A view on phosphate ester photochemistry by time-resolved solid state NMR. Intramolecular redox reaction of caged ATP

Article abstract of DOI:10.1039/b806677a

The light-driven intramolecular redox reaction of adenosine-5′- triphosphate-[P³-(1-(2-nitrophenyl)-ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the α-, β-, and γ-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is 2.4 ± 0.2 h^-1 at 245 K. The proton released in the reaction binds to the triphosphate moiety of the nascent ATP, causing the upfield shifts of the ³¹P resonances. Analyses of the reaction kinetics indicate that bond cleavage and proton release are two sequential processes in the solid state, suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the reaction. The β-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are interpreted in terms of chemical exchange processes, hydrogen bonding of the β-phosphate oxygen atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP. Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis of ATP-dependent enzymes by NMR spectroscopy in rotating solids. the Owner Societies.

Full text of DOI:10.1039/b806677a

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A view on phosphate ester photochemistry by time-resolved solid state
NMR. Intramolecular redox reaction of caged ATP
a
b
Alexey V. Cherepanov,*ab Elena V. Doroshenko,ab Jorg Matysik, Simon de Vries
¨
and Huub J. M. De Groota
Received 21st April 2008, Accepted 11th August 2008
First published as an Advance Article on the web 8th October 2008
DOI: 10.1039/b806677a
The light-driven intramolecular redox reaction of adenosine-50-triphosphate-[P3-(1-(2-nitrophenyl)-
ethyl)]ester (caged ATP) has been studied in frozen aqueous solution using time-resolved solid state
NMR spectroscopy under continuous illumination conditions. Cleavage of the phosphate ester
bond leads to 0.3, 1.36, and 6.06 ppm downfield shifts of the a-, b-, and
g-phosphorus resonances of caged ATP, respectively. The observed rate of ATP formation is
2.4 ꢀ 0.2 hꢁ1 at 245 K. The proton released in the reaction binds to the triphosphate moiety of the
nascent ATP, causing the upfield shifts of the 31P resonances. Analyses of the reaction kinetics
indicate that bond cleavage and proton release are two sequential processes in the solid state,
suggesting that the 1-hydroxy,1-(2-nitrosophenyl)-ethyl carbocation intermediate is involved in the
reaction. The b-phosphate oxygen atom of ATP is protonated first, indicating its proximity to the
reaction center, possibly within hydrogen bonding distance. The residual linewidth kinetics are
interpreted in terms of chemical exchange processes, hydrogen bonding of the b-phosphate oxygen
atom and evolution of the hydrolytic equilibrium at the triphosphate moiety of the nascent ATP.
Photoreaction of caged ATP in situ gives an opportunity to study structural kinetics and catalysis
of ATP-dependent enzymes by NMR spectroscopy in rotating solids.
NMR) was used to monitor chemical transformations in real
time. The alternative way to trigger DNA ligase catalysis is to
use caged ATP, adenosine-50-triphosphate-[P3-(1-(2-nitro-
phenyl)-ethyl)]ester (NPE-ATP), which contains the 2-nitro-
benzyl phototrigger derivative at the g-phosphate.15 Caged
ATP is a versatile tool in mechanistic studies of a broad range
of cellular processes, the actin-activated ATPase reaction of
myosin during muscle contraction and relaxation,16–18
molecular rearrangements in protein chaperones,19,20
kinesin-microtubule motility,21,22 ATP hydrolysis in P-type
ATPases,23,24 action of mitochondrial ATP/ADP carrier,25
in vivo intracellular Ca2+ mobilization,26,27 and more. The
goal of the present study is to show that caged ATP can be
used in time-resolved LT-MAS NMR experiments for the
photocontrolled release of ATP.
Introduction
Caged compounds are the photolabile probes, which contain
biomolecules in the ‘‘chemically protected’’ inactive form.
Irradiation of the probe with UV/visible light cleaves the cage
moiety, releasing the biomolecule and triggering the process of
interest. Phototriggering is a powerful experimental approach
for targeting mechanistic problems in cellular systems biology,
biochemistry and signal transduction.1–10 The role of acetyl-
cholinesterase in nerve-impulse transmission at cholinergic
synapses has been studied by kinetic X-ray diffraction spectro-
scopy using caged arsenocholine as a product analogue.11
Caged NADP and isocitrate derivatives have been employed
in time-resolved Laue diffraction experiments for the spatio-
temporal characterization of a transient enzyme-substrate
complex of isocitrate dehydrogenase.12 Very recently, we have
applied phototriggering in the studies of molecular kinetics of
Mg2+-dependent adenylyl transfer catalyzed by DNA ligase
from bacteriophage T4.13,14 The reaction was initiated by
release of Mg2+ from its complex with the photolabile EDTA
cage derivative, DM-nitrophen. Low-temperature (LT) solid
state NMR spectroscopy with magic angle spinning (LT-MAS
Light-driven hydrolysis of NPE-ATP proceeds via the intra-
molecular oxidation of a nitro benzyl ester forming a nitroso-
ketone, and involves 3 key reaction intermediates (Scheme 1).
Excitation of the caged nucleotide 1a leads to the formation of the
aci-nitro intermediate 2a, subsequent redox cyclization yields
benzisoxazole 3a, opening of the oxazole ring forms the hemiacetal
intermediate 4a, collapse of the hemiacetal releases nitrosoketone
6a, ATP 7a, and the proton. In solution, neutral 2a and 3a are in
protolytic dissociation equilibrium with the anion species 2c and
3c. Ab initio, DFT,28 semiempirical AM1 calculations29 and
experimental data30 suggest that aci-nitro anion 2c does not
participate in cyclization. Using nanosecond laser flash photolysis
and time-resolved infrared spectroscopy, it was shown that the
rate of ATP release in solution at pH 4 6 and ambient tempera-
ture is determined by the decay of the aci-nitro compound: the
a Biophysical Organic Chemistry/Solid State NMR group, Leiden
Institute of Chemistry, Faculty of Mathematics and Natural
Sciences, Leiden University, Einsteinweg 55, 2333 CC Leiden,
The Netherlands. E-mail: a.cherepanov@chem.leidenuniv.nl;
Fax: +31715274603; Tel: +31715274539
b Section of Enzymology, Department of Biotechnology, Faculty of
Applied Sciences, Delft University of Technology, Julianalaan 67,
2628 BC Delft, The Netherlands. E-mail: a.v.cherepanov@tudelft.nl;
Fax: +31152782355; Tel: +31152781625
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Scheme 1 Photoreaction of caged ATP: a view by 31P time-resolved cryo-MAS NMR. Putative hydrogen bonds are indicated with dashed lines.
ATP moiety is shown schematically, indicating positions of the oxygen atoms at b- and g-phosphate and the formal charge of the triphosphate
function. Apparent rate constants and the Ea value are shown for the reaction at 245 K in the solid state. Intermediates 1a–4a and 6a have been
previously characterized in time-resolved studies of the reaction in solution as well as by molecular modeling. Putative intermediate 5a and the Trisꢃ
Na+-bound nucleotide species 1b–4b and 7b are observed in the present work. Scission of the phosphate ester bond is indicated with the red arrow.
It causes downfield shifting of the phosphorus resonances of the nucleotide (Fig. 1). The proton release step is indicated with the green arrow. It
causes the upfield shifts of the resonances of the nascent ATP. Chemical steps that are invisible by 31P NMR are indicated with the gray arrows.
Binding of TrisꢃNa+ to the nucleotide is indicated with the blue arrows. It occurs in the slow exchange regime, with the estimated exchange rate
kex B 0.5-1 ꢄ 10ꢁ3
s
ꢁ1. Under these conditions, the exchanging species are observed as two separate sets of signals. The yellow arrows indicate
proton transfer steps, which occur under the fast exchange conditions, with kex B 300 sꢁ1. In this case, the signals of the four exchanging species
(7a, 7d, 7f, 7g) converge to a single set of resonances after completion of the reaction. During the reaction, these resonances shift upfield following
an increase in [H+]. The brown arrows indicate the weak hydrogen bonding between the Ob and the hydroxyl hydrogen in 4c and 6c. Broadening
of the bP resonance can be interpreted in terms of this exchange process. The violet arrow indicates formation of a stronger hydrogen bond
between the Ob of 7c and the hydroxyl hydrogen of 5c. The B0.1 ppm upfield ‘‘burst shift’’ of the bP resonance of the nascent ATP can be
explained by this process. The colors of the 31P-containing species correspond to different sets of chemical shift values summarized in Table 1.
oxidative cyclization step 2a
-
3a is rate-limiting;28
used without further purification. Solid ATPꢃNa2 and 0.5 M
solution of Tris(2-carboxyethyl)phosphine (TCEP), pH = 7
were obtained from Sigma Aldrich. For MAS NMR experi-
ments, the 4-mm zirconia rotors from Bruker were used. Care
was taken to avoid the contact of the nucleotide solutions with
the partially stabilized zirconia ceramics (MgO ZrO2) of the
rotor. Diluted HCl causes corrosion and disintegration of
zirconia at elevated temperature and pressure, dissolving
MgO.32 Mg2+ in solution binds ATP and subsequently the
proton, forming the HꢃATPꢃMg1ꢁ complex, which has the
apparent ionization constant pKa1 B 3.6, similar to the ATP
complexes with the other divalent cations.33 To separate
zirconia from the solution, the inner surface of the rotor was
coated with parafilm (Pechiney Plastic Packaging Company)
prior to loading the sample. The rotor was placed vertically in
the solid aluminium holder in the furnace and heated to
420–430 K. A tightly compressed parafilm roll (2–3 mm
diameter, 10–15 mm length) was inserted in the rotor and
allowed to melt for 5–10 min. The excess of parafilm was
forced out of the rotor with the 3.8-mm-diameter stainless steel
rod, leaving a thin glue-like layer of melted parafilm on the
inner surface of the rotor. The hot rotor with the holder was
removed from the furnace and left at room temperature for
both benzisoxazole and hemiacetal intermediates are transient,
hydrolyzing as quickly as they are formed.31
In this work, we have used LT-MAS 31P NMR to study the
photoreaction of caged ATP at 245 K in the solid state. The
inherently high NMR chemical shift sensitivity allowed
resolving collapse of the hemiacetal intermediate with con-
comitant release of the proton and observing the chemical
exchange processes, which include hydrogen bonding interac-
tions and evolution of the protolytic equilibrium. The detailed
kinetic analyses of the isotropic chemical shift, residual line-
width and intensity changes lead to a model where the initial
light excitation step is followed by two parallel cascades of dark
reactions interconnected by chemical equilibria. The present
study opens a new field of application of NPE-ATP in protein
chemistry and catalysis, where it can be used for the structural
characterization of enzyme-catalyzed chemical reactions by
time-resolved solid state NMR spectroscopy.
Experimental
Adenosine-50-triphosphate-[P3-(1-(2-nitrophenyl)-ethyl)]ester
disodium salt was purchased from Calbiochem (#119127) and
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slow cooling. A solution of 10 mM caged ATP was prepared in
77 mM Tris-HCl, pH = 7.5 (293 K) and 2 mM Tris-
(2-carboxyethyl)phosphine (TCEP) at 277 K in the dark,
transferred to the rotor (ca. 0.7 mg of solid ATP, 1 mMol),
stored on ice and loaded in the spectrometer within 2 h after
preparation. The sample was freeze-quenched in the spectro-
meter at 1 kHz spinning frequency (12.5 m sꢁ1 linear rate) and
a temperature ramp rate of 10–20 K minꢁ1, providing
complete solidification. Proton-decoupled 31P MAS NMR
spectra were recorded at a frequency of 303.606 MHz with a
wide bore Bruker Avance DMX 750 spectrometer using LT
MAS probe modified for the light-driven experiments.34,35 The
chemical shift positions of the nucleotides were measured
relative to (NH4)3PO4. Each spectrum contained 512 scans
recorded at 245 K and 8 kHz spinning rate with the recycle
delay of 0.25 s (spectrum acquisition time, expt = 2.4 min).
The optical setup, which was used in this work is equipped
with a 1 kW Xenon arc light source. The measured illuminance
in the MAS NMR rotor is 100–120 kLux: 1 mE of photons (in
the 380–780 nm wavelength range) is delivered every 30 sec per
1 mMol of NPE-ATP. The light was turned on after acquisi-
tion of the 5th spectrum. Aqueous solutions of 10 mM ATP,
30 mM NaOH, 2 mM TCEP (A), 10 mM ATP, 77 mM Tris
base, 2 mM TCEP (B), 77 mM Tris base, 2 mM TCEP (C),
and 77 mM Tris base (D) were titrated by addition of HCl.
For NMR experiments, the aliquots of solution (B) were
withdrawn at different pH values and stored at 77 K until
use. The NMR measurements were performed at 225 and
245 K; the sample was equilibrated at each temperature for
30 min before starting the acquisition. Each spectrum
Fig. 1 The 31P signal-to-noise ratio (SNR)-time spectral density plot (SDP) of the photoreaction of caged ATP. The spectral changes are
correlated to the reaction intermediates abbreviated in Scheme 1. The resonances of TrisꢃNaꢃnucleotide complexes are marked with an asterisk.
The color scale shows the SNR of the proton decoupled 31P MAS NMR resonances.
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contained 5400 scans (expt = 25 min). Igor Pro software
(Wavemetrics) was used for data analysis, including deconvo-
lution of the spectrum in Fig. 1.
scale of the NMR experiment. Assuming that the relaxation
times of the conformers are similar, the estimated conformer
ratio is 0.2:0.8 at 245 K and pH = 7.5. The resonances of the
minor species are shifted downfield in both caged and nascent
ATP (Fig. 1, peaks marked with the asterisk). The conformer
ratio decreases with both pH and temperature and the minor
species dissapear at pH o 3 and/or T o 225 K. The exchange
rate of kex B 0.5–1 ꢄ 10ꢁ3 sꢁ1 could be estimated in the
temperature jump experiment by following the redistribution
of the conformers after a rapid drop of the temperature by
10 degrees.
Results and discussion
Time-resolved solid state NMR studies were performed at low
temperatures with magic angle spinning. The experiment
included: (i) preparing a liquid sample of caged ATP; (ii)
freeze-quenching the sample in the NMR spectrometer at
245 K; (iii) triggering the reaction by illumination; and (iv)
monitoring chemical transformations in real time. The reac-
tion chemistry at the g-phosphorus of NPE-ATP is reflected in
the proton-decoupled 31P MAS NMR spectral density plot
(SDP) shown in Fig. 1. The spectral changes are correlated to
the reaction intermediates in Scheme 1. The isotropic chemical
shift values (d) of the intermediates are summarized in Table 1.
The resonances of NPE-ATP shift downfield during cleavage
of the NPE ester bond, release of the photoactive protecting
group (ppg) and formation of ATP. The shift of the
g-phosphorus atom harboring the cage moiety is the largest,
6.06 ppm. The b-phosphorus atom, which is located further
away from ppg shifts by 1.12 ppm; the shift of the remote
a-phosphorus atom is the smallest, 0.3 ppm. Four distinct
species are observed throughout the reaction, indicating that
the triphosphate moieties of NPE-ATP and ATP both exist as
mixtures of two conformers that exchange slowly on the time
The origin of the minor species derives from the Tris buffer,
which readily forms binary and ternary complexes with metal
cations and ATP. The dissociation constants vary in the range
between 280 mM and 0.2 M, depending on the metal type, pH,
ionic strength and temperature.36 In Fig. 1, the minor species
can be attributed to the ternary TrisꢃNaꢃnucleotide complexes.
Even without Tris, Na+ forms complexes with ATP, causing
downfield shifts of the a-, b- and g-phosphorus resonances by
0.05, 0.16 and 0.26 ppm, respectively.37 From the LT-MAS
NMR data of the TrisꢃNa complexes with NPE-ATP or ATP
presented here, it is difficult to estimate their dissociation
constants in solution: at low temperature the Bjerrum ion-
pairing and aggregation of ionic species increases, which is
often enhanced by the water–ice phase transition and the
structural mobility of the ice lattice. At the conditions of our
NMR experiment (frozen aqueous solution at 245 K and pH
= 7.5), the dissociation constants for the TrisꢃNa complexes
with NPE-ATP or ATP can be estimated as Kd B 0.3 M.
For NPE-ATP, the b-phosphate moiety is the main point of
contact of the nucleotide with the buffered sodium cation: the
0.68-ppm difference between the chemical shift of the bP
resonance (dbP) in TrisꢃNaꢃNPE-ATP2ꢁ and NPE-ATP3ꢁ is
the largest compared to the similar Dds of the a- and
g-phosphorus resonances, 0.3 and 0.23 ppm, respectively
(cf. Table 1). The NPE cage sterically hinders the g-phosphate
Table 1 The isotropic chemical shifts for the observed 31P signals of
the reacting species
Intermediate
daP
dbP
dgP
1a, 2a, 3a, 4a, 4c (green)
1b, 2b, 3b, 4b (blue)
2c, 3c (black)
ꢁ13.32
ꢁ13.02
ND
ꢁ25.08
ꢁ24.4
ꢁ14.23
ꢁ14
7a (dark green)
7b (light blue)
7c (violet)
7d, 7e, 7f, 7g (yellow)
ꢁ13.02
ꢁ12.72
ꢁ13.02
ꢁ13.05
ꢁ23.72
ꢁ23.07
ꢁ23.8
ꢁ8.17
ꢁ7.71
ꢁ8.17
ꢁ8.77
group from interacting with TrisꢃNa1+
: DdgP increases
almost two-fold upon the release of ppg, 0.23 ppm for
TrisꢃNaꢃNPE-ATP2ꢁ vs. 0.38 ppm for TrisꢃNaꢃATP3ꢁ. The
interaction with a-phosphate remains the same, while the
ꢁ23.96
ND—not detected; colours refer to Scheme 1.
Fig. 2 Kinetic traces of the photoreaction of caged ATP obtained from deconvolution of the SDP in Fig. 1 and integration of the individual
spectral components. (K) – the intensity values of the 31P resonances of caged ATP; (J) – ibid. of the nascent ATP. aP. (A) – 1a–4a, 4c at
ꢁ13.32 ppm. (B) – 7a, 7c, 7d–7g between ꢁ13.02 and ꢁ13.05 ppm. (C) – 7b at ꢁ12.72 ppm. bP. (A) – 1a–4a, 4c at ꢁ25.08 ppm. (B) – 1b–4b at
ꢁ24.4 ppm. (C) – 7a, 7c, 7d–7g between ꢁ23.72 and ꢁ23.96 ppm. (D) – 7b between ꢁ23.07 and ꢁ23.1 ppm. cP. (A) – 1a–4a, 4c at ꢁ14.23 ppm.
(B) – 1b–4b at ꢁ14 ppm. (C) – 7a, 7c, 7d–7g between ꢁ8.17 and ꢁ8.77 ppm. (D) – 7b between ꢁ7.71 and ꢁ7.79 ppm.
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interaction with b-phosphate slightly weakens, with the
DdbP = 0.61 ppm for TrisꢃNaꢃATP3ꢁ vs. 0.68 ppm for
TrisꢃNaꢃNPE-ATP2ꢁ species.
regime; otherwise, the product formation traces in Fig. 2
would be be linear, following the zero-order steady-state
conditions. To verify that the reaction kinetics does not
depend on the light intensity, we have decreased the illumi-
nance 7.1-fold and repeated the measurements. The
ko1bs decreased 1.7-fold, to 1.4 ꢀ 0.2 hꢁ1 and the product
formation trace linearized. From this value, the rate of photo-
excitation under full illuminance conditions was estimated as
kphot B 10 hꢁ1, 4.2-fold higher than the ko1bs of ATP release at
245 K. The decrease of the temperature to 240 K under full
illuminance conditions slowed down the reaction twofold, to
1.2 ꢀ 0.2 hꢁ1. Assuming the Arrhenius reaction behavior, the
activation energy for ATP release in ice can be estimated
Deconvolution of the SDP to the individual spectral com-
ponents shows a monophasic exponential process with the
observed rate constant ko1bs = 2.4 ꢀ 0.2 hꢁ1 (Fig. 2). Both free
and buffered sodium-nucleotide complexes display the same
rate of conversion, indicating that the configuration of the
triphosphate chain does not influence the light-induced bond
alteration at the NPE moiety. The exponential kinetics indi-
cate that the reaction does not occur in the light-limiting
as Ea B 15 kcal molꢁ1
, similar to that in solution,
14.8 kcal molꢁ1 38,39
.
Taking the ko1bs = 218 ꢀ 33 sꢁ140,41 at
pH = 7 and 295 K and accounting for the rate dependence on
pH28 and temperature,38,39 the rate constant in solution at pH
= 8 and 273 K can be estimated as ko1bs B 2.8 sꢁ1. At 273 K in
ice, the same value can be calculated as ko1bs B 64 hꢁ1, taking
the k1obs = 2.4 ꢀ 0.2 hꢁ1 at 240 K and the 15-kcal molꢁ1
activation energy. It is unlikely that the mechanism of the rate-
limiting oxidative cyclization step 2a - 3a or the structure of the
reaction intermediates would differ in ice from that in solution: the
enthalpic component (Ea) of the rate constant is similar in both
phases. On the other hand, ice lattice might impose an entropic
penalty on the functional motions of the reacting groups. Such a
loss of activation entropy could lead to the estimated B160-fold
drop of the ko1bs value during water–ice phase transition.
The analyses of the 31P LT-MAS NMR data recorded for
ATP in the pH range between 1 and 9 demonstrate the
sensitivity of the ATP phosphorus nuclei to the protonation
state of the phosphate oxygen atoms (Fig. 3, Table 2). ATP
has four ionizable phosphate groups and the adenine ring,
which can be protonated at the N1 position.42 In strongly
acidic solutions ATP exists as a cation H5ꢃATP1+. At pH B 9,
the starting point in our titration experiments, ATP is present
in the fully ionized form ATP4ꢁ. Binding of the first proton to
the triphosphate moiety of ATP4ꢁ results in the upfield shifts
Fig. 3 pH titration curves recorded at 293 K in solution by using the
glass electrode (I) and at low temperatures in the solid state by using
31P LT-MAS NMR (aꢁ, bꢁ, cP). NMR spectra were recorded as
described in the Experimental section. Empty circles in panels aꢁ, bꢁ,
cP refer to the isotropic chemical shifts (d) of the corresponding ATP
31P resonances determined at 225 K; solid circles—ibid. at 245 K. Dd
and apparent pKa values were calculated by treating the titration curve
of a polyprotic acid (H5ATP1+) as a superposition of those for
monoprotic acids having the corresponding Kas. Traces in
panels aꢁ, bꢁ, cP were fitted with a linear sum of the modified
maxꢁd
Henderson–Hasselbalch equation, pH ¼ pKa ¼ log d
where d is
dꢁdmin
the observed chemical shift, dmax is the shift under acidic conditions
and dmin is the shift under basic conditions for each protonation site.
For fitting of the titration curve in panel I, the log part of the equation
þ
was rewritten as log DꢁH where H+ is the equivalent fraction of
Hþeqeq
eq
added acid and D is the fraction required to pass through a single
equivalence point (D = 1 for pKa1,2 and D = 3 for pKa3–5). Fitting
residuals are shown above the titration curve. To exclude the proto-
nation of the Tris base for clarity of the presentation, the difference
trace B minus C (cf. Experimental section) is shown in panel I, which
reflects only the ionizable groups of ATP.
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Table 2 The isotropic chemical shifts and the apparent pKa values for
the ionized forms of ATP and TrisꢃNaꢃATP
(Fig. 4-II aP-1, gP-1). The fractional protonation values are
calculated from the Dd values shown in Fig. 3. After the release
of the proton, the pH in the frozen reaction mixture decreases
from 7.5 to 6.8, which can be calculated from the shifts of the
g- and aP resonances and the titration curves in Fig. 3. The
same decrease for 0.7 pH units occurs in solution B at 293 K
and pH = 7.5 after addition of one proton equivalent. This
relatively small value reflects the buffering capacity of the Tris
base. In the presence of ATP alone, pH would decrease for
2.5 units, from 7.5 to 5 (Fig. 3-I). The 0.29-ppm upfield shift of
the bP resonance is higher than expected from the pH-jump
only, indicating the presence of an additional polarizing
interaction between the bP oxygen atom and ppg. The residual
interaction of the nascent ATP4ꢁ (7a) with the cleaved ppg in
the solid state is evident from the difference between the
chemical shifts of 7a (Table 1) and ATP4ꢁ prepared in the
absence of ppg (Table 2). All resonances of 7a are shifted
upfield compared with ATP4ꢁ. The size of the shifts decreases
in the sequence gP 4 bP 4 aP.
Upon release of the proton, the phosphate resonances of the
nascent TrisꢃNaꢃATP3ꢁ complex display minor upfield shifts,
the gP B 3-fold more than the bP (Fig. 4-II bP-2, gP-2),
indicating that TrisꢃNa1+ shields the triphosphate moiety
from the proton, perhaps by sandwiching between the NPE
phototrigger and the phosphate groups. The shifts match
the values estimated by using the pKa1 = 5 ꢀ 0.3 for
TrisꢃNaꢃATPꢃH2ꢁ (Table 2) and the pH decrease from 7.5 to
6.8. The phosphorus resonances of caged ATP do not shift
significantly during the reaction (Fig. 4-I), indicating that in
the solid state at 245 K 2c and 3c are not formed, i.e., that
the proton is not released before the ester bond is broken
(Scheme 1).
Compound
daP
dbP
dgP
pKa
ATP4ꢁ
ꢁ12.99
ꢁ13.19
ꢁ23.66
ꢁ24.76
ꢁ7.98
HꢃATP3ꢁ
ꢁ12.48
ꢁ12.48
a6.15 ꢀ 0.08/
b6.21 ꢀ 0.02
4.05 ꢀ 0.01
H2ꢃATP2ꢁ
ꢁ13.19
ꢁ24.76
H3ꢃATP1ꢁ
oꢁ13.22c oꢁ25.1c oꢁ12.6c o1c
TrisꢃNaꢃATP3ꢁ
ꢁ12.72
ꢁ23.09
ꢁ7.68
ꢁ11.3
b
TrisꢃNaꢃATPꢃH2ꢁ ꢁ12.86
ꢁ23.78
5 ꢀ 0.3
a
Determined at 225 and 245 K in the solid state. Measured at 293 K
c
in solution. Upper limit values.
of the g- and bP resonances by 4.48 and 1.1 ppm, respectively;
the upfield shift of the remote a-phosphorus atom is the
smallest, 0.19 ppm. These values are essentially the same at
225 and 245 K. The situation resembles the case of NPE-ATP,
where a bound proton, similarly to the covalently attached
NPE moiety increases the electronic polarization at the phos-
phorus nucleus, thereby decreasing its resonance frequency.
Polarization decays with the inverse square of the distance
from the proton binding site, which can be noted by compar-
ing the Dd values of the individual 31P resonances (Fig. 3).
Judging these values, the first proton should bind to the gP
phosphate of ATP4ꢁ, which is also electrostatically favorable,
since it carries the highest negative charge. The apparent
ionization constant for HꢃATP3ꢁ is the same at 225 and
245 K, pKa1 = 6.15 ꢀ 0.08. It is similar to the value
determined at 293 K in solution, pKa = 6.21 ꢀ 0.02
(Fig. 3-I) and the values reported in the literature.43,44
TrisꢃNaꢃATP3ꢁ also binds the proton to the g-phosphate,
forming TrisꢃNaꢃATPꢃH2ꢁ with the pKa1 = 5 ꢀ 0.3. The
titration cannot be completed; the proton seem to compete
The sequence of ATP protonation events is somewhat
puzzling; the first phosphate group to encounter the proton
is beta: the bP resonance experiences a ‘‘burst’’ upfield shift
with TrisꢃNa+ in the dynamic equilibrium TrisꢃNaꢃATPꢃH2ꢁ
=
TrisꢃNa+ + ATPꢃH3ꢁ; TrisꢃNa+ + H+ = TrisꢃH+ + Na+,
leading to the gradual decrease of the 31P resonances of
TrisꢃNaꢃATPꢃH2ꢁ and dissapearance of the signal at pH o 3.
The second proton binds to the N1 atom of the adenine ring
with the pKa2 = 4.05 ꢀ 0.01 (Fig. 3-I, Table 2), which matches
the earlier reported values.42–44 Binding of this proton is
invisible in 31P NMR experiments (Fig. 3), indicating no
interaction between the N1–H+ form and the ATP phosphate
oxygen atoms. The three remaining proton-binding sites at the
triphosphate chain ionize at pH below 1. The corresponding
equivalence points could not be resolved with the glass
electrode, the response of which does not follow that of
the hydrogen electrode at low pH. In the NMR experiments,
we could observe weak upfield shifts of all three 31P reso-
nances of ATP at low pH values, which imply binding of the
third proton, forming H3ꢃATP1ꢁ. Judging by the size of
the shifts (Fig. 3, Table 2), the third proton binds to the
b-phosphate-oxygen anion.
during the first 10 min of the reaction (Dd = 0.08 ppm, k2obs
=
2.1 ꢀ 0.2 hꢁ1, Fig. 4-II bP-1). Subsequently, the shifting rate
slows down B2-fold, entering a single-exponential phase
(Dd = 0.21 ppm, k3obs = 1.1 ꢀ 0.1 hꢁ1), during which the
protolytic equilibrium at the triphosphate moiety is estab-
lished (Scheme 1). The ‘‘burst’’ shift indicates that the
b-phosphate oxygen atom interacts with ppg, possibly by
forming a hydrogen bond with the hydroxyl hydrogen at the
exocyclic Ca position (Scheme 1, 5c,7c). The sigmoidal proto-
nation kinetics at the g-phosphate, with a distinctive lag phase
at the time of the bP protonation burst, supports this conclu-
sion (Fig. 4-II gP-1). On the other hand, hydrogen bonding of
the b-phosphate oxygen is not essential for the bond making-
and-breaking: the TrisꢃNaꢃNPE-ATP complex shows the same
rate of ATP release (Fig. 2), even though the triphosphate
moiety is separated from ppg by TrisꢃNa1+. Thus, the
b-phosphate oxygen atom of ATP appears to be a mere
NMR sensor of the reaction events, rather than an active
participant.
In the case of NPE-ATP, cleavage of the NPE ester bond is
accompanied by a release of one proton equivalent. This
process is detected by 31P NMR as a shift of the 31P signals
of the nascent ATP (Fig. 4-II). The a- and gP resonances shift
upfield during the reaction by 0.03 and 0.71 ppm, respec-
tively, corresponding to an effective protonation of 0.16 H+
The k3obs for the protonation of ATP (1.1 ꢀ 0.1 hꢁ1) is
smaller than the ko1bs for the cleavage of the NPE ester bond
(2.4 ꢀ 0.2 hꢁ1). Binding of the proton is observed as a shift of
the resonances of the nascent ATP and not of its caged
precursor. The overall protonation rate is determined by two
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processes, proton release from the benzylic alcohol and diffu-
sion of the proton to the triphosphate moiety. The intersite
diffusion time can be estimated as t = d2/D, where d is the
separation distance and D is the proton diffusion coefficient.
At 245 K, the self-diffusion coefficient for the proton in
hexagonal ice Ih is B3 ꢄ 10ꢁ16 m2 sꢁ1 45
.
The maximal
separation distance is B10 A, taking the total length of four
P–O, two C–O and one O–H bonds. The resulting
t B 3 ꢄ 10ꢁ3 s gives a diffusion rate constant, which is 6
orders of magnitude higher than ko3bs. It is clear that the rate of
Fig. 5 31P residual linewidth kinetic traces of the photoreaction of
caged ATP. The gray shading of each data point shows the SNR value
of the corresponding peak represented on a gray scale from 0 to 93.
The values at the beginning and end of each trace show the SNR value
for the first and last point, respectively. Kinetic traces are correlated to
the chemical exchange events shown in Scheme 1.
protonation of ATP is determined by the formation of nitroso-
ketone 6a and not by proton diffusion. This implies a
sequential order of reaction events: first, the bond is
cleaved (2.4 ꢀ 0.2 hꢁ1) and second, the proton is released
(1.1 ꢀ 0.1 hꢁ1). An additional reaction intermediate, the
doubly stabilized 1-hydroxy,1-(2-nitrosophenyl)ethyl carbo-
cation 5a, in which the ester bond is broken, but the proton is
not yet released, is proposed to participate in the reaction. This
intermediate is 31P NMR silent, but the co-formed ATP4ꢁ (7a)
has the characteristic b- and gP resonances at ꢁ23.72 and ꢁ8.17
ppm, respectively (Fig. 1, Table 1). In our setup, the photo-
reaction is driven by a continuous illumination of the NMR
sample. Under these conditions, hemiacetal 4a might
Fig. 4 31P chemical shift kinetic traces of the photoreaction of caged
ATP. The gray shading of each data point shows the SNR value of the
corresponding peak represented on a gray scale from 0 to 93. The
values at the beginning and end of each trace show the SNR value for
the first and last point, respectively. Panel I—NPE-ATP. The scale of
each graph is 0.1 ppm. aP. 1a–4a, 4c; bP. 1—1a–4a, 4c; 2—1b–4b; cP.
1—1a–4a, 4c; 2—1b–4b; Panel II—nascent ATP. aP. 1—7a, 7c, 7d–7g;
2—7b. The scale of the graphs is 0.1 ppm. bP. 1—7a, 7c, 7d–7g; 2—7b.
The scale of II bP-2 is 0.1 ppm. cP. 1—7a, 7c, 7d–7g; 2—7b. The scale
of II gP-2 is 0.1 ppm.
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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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