Helvetica Chimica Acta ± Vol. 86 (2003)
2831
A phosphorylating extract was prepared from commercial baker×s yeast with slight modification of the
procedure described in [9]. The yeast cells were dried at 288 for 24 h and then treated with 0.3m K-phosphate
buffer (pH 7.0; 10 ml/g of dry yeast) containing 15 mm MgCl and 3% glucose. The suspension was kept for 3 h
2
at 288 and then centrifuged (8000 Â g, 5 min). The supernatant was used as the source of phosphorylating
enzymes.
3. Assay of NPase Activity. The progress of the NMPS×s and NMP×s synthesis with the use of the whole E.
herbicola cells was monitored by HPLC and TLC [9]. The reaction mixture (1 ml) containing donor (10 mm) of
the thiophosphate group, AMPS, or phosphate group, AMP, nucleoside acceptor (50 mm), sodium acetate
buffer (0.2m, pH 4.5), and wet paste of intact E. herbicola cells (6.0 mg, calculated as abs. dry weight) was
incubated at 508 with gentle stirring. Aliquots of the reaction medium were analyzed by the TLC on Silufol UV-
2
54 plates (Serva, Germany; i-PrOH/25% aq. NH
coefficients for long-wave maximum for nucleoside under investigation. The amount of the enzyme producing
nmol of NMPS per min was taken to be one unit.
. Adenosine-5'-O-(1-thiophosphate). A soln. of adenosine (0.257 g, 0.96 mmol) in trimethyl phosphate
2.5 ml) was heated on the glycerin bath from 208 to 1008 during 20 min and then rapidly cooled to 08. To this
soln., 2,6-lutidine (0.4 ml, 3.5 mmol) and then PSCl (0.3 ml, 3 mmol) were added, and the mixture was stirred
3 2
/H O 7:2 :1 (v/v)) and/or by HPLC with a molar extinction
1
4
(
3
for 3 h at 08. The mixture was poured into 0.5m TEAB (20 ml), stirred at 208 for 3 h, and evaporated. The
À
residue was chromatographed on a DEAE-Sephadex A-25 (HCO
3
-form, 2.5 Â 30 cm) column with a linear
gradient (0.001 ! 0.6m, p H 7.6; 2Â 500 ml) of TEAB buffer. The product-containing fractions were collected,
evaporated to dryness, and transformed into Na salts [10] to give, in order of elution, adenosine-5'-O-
monophosphate (AMP; 106 mg, 28%) and AMPS (188 mg, 48%) as amorphous powders; TLC: R
.56, resp. HPLC of AMPS: t 7.58 min (lmax 258 nm; 96.7%); this product contained AMP according to the
HPLC, t 5.31 min (lmax 258 nm; 2.6%). H-NMR ((D
C(6)ÀNH )); 5.92 (d, J(1',2') 6.5, HÀC(1')); 4.68 (t, J(2',3') 6.5, HÀC(2')); 4.24 (br. s, HÀC(3')); 4.06 (br. s,
HÀC(4')); 3.84 (br. s, HÀC(5')).
. Synthesis of Adenosine-5'-O-(1-thiotriphosphate). a) Enzymatic Synthesis. A mixture containing AMPS
f
0.51 and
0
R
1
R
6
)DMSO): 8.50 (s, HÀC(8)); 8.16 (s, HÀC(2)); 7.32 (br. s,
2
5
(
Na salt; 56.7 mg, 0.14 mmol) and the phosphorylating extract (6 ml) was incubated at 288 for 8 h with gentle
stirring. The reaction was stopped by boiling for 3 min with subsequent clarification of the mixture by
À
centrifugation (5000 Â g, 10 min). The supernatant was chromatographed on a DEAE Sephadex A-25 (HCO
3
form, 1.8 Â 27 cm) column with a linear gradient (0.001 ! 1.0m, p H 7.6; 2Â 400 ml) of TEAB buffer. In order of
elution, the following products were isolated and transformed to their Na salts: AMP (4 mg, 8%), AMPS
(
4
13 mg, 23%), ATP (9 mg, 11%; HPLC: t
.21 min (lmax 258 nm); 37%; 48% based on the consumed AMPS).
)-ATPaS: 1H-NMR (D
O): 8.62 (s, HÀC(8)); 8.21 (s, HÀC(2)); 6.10 (d, J(1',2') 5.92, HÀC(1'));
HÀC(2') and HÀC(3') resonances are overlapped by the intense HOD signal; 4.38 (m, HÀC(4')); 4.30 (ddd,
R p R
3.67 min (lmax 258 nm)), and (S )-ATPaS (30.5 mg, HPLC: t
(
S
p
2
3
1
J(5',4') 2.49, J(5',P) 7.79, J(5',5'') 11.83, HÀC(5')); 4.23 (ddd, J(5',4') 3.27, J(5',P) 5.61, HÀC(5')). P-
NMR (H
2
O): 43.85 (d, J(Pa,Pb) 27.46, Pa); À8.52 (d, J(Pg,Pb) 19.33, Pg); À22.93 (dd, Pb).
b) Chemical Synthesis. A soln. of adenosine (0.267 g, 1.0 mmol) in trimethyl phosphate (2.6 ml) was heated
on a glycerin bath from 208 to 1008 during 20 min and then rapidly cooled to 08. 2,6-Lutidine (0.46 ml, 4.0 mmol)
and then PSCl
3
(0.2 ml, 2.0 mmol) were added to the soln., and the mixture was stirred at 08 for 5 h. A mixture of
N (0.5 ml) was then added, and
0
.5m bis(tributylammonium) pyrophosphate in anh. DMF (3.5 ml) and Bu
3
stirring was continued at r.t. for 30 min. The mixture was poured into 0.5m TEAB (10 ml), stirred at 208 for 3 h,
À
and evaporated. The residue was chromatographed on a DEAE-Sephadex A-25 (HCO
3
form, 2.5 Â 30 cm)
column with a linear gradient (0.001 ! 1.0m, p H 7.6; 2Â 700 ml) of TEAB buffer. The product-containing
fractions were collected, evaporated, dissolved in MeOH, and treated with a NaI soln. in acetone [10]. The
precipitate was centrifuged (4000 Â g, 5 min), washed twice with acetone and centrifuged, dried in vacuo over
CaCl
2
at r.t. overnight to give 0.143 g (24%) of ATPaS as Na salt. This product showed one spot on TLC with R
f
0
4
.35. HPLC Analysis displayed two main peaks with the following t
R
values: 4.21 min (lmax 258 nm; 53%) and
) diastereoisomers, respectively.
.94 min (lmax 258 nm; 44%), which correspond to the (S
p
) and (R
p
1
(
S
p
)-ATPaS: H-NMR (D
2
O): 8.60 (s, HÀC(8)); ca. 8.16 (s, HÀC(2)); ca. 6.10 (d, J(1',2') 5.80, HÀC(1'));
HÀC(2') and HÀC(3') resonances are overlapped by the intense HOD signal; 4.25 ± 4.40 (m, HÀC(4'),
3
1
2
Pb).
HÀC(5')). P-NMR (H
2
O): 43.81 (d, J(Pa,Pb) 27.46, Pa); À7.99 (d, J(Pg,Pb) 19.65, Pg); À22.73 (dd,
)-ATPaS: 1H-NMR (D
O): 8.53 (s, HÀC(8)); 8.17 (s, HÀC(2)); 6.07 (d, J(1',2') 5.92, HÀC(1'));
(
R
p
2
HÀC(2') and HÀC(3') resonances are overlapped by the intense HOD signal; 4.25 ± 4-40 (m, HÀC(4'),