Microsynthesis of photolabile 8‐[2‐³H]azidoadenosine nucleotides

Photolabeling of Mitochondrial F1-H+ATPase by 2-Azido[3H]ADP and 8-Azido[3H]ADP Entrapped as Fluorometal Complexes into the Catalytic Sites of the Enzyme

“…This was followed by addition of 150 µ of a solution of 1 M tetraethylammonium azide in anhydrous DMF, resulting in the synthesis of 8-azido[3H]AMP (Boos et al, 1978). After one night at 70 °C, the reaction mixture was applied to a DE 52 column.…”

“…The gumlike triethylammoniumsalt of 8-Br[2-3H]AMP (1 5 pmol) was dried twice with anhydrous DMF in a rotary evaporator. This was followed by addition of 150 pL of a solution of 1 M tetraethylammonium azide in anhydrous DMF, resulting in the synthesis of 8-a~ido[~H]AMP (Boos et al, 1978). After one night at 70 "C, the reaction mixture was applied to a DE 52 column.…”

“…Elution was carried out with a linear gradient of 0-0.4 M TEA-HCO3 (pH 7.4), 1-L total volume. The presence of 8-azid0[~H] AMP in the eluates was characterized spectrophotometrically by absorbance at 28 1 nm (Boos et al, 1978). For phosphorylation of 8-a~ido[~H]AMP to 8-azido-[3H]ADP, the same protocol as that described for 2-azido-[3H]ADP was used.…”

Mitochondrial adenine nucleotide carrier. Investigation of principal structural, steric, and contact requirements for substrate binding and transport by means of ribose-modified substrate analogs

“…Separation of the reaction mixture was achieved on a DEAE-52-cellulose column (3 X 80 cm) using a linear gradient of 0-0.3 M (Et)3NH+HC03~, pH 7.5, 4 °C, and 2.4-L total volume. The a-32P-labeled nucleoside triphosphates were prepared starting with 20 µ of the corresponding [32P]-5,-monophosphate according to Hoard & Ott (1965) with the following modifications (Boos et al, 1978a): the tri-n-octylammonium salts of nucleoside S'-monophosphate as well as pyrophosphate were used instead of the less soluble pyridinium salts. During activation with l,l'-carbonyldiimidazole, the reaction mixture was stored under vacuum (over P205/Na0H), thus leading to higher yields.…”

Identification of amino acid residues photolabeled with 8-azidoadenosine 5'-diphosphate in the catalytic site of sarcoplasmic reticulum calcium-ATPase

“…The gumlike triethylammonium salt of 8-Br-[3H]-AMP (15 /tmol) was dried twice with anhydrous dimethylformamide on a rotavapor. AMP was synthesized as described by Boos et al (1978) by adding 150 pL of a 1 M solution of tetraethylammonium azide in dry DMF. After 1 night at 70 °C, the reaction mixture was applied to a DE 52 column and eluted using a linear gradient of 0-0.4 M triethylammonium bicarbonate, 1-L total volume.…”