decompose to 5a and 7a via heterolytic photofragmentation,
similar to the reaction of the arylmethyl phosphate esters, where
the electron-deficient benzyl cation paired with the departing
phosphate ion is a key photolytic intermediate.46,47
in frozen aqueous solution under conditions of continuous
illumination by time-resolved 31P LT MAS NMR spectro-
scopy. ATP with the 31P resonances at ꢁ8.77 (g), ꢁ13.05 (a)
and ꢁ23.98 ppm (b) is formed with 100% yield from its caged
precursor at ꢁ13.32 (a), ꢁ14.23 (g) and ꢁ25.08 ppm (b) with
an observed rate constant ko1bs = 2.4 ꢀ 0.2 hꢁ1 at 245 K. The
reaction is accompanied by a release of the proton equivalent,
which can be monitored by 31P NMR as the induced shift of
the phosphorus resonances of ATP. Analyses of the chemical
shift and residual linewidth kinetics indicate that the NPE-
phosphate ester bond is broken prior to the proton release,
suggesting the involvement of the 1-hydroxy,1-(2-nitroso-
phenyl)-ethyl carbocation intermediate. The detailed kinetic
analyses of the isotropic chemical shift, residual linewidth and
intensity changes indicate that the initial light excitation step is
followed by two parallel cascades of dark reactions inter-
connected by chemical equilibria. The photoactivation of
caged ATP is accompanied by a pH jump, which, in the
absence of the appropriate proton sink can reach several pH
units. This has to be taken into account when using caged ATP
in studies of the ATP-dependent enzymatic reactions. In many
cases, protonation of the functional residues in the active site
after cage release may modulate the catalytic properties of the
enzyme.
The 31P residual linewidth (W1/2) kinetics of the photoreaction
are shown in Fig. 5. The most affected resonance is gP of ATP.
In the first 40 min its linewidth increases nearly two-fold
gP
1/2
(Fig. 5 gP-A). The W trace has a sigmoid shape, which is
more pronounced than for the gP chemical shift trace. The main
gP line-broadening event occurs between 20 and 40 minutes of
the reaction. Subsequently the line narrows, but not more than
bP
1/2
by 5%. The W trace of ATP shows more complex behavior;
within 100 min it makes a full oscillation about the 0.26 ppm
value (Fig. 5 bP-A). In the first 10–15 min, the bP resonance
narrows by 10%. Between 20 and 40 min, it broadens by 20%
(in concert with gP) and narrows again afterwards. During the
first 40 min, the W value of caged ATP does not change;
bP
1/2
subsequently, it increases by 20% (SNR values between 2.1 and
19.8) (Fig. 5 bP-B). The aP resonances of caged and nascent
ATP are separated by 0.3 ppm, which is only slightly larger than
their linewidths. Thus, the fitted Wa1/P2 data are likely to be biased
by the spectral overlap and will not be discussed.
The observed residual linewidth kinetics of b- and gP
resonances can be interpreted in terms of weak hydrogen
bonding 4a 2 4c; 5a,7a 2 5c,7c; 6a,7d 2 6c,7e and proton
exchange between 7a, 7d, 7g, 7f, illustated in Scheme 1. The gP
resonance can be additionally broadened by the fast proton
exchange between the two almost identical oxygen anions at
the g-phosphate, 7g 2 7g.
Acknowledgements
The support of J. Hollander with the light-induced experi-
ments is gratefully acknowledged. The research was supported
by a VENI grant to A.V.C. (700.53.406) from the Netherlands
Organization for Scientific Research (NWO). A.V.C. thanks
G. Lodder and D. V. Fillipov for insightful discussions.
Chemically exchanging 4a and 4c are formed in the course of
the reaction. Their bP resonances overlap, which results in
apparent line broadening. The dP values of the caged inter-
mediates 1a–3a are identical. Initially, the low-intensity, broad
bP line of 4a,b is hidden under the high-intensity, narrow bP
signal of 1a–3a. In the course of the photoreaction, the signal
intensity of 1a–3a decreases to the level of 4a,b, from SNR =
86.4 to 19.8. As a result, the line broadening becomes apparent
after B50 min (Fig. 5 bP-B). Accordingly, chemical exchange
5a,7a 2 5c,7c can be observed only during the initial period of
the reaction, when the product species 7e are just being formed
(SNR values between 2.9 and 33.2). After 10–20 min, the low-
intensity broad bP signal of 7a,7c is covered by the high-intensity,
narrow bP resonance of 7e, leading to the apparent narrowing of
the line (Fig. 5 bP-A). The second bP line-broadening event
occurs after 40–50 min of the reaction, during evolution of the
protolytic equilibrium at 7a, 7d, 7g, 7f as the proton exhanges
between the phosphates (Fig. 5 bP-A, gP-A). For the bP
resonance of ATP, an additional exchange 6a,7d 2 6c,7e can
be envisaged in the ‘‘post-reaction’’ hydrogen bonding between
the keto-oxygen of 6c and the bP-bound proton of 7e, which
would additionally contribute to the bP line broadening after
40–50 min of the reaction. The exchange vanishes as 6a and 7d
diffuse from each other, and the bP line narrows again.
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Conclusions
Chemical kinetics of the photoinduced intramolecular redox
reaction of the adenosine-50-triphosphate-[P3-(1-(2-nitro-
phenyl)-ethyl)]ester (caged ATP, NPE-ATP) has been studied
ꢂc
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