Monomolecular pyrenol-derivatives as multi-emissive probes for orthogonal reactivities

Article abstract of DOI:10.1039/c6pp00290k

Photoacids on the basis of pyrenol have been extensively studied in the past 60 years. As their photophysical properties strongly depend on the substituents at the aromatic scaffold, we introduced two reactive moieties with different electronic coefficients thus creating multi-wavelength fluorescent probes. One probe is capable of monitoring two orthogonal transformations by four fluorescence colors, distinguishable even by the naked human eye. Another derivative can act as a three-color sensor for a wide range of different pH values. Both the presented compounds allow for mimicking of fundamental and advanced two-input logic operations due to the multi-wavelength emission. Furthermore, these compounds can process information in a logically reversible way (Feynman gate).

Full text of DOI:10.1039/c6pp00290k

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Monomolecular PyrenolꢀDerivatives as Multiꢀ
Emissive Probes for Orthogonal Reactivities
Björn Finkler[a], Iris Riemann[b], Michael Vester[a], Andreas Grüter[a], Frank
Stracke[b], Gregor Jung[a]*
[a] Biophysical Chemistry, Saarland University, Campus B2 2, 66123 Saarbrücken, Germany.
[b] FraunhoferꢀIBMT, Ensheimer Straße 48, 66386 St. Ingbert, Germany.
*Eꢀmail: g.jung@mx.uniꢀsaarland.de. Fax: +49 (0)681 302 64846. Phone: +49 (0)681 302 64848.
Abstract: Photoacids on basis of pyrenol have been extensively studied in the past 60 years.
As their photophysical properties strongly depend on the substituents at the aromatic scaffold,
we introduced two reactive moieties with different electronic coefficients thus creating multiꢀ
wavelength fluorescent probes. One probe is capable of monitoring two orthogonal
transformations by four fluorescence colors, distinguishable even by the naked human eye.
Another derivative can act as threeꢀcolor sensor for a wide range of different pH values. Both
presented compounds allow for mimicking fundamental and advanced twoꢀinput logic
operations due to the multiꢀwavelength emission. Furthermore, these compounds can process
information in a logically reversible way (Feynman gate).
Introduction
Information processing with optical probes enables rapid and sensitive detection of different
analytes in biochemical and supramolecular systems.1–3 Fluorogenic substrates, i.e. a logic
YESꢀgate, are widely used for the detection and activity determination of enzymes due to the
high sensitivity of fluorescence based methods.1,4–9 Yet, measurements with onꢀoff probes
impose complications in quantification10,11, which are largely overcome by use of dualꢀ
emissive probes, i.e. a YES and a NOT gate in a single molecule.2,3,11–16 Enzymes with their
fundamental importance for the function of biological systems are one of the key objectives of
these probes. Since multianalyte systems are promising tools for “smart” medical
diagnostics17–20, probes for multiple enzymes are of particular interest. So far, only few single
probes for two enzymes were reported.17,21–23 Most of these examples are fluorogenic since
they emit only fluorescence if both enzymes are present and can monitor only a combined
activity of both enzymes. When only one or the other enzyme is present, these systems
provide no information. A separate single enzyme probe would be necessary to address this
1

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scenario. One notable exception that enables resolution of individual enzyme activity with a
cleavable FRETꢀpair is presented by Li et al..21 While the fluorogenic probes can be seen as
AND gates, this latter example “has to be regarded as an OR logic gate”22, since a clear
assignment of the corresponding metabolization states to the emission spectra is demanding
for this probe. Here we present the ratiometric, monomolecular dual enzyme probe 1. The
probe utilizes the spectral differences of photoacid and corresponding base as well as the
sensitivity of the pyrene based photoacids to modifications which alter the electronꢀ
withdrawing strength of the aromatic substituents. Our system exhibits four distinct
metabolization states denoted by four easily distinguishable fluorescence colors. Since
phosphatase activity is an important marker for several human diseases13,24,25 and carboxyl
esterase plays a key role in detoxification processes and drug targeting12,16,26, these enzymes
were chosen as target. Furthermore, a multiꢀwavelength sensor for a wide range of different
pH values is introduced which is also based on the same system as probe 1. On top, both
derivatives can mimic Boolean operations like AND or INHIB27–29 and even more advanced
logic operations.
Results and Discussion
Synthesis
Scheme1
Scheme 1 illustrates the synthetic pathway yielding compounds 1ꢀ4. In the first step 1ꢀpyrenol
was selectively monoꢀbrominated at the distal ring. The mixture of both regioisomers 5 was
subsequently transformed into the corresponding disulfonamides 6. After reaction with benzyl
bromide, the two isomers could be separated by preparative column chromatography. For
further synthesis, solely derivative 7 was used, of which the substitution pattern was
confirmed by Xꢀray crystallographic analysis (see ESI S17ꢀS20). Subsequently, the bromine
atom was transformed in a palladiumꢀcatalyzed carbonylation to the carboxylic ethyl ester 8.
The hydroxyl group was then deprotected via palladiumꢀcatalyzed transfer hydrogenation
yielding 2. The dual enzyme sensor 1 was synthesized by reaction of 2 with phosphoroxy
chloride and subsequent hydrolysis of the resulting dichloride in a water/acetone mixture in an
overall yield of 10% over six steps. Additionally, the expected products of an enzymatic
hydrolysis (3 and 4) were synthesized for comparison. The multiꢀwavelength pHꢀsensor
compound 4 was obtained upon alkaline hydrolysis of 2 with potassium tertꢀbutoxide in
2

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presence of water according to the procedure of Gassman et al.30, and compound 1 was
hydrolyzed in a weakly alkaline aqueous solution to yield 3.
Since the hydroxyl group and the opposite position of the aromatic system are the substitution
sites inducing spectral changes to distinctly different extents, derivatives for other analytes
can be created by variation of two of the six reaction steps (Scheme 1). Furthermore,
compounds 1ꢀ4 exhibit high photostability (see ESI S38), long fluorescence lifetimes and
quantum yields of at least 85% (Table 1 and 3) which qualifies them as powerful tools for
ultrasensitive fluorescence spectroscopy.
Dual enzyme substrate
Pyrenol and especially its sulfonated derivatives are classified as photoacids.31–33 If these
molecules are electronically excited, the observed fluorescence mainly results from the
conjugated base formed by excitedꢀstate proton transfer (ESPT) and is bathochromically
shifted compared to the conjugated acid for electronic reasons. The corresponding base can
also be excited directly at pH values above the ground state pKa. Introduction of electronꢀ
withdrawing groups into the aromatic system is a common way to tune the photophysical
properties of photoacids.34–43 Hence, we introduced a cleavable carboxylic ester into the
pyrene scaffold (1 and 2). The carboxylic ethyl ester (RꢀCO2Et) exhibits a Hammett σpꢀvalue,
a convenient measure of electronꢀwithdrawing property, of 0.4544 whereas carboxylate (Rꢀ
ꢀ
CO2 ) at a physiological pH is less electronꢀwithdrawing (σp = 0.00).44 Thus, fluorescence
emission of the hydrolysis products (3 and 4) is blue shifted compared to 1 and 2, respectively
(Figure 1). A substrate for phosphatases is obtained by conversion of the hydroxylꢀgroup to a
phosphate monoester (1 and 3). At physiological pH, hydrolysis of this group results in a
strongly red shifted emission since the emission arises from the corresponding base of
compound 2 or 4, respectively (Figure 1, Table 1). Remarkably, the effects of phosphorylation
and esterification are roughly additive on the wavelength scale.
Figure1
Table 1
Enzymatic hydrolysis of 1 by porcine liver esterase (PLE) shifts the emission from λem = 472
nm to λem = 448 nm. Upon dephosphorylation by alkaline phosphatase (AlP), fluorescence is
shifted from λem = 472 to λem = 558 nm. When both enzymes are present, their combination
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induces an emission maximum at λem = 536 nm. Precise comparison of the spectroscopic data
with the reference compounds 2ꢀ4 confirms that indeed these products are formed by the
intended enzymatic reactions. Kinetic parameters of the enzyme reactions were determined
via fluorescence spectroscopy using a LineweaverꢀBurk plot (see ESI S39ꢀS44). As reference
substrates, commercial available 4ꢀmethylumbelliferyl phosphate (4ꢀMUP)45 resp. butyrate
(4ꢀMUBu)46 were used. The determined Km values for the enzymatic hydrolysis of 4ꢀMUP by
AlP (Table 2) under the used assay conditions are in a similar range as those determined by
Fernley et al..45
Table 2
The determined Km values (Table 2) characterize 1 (Km = 3.0 x 10ꢀ6 M) and 3 (Km = 2.9 x 10ꢀ6
M) as good substrates for alkaline phosphatase comparable to the fluorogenic reference
compound 4ꢀMUP under the same assay conditions (Km = 2.2 x 10ꢀ6 M). The enzymatic
efficiency of these probes, i.e. kcat/Km are also in a similar range as that found for 4ꢀMUP
(Table 2). Furthermore, the phosphate hydrolysis of 1 and 3 can also be achieved by acid
phosphatase (AP). Km generally about ten times higher compared to enzymatic hydrolysis
with alkaline phosphatase, but kcat/Km is reduced by several orders of magnitude (Table 2).
Km values for the ester hydrolysis of 2 by porcine liver esterase are lower by the factor 5
whereas kcat/Km is roughly 1.3 times higher compared to the 4ꢀmethylumbelliferyl substrate 4ꢀ
MUBu (Table 2), which suggests that 2 is a somewhat better substrate than the commercial
fluorogenic probe. While the hydrolysis of 2 to 4 by PLE is rather fast, hydrolysis of 1 to 3 by
PLE is decelerated, thus disabling determination of Km. Hydrolysis rates were similarly slow
with carboxylic acid esterase from Paenibacillus barcinonensis, which suggests that 1 is not
an ideal substrate for carboxylesterases. According to the widely used empirical model
proposed by Jones et al.47,48, only the large hydrophobic pocket provides enough space for
binding the aromatic core of 1 and 2. Compared to 2, 1 is not only sterically more bulky, but
also twice negatively charged, which likely causes repulsion from the hydrophobic pocket. As
a result, substrate 1 might not be optimally coordinated at the active site and hence, the
enzymatic hydrolysis may not be very effective. However, kcat/Km of AlP and PLE are ordered
in such a way that the conversion of 1ꢀ2ꢀ4 can be easily tracked by fluorescence
spectroscopy.
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Liveꢀcell application
The dual enzyme sensor 1 was applied to L929 cells (Figure 2; for T 84 cells, see ESI S45).
Excitation of 1 and the corresponding metabolites was performed, as previously, with twoꢀ
photon excitation (λex = 800 nm).43
Figure 2
As control, cells were incubated with the different possible products of the enzymatic
transformation (see ESI S46, S47). Even 24 hours after incubation, no permeation in cells was
found for compounds with the free carboxylate group (3 and 4), presumably due to the
negative charge at this substituent. Besides the absence of membrane penetration of 4, the
corresponding image further shows that no significant autofluorescence was detectable under
the used setup conditions. Interestingly, uptake of the negatively charged phosphoric acid
monoester 1 into the cell plasma was detectable in each measurement. The transport inside
might be facilitated by transporter proteins which are naturally specialized in the exchange of
nucleosides49,50 through the cell membranes.
Fluorescence filters in Figure 2(a, b) and (c, d) were selected to exclusively monitor the
phosphatase activity. In the extracellular medium only emission of 1 is detected. After three
hours, fluorescence emission of 1 is still detectable in the medium outside the cell, indicating
an incomplete uptake and, moreover, the absence of segregated phosphatase. Detection of 1
within the cells suggests that metabolization only occurs after internalization. Emission of the
dephosphorylated product 2 measured in the cytosol rises over time and reflects the
intracellular phosphatase activity.
After conversion of 1 to 2, esterase activity was monitored by observation of the hydrolysis of
2 (λdet = 565ꢀ615 nm) to 4 (λdet = 500ꢀ530 nm). One hour after incubation, fluorescence from
2 is clearly discernible and emission of the hydrolyzed product 4 is only weakly detectable.
While emission intensity of 2 was about the same after 3 hours (Figure 2(g, h)), the
fluorescence emission of 4 is much more pronounced (Figure 2(e, f)). Similar behavior was
previously observed in HeLa cells by use of a carboxyl BODIPY derivative.16 Beneficially,
kcat/Km of AlP and PLE (Table 2, Figure 1(b)) are ordered in such a way that the uptake and
the subsequent conversion of 1ꢀ2ꢀ4 can be easily monitored by microscopy. It should be
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mentioned that 3 can only be detected when phosphatase activity is completely lacking. This
is observed in selected areas of T84 cells (ESI Figure S74).
The possible monitoring of the four distinct fluorescent products in the cell clusters enables
the application of the above described concepts of molecular logic. For example, after
incubation with 1, the output of the green channel (λdet = 500ꢀ530 nm, Figure 2(e, f)) in
combination with a suitable threshold value and esterase/phosphatase activity as inputs
represents an AND gate within the cell cluster similarly as previously shown.22 In contrast to
fluorogenic systems22,51 our multiꢀwavelength probe additionally enables the readꢀout of more
gates like INHIB (λdet = 565ꢀ615 nm, Figure 2(g, h)) without further effort, thus providing
additional information. The use of all four detection wavelengths enables distinction of each
metabolization state and allows for sensitive multiparameter sensing. As physicians often
evaluate clinical parameters by binary readꢀout to draw a first diagnosis, the range of different
molecular logic gates qualifies our device for multipurpose use in medical diagnostics.52 This
is actually possible without further technical tools as color changes are visible even by the
naked human eye. In summary these first preliminary tests hint to the use of 1 as a valuable
tool for liveꢀcell experiments, and other substrates with a broader application range are
conceivable on the basis of scheme 1.
Fluorescent multiꢀwavelength pHꢀsensor 4
Compound 4 contains two pHꢀsensitive groups which affect its spectral properties in a
specific way (Figure 3(b) and 5, Table 3). The spectral variation is caused by the different
emission of the photoacid (ROH) and corresponding base (ROꢀ) and by the change of the
electronꢀwithdrawing properties by protonation/deprotonation of the carboxylic acid group (σp
ꢀ
(RꢀCO2H) = 0.4544; σp (RꢀCO2 ) = 0.0044). In contrast to compound 1, where all the mutual
combinations of substituents at the two orthogonal reaction sites are easily discernible (Figure
1(a, c)), only three states are visible in the emission spectra of 4 (Figure 3(a)). The assignment
of protonation states of 4aꢀd to the emissive species relies on similar spectroscopic features of
compounds 1ꢀ3, where the substituents exhibit similar Hammettꢀcoefficients. Moreover, our
interpretation also takes into account that the ordering of the acidities of the ꢀOH and the ꢀ
CO2H group likely is reversed upon excitation due to the pronounced photoacidity of aromatic
alcohols and the photobasicity of aromatic carboxylates.53–56 This peculiarity leads to the
situation that 4d is the stable monovalent form in the ground state whereas isomeric 4b is only
found in the excited state. Försterꢀcalculations to estimate lacking acidity constants e.g. of 4d
or 4b are therefore not yet possible.
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Figure 3
Table 3
At pH << 0, fluorescence emission of compound 4 at λem = 482 nm (Figure 3(b) and 5) results
from the completely protonated excited species (4a). The excitedꢀstate acidity behind (pKa*=
ꢀ0.9) agrees well with the value of 2 (pKa*,= ꢀ0.4; Table 1 and 3), evaluated by fluorescence
titration (ESI S32, S30)57,58, and also lies in the range of that of HPTA (N,N,N’,N’,N’’,N’’ꢀ
hexamethylꢀ1,3,6ꢀtrisulfonamide) (pKa* = ꢀ0.3).43 Ongoing ESPT at pH values > 0 delivers
the red shifted spectrum of the corresponding base (4b, λem = 557 nm). In this pHꢀrange, i.e. 0
< pH < 4, no color change in the emission by conversion of 2 ꢀ 4 would be distinguishable
because of the identical Hammettꢀcoefficients of the ꢀCO2H and ꢀCO2Et substituents. At pH >
5, the fluorescence emission shifts hypsochromically due to deprotonation of the carboxylic
acid thus reducing the electronꢀwithdrawing strength (4c, λem = 536 nm). Please note, that the
exact titration of the latter pKa*ꢀvalues using fluorescence intensities is hampered by
convolution of the respective molar extinction coefficients with the ground state equilibria.
Anyway, the two mentioned underlying pKa*ꢀvalues are experimentally accessible and are
obtained from the trend of the depicted fluorescence titrations (Figure 3(a); see also ESI
Figures S54, S55, S59, S60), whereas the pKa*ꢀvalues of the unstable isomeric form 4d are
not yet experimentally accessible.
Conventional absorption titration experiments allow for the determination of the ground state
pKaꢀvalues (ESI Figures S57, S58). Above pH 6.8, 4 is primarily present in the double
deprotonated form (4c) and the excitation maximum shifts from λex = 415 nm (4d) to λex
=
463 nm (4c, Table 3). The fluorescence intensity at λdet = 548 nm therefore drops from pH 6.5
to 11 due to weak excitation with light of the wavelength λex = 395 nm (Figure 5). A
comparison with other pyrenolꢀderivatives provides insight into the electronic effects and
supports our interpretation: structurally and electronically, 4 (as well as 2) is a derivatives of
the photoacid HPTA.42,43,59–62 This molecule contains a third N,N’ꢀdimethyl sulfonamide
group (σp = 0.65) which is more electronꢀwithdrawing than the carboxylate resp. the ester in
dye 4d and 2. As the influence of σp is reflected in ground state acidity, the hydroxyl’s pKa of
2 and 4d (pKa = 6.6 and 6.8; Table 3) is about one logarithmic unit higher than that found for
HPTA (pKa = 5.6) but almost one unit below that of HPTS (8ꢀHydroxypyreneꢀ1,3,6ꢀ
trisulfonic acid).43,63 It should be mentioned that the difference of hydroxyl’s pKaꢀvalues
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between 2 and 4d is less than expected, since the Hammett coefficients of the substituents (σp
ꢀ
(RꢀCO2Et) = 0.45; σp (RꢀCO2 ) = 0.00) point to higher distinction. Indeed, the slightly lower
pKaꢀvalues obtained from FCSꢀexperiments provide a larger difference (see ESI S37).
The second protonation equilibrium of 4a due to deprotonation of the ꢀCO2H moiety is less
clearly defined in the absorption spectra. Only a minor spectral shift between pH 0 and 3 from
λabs = 418 to 415 nm (ESI Figure S56) prevents a reliable pKa evaluation with absorption
titration and provides only an estimate for the ground state pKaꢀvalue of 4a. Further
experiments, including less ambiguous NMRꢀtitrations with their demand for larger amounts
of 4 and following the model by Ullmann64, represent current research. Especially detection of
the isomeric form 4b, which appears to be less populated than in other systems65, and the
associated protonation equilibria in the electronic ground state deserve exhaustive
investigations. It is, however, yet unclear whether the excitedꢀstate acidity constant of 4d can
be experimentally found by timeꢀresolved spectroscopy.
As the pKa and pKa* of the hydroxyl and carboxyl group are significantly different and the
prevalent protonation states can be identified via fluorescence and/or absorption spectroscopy,
compound 4 can be used as optical sensor for a wide of range different pH values from highly
acidic (pH < ꢀ1) to basic conditions (pH > 7). Four different pHꢀstages can be discriminated
by optical spectroscopy due to the different spectral properties of species 4aꢀd. While the pH
values below ꢀ1 and between ꢀ0.5 and 4 resp. 5 and 6 can be identified via fluorescence
spectroscopy, the pHꢀvariation from 6 to 7 can be followed by a drop in fluorescence intensity
(Figure 5). This idea can also be transferred to the concept of molecular logic.
Molecular logic
Although many examples of molecular logic are already known, we demonstrate the diversity
of our systems which can act as different gates or mimic more complex operations. As each
metabolization state of 1 is characterized by a distinct fluorescence color and thus several
different output channels can be observed, probe 1 in combination with different enzyme
inputs represents a versatile tool for molecular logic. This system is able to perform simple
Boolean logic operations like AND (O4), NOR (O1) and INHIB (O2, O3) (Figure 4(a), Table
4, for details see ESI S50ꢀS58). 27–29,66–68 Additionally, OR and XOR gates can be constructed
by use of negative logic (see ESI S51, S57).69 The logic gate which is mimicked by the
system is determined by selection of a suitable output channel wavelength and threshold
value. In any of the mentioned examples, a buffer solution of 1 without any enzyme is defined
as initial state. By combination of several output channels and the two enzymes as input a 2:4
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decoder70–72, 1:2 demultiplexer70,73,74, a halfꢀadder75–79 and ꢀsubtractor69,78,79 and a transfer
gate70 (see ESI S49, S48, S59, S60, S56) can be implemented. Furthermore, a molecular
keypad lock70,80–86 is realized by use of acid phosphatase, potassium hydroxide and excitation
light as input (see ESI S62).
Figure 4
To exemplify some logic operations, use of 1 as 2:4 decoder is explained in more detail. A 2:4
decoder translates binary information from two encoded (In1, In2) inputs to four unique
outputs (O1ꢀO4).71 The truth table for this information processor is shown in Table 4. In the
initial state, only O1 is on due to the fluorescence emission of 1. When either AlP (In1) or
PLE (In2) is added, the substrate is converted into the corresponding product, which means
that the substrate (1) fluorescence vanishes (O1 = off) and solely the fluorescence of the
formed dye is monitored (O2 = on resp. O4 = on). If both inputs are present, compound 4 is
generated and the fluorescence at output channel O4 exceeds the threshold value, while all
other output channels are off.
Table 4
Reversible molecular logic was achieved with a Feynman gate (Figure 4(b, c), Table 4, see
ESI S61). Here, O5 represents the control output for the AlP input (YESꢀgate) and O6 acts as
XOR gate for AlP and PLE (target output). While the fluorescence emission at O5 only
exceeds the corresponding threshold value when AlP is present, simultaneous absence or
presence of both PLE and AlP yields an emission at O6 above the corresponding threshold.
As output of O6 is treated as negative logic, the XORꢀlike behavior is realized. The
combination of these both gates to the Feynman gate generates a unique output pattern for
each input.
Despite the presented system can process information in a logically reversible way, it is not
resettable since 1 is irreversibly metabolized by the enzymes. Many resettable systems with a
chelating motif use ions as input. However, resetting the system in its initial state usually
requires removal of the ions with additional effort.78,87 Alternative resettable systems use
photons70,71,81,88 or pHꢀvariation as input.77–79,89 In the latter case, H+ and OHꢀ are used as
inputs and the system becomes resettable, since these inputs can annihilate each other.78
Figure 5
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In combination with definite amounts of H+ and/or OHꢀ as input and a particular output
wavelength, the pHꢀdependent fluorescence changes of 4 allow for mimicking nearly every
simple logic operation (see ESI S63ꢀS71). Moreover, the system can be applied as halfꢀadder,
halfꢀsubtractor and comparator80,90 (see ESI S72ꢀS74). In each of these applications, the
system can be reset by addition of acid or base.
In the mentioned operation examples, the inputs are either chemically indistinguishable (same
amounts of H+ or OHꢀ) or can fully annihilate each other (same amounts of H+ and OHꢀ).
However, a reversible logic operation is achieved by choosing two different concentrations of
base as input to create a Feynman gate (Figure 5, Table 5). While O I in the negative logic
mode represents the YES gate for In I (low amount of OHꢀ), O II mimics a XOR gate for In I
and In II (higher amount of OHꢀ). In the initial state at pH < 0, the emission above the
threshold value at O I causes an onꢀresponse which is inverted by the negative operation to
off. The same is true for the addition of In II, which sets the system to pH ~ 6.5. Each other
scenario causes an off response which is converted to on. O II only processes an onꢀsignal
after In I or In II addition (pH ~ 3.5 or ~ 6.5, Figure 5, Table 5).
Conclusion and outlook
The dual enzyme probe 1 constitutes an efficient tool for monitoring orthogonal enzymatic
activities producing four distinct emission colors which are distinguishable even by the naked
human eye. It can act as individual probe for two different enzymes, allowing for their
identification on basis of different fluorescence colors, and can as well indicate the presence
of both enzymes. Internal referencing in a ratiometric way is enabled by the fluorescent
substrate.
Compound 4 can be used as multiꢀwavelength sensor for a wide range of different pH values
as the protonation states can be distinguished by their spectral properties. Furthermore, the
two multiꢀwavelength ratiometric pyrene derivatives can be used for different applications of
molecular logic. Beyond simple Boolean logic, both derivatives 1 and 4 are capable of more
complex operations. Moreover, both systems can operate in a logically reversible way thus
preventing information loss, and 4 can even be reset. Suchlike operation of 4 as nonꢀtrivial
Feynman gate appears promising in view of our own attempts to combine chemistry with
quantum optics.91,92 The described synthesis allows for a modification of pyrenol into a multiꢀ
wavelength probe for other analytes or macromolecules. Since the color of the fluorescence
emission strongly relies on the substitution of the hydroxyl group (position 6) by enabling or
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disabling deprotonation and to a minor extent on the opposite position (position 1), more
derivatives with orthogonal reactivities will be available. In summary, the described pyrenol
derivatives present a versatile, flexible and chameleonic platform for the multianalyte probing
and molecular logic in life science and chemistry.
Experimental section
UV/Vis and Fluorescence Spectroscopy
Absorption spectra were recorded with Jasco Spectrophotometer Vꢀ650, fluorescence
emission and excitation spectra with Jasco Spectrofluorometer FPꢀ6500. Concentrations of the
measured solutions were in micromolar range if not stated otherwise.
Enzyme kinetics
All enzymatic in vitro reactions were examined at 25°C in aqueous buffer solution (0.1 M
TrisꢀHCl at pH 8.0 for alkaline phosphatase (AlP) and porcine liver esterase (PLE); 0.1 M
acetate at pH 5.0 for acid phosphatase (AP)). Esterase from porcine liver (activity = 18
units/mg solid) and acid phosphatase from potato (3.3 units/mg solid) were obtained as
lyophilized powder from SigmaꢀAldrich. Alkaline phosphatase from bovine intestinal mucosa
in buffered aqueous solution (3.0 M NaCl, 0.2 mM MgCl2, 30 mM triethanolamin, pH 7.6)
with an activity of 3408 units/mg protein was also obtained from SigmaꢀAldrich. Kinetic
parameters were determined via fluorescence spectroscopy using a LineweaverꢀBurk plot.
Substrate concentrations were in the range of 0.1ꢀ25 ꢁM and enzyme concentration was in the
picomolar range in the case of AlP and in nanomolar range in that of PLE and AP. For
determination of the kinetics, the decrease of the substrate fluorescence was monitored. When
evolution of the product fluorescence is recorded, the observed kinetics were found to be
nearly the same. Details can be found in the ESI S39ꢀS45.
Twoꢀphoton excitation laser scanning microscopy
Laser scanning microscopy was performed with a confocal laser microscope (LSM510
META, Zeiss; Objective: PlanꢀNeofluar 40x/1.3, Zeiss). Excitation was performed with a
Ti:Sa laser (Chameleon XR, Coherent) operating at λ = 800 nm.
Cell cultures
T 84 cells were grown in a 1:1 mixture of Ham's F12 medium and Dulbecco's modified
Eagle's medium with 2.5 mM 95% Lꢀglutamine and 5% fetal bovine serum before incubation
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at 37°C, 5% CO2 for 1ꢀ2 days. Before the experiment, the cells were washed with 0.25%
(w/v) Trypsinꢀ 0.53 mM EDTA solution to remove all traces of serum that contains trypsin
inhibitor. L929 cells were grown in Eagle's Minimum Essential Medium modified to contain
Earle's Balanced Salt Solution with nonꢀessential amino acids, 2 mM Lꢀglutamine, 1 mM
sodium pyruvate, and 1500 mg/L sodium bicarbonate containing horse serum to a final
concentration of 10% prior incubation at 37°C, 5% CO2 for 1ꢀ2 days. Before the experiment,
the cells were washed with 0.25% (w/v) Trypsinꢀ 0.53 mM EDTA solution to remove all
traces of serum that contains trypsin inhibitor. For incubation, compounds 1 and 3 were
dissolved in water and dyes 2 and 4 were dissolved in DMSO.
Synthetic Procedures: General
All reagents and solvents were obtained from SigmaꢀAldrich, Merck or Acros organics and
used without further purification. For the chromatographic purification of compound 4, Silica
gel was washed prior to use with an 8:2 (v/v) mixture of methylene chloride / methanol and
then dried in vacuo. Pyrenol was synthesized according to a modified procedure of Sehgal et
al.93
1Hꢀ and 13CꢀNMR Spectra were recorded with a Bruker AM 400 spectrometer operating at
400 and 100 MHz, respectively. Alternatively, a Bruker AM 500 spectrometer operating at
500 resp. 125 MHz was used.
HRMS spectra were recorded with a Bruker solariX 7 Tesla instrument equipped with an
Infinity Cell.
Data for XꢀRay structure determination were collected on a X8 ApexII Xꢀray diffractometer
operating with MoK/α radiation. Further details of the crystal data and structure refinement
are gathered in Table S1 and S2 in the ESI.
6ꢀ and 8ꢀ bromopyrenꢀ1ꢀol (5). Pyrenol (120.0 mg, 0.55 mmol) was dissolved in absolute
acetonitrile (20 mL) and cooled to ꢀ35°C. Subsequently, tetrafluoroboric acid diethyl ether
complex (98.2 mg, 0.61 mmol) was introduced to the solution, and Nꢀbromosuccinimide
(107.6 mg, 0.61 mmol) was added in small portions. After stirring for 18 hours and warming
to room temperature, the resultant black mixture was diluted with NaHSO3 solution (38%, 30
mL) and stirred for additional 15 minutes. Diethylether (40 mL) was added and the resulting
mixture was washed three times with water (40 mL) and once with saturated NaCl (40 mL)
solution, before the organic phase was dried over sodium sulfate. After evaporation of the
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solvent, the crude product was purified via column chromatography (diethylether/petrolether
40ꢀ65 = 4:6 (v/v)) and a mixture of 6ꢀ and 8ꢀbromopyrenꢀ1ꢀol was obtained as white solid
(142.0 mg, 0.48 mmol, 86%). UV/Vis (DMSO): λmax = 358 nm, (DMSO+NaOH): λmax = 472
nm; fluorescence (DMSO): λmax = 399 nm, (DMSO+NaOH): λmax = 477 nm. 6ꢀbromopyrenꢀ1ꢀ
3
ol: 1HꢀNMR (400 MHz, acetoneꢀd6, 25°C): δ = 9.68 (1H, s, OH), 8.58 (1 H, d, J(H,H)= 9.6
3
Hz, ArꢀH), 8.30 (1 H, d, J(H,H) = 9.6 Hz, ArꢀH), 8.22 – 8.14 (2 H, m, ArꢀH), 8.05 – 8.01 (2
H, m, ArꢀH), 7.88 (1 H, d, 3J(H,H) = 9.0 Hz, ArꢀH), 7.69 ppm (1 H, d, 3J(H,H) = 8.5 Hz, Arꢀ
H). 13CꢀNMR (100 MHz, acetoneꢀd6, 25°C): δ = 153.5, 132.6, 131.1, 131.0, 130.3, 128.9,
128.1, 127.2, 126.3, 125.8, 124.8, 124.7, 124.3, 119.5, 118.6, 114.8 ppm. 8ꢀbromopyrenꢀ1ꢀol:
1HꢀNMR (400 MHz, DMSOꢀd6, 25°C): δ = 9.67 (1H, s, OH), 8.48 (1 H, d, 3J(H,H)= 9.3 Hz,
3
ArꢀH), 8.22 – 8.14 (4 H, m, ArꢀH), 8.05 – 8.01 (2 H, m, ArꢀH), 7.68 ppm (1 H, d, J(H,H) =
8.3 Hz, ArꢀH). 13CꢀNMR (100 MHz, DMSOꢀd6, 25°C): δ = 153.7, 132.5, 131.1, 131.0, 127.8,
127.1, 126.4, 126.3, 125.6, 125.5, 125.4, 123.2, 122.8, 119.7, 118.9, 114.8 ppm. MS (ESI):
m/z calc. for C16H9BrO: 294,975852 [MꢀH]ꢀ, found: 294,97555.
3ꢀbromoꢀ8ꢀhydroxyꢀN,N,N’,N’ꢀtetramethylpyreneꢀ1,6ꢀdisulfonamide and 3ꢀbromoꢀ6ꢀ
hydroxyꢀN,N,N’,N’ꢀtetramethylpyreneꢀ1,8ꢀdisulfonamide (6). The mixture of regioisomers
5 (100.0 mg, 0.34 mmol) was dissolved in dry methylene chloride (20 mL) and cooled to ꢀ
10°C. Chlorosulfonic acid (392.1 mg, 3.37 mmol) was added slowly and the resultant mixture
was stirred for 18 hours upon warming up to room temperature. After addition of thionyl
chloride (200.5 mg, 1.69mmol), the black slurry was heated to reflux for five hours. Before
removal of the solvent and excessive thionyl chloride in vacuo, the resulting solution was
cooled down to room temperature. After addition of dry THF (20 mL) to the residue and
cooling to ꢀ10°C, dimethylamine dissolved in THF (1M, 10 mL) was added to the resulting
solution. The reaction mixture was stirred for 18 hours, ethyl acetate was added and the
organic phase was washed three times with hydrochloric acid (1M, 40 mL) and saturated
NaCl solution (40 mL) before being dried over sodium sulfate. Subsequently, the solvent was
evaporated and the crude product was purified by column chromatography (ethylacetate /
petrolether 40ꢀ65 = 6:4 (v/v)) to yield the isomer mixture 6 (96.1 mg, 0.19 mmol, 56%) as
yellow powder. UV/Vis (DMSO+TFA): λmax = 432 nm, (DMSO): λmax = 532 nm;
fluorescence (DMSO; DMSO+TFA): λmax = 450 nm, (DMSO): λmax = 532 nm. 3ꢀbromoꢀ6ꢀ
1
hydroxyꢀN,N,N’,N’ꢀtetramethylpyreneꢀ1,8ꢀdisulfonamide: HꢀNMR (400 MHz, acetoneꢀd6,
25°C): δ = 10.54 (1H, s, OH), 9.36 (1 H, d, 3J (H,H) = 9.6 Hz, ArꢀH), 9.23 (1 H, d, 3J (H,H) =
3
3
9.6 Hz, ArꢀH), 8.86 (1 H, s, ArꢀH), 8.79 (1 H, d, J (H,H) = 9.6 Hz, ArꢀH), 8.50 (1 H, d, J
(H,H) = 9.6 Hz, ArꢀH), 8.38 (1 H, s, ArꢀH), 2.90 (6 H, s, 2 CH3), 2.87 ppm (6 H, s, 2 CH3).
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13CꢀNMR (100 MHz, acetoneꢀd6, 25°C): δ = 153.9, 134.1, 133.8, 133.3, 131.5, 130.3, 129.4,
127.3, 126.8, 125.7, 125.1, 124.4, 122.7, 122.5, 119.5, 116.9, 38.1 (2C), 38.0 (2C) ppm. 3ꢀ
bromoꢀ8ꢀhydroxyꢀN,N,N’,N’ꢀtetramethylpyreneꢀ1,6ꢀdisulfonamide: 1HꢀNMR (400 MHz,
acetoneꢀd6, 25°C): δ = 10.54 (1H, s, OH), 9.27 (1 H, d, 3J (H,H) = 10.0 Hz, ArꢀH), 9.08 (1 H,
3
3
d, J (H,H) = 10.0 Hz, ArꢀH), 8.86 (1 H, s, ArꢀH), 8.85 (1 H, d, J (H,H) = 9.6 Hz, ArꢀH),
8.61 (1 H, d, 3J (H,H) = 9.6 Hz, ArꢀH), 8.38 (1 H, s, ArꢀH), 2.91 (6 H, s, 2 CH3), 2.88 ppm (6
13
H, s, 2 CH3). CꢀNMR (100 MHz, acetoneꢀd6, 25°C): δ = 153.9, 134.6, 134.3, 133.9, 131.5,
133.4, 131.7, 129.3, 127.5, 127.3, 126.8, 126.5, 123.5, 122.1, 119.5, 116.9, 38.1 (2C), 38.0
(2C) ppm. MS (ESI): m/z calc. for C20H19BrN2O5S2: 508,984052 [MꢀH]ꢀ, found: 508,98384.
3ꢀ(benzyloxy)ꢀ8ꢀbromoꢀN,N,N’,N’ꢀtetramethylpyreneꢀ1,6ꢀdisulfonamide (7). Sodium
carbonate (329.4 mg, 2.28 mmol) and tetrabutylammonium iodide (TBAI) (17.2 mg, 0.05
mmol) were added to a solution of the isomer mix 6 (221.6 mg, 0.43 mmol) in dry acetone (50
mL). After addition of benzyl bromide (96.5 mg, 0.56 mmol), the orange solution was stirred
for 42 hours at room temperature. The solvent was removed, methylene chloride (40 mL) was
added and the organic phase was washed three times with hydrochloric acid (1M, 40 mL) and
saturated NaCl solution (40 mL) before drying over sodium sulfate. Column chromatography
(methylene chloride / petrolether 40ꢀ65 = 9:1 (v/v)) was used to purify the crude mixture of
the resulting regioisomers and yielded 7 as yellow powder (116.7 mg, 0.19 mmol, 45 %).
UV/Vis (DMSO): λmax = 420 nm; fluorescence (DMSO): λmax = 436 nm. 1HꢀNMR (400 MHz,
3
3
chloroformꢀd, 25°C): δ = 9.26 (1 H, d, J (H,H) = 9.8 Hz), 9.27 (1 H, d, J (H,H) = 9.19 Hz,
3
3
ArꢀH), 8.93 (1 H, s, ArꢀH), 8.82 (1 H, d, J (H,H) = 9.8 Hz, ArꢀH), 8.49 (1 H, d, J (H,H) =
9.8 Hz, ArꢀH), 8.32 (1 H, s, ArꢀH), 7.5 (5 H, m, ArꢀH), 5,58 (2 H, s, CH2), 2.89 (6 H, s, 2
CH3), 2.72 ppm (6 H, s, 2 CH3). 13CꢀNMR (100 MHz, chloroformꢀd, 25°C): δ = 152.8, 135.8,
132.9, 132.8, 131.8, 130.6, 128.9 (2 C), 128.8, 128.5, 127.9, 127.5 (3C), 126.2, 125.6, 125.4,
123.5, 123.3, 122.7, 119.8, 112.9, 71.3, 37.6 (2C), 37.5 (2C) ppm. MS (ESI): m/z calc. for
C27H25BrN2O5S2: 623,028043 [M+Na]+, found: 623,02891.
Ethyl 6ꢀ(benzyloxy)ꢀ3,8ꢀbis(N,Nꢀdimethylsulfamoyl)pyreneꢀ1ꢀcarboxylate (8). Compound
7 (64.5 mg, 0.11 mmol) was dissolved in a reaction tube with a mixture of benzene (9.29 mL),
triethyamine (3.44 mL) and ethanol (1.82 mL). Before the tube was introduced in the
autoclave, [Pd(PPh3)2Cl2] was added to the reaction mixture which then was heated to 65°C
for 12 hours under a CO pressure of 16 bar. The solution was cooled the room temperature
and transferred to a 100 mL flask, the solvent was removed and the residue was dissolved in
methylene chloride (30 mL). Before being dried over sodium sulfate and evaporation of the
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solvent, the organic phase was washed three times with water (30 mL) and saturated NaCl
solution (30 mL). Compound 8 was obtained as yellow powder after column chromatography
(methylene chloride / petrolether 40ꢀ65 = 9:1 (v/v)) in a yield of 80% (48.9 mg, 0.09 mmol).
UV/Vis (DMSO): λmax = 420 nm; fluorescence (DMSO): λmax = 462 nm. 1HꢀNMR (400 MHz,
chloroformꢀd, 25°C): δ = 9.24 (4 H, m, ArꢀH), 8.91 (1 H, 3J (H,H) = 9.6 Hz ArꢀH), 8.32 (1 H,
3
s, ArꢀH), 7.47 ppm (5 H, m, ArꢀH), 5,59 (2 H, s, CH2), 4,61 (2H, q, J (H,H) = 7.3 Hz, CH2)
3
2.90 (6 H, s, 2 CH3), 2.73 (6 H, s, 2 CH3), 1,55 ppm (3H, t, J (H,H) = 7.3 Hz, CH3). 13Cꢀ
NMR (100 MHz, chloroformꢀd, 25°C): δ = 166.6, 153.2, 135.7, 135.0, 133.9, 132.5, 131.9,
131.1, 130.5, 129.5, 128.9, 128.5, 128.0, 127.5, 125.8, 125.7, 125.2, 125.0, 124.2, 123.9,
123.2, 122.1, 112.7, 71.3, 62.0, 37.5 (4C), 14.4 ppm. MS (ESI): m/z calc. for C30H30N2O7S2:
617,138661 [M+Na]+, found: 617,13953.
Ethyl 3,8ꢀbis(N,Nꢀdimethylsulfamoyl)ꢀ6ꢀhydroxypyreneꢀ1ꢀcarboxylate (2). In a 100 mL
flask equipped with reflux condenser and a gas bubbler, ammonium formate (223.4 mg, 63.1
mmol) and palladium on carbon (10% w/t, 360 mg) were added to a solution of 8 (202.2 mg,
0.35 mmol) in acetone (40 mL). The reaction suspension was heated to reflux. When the
evolution of gas ceased, the mixture was stirred for an additional hour. After cooling to room
temperature, the suspension was filtered through a plug of celite and the solvent of the
resulting solution was evaporated. The residue was dissolved in ethyl acetate (40 mL), washed
three times with hydrochloric acid (1M, 40 mL) and saturated NaCl solution (40 mL) and
dried over sodium sulfate. Column chromatography (ethylacetate / petrolether 40ꢀ65 = 6:4
(v/v)) of the raw product yielded 2 as yellow powder (143.0 mg, 0.30 mmol, 85%). UV/Vis
(DMSO+TFA): λmax = 426 nm, (DMSO): λmax = 559 nm, (H2O+HCl): λmax = 419 nm, (H2O):
λmax = 494 nm; ; ε(494) (H2O, ROꢀ) = 25000 L molꢀ1 cmꢀ1; fluorescence (DMSO+TFA): λmax
=
482/576 nm, (DMSO): λmax = 576 nm, (H2O+HCl): λmax = 478 nm, (H2O): λmax = 558 nm. 1Hꢀ
3
NMR (500 MHz, acetoneꢀd6, 25°C): δ = 9.34 (1 H, d, J (H,H) = 10.0 Hz, ArꢀH), 9.28 (1 H,
d, 3J (H,H) = 9.4 Hz, ArꢀH), 9.25 (1 H, 3J (H,H) = 10.0 Hz ArꢀH), 9.21 (1 H, s, ArꢀH), 8.88 (1
3
3
H, d, J (H,H) = 9.4 Hz, ArꢀH), 8.38 (1 H, s, ArꢀH), 4,60 (2H, q, J (H,H) = 7.3 Hz, CH2),
3
2.89 (6 H, s, 2 CH3), 2.88 (6 H, s, 2 CH3), 1,53 ppm (3H, t, J (H,H) = 7.3 Hz, CH3). 13Cꢀ
NMR (125 MHz, acetoneꢀd6, 25°C): δ = 167.3, 154.3, 134.7 (2C), 132.2, 131.8, 130.4, 129.2,
127.1, 126.8, 126.0, 125.6, 124.6, 124.0, 122.4, 122.1, 116.8, 62.6, 38.1 (2C), 38.0 (2C), 14.8
ppm. MS (ESI): m/z calc. for C23H24N2O7S2: 503,09467 [MꢀH]ꢀ, found: 503,09282.
3,8ꢀbis(N,Nꢀdimethylsulfamoyl)ꢀ6ꢀhydroxypyreneꢀ1ꢀcarboxylic acid (4). Compound 2
(17.8 mg, 0.04 mmol) was dissolved in dry THF (10 mL) to which was then added water
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(3.06 mg, 0.17 mmol) and potassium tertꢀbutoxide (83.04 mg, 0.74 mmol). The reaction
mixture was stirred 24 hours at room temperature, before the addition of ethyl acetate (20
mL). Subsequently, the organic phase was washed three times with hydrochloric acid (1M, 20
mL) and saturated NaCl solution (20 mL) and dried over sodium sulfate. 4 was purified with
column chromatography (methylene chloride / methanol = 9:1 (v/v)) to give a yellow powder
(12.6 mg, 0.03 mmol, 72%). UV/Vis (DMSO+TFA): λmax = 425 nm, (DMSO): λmax = 424/557
nm, (H2O+HCl): λmax = 415/418 nm, (H2O): λmax = 463 nm; ε(463) (H2O, ROꢀ) = 17000 L molꢀ1
cmꢀ1; fluorescence (DMSO+TFA): λmax = 476/573 nm, (DMSO): λmax = 573 nm, (H2O+HCl):
1
λmax = 482 nm, (H2O): λmax = 536/557 nm. HꢀNMR (500 MHz, acetoneꢀd6, 25°C): δ = 9.38
3
3
3
(1 H, d, J (H,H) = 10.0 Hz, ArꢀH), 9.24 (1 H, d, J (H,H) = 9.6 Hz, ArꢀH), 9.20 (1 H, J
3
(H,H) = 10.0 Hz ArꢀH), 9.16 (1 H, s, ArꢀH), 8.79 (1 H, d, J (H,H) = 9.6 Hz, ArꢀH), 8.34 (1
H, s, ArꢀH), 2.88 (6 H, s, 2 CH3), 2.85 ppm (6 H, s, 2 CH3). 13CꢀNMR (125 MHz, acetoneꢀd6,
25°C): δ = 179.3, 153.8, 134.1, 133.7, 132,1, 131.7, 130.0, 128.8, 127.9, 127.2, 126.0, 125.6,
125.4, 125.0, 122.4, 122.2, 116.3, 38.1 (2C), 38.0 (C2). MS (ESI): m/z calc. for
C21H20N2O7S2: 475,06337 [MꢀH]ꢀ, found: 475,06494.
Ethyl 3,8ꢀbis(N,Nꢀdimethylsulfamoyl)ꢀ6ꢀ(phosphonooxy)pyreneꢀ1ꢀcarboxylate (1). After
dissolving phosphoroxy chloride (140 ꢁL) in dry methylene chloride (40 mL) and cooling to ꢀ
10°C, a solution of 2 (48.1 mg, 0.10 mmol) and trimethylamine (40 ꢁL) in dry methylene
chloride (20 mL) was added slowly. The resulting, nearly colorless, solution was stirred for 18
hours upon warming up slowly to room temperature. Water (3 mL) was added slowly and the
mixture was stirred for additional 10 minutes. The organic phase was washed three times with
cooled water (~5°C, 40 mL) and one time with saturated NaCl solution (~5°C, 40 mL) before
drying over sodium sulfate. After evaporation of the solvent, the raw dichlorophosphate was
dissolved in a mixture of water (5 mL) and acetone (5 mL). Hydrolysis was monitored by RPꢀ
TLC and was complete after 48 hours. The reaction mixture was extracted three times with
ethyl acetate and the aqueous phase was dried in vacuo. Reverse phase column
chromatography (water / methanol = 1:1 (v/v)) of the crude product yielded 1 as yellow
powder (37.6 mg, 0.07 mmol, 68%). UV/Vis (H2O): λmax = 410 nm; ε(410) (H2O) = 19300 L
molꢀ1 cmꢀ1; fluorescence (H2O): λmax = 472 nm. 1HꢀNMR (500 MHz, methanolꢀd4, 25°C): δ =
9.35 (1 H, d, 3J (H,H) = 10.0 Hz, ArꢀH), 9.30 (1 H, d, 3J (H,H) = 9.6 Hz, ArꢀH), 9.24 (1 H, 3J
(H,H) = 10.0 Hz ArꢀH), 9.19 (1 H, s, ArꢀH), 8.92 (1 H, d, 3J (H,H) = 10.0 Hz, ArꢀH), 8.86 (1
H, s, ArꢀH), 4.61 (2H, q, 3J (H,H) = 7.0 Hz, CH2), 2.93 (6 H, s, 2 CH3), 2.86 (6 H, s, 2 CH3),
3
1,55 ppm (3H, t, J (H,H) = 7.0 Hz, CH3). 13CꢀNMR (125 MHz, methanolꢀd4, 25°C): δ =
168.0, 150.2, 134.9, 134.7, 133.3, 132.1, 131.1, 129.4, 127.1, 127.0, 126.9, 126.6, 125.8,
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125.5, 124.9, 121.3 (C2), 63.3, 38.4 (C2), 38.0 (C2), 14.9 ppm. MS (ESI): m/z calc. for
C23H25N2O10PS2: 583,06155 [MꢀH]ꢀ, found: 583,06245.
3,8ꢀbis(N,Nꢀdimethylsulfamoyl)ꢀ6ꢀ(phosphonooxy)pyreneꢀ1ꢀcarboxylic
acid
(3).
Compound 1 (4.0 mg, 0.007) was dissolved in an aqueous solution of NaOH (0.05 M, 10
mL). Reaction progress was monitored with RPꢀTLC and found to be complete after 2 hours.
The pH of the reaction solution was neutralized, and the water was evaporated from the
aqueous phase after extraction with ethyl acetate. The crude product was purified by reverse
phase column chromatography (water / methanol = 7:3 (v/v)) and yielded 3 as yellow powder
(3.1 mg, 0.006 mmol, 81%). UV/Vis (H2O): λmax = 407 nm; ε(407) (H2O) = 15000 L molꢀ1 cmꢀ1;
1
fluorescence (H2O): λmax = 448 nm. HꢀNMR (500 MHz, methanolꢀd4, 25°C): δ = 9.25 (1 H,
3
3
3
d, J (H,H) = 9.8 Hz, ArꢀH), 9.24 (1 H, d, J (H,H) = 9.8 Hz, ArꢀH), 8.97 (1 H, J (H,H) =
10.0 Hz ArꢀH), 8.89 (1 H, s, ArꢀH), 8.87 (1 H, s, ArꢀH), 8.86 (1 H, d, 3J (H,H) = 10.0 Hz, Arꢀ
H), 2.90 (6 H, s, 2 CH3), 2.86 ppm (6 H, s, 2 CH3). 13CꢀNMR (125 MHz, methanolꢀd4, 25°C):
δ = 175.5, 149.7, 136.9, 133.1, 132.4, 130.9, 130.7, 129.8, 127.6, 127.5, 127.1, 127.0, 126.8,
126.7, 125.4, 125.3, 121.1, 38.4 (C2), 38.1 (C2) ppm. MS (ESI): m/z calc. for
C21H21N2O10PS2: 555,03025 [MꢀH]ꢀ, found: 555,03196.
Acknowledgements
Financial support by the German Science Foundation (DFG, JU650/7ꢀ1) is gratefully noticed.
Furthermore we thank Tobias Dier for recording the mass spectra, Volker Huch for Xꢀray
crystallography measurements and Alexander Schiller for the helpful discussion of our
manuscript.
Electronic Supplementary Information (ESI)
Further compound characterization, determination of enzyme kinetics, further preliminary
liveꢀcell experiments and details for molecular logic are provided in the Electronic
Supplementary Information (ESI).
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Tables
Table 1: Spectroscopic properties of 1ꢀ3 at physiological pH (pH = 8), for spectroscopy of 4
see Table 3, for other forms of 2, see ESI S35.
2[a]
1
λmax,abs [nm] 410
λmax,em [nm] 472
3
484
558
407
448
1.00
3.73
0.98
3.89
0.86
5.81
Ф
τfl, ROꢀ [ns]
[a] ROꢀꢀform
Table 2: Kinetic parameters for reactions of the dye system with different enzymes.
Compound
Enzyme
1
3
4ꢀMUP
1
3
2
4ꢀMUBu
Alkaline Phosphatase
Acid Phosphatase
Porcine Liver Esterase
Km [ꢁM]
kcat/Km
[1/M/s]
3.0
6.9 x
108
2.9
1.1 x
108
2.2
1.8 x
107
40.8
6.0 x
102
48.6
3.9 x
104
3.7
7.1 x 103
16.9
5.7 x 103
Table 3: Spectroscopic data of compound 4.
4
λmax,abs [nm]
λmax,em [nm]
418 (4a); 415 (4d); 463 (4c)
482 (4a); 557 (4b); 536 (4c)
[a]
ꢀ
pKa
6.8 (ROHCO2 ); ~ 2.0 (ROꢀCO2H)
pKa*[b]
4.5 (ROHCO2H); ꢀ0.9 (ROHCO2H)
0.85 (4c)
Ф
τfl, ROꢀ [ns]
5.58
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[a]
[b]
determined via absorption titration (for details, see ESI S32ꢀS35),
determined by
fluorescence titration (for details, see ESI S32ꢀS35).
Table 4: Truth table for simple logic operations, 2:4 decoder (O1ꢀO4) and Feynman gate
(O5, O6) (λex = 395 + 460 nm).
O1
(λ = 498
nm)
O3
(λ = 436
nm)
O2
(λ = 576
nm)
O4
(λ = 528
nm)
O5
(λ = 553
nm)
O6
In1
(AlP)
In2
(PLE)
(λ = 509 nm,
negative logic)
0
1
0
1
0
0
1
1
1
0
0
0
0
1
0
0
0
0
1
0
0
1
1
1
0
0
0
0
0
1
1
0
gate (isolated)
NOR
INHIB2
INHIB2
AND
YES
XOR
Table 5: Truth table for operation of 4 as Feynman gate (λex = 395 nm).
In I (low In II (high
O I
O II
amount
amount
(λ = 514 nm, (λ = 548
OHꢀ)
OHꢀ)
neg. logic)
nm)
0
1
0
1
0
0
1
1
0
1
0
0
1
1
1
0
gate (isolated)
YES
XOR
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Figure and scheme captions
Scheme 1: Synthesis of 1ꢀ4.
Figure 1: (a) Normalized fluorescence emission of 1 (10ꢀ5 M, pH = 8) recorded 3 hours after
incubation (enzymes in nanomolar concentration); (b) Schematic overview of spectral and
molecular changes upon enzymatic conversion and corresponding Km and kcat/Km values; (c)
Different metabolic states of 1, visualized by blacklight fluorescence excitation (λex = 365
nm).
Figure 2: Pseudo colored fluorescence microscopic images of several cell clusters incubated
with 1 and excited at λex = 800 nm: (aꢀd) L929 cells, 1 h (a, c) and 3 h (b, d) after incubation,
blue channel (a, b): λdet = 435ꢀ485 nm (detection of compound 1), yellow channel (c, d): λdet
=
535ꢀ612 nm (detection of compound 2); (eꢀh) L929 cells, 1 h (e, g) and 3 h (f, h) after
incubation, green channel (e, f): λdet = 500ꢀ530 nm (detection of compound 4), yellow channel
(g, h): λdet = 565ꢀ615 nm (detection of compound 2).
Figure 3: (a) pHꢀtitration of compound 4 (λex = 395 nm); (b) Different protonation states of 4
in the ground and excited state. The color refers to the spectra in (a).
Figure 4: (a) Fluorescence spectra (normalized to substrate fluorescence, λex = 395 + 460 nm
(see ESI S48), substrate concentration: 10ꢀ5 M, trisꢀHCl buffer solution at pH = 8) with
corresponding output channels and threshold levels for (a) several logic operations (O1ꢀO4)
and (b) Feynman gate (O5, O6); (c) Logic circuit for the Feynman gate.
Figure 5: Fluorescence emission of 4 at different pH values (λex = 395 nm), output channels
and corresponding threshold values for molecular logic.
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Scheme 1
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Figures
Figure 1
(a)
(b)
(c)
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Figure 2
29