Chemo-enzymatic synthesis and biological evaluation of photolabile nicotinic acid adenine dinuclotide phosphate (NAADP+)

Abstract

A chemo-enzymatic synthesis of novel caged NAADP+ without the formation of multiple cage compounds has been achieved. The biological activity of the caged NAADP+ was demonstrated by its fast uncaging in intact sea-urchin eggs.

Figure 1

³¹P NMR spectrum of DMNPE-caged NAADP. The signal for 2′-phosphate is shifted upfield by about 2 ppm compared to NAADP.

Figure.2

HPLC chromatogram of the DMNPE-caged and uncaged NAADP. As can be seen, the uncaging reaction is quite labile and leads to a quantitative production of NAADP (black trace).

Figure 4

Effect of photoreleasing DMNPE-caged NAADP on Ca2+ signalling in the intact sea-urchin eggs. (A) Injecting sub-threshold amounts of NAADP (10 nm), prior to photoreleasing DMNPE-caged NAADP failed to illicit a response suggesting the inactivation of NAADP receptors. (B) Images are self-rations with colors representing the ratio according to the calibration scale. Traces are of average ratio over times. Eggs contained Oregon Green 488 BAPTA Dextran (10 μM) and DMNPE-caged NAADP (0.5 μM) as labeled. Caged was photoreleased as indicated by the arrows.

Scheme1

Chemical structure and synthesis of DMNPE-Caged-NAADP. (a) CHCl₃-H₂O, pH = 4; 60 %, 14 h. (b) ADP-ribocyclase, acetate buffer pH = 4.4, 5 h, 80%.