Di(1,N⁶‐ethenoadenosine)5′, 5‴‐P¹,P⁴‐tetraphosphate, a fluorescent enzymatically active derivative of Ap₄A

Abstract

Di(1,N6‐ethenoadenosine) 5′, 5‴‐P1, P4‐tetraphosphate, ϵ‐(Ap4A), a fluorescent analog of Ap4A has been synthesized by reaction of 2‐chloroacetaldehyde with Ap4A. At neutral pH this Ap4A analog presents characteristic maxima at 265 and 274 nm, shoulders at ca 260 and 310 nm and moderate fluorescence (λexc 307 nm, λem 410 nm). Enzymatic hydrolysis of the phosphate backbone produced a slight hyperchromic effect but a notorious increase of the fluorescence emission. Cytosolic extracts from adrenochromaffin tissue as well as cultured chromaffin cells were able to split ϵ(Ap4A) and catabolize the resulting ϵ‐nucleotide moieties up to ϵ‐Ado.

Adenosine Tetraphosphate, Ap4, a Physiological Regulator of Intraocular Pressure in Normotensive Rabbit Eyes

“…Samples were chromatographed through these cartridges, and the elution of these compound was obtained by applying 500 l of a mixture containing 0.1 N HCl, 0.2 N KCl. Eluates were neutralized with 10 N KOH before HPLC analysis (Rotllá n et al, 1991).…”

Presence of ε‐adenosine tetraphosphate in chromaffin granules after transport of ε‐ATP

“…The column used was a Nova-Pak C-18 from Waters and, in some experiments, an RSiL C18 HL column from Bio-Rad. The eluents from the column were excited at 306 nm and the emission was recorded at 410 nm for all e-nucleotides [12,13]. The peak areas were transformed to concentrations by correlation with commercial standards of nucleotides and e-nucleotides (Sigma, St. Louis, Mo).…”

“…phates [13] and purified by HPLC under the same elution conditions as described above. Non-fluorescent nucleotides were detected using a Xmax 481 Spectrophotometer (Waters) at 260 nm wavelength.…”

Novel fluorescent labelled affinity probes for diadenosine-5′,5‴-P1,P4-tetraphosphate (Ap4A)-binding studies

“…Therefore, there is a clear need for appropriate probes for further extensive Ap n A-molecular recognition and binding studies. 3 Previously, we reported the use of a tandem synthetic-biosynthetic procedure to synthesise a variety of novel fluorescent labelled diadenosine-5 0 ,5 000 -P 1 ,P 4 -tetraphosphate (Ap 4 A) analogues. 4 Here, we report how this procedure has been extended for the synthesis and initial testing of two fluorescent labelled Ap 4 A analogue affinity probes, 2 0 ,3 0 -dial-adenosine(2 0 /3 0 -O-[N-methylanthraniloyl]adenosine)-5 0 ,5 000 -P 1 ,P 4 -tetraphosphate (dial-mant-Ap 4 A 1) and adenosine(8-azido-2 0 /3 0 -O-[N-methylanthraniloyl]-adenosine)-5 0 ,5 000 -P 1 ,P 4 -tetraphosphate (azido-mant-Ap 4 A 2) (see Fig.…”