rotovaporator. The material is dissolved and coevaporated four times with methanol to remove excess triethylammonium bicarbonate. An ultraviolet spectrum of the product has a Amax of 264 as expected for 8-Br-cAMP. The [ P]8-Br-cAMP is placed in a 25-ml round-bottomed flask with 25 ml of dimethylformamide containing 10 mmoles of triethylammonium azide. The last solution is prepared by passing 500 mg of LiNa (10 mmoles), dissolved in dimethylformamide, through a 20-ml Dowex 50 column in the triethylammonium form and also in dimethylformamide as solvent. 

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