Fluorescence-Lifetime-Sensitive Probes for Monitoring ATP Cleavage

Article abstract of DOI:10.1002/chem.201803234

Adenosine triphosphate (ATP) probes modified with fluorescence dyes that change their fluorescence properties upon cleavage are an interesting tool for monitoring enzymatic ATP turnover. As a readout parameter, fluorescence lifetime is attractive because it is nearly independent of concentration. In our study, we synthesised and investigated fifteen different ATP analogues, in which the fluorophores were attached to the γ-phosphate of ATP. All analogues showed distinctly different fluorescence lifetimes compared to the corresponding values of the free fluorophores. Both increases and decreases in fluorescence lifetime were observed upon attachment to ATP. To shed light on the photophysical processes governing the lifetime changes, we performed photoelectron spectroscopy in air (PESA) to determine HOMO energy levels and time-resolved fluorescence spectroscopy to obtain rate constants. We present evidence that fluorescence quenching in the compounds tested is dynamic and attributed to photoinduced electron transfer (PET), whereas fluorescence lifetime increases are caused by stacking interactions between chromophore and the nucleobase reducing non-radiative relaxation. Finally, we demonstrate that enzymatic cleavage of the ATP analogues presented can be followed by continuous monitoring of fluorescence lifetime changes.

Full text of DOI:10.1002/chem.201803234

A Journal of
Accepted Article
Title: Fluorescence lifetime sensitive probes for monitoring ATP
cleavage
Authors: Andreas Marx, Andreas Zumbusch, and Daniel Hammler
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To be cited as: Chem. Eur. J. 10.1002/chem.201803234
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10.1002/chem.201803234
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Fluorescence lifetime sensitive probes for monitoring ATP
cleavage
Daniel Hammlera, Andreas Marxa,* and Andreas Zumbuschb,*
Abstract: Adenosine triphosphate (ATP) probes modified with
fluorescence dyes that change their fluorescence properties upon
cleavage are an interesting tool for monitoring enzymatic ATP
turnover. As a readout parameter, fluorescence lifetime is attractive
since it is nearly independent of concentration. In our study we
synthesised and investigated eleven different ATP analogues, in
which the fluorophores were attached to the γ-phosphate of ATP. All
analogues showed distinctly different fluorescence lifetimes
compared to the corresponding values of the free fluorophores. Both
fluorescence lifetime increases and decreases upon attachment to
ATP were observed. In order to shed light on the photophysical
processes governing the lifetime changes we performed
photoelectron spectroscopy in air (PESA) to determine HOMO energy
levels and time-resolved fluorescence spectroscopy to obtain rate
constants. We present evidence that fluorescence quenching in the
compounds tested is dynamic and due to photoinduced electron
transfer (PET), whereas fluorescence lifetime increases are caused
by stacking interactions between chromophore and the nucleobase
reducing non-radiative relaxation. Finally, we demonstrate that
enzymatic cleavage of the ATP analogues presented can be followed
by continuous monitoring of fluorescence lifetime changes.
This assay is based on doubly labelled ATP analogues, where
one fluorescence dye (donor) is attached to the γ-phosphate and
the second fluorescence dye (acceptor) is covalently attached to
the nucleoside. Before enzymatic conversion of the TRASE, the
two different dyes undergo Förster resonance energy transfer
(FRET), such that excitation of the donor dye results in the
emission of the acceptor dye. Upon cleavage, emission of the
donor can be observed which makes this assay suited for direct
measurements of enzymatic activity. However, since the second
modification is attached to the nucleoside, for each experiment it
has to be considered which modification is enzymatically
accepted. While it was shown that different classes of ATP
consuming enzymes accept various modifications on the
nucleoside of ATP[12]
, there is no universal position for
modification on the nucleoside scaffold that would allow a general
application for a variety of enzymes. Moreover, it is obvious that
the higher the number of modifications of ATP, the less likely it
will be accepted by a broad variety of enzymes.
In this work, we report the synthesis and spectroscopic
characterisation of ATP probes which carry only one fluorophore
(Fig. 1a). These novel probes shall enhance the acceptance of
the modified ATP probes by the target enzymes. As a read-out for
hydrolysis, we employed fluorescence lifetime, τfl, measurements
of the label (Fig. 1c). Fluorescence lifetime measurements are
more robust than intensity or absorption read-outs because they
are independent of the fluorophore concentration, the sample
absorption, the sample thickness, the method of measurement,
the fluorescence intensity, photo bleaching, and the excitation
intensity[13]. Accordingly, it is not surprising that fluorescence
lifetime experiments have become an important tool in the
discovery of new drug candidates[14] and inhibitors[15] in recent
years. Fluorescence lifetime changes are also increasingly used
Introduction
Adenosine triphosphate is the main energy transporter in cells
and plays a central role for the function of a variety of enzymes,
such as kinases[1], ligases[2], RNA polymerases[3], motor proteins
like kinesins[4], and ATPases[5]. The energy is released by the
hydrolysis of the α/β- and, or β/γ-phosphate bond and can be
enzymatically coupled to endergonic processes to drive them
thermodynamically uphill[6]. This makes the number of enzymes
using ATP as a substrate almost uncountable[7]. To investigate
processes in which ATP hydrolysis is involved, several methods
exist, e.g. the use of radioactively labelled ATP[8], spectroscopic
detection of released phosphate or pyrophosphate[9], or enzyme-
coupled assays[10]. These methods are laborious or do not allow
direct and continuous measurements. As an alternative, we have
recently reported the development of a time-resolved ATPase
sensor (TRASE) assay that can be used for the continuous
a)
τ
dye/adenine
change of
Dye
fl
interaction
HN
τ
O
O
O
no
fl
Dye
interaction
P
3N
H
O
3-n
NH2
NH2
N
N
N
N
N
N
O
P
O
O
O
enzymatic
cleavage
O
O
O
O
O
O
N
P
P
N
O
P
O
O
O
O
O
n
OH OH
OH OH
monitoring of the ATP consumption of single enzymes in vitro[11]
.
b)
O
N
OH
O
[a]
[b]
D. Hammler, Prof. Dr. A. Marx
Department of Chemistry, University of Konstanz
Universitaetsstraße 10, 78457 Konstanz (Germany)
E-mail: andreas.marx@uni-konstanz.de
Prof. Dr. A. Zumbusch*
Department of Chemistry and Center for Applied Photonics
University of Konstanz
H2N
O
NH2
SO3
SO3
ATTO488
Universitaetsstraße 10, 78457 Konstanz (Germany)
E-mail: andreas.zumbusch@uni-konstanz.de
Figure 1. a) General scheme of dye-linked ATP: fluorescence lifetime of the
attached fluorophore changes when it is in close proximity to the nucleobase.
b) Structure of the rhodamine dye ATTO488-COOH. c) Proposed change of τfl
when modified ATP analogues are quenched by the nucleobase bevor
cleavage.
Supporting information for this article is given via a link at the end of
the document
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10.1002/chem.201803234
Chemistry - A European Journal
FULL PAPER
as a contrast in fluorescence lifetime imaging (FLIM)[16]. Moreover, (2) yielded compound 3 in 82% yield, followed by saponification
there are also examples for fluorescence lifetime based
nucleotide probes[17]. For fluorescence lifetime based assays, a
dynamic quenching mechanism is preferred over a static complex
formation on which would reduce the overall intensity without
having an effect on the fluorescence lifetime[18]. In many instances
both mechanisms are present, however, the contribution of the
static complex can be diminished when stacking to a suitable
quencher is supressed. An additional requirement is that the
fluorescence lifetime of the dye must be long enough to be
measured when quenched. We therefore based our experiments
on derivatives of the highly hydrophilic fluorescent rhodamine dye
4’,5’-disulforhodamine ATTO488 (Fig. 1b) which has a fluores-
cence lifetime in aqueous solution of 4.1 ns[19] independent of the
pH value over a broad range[20], and has got an exceptionally high
photo- and thermal stability[21]. Furthermore, it is known that
rhodamine derivatives are sensitive to their microenvironment
due to the fact that polarity influences the equilibrium of planar
and twisted conformations of the two amino functions which have
different nonradiative relaxation rates[22]. These properties make
ATTO488 a well-suited fluorescence dye for the systematic
investigation of fluorescence lifetime sensitive mono labelled ATP
analogues.
with lithium hydroxide to give ATTO488 (4). The choice of LiOH as
a base was crucial in this step, since the use of sodium hydroxide
led to the starting material 4’,5’-disulforhodamine 110 (1) due to
amide hydrolysis. The activation to the NHS ester was achieved
with N,N,N’,N’-tetramethyl-O-(N-succinimidyl)uronium tetrafluoro-
borate (TSTU). After freeze-drying and storage at - 20 °C we did
not observe decomposition over extended time periods (> 12
months). More synthetic details and the spectroscopic data of the
products are contained in the Supplementary Information. With
the ATTO488-NHS ester in hand we completed the synthesis of the
nucleoside modified ATP analogues (Fig. 2 (6 – 9), syntheses
details shown in the Supplementary Information).
(6): R1 = -NH-(CH2)6-NH-ATTO488
,
R2, R3, R4, R5 = H
R2
NH
(7): R2 = -(CH2)6-NH-ATTO488, R1,
N
N
R3, R4, R5 = H
N
O
P
O
P
O
P
R3
O
O
O
O
(8): R3 = NH-(CH2)6-NH-ATTO488
R1, R2, R4, R5 = H
,
R5
N
R1
O
O
O
O
OH
O
(9): R4 = -(CH2)6-NH-ATTO488, R1,
R2, R3, R5 = H
R4
6 10
(10): R5 = -(CH2)6-NH-ATTO488, R1,
R2, R3, R4 = H
Figure 2. Five synthesised ATTO488 labelled ATP analogues.
Fluorescence lifetime measurements of compounds 6 – 9 reveal
that in all cases, the lifetimes are lower (ranging
from 2.07 – 3.33 ns) than that of the unbound dye, for which a
fluorescence lifetime of 4.28 ns was determined. This means that
the fluorescence of ATTO488 is significantly quenched when
bound to any of the positions at the nucleoside tested. The
observation of multi-exponential fluorescence decays for all
compounds suggests the existence of various conformations with
different quenching rates. To investigate further whether the
triphosphate moiety has an influence on the fluorescence lifetime,
compounds 6 – 9 were treated with the α/β anhydride cleaving
phosphodiesterase I from Crotalus adamanteus (Snake Venom
Phosphodiesterase, SVPD), before the determination of
fluorescence lifetimes (Fig. S1, left). We found that the cleavage
of the anhydride bond does not have any influence on the
fluorescence lifetimes. Therefore, attachment of the label
ATTO488 to the nucleoside appeared not to be suited for the
development of a general strategy to obtain fluorescence lifetime
probes for ATP hydrolysis.
Results and Discussion
In order to design an efficient lifetime probe, we first investigated
the fluorescence variations as a function of labelling position in
mono labelled ATP analogues. For this purpose, we initially
synthesised five modified ATP analogues bearing an ATTO488
dye on different positions of the nucleobase, sugar, and γ-
-
phosphate residue (Fig. 2).
The target molecules were obtained by transferring a synthesis
strategy developed for modified rhodamine 6G[23] to a five step
synthesis of the commercially available fluorescence dye
ATTO488-NHS ester which could be coupled to the corresponding
modified ATP analogues (scheme 1). The first two steps were
already described in the literature by Ioffe et al.[24] and Mao et
al.[25]. The amide formation with the peptide coupling reagent O-
(7-azabenzotriazol-1-yl)-N,N,N’,N’-tetra-methyluronium hexaflu-
orophosphate (HATU) and the methyl 3-(N-methylamino)butyrate
SO3
SO3
SO3
SO3
H2N
O
NH2
H2N
O
NH2
Since the phosphate groups had no influence on the fluorescence
lifetimes, while ATTO488 was strongly quenched when attached to
the nucleoside, we next attached the ATTO488-dye via
phosphoester linkage to the γ-phosphate of ATP and measured
the fluorescence lifetime. The synthesis was built upon commer-
cially available ATP which was alkylated[26], followed by azide
reduction and subsequent NHS ester coupling (scheme 2,
synthetic details described in the Supplementary Information).
a
b
O
O
82%
O
64%
O
H
OH
N
N
O
O
1
2
3
-
-
-
-
SO3
SO3
SO3
SO3
H2N
O
NH2
H2N
O
NH2
c
66%
O
O
O
As for the compounds 6 – 9, the fluorescence lifetime was also
reduced for ATTO488-γATP (10) before phosphodiesterase treat-
ment (from 4.28 ns to 3.64 ns). However, for compound 10 we
found that cleavage of the α/β anhydride bond has a strong effect
on the fluorescence lifetime, which raised to the value of the free
dye due to the spatial separation after cleavage (Tab. 1).
OH
O
N
N
N
O
O
O
4
5
Scheme 1. Synthesis of ATTO488-NHS: a) methyl 4-(N-methylamino) butyrate,
HATU, DMF, Et3N, 0 °C, 3 h, b) 2.5 eq. LiOH, 4 °C, 48 h, c) TSTU, Et3N, DMF,
rt., 2.5 h.
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NH2
NH2
N
N
N
N
N
N
a
O
P
O
P
O
P
O
P
O
P
O
P
O
3
O
O
O
Rx
O
3
O
O
O
H2N
N
H
N
N
24-75%
O
O
O
O
O
O
O
O
OH OH
OH OH
11
10, 12 21
(12) R1: 2',7'-DifluoroATTO488
(13) R2: 2',7' DichloroATTO488
(10) R3: ATTO488
36% (two steps)
24% (two steps)
50%
(15) R5: ATTO532
(16) R6: Bodipy R6G
(17) R7: ATTO465
(18) R8: ATTO520
55%
57%
46%
75%
(19) R9: Bodipy FL
(20) R10: rhodamine R6G
(21) R11: ATTO495
21% (two steps)
60%
68%
(14) R4: ATTO514
67%
Scheme 2. Synthesis of γ-phosphoester- (dye amidohexyl)-adenosinetriphosphates: a) Dye-NHS ester, aqueous 0.1 M NaHCO3, pH = 8.7, 1 h, rt. Detailed yields
are given next to the compound list.
Attachment of the chromophore to the γ-phosphate therefore
presents a way to generate mono-labelled ATP analogues which
exhibit pronounced fluorescence lifetime changes upon ATP
hydrolysis. In order to study the quenching effect in more detail,
we prepared a series of ATP analogues in which different
chromophores were attached to the γ-phosphate (Scheme 2,
Fig. 3). With the exception of two BODIPY derivatives, the
chromophores chosen are cyanine dyes similar to ATTO488, but
substituted with different electron withdrawing and donating
groups.
groups is photoinduced electron transfer (PET). In the first step of
this process, an acceptor chromophore is electronically excited,
which leads to singly occupied highest occupied and lowest
unoccupied molecular orbitals (HOMO and LUMO, respectively,
cf. Fig. 4). If the HOMO of a neighbouring donor molecule is
higher in energy than the HOMO of the acceptor, an electron of
the donor’s HOMO can be transferred to the excited acceptor’s
singly occupied HOMO. The cycle is closed by transferring the
electron from the acceptor’s LUMO to the donor’s HOMO. PET
competes with radiative relaxation and quenches the
fluorescence emission of the dye. It is only efficient, if the organic
fluorophore and the quenching moiety come into close contact,
typically on the scale of a few nanometer[27]. PET has been widely
used for fluorescence sensors in sensing of e.g. inorganic salt[28]
and nucleotides[29]. Until now, tryptophan and guanine are the
only amino acid and nucleobase described to be potent
quenchers which are able to reduce, e.g. cumarine[30] and
Fluorescence lifetime measurements for all the synthesised ATP
analogues with different chromophores at the γ-phosphate are
summarised in table 1. For all compounds, we determined the
fluorescence lifetimes of the intact ATP analogues and the
fluorescence lifetimes after cleavage of the anhydride bond with
SVPD. The latter values are referred to as the values of the
unbound chromophore. The full cleavage of every single ATP
probe was monitored by analytical HPLC and the cleavage
products verified by MS.
The data show that attachment of the chromophores to the γ-
phosphate of ATP can lead to both, reductions but also increases
in fluorescence lifetimes. A closer inspection of the data reveals,
that an increase in fluorescence lifetime was only observed for
chromophores which did not obtain any sulfonate groups. All the
respective ATP analogues also exhibited slight bathochromic
rhodamine fluorophores[31]
.
Several fluorescently modified guanosine triphosphate (GTP)
analogues are commercially available[32]. To the best of our
knowledge, however, a fluorescently modified ATP instead of
GTP for monitoring enzyme activity based on PET has not been
reported yet. This is most likely due to the fact that the oxidation
potential for adenine is higher than that of guanine[33]. However, a
couple of the modified ATP analogues described in this work are
commercially available too and may be directly used for cleavage
shifts as compared to the unbound chromophores. For this reason, experiments.
the ATP analogues with sulfonate substituted chromophores are
discussed separately from those without sulfonate groups.
As outlined above, a pivotal parameter for PET to occur is the
relative energetic position of the HOMOs of acceptor and donor.
Most often, the respective energies are derived from cyclic
voltammetry (CV) experiments. In our studies, however, the poor
A possible mechanism explaining the observed fluorescence
lifetime reductions of the chromophores containing sulfonate
O
O
O
O
O
N
N
N
N
N
O
O
O
O
O
F
F
Cl
Cl
H2N
O
NH2 H2N
O
NH2
H2N
O
NH2
N
H
O
N
H
F3C
N
O
N
CF3
H
H
SO3
SO3
SO3
SO3
SO3
SO3
SO3
SO3
SO3
SO3
ATTO488
ATTO532
ATTO514
2',7'-DifluoroATTO488
2',7'-DichloroATTO488
O
O
O
O
O
O
N
F
F
O
F F
B-
H2N
N
NH2
N
N
N
B-
N+
N
N+
N
N
O
N
N
O
N
H
H
H
H
ATTO520
ATTO465
ATTO495
Bodipy FL
Bodipy R6G
rhodamine 6G
Figure 3. Hydrophilic ATTO488 core structure (top blue) and hydrophobic (bottom red) fluorescence dyes.
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PET most likely is the mechanism responsible for the reduction of
fluorescence lifetimes in ATP analogues with sulfonate groups
containing chromophores. In order to investigate whether this
quenching is dynamic or static, one can compare the ratios of
fluorescence intensities with the free or nucleoside-bound
chromophore, I0 and I, respectively, and the corresponding
fluorescence lifetimes τ0 and τ. For purely dynamic quenching,
one expects that I0/I = τ0/τ, whereas I0/I >> τ0/τ for static quenching.
We find that τ0/τ lies between 0.67 to 0.82 times I0/I, indicating that
the quenching is mostly dynamic. This finding is further
corroborated by the fact, that no spectral shifts between bound
and unbound chromophores have been observed (data shown in
SI). This hints at very little interaction between the chromophore
and the π-electron system of the nucleobase most likely due to
the high hydrophilicity of the sulfonate substituted chromophores.
LUMO
LUMO
h
e-
ν 1.
abs
2.
HOMO
HOMO
acceptor
donor
Figure 4. The efficiency of PET is governed by the relative energies of donor
and acceptor orbitals.
solubility of the ATP analogues in suitable solvents prevented us
from obtaining reproducible results for the redox potentials from
CV experiments. We therefore employed photoelectron spec-
troscopy in air (PESA) to determine the HOMO energies of the
chromophores from dry thin films (Fig. 5). As a reference, we first
determined the ionisation energy of ATP by PESA with a value of
-5.78 ± 0.02 eV (dashed line in Fig. 5).
The picture is more complicated for the group of analogues in
which the chromophores contain no sulfonate groups. We
interpret the fact that all these compounds exhibit slight
bathochromic shifts as a sign for a weak interaction between the
π-systems of the chromophore and the adenine. Compared to
their sulfonated counterparts, the chromophores in these ATP
analogues are more hydrophobic. It can therefore be expected
that the hydrophobic effect will lead to a stacking interaction
between the similarly hydrophobic adenine and the chromophore.
While this interaction only leads to minor spectral changes, it
seemingly has a stronger influence on the relaxation rates of the
excited chromophores. In order to get more detailed insight into
this phenomenon, we determined fluorescence quantum yields Φ
for all the compounds exhibiting an increase in the fluorescence
lifetimes upon attachment of the chromophore to ATP (cf. Tab. 1).
Only chromophores with HOMO energies below this level should
be able to undergo PET. The data obtained for the HOMO levels
of the different chromophores indeed show that the reduction in
fluorescence lifetimes are directly related to the differences in
HOMO energies relative to the HOMO of ATP. This shows that
Table 1. Spectroscopic properties and intrinsic fluorescence lifetimes of γ-O-
(Dye amidohexyl)-adenosine triphosphate (scheme 2) before and after
treatment with SVPD. All fluorescence lifetimes represent mean ± standard
error of triplicates.
EHOMO
[eV]
λabs λem
[nm] [nm]
kr
Φ
[ms-1][ms-1]
knr
τfl [ps]
Rx-γ-ATP
Δτ[ps]
SVPD
Again, these data were determined before and after application of
SVPD as an agent for the hydrolysis of the anhydride bond. From
the quantum yield and fluorescence lifetime measurements, we
calculated the radiative and non-radiative relaxation rates kr and
knr, respectively, of the excited chromophore states using
Φ=kr/(kr+knr) and τ=1/(kr+knr). We find that in all cases, the
attachment of the chromophore to ATP leads to no or only a slight
decrease of kr (+1% to -21%). This change in the radiative rate
alone would thus lead to a small increase in the fluorescence
lifetimes. However, as pointed out above, the latter also depends
on the non-radiative rates. Here, we found a non-uniform
behaviour with changes in knr ranging from +109% to -58% upon
attachment of the chromophore to ATP. Interestingly, among the
chromophores exhibiting an increase in fluorescence lifetime, the
largest decrease in knr was observed for ATTO495 which by far has
the smallest fluorescence quantum yield of all chromophores
used. The data can be rationalised in the following manner: for
chromophores which in the unbound states have a high quantum
yield, small decreases in the radiative rates lead to an increase in
the fluorescence lifetimes upon binding to ATP. Since in these
cases the non-radiative rates are much smaller, the minor
changes in the latter will not have an important influence on the
fluorescence lifetimes. For chromophores with low fluorescence
quantum yields, however, the interaction of the chromophore with
adenine will lead to an important reduction of the non-radiative
rate knr. Since for these compounds knr is larger than kr, the
reduction of knr is the dominating effect explaining the increase of
the fluorescence lifetime upon binding to ATP.
2’,7’-Difluoro
ATTO488 (12)
-
+
-
3438 ± 49 501 520 0.57 167 124
4255 ± 17 502 520 0.90 210 24
3350 ± 29 511 527 0.55 164 135
4016 ± 48 511 527 0.84 210 39
3661 ± 16 502 520 0.54 148 126
4225 ± 39 501 521 0.86 203 33
3554 ± 32 513 531 0.87 243 38
4088 ± 41 512 530 0.71 173 72
3995 ± 19 534 552 0.82 205 46
3998 ± 22 533 552 0.84 210 41
5877 ± 35 529 550 0.84 143 27
5603 ± 20 525 546 0.79 141 38
- 817 - 5.92
± 52 ± 0.01
2’,7’-Dichloro
ATTO488 (13)
-666 - 5.88
± 57 ± 0.03
+
-
- 576 - 5.87
± 43 ± 0.02
ATTO488 (10)
ATTO514 (14)
ATTO532 (15)
+
-
- 534 - 5.78
± 52 ± 0.02
+
-
+ 4
- 5.71
± 29 ± 0.02
+
-
+ 274 - 5.75
± 40 ± 0.01
Bodipy R6G
(16)
+
-
5609 ± 11 460 508 0.54 96
5115 ± 27 454 507 0.50 98
82
98
+ 494 - 5.68
± 29 ± 0.02
ATTO465 (17)
ATTO520 (18)
+
-
4260 ± 29 527 555 0.87 203 32
3895 ± 47 523 552 0.87 223 33
6544 ± 15 504 514 0.86 131 21
6030 ± 48 503 510 0.94 156 10
4731 ± 64 539 559 0.86 143 27
4262 ± 20 534 556 0.94 141 38
2094 ± 55 502 525 0.33 156 323
1067 ± 44 498 525 0.18 164 771
+ 365 - 5.56
± 55 ± 0.02
+
-
+ 513 - 5.55
± 50 ± 0.01
Bodipy FL
(19)
+
-
+ 470 - 5.47
± 67 ± 0.03
Rhodamine
6G (20)
+
-
+ 1028 - 5.32
± 70 ± 0.02
ATTO495 (21)
+
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measurements, as indicated by the constant fluorescence
lifetimes. For the SVPD experiments, the ATP analogues are
rapidly processed, resulting in a change of the fluorescence
lifetimes to the corresponding free uncoupled dyes (Fig. 6 a) and
b)).
Conclusions
In conclusion, we synthesised 15 fluorescently labelled ATP
analogues and determined their absorption spectra, emission
spectra and fluorescence lifetimes. For the four nucleoside
modified probes (6 – 9), we show that there are no influences of
the polyphosphates on the fluorescence lifetime of ATTO488
.
However, all four fluorescence lifetimes were lower than for the
uncoupled dye. In contrast, when attaching ATTO488 to the γ-
phosphate of ATP, the fluorescence lifetime is still lower. We give
evidence that the observed reduced fluorescence lifetime is
dynamic in nature and occurs due to photoinduced electron
transfer, where the fluorophores used are quenched by the
adenosine moiety of the ATP. On the one hand, by introducing
electron withdrawing groups into the fluorescence dye, it was
possible to lower the HOMO energy level, which resulted in
reduced fluorescence lifetimes which makes the probes more
sensitive and supports our PET hypothesis. On the other hand,
when attaching dyes like the acridines ATTO465 and ATTO495 to
the γ-phosphate of ATP, longer fluorescence lifetimes were
observed. Our fluorescence lifetime-sensitive γ-modified ATP
probes can be used in monitoring enzyme activity in a continuous
manner. These sensors work without any modification on the
nucleoside which renders them more promising for applications
compared to doubly labelled ATP probes. All these properties and
the fact that the used fluorescence lifetime read-out is robust and
less affected make the fluorescence lifetime sensitive γ-modified
ATP probes a promising tool that has the potential to complement
existing methods to study enzyme activity continuously.
Figure 5. Measured ionisation potentials for eleven different carboxylic acid
fluorescence dyes. The LUMO potentials are calculated by adding the absor-
ption maximum. The dashed line corresponds to the HOMO of ATP.
Afterwards, we compared the cleavage kinetics of the hydrophilic
dye ATTO488 attached to ATP (10), the unsulfonated hydrophobic
dye rhodamine B attached to ATP (20) and natural ATP with
SVPD and monitored the ratio between cleaved and uncleaved
probes by analytical HPLC over time. It is worthwhile to mention
that the hydrophilic ATTO488 analogue (10) is hydrolysed
significantly faster than the hydrophobic rhodamine B (20) (results
are shown in the SI, Fig. 2). This can be explained by the already
mentioned stacking behavior between hydrophobic dyes and the
nucleobase adenine leading to
a stacked complex. This
conformation is less tolerated and/or has to rearrange to an open
conformation to be accepted by SVPD. Natural ATP is slower
processed by SVPD than compound 10 but faster than compound
20, thus taking an intermediate positon in this cleavage assay.
As a first test, whether the concept of using mono functionalised
ATPs as more widely applicable fluorescence lifetime probes for
enzyme dynamics can be realised, we continuously measured the
hydrolysis of fluorescence lifetime sensitive mono labelled ATP
Acknowledgements
We thank Prof. Dr. E. Daltrozzo for helpful discussions. We are
indebted to Prof. Dr. L. Schmidt-Mende for the use of the PESA
equipment. Funding by Deutsche Forschungsgemeinschaft, SFB
969, TP B8 is gratefully acknowledged.
analogues on
microscope (FLIM). ATTO488-γATP (10) with
a
wide-field fluorescence lifetime imaging
reduced
a
fluorescence lifetime and ATTO495-γATP (21) with an increased
fluorescence lifetime before cleavage were both separately
incubated with and without SVPD. Both control experiments
without SVPD showed that the compounds are stable during the
Keywords: ATP • modified nucleotide • photoinduced electron
transfer • fluorescence lifetime • molecular probe
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Daniel Hammler, Andreas Marx* and
Andreas Zumbusch*
no
PET
PET
NH2
N
NH2
N
Page No. – Page No.
N
N
N
N
O
P
O
P
O
P
O
O
O
P
enzymatic
turnover
O
O
O
O
O
O
O
Fluorescence lifetime sensitive ATP
probes for monitoring enzyme activity
Dye
Dye
N
P
N
N
N
3
3
O
O
H
H
O
O
O
O
O 3-n
n
τ
τ
fl
OH OH
OH OH
fl
Monitoring of ATP consumption in real time: Fluorescently modified adenosine
triphosphate (ATP) probes that change their fluorescence properties upon enzymatic
turnover have been synthesised and applied in a simple enzymatic cleavage reaction.
Both, fluorescence lifetime increases and decreases upon attachment to ATP were
observed and we present evidence that fluorescence quenching in the compounds
tested is dynamic and due to photoinduced electron transfer (PET), whereas
fluorescence lifetime increases are caused by stacking interactions between
chromophore and the nucleobase reducing non-radiative relaxation.
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