Synthesis of (Rp,Rp)-P1,P4-Bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2] tetraphosphate from (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2], 4[thio-18O2]tetraphosphate with retention at phosphorus and the stereochemical course of hydrolysis by the unsymmetrical Ap4A phosphodiesterase from lupin seeds.

The three stereoisomers of P1,P4-bis(5'-adenosyl)-1,4-dithiotetraphosphate have been synthesized and their 31P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (Sp,Sp)-stereoisomer shows that unstacking of the molecule occurs as the temperature is raised. Treatment of the (Sp,Sp)-stereoisomer with cyanogen bromide in [18O]water leads to substitution of sulfur by 18O with predominant retention of configuration at P1 and P4. (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-18O2],4[thio-18O2]tetraphosphate was synthesized and on treatment with cyanogen bromide in [17O]water gave (Rp,Rp)-P1,P4-bis(5'-adenosyl)-1[17O,18O2],4[17O,18O2]tetraphosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-[16O,17O,18O]AMP. The reaction therefore proceeds with inversion of configuration at phosphorus, indicating that the enzyme-catalyzed displacement by water occurs by a direct "in-line" mechanism.