Synthesis of (Rp,R,)-~1,P4-Bis(5'-adenosyl)-1[170,'802],4[170,180~] tetraphosphate from (Sp,Sp)-P1,P4-Bis(5'-adenosyl)-1[thio-1802], 4[thi0-~~0~]tetraphosphate with Retention at Phosphorus and the Stereochemical Course of Hydrolysis by the Unsymmetrical Ap4A Phosphodiesterase from Lupin Seeds*

The three stereoisomers of P',P4-bis(5'-adenosy1)-1,4-dithiotetraphosphate have been synthesized and their "P NMR spectra investigated. The effect of temperature on the circular dichroic spectrum of the (S,,S,)-stereoisomer shows that unstacking of the mol-ecule occurs as the temperature is raised. Treatment of the (S,,,S,)-stereoisomer with cyanogen bromide in ["Olwater leads to substitution of sulfur by "0 with predominant retention of configuration at P' and P4. (S,,Sp)-P',P4-Bis(5'-adenosyl)-l[thio-'E0~],4[thio- 'EOz]tetraphosphate was synthesized and on treatment with cyanogen bromide in ["Olwater gave (Rp,Rp)-P',P4-bis(5'-adenosyl)-l[170,'E02],4['70,'E02]tetra- phosphate. Hydrolysis by unsymmetrical Ap4A phosphodiesterase from lupin seeds gave (Rp)-5'-['60,'70,'80]AMP.

been found which hydrolyze Ap4A to ATP and AMP (9, 10, 24-26), and ADP (11). With a view to investigating the stereochemical course of unsymmetrical Ap4A phosphodiesterases, Ap4A made chiral at P1 and P was required.
Analysis of nucleotides was performed by ion-exchange (Mono Q) chromatography on a fast protein liquid chromatography system (Pharmacia, Hounslow, U.K.). This system was used for the separation of the diastereoisomers of P1,P-bis(5'-adenosyl)-1,4-dithiotetraphosphate.
NMR spectra were recorded with quadrature detection at 500 MHz ('H) or 202 MHz (31P) on a Bruker AM 500 Fourier transform spectrometer under Aspect 3000 control. Field frequency locking was provided by the deuterium resonance of D20. Chemical shifts are referred to internal 3-(trimethylsily1)propionate ('H) or external trimethyl phosphate in D,O The measured chemical shifts and coupling constants were confirmed by spectral simulation with PANIC, a microcomputer version of the LAOCOON program.
Circular dichroic spectra were measured on a JASCO J40C instrument by Dr. Alex S. Drake and Dr. P. M. Udvarhelyi of the National CD Service, Birbeck College, London.

(S,,S,)-P',P4-Bis(5'-adenosyl)-1,4-dithiotetraphosphate
(2)-(S,)-ADP& (1) (340 pmol), prepared by the method of Sheu and Frey (12) was converted into its tri-n-octylammonium salt and dried by coevaporation of dioxan (3 X 1 ml) and pumped at high vacuum (0.1 mm, 2 h). It was dissolved in dioxan (8 ml) under a dry Nz atmosphere. Tri-n-butylamine (0.162 ml, 680 pmol) and diphenyl chlorophosphate (0.035 ml, 170 pmol, 0.5 equivalent) were added and stirred for 3 h, protected from moisture. The dioxan was evaporated, and dry pyridine (2 ml) was added and evaporated. Dry pyridine (8 ml) was added and the mixture stirred for a further 3 h. The pyridine was evaporated and the residue dissolved in triethylammonium bicarbonate buffer (0.1 M , pH 7.5). The solution was washed with ether and the ether backwashed with water. The combined aqueous extracts were adjusted to pH 7.5 with triethylamine and diluted. The solution was applied to a column (1.5 X 25 cm) of DEAE-Sephadex A-25 that had been equilibrated with triethylammonium bicarbonate buffer (TEAB; 0.5 M, pH 7.5). The column was eluted with a linear gradient of TEAB (0.5-1.0 M , pH 7.5). The product (60 pmol, 35%) appeared at about 0.8 M TEAB. The combined fractions were evaporated and freed from buffer salts by the addition and evaporation of methanol.
The (S,,S,)-isomer was found to be contaminated with up to 20% of the (R,,R,)-isomer. This could be removed by a second chromatographic separation or by discarding the last 20% of the peak eluted from the column.
When a mixture of (Rp)and (Sp)-ADPaS (13)  Ap4A contained 70% of the label at P' and P and 30% at p2 and P. The P1,P-bis(5'-adenosyl)-['*0]-1-thiotetraphosphate was found to contain 78% label at P, 8% at P", and 14% at p2. Clearly, the central phosphate residues can participate in the reaction leading to cyclo-triphosphate and cyclodiphosphate as transient intermediates. If the label at P' and P was introduced exclusively by way of these cyclic intermediates overall retention of stereochemistry would be expected (14).
The   Procedures") and the ApA was 70% "0 at P' and P, so all four isotopomers containing I 6 0 and "0 were observed. The stereochemical evidence is provided by the ratio of the two mono-"O esters. Since an unknown amount of [160]water was introduced with the snake venom phosphodiesterase so changing the composition of the "[170]water," it was not possible to quantify the stereochemical analysis. However, since snake venom phosphodiesterase is known to hydrolyse phosphate diesters with retention of configuration at phosphorus (17, 29), the sulfur displacement has occurred with participation of the neighboring phosphate groups leading to predominant retention of configuration, as was observed with adenosine  Table I together with those of Ap4A. positive ellipticity at shorter wavelengths and a negative ellipticity at longer wavelengths. This has been attributed to it adopting a conformation in which the adenine rings are stacked with their a-faces facing each other. The CD spectra of Ap4A and the stereoisomers of P ' , Pbis(5'-adenosyl)-1,4-dithiotetraphosphate are independent of pH in the range 6.0-9.0. The (Sp,Sp)-P',P-bis(5'-adenosyl)-1,4-dithiotetraphosphate stereoisomer at pH 9.0 showed a loss of amplitude as the temperature was raised from 9 to 89 "C (Fig. 3); an isosbestic point occurs at 270 nm. This suggests, as expected, that the stacking of the adenine rings is disrupted as the temperature is raised. A plot of the change in ellipticity at 280 nm against 1/T (K) gives a straight line.

Replacement of Sulfur by "0 in (S,S~-P1,P-Bis(5'-adeno-sy1)-1 [tthi0-'~0J,4[thio-'~OJtetraphosphate-
Ap4A and (Sp,Sp)-P',P-bis(5'-adenosyl)-1,4-dithiotetraphosphate show different behavior in their CD spectra when titrated with Cd". When Ap,A was titrated with Cd2+ over the range 0-250 PM CdC12, a loss of amplitude of the peak and trough was observed but their positions did not alter. pH 9.0 is titrated with ZnC12, again the CD spectrum becomes less intense but the position of the peak and trough does not alter. NMR spectroscopy (Fig. 4) after cyclization and methylation (15). From the ratio of the intensities of the two mono-80 isotopomers of the axial and equatorial triesters it is clear that the ['60,'70,'sO]AMP haspredominantly the (R,) configuration. Comparison of the relative intensities from Fig. 4 with those from Fig. 2 (see Table 11) shows that Ap4A phosphodiesterase catalyzes the hydrolysis of Ap4A with inversion of configuration at Pa, since snake venom phosphodiesterase is known to catalyse the hydrolysis of phosphodiesters with retention (17,29). This result indicates that the Ap,A phosphodiesterase catalyzes the hydrolysis of Ap4A at P' by a direct "in-line" mechanism.  Fig. 4)   . .