Detailed Mechanism of Phosphoanhydride Bond
Hydrolysis
Promoted
by
a
Binuclear
ZrIV-
Substituted Keggin Polyoxometalate Elucidated by a
Combination of 31P, 31P DOSY and 31P EXSY NMR
Spectroscopy
Thi Kim Nga Luong,† Pavletta Shestakova,*,†, ‡ Gregory Absillis,† and Tatjana N. Parac-Vogt*,†
†
Laboratory of Bioinorganic Chemistry, Department of Chemistry, KU Leuven,
Celestijnenlaan 200F, 3001 Leuven, Belgium
‡
NMR Laboratory, Institute of Organic Chemistry with Centre of Phytochemistry
Bulgarian Academy of Sciences, Acad. G. Bontchev Str., B1.9, 1113 Sofia, Bulgaria
Supporting Information
Figure S1. 1H NMR spectra of the hydrolysis reaction: (a) 20.0 mM ATP at pD 6.4, no heating, (b) 20.0 mM
ATP in the presence of 3.0 mM ZrK 2:2 at pD 6.4 measured after pD adjustment, (c) after 3 days, (d) 20 days,
(e) 27 days, and (f) 54 days at pD 6.4 and 50 °C. (600 MHz, D2O, 298 K, NS = 64)
Figure S2. 31P NMR spectra of 20.0 mM of ATP in the presence of 3.0 mM of ZrK 2:2 at different time intervals
during the course of the hydrolysis reaction at pD 6.4 and 50 °C. (400 MHz, D2O, 293 K, NS = 256, TMP)
Figure S3. Expanded area of 31P DOSY spectrum of 20.0 mM of ATP in the presence of 3.0 mM of ZrK 2:2
after adjusting pD 6.4. The presence of additional broad signal assigned as I1B, partially overlapped with the
main ATP resonance at around −9.23 ppm is clearly seen.
Figure S4. 31P NMR spectra of (a) 3.0 mM ZrK 2:2 at pD 6.4, (b) 20.0 mM AMP at pD 6.4, a mixture of 20.0
mM AMP and 3.0 mM ZrK 2:2 (c) after sample preparation, (d) after 1 day, (e) after 3 days and (f) after 6 days
at pD 6.4 and 50 °C. (400 MHz, D2O, 293 K, NS = 256, TMP)
Figure S5. 31P NMR spectra of a mixture of (a) 3.0 mM ZrK 2:2 (pD 6.4), (b) 20.0 mM Na2HPO4 in the presence
of 3.0 mM ZrK 2:2 after adjusting to pD 6.4 ,(c) 20.0 mM Na2HPO4 in the presence of 3.0 mM ZrK 2:2 after 4
days at pD 6.4 and 50 °C, (d) 6.0 mM Na2HPO4 in the presence of 6.0 mM ZrK 2:2 after adjusting pD 6.4 and
(e) 31P NMR spectrum of 20.0 mM of Na2HPO4 and 3.0 mM of ZrK 1:2 at pD 6.4 is added for comparison (400
MHz, D2O, 293 K, NS = 256, TMP).
Figure S6. 31P NMR spectra of (A) 3.0 mM ZrK 2:2(a), 20.0 mM ADP (b) and a mixture of 20.0 mM ADP and
3.0 mM ZrK 2:2 (c), (B) 31P DOSY spectrum and (C) 31P EXSY spectrum of 20.0 mM of ADP in the presence of
3.0 mM of ZrK 2:2 after adjusting pD 6.4.
Because during the course of the ATP hydrolysis ADP and I2 were detected, in the next step
we demonstrated the interaction between ADP and ZrK 2:2 by measuring 31P, 31P DOSY, 31P
EXSY NMR for the mixture of 20.0 mM ADP and 3.0 mM ZrK 2:2 at room temperature after
adjusting pD to 6.4 to confirm the formation of I2. Figure S6A shows that the addition of
ADP to ZrK 2:2 leads to the formation of the intermediate I2 at −7.65 ppm and −11.20 ppm.
31
P DOSY spectra of this mixture was also recorded to provide further evidence for the
presence of I2. Figure S6B shows that I2 has the lower diffusion coefficient of 2.75×10-10 m2s1
in comparision with that of 4.63 × 10-10 m2s-1 of ADP (δ = −9.26 ppm and −10.36 ppm),
proving that I2 is a complex of ADP and ZrK. Figure S6C shows the exchange cross-peaks
between ADP and I2.
Figure S7. 31P NMR spectra of (A) 3.0 mM ZrK 2:2(a), 20.0 mM PP (b) and a mixture of 20.0 mM PP and 3.0
mM ZrK 2:2 (c), (B) 31P DOSY NMR spectrum and (C) 31P EXSY NMR spectrum of 20.0 mM of PP in the
presence of 3.0 mM of ZrK 2:2 after adjusting pD 6.4.
To confirm the interaction between PP and ZrK 2:2 that occurred during the course of the
ATP hydrolysis in the presence of ZrK 2:2, were recorded 31P, 31P DOSY and 31P EXSY
spectra of the mixture of 20.0 mM PP and 3.0 mM ZrK 2:2 after adjusting pD to 6.4 at room
temperature. Figure S7 shows that the addition of PP to ZrK 2:2 solution results in the
formation of the intermediate I5 at −7.25 ppm. At pD 6.4 PP is characterized by a peak at
−8.53 ppm (half-width 4.18 Hz). In the presence of 3.0 mM of ZrK 2:2 the peak of PP is
shifted by 0.41 ppm upfield and the half-width increased to 46.52 Hz (Figure S7A). Both the
change in chemical shift and the line-broadening prove that interaction between PP and ZrK
2:2 takes place.
31
P DOSY spectrum of the mixture between 20.0 mM PP and 3 mM ZrK 2:2 was also
recorded to provide more evidence for the presence of I5. Figure S7B shows that I5 (δ =
−7.25 ppm) has lower diffusion coefficient of 2.67×10-10 m2s-1 in comparison with that of PP
which is 6.76×10-10 m2s-1 (δ = −8.94 ppm), suggesting that I5 is a complex of PP and POM.
Figure S7C shows the exchange cross-peaks between PP and I5.
Figure S8. 31P EXSY NMR spectrum of 20.0 mM of ATP in the presence of 3.0 mM of ZrK 2:2 after 20 days at
pD 6.4 and 50 °C.
Figure S9. 31P EXSY NMR spectra of 20.0 mM of ATP in the presence of 3.0 mM of ZrK 2:2 after 27 days at
pD 6.4 and 50 °C.
Figure S10. 31P NMR spectra of a mixture of 20.0 mM ATP and 3.0 mM ZrK 2:2 after (a) adjusting pD to 6.4 ,
(b) 3 days, (c) 20 days (d) 27 days, and (e) 54 days after heating at 50°C; a mixture of (f) 20.0 mM ADP and 3.0
mM ZrK 2:2, (g) 20.0 mM AMP and 3.0 mM ZrK 2:2, (h) 20.0 mM PP and 3.0 mM ZrK 2:2, (k) 20.0 mM P and
3.0 mM ZrK 2:2 at pD 6.4. (600 MHz, D2O, 298 K, NS = 128).
Figure S10 demonstrates that the chemical shifts of ADP and I2, which were observed during
the hydrolysis of ATP in the presence of ZrK 2:2, are identical with the chemical shift of ADP
and I2 in the mixture of ADP and ZrK 2:2. Similarly, the chemical shifts of I5 as well as AMP
and P formed during this hydrolysis as reaction products are identical with that in the mixtures
of AMP, P, PP respectively and ZrK 2:2.
Figure S11. Structures of substrates structurally related to ATP.
Figure S12. 31P NMR spectra of (a) 3.0 mM ZrK 2:2, (b) a mixture of 20.0 mM adenine and 3.0 mM ZrK 2:2,
(c) a mixture of 20.0 mM adenosine and 3.0 mM ZrK 2:2 and (d) a mixture of 20.0 mM ribose phosphate and 3.0
mM ZrK 2:2 after adjusting pD 6.4. (400 MHz, D2O, 293 K, NS = 256, TMP)
Since ATP molecule is structured from three chemically different components, including a
ribose sugar, a nucleoside base adenine and three phosphate groups, the interaction of other
substrates bearing one or two of the components found in ATP was studied with the aim to
give further information for the proposed mechanism of ATP hydrolysis in the presence of
ZrK 2:2. The following compounds adenine, adenosine, ribose phosphate with their structures
shown in Figure S12 are used in this study. The interaction between each compound and ZrK
2:2 was followed by 31P NMR of the solution containing 20.0 mM of each compound and 3.0
mM ZrK 2:2. Figure S12 shows that in the case of adenine and adenosine, no changes in the
chemical shift of ZrK 2:2 (−12.89 ppm) were observed in 31P spectra upon addition of ZrK
2:2. The interaction between ribose phosphate and ZrK 2:2 leads to the formation of a new
complex at −13.34 ppm. This interaction is similar to the interaction of P or AMP to ZrK 2:2.
These observations suggest that ZrK 2:2 interacts preferentially with phosphate group of ATP
rather than with the ribose sugar or the nucleoside base adenine of ATP.
Figure S13. ln[ATP] as a function of time (R2 = 0.98) for the reaction between 20.0 mM of ATP and 3.0 mM of
ZrK 2:2 at pD 6.4 and 50 °C.
Figure S14. 31P NMR spectra of the hydrolysis reaction of 3.0 mM of ATP in the presence of 3.0 mM of ZrK
2:2 at different time intervals during the course of the hydrolysis reaction at pD 6.4 and 50 °C. (400 MHz, D2O,
293 K, NS = 256, TMP)
Figure S15. ln[ATP] as a function of time (R2 = 0.97) for the reaction between 3.0 mM of ATP and 3.0 mM of
ZrK 2:2 at pD 6.4 and 50 °C.
(1)
Luong, T. K. N.; Shestakova , P.; Mihaylov, T. T.; Absillis, G.; Pierloot, K.; Parac-Vogt, T. N. Chemistry – A
European Journal 2015, 21, 4428.