Hydrodabcyl: A Superior Hydrophilic Alternative to the Dark Fluorescence Quencher Dabcyl

Article abstract of DOI:10.1021/acs.analchem.7b03488

Dark fluorescence quenchers are nonfluorescent dyes that can modulate the fluorescence signal of an appropriate fluorophore donor in a distance-dependent manner. Dark quenchers are extensively used in many biomolecular analytical applications, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, especially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenzene-based dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the much-used dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hydrodabcyl-Ser-Phe-EDANS by the proteases thermolysin and papain.

Full text of DOI:10.1021/acs.analchem.7b03488

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Letter
Hydrodabcyl: a superior hydrophilic alternative
to the dark fluorescence quencher dabcyl
Oxana Kempf, Karl Kempf, Rainer Schobert, and Elisa Bombarda
Anal. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.analchem.7b03488 • Publication Date (Web): 26 Oct 2017
Downloaded from http://pubs.acs.org on October 28, 2017
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Analytical Chemistry
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Hydrodabcyl: a superior hydrophilic alternative to the dark fluores-
cence quencher dabcyl
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Oxana Kempf,† Karl Kempf, ‡ Rainer Schobert‡ and Elisa Bombarda*,†
†Department of Biochemistry and ‡Organic Chemistry Laboratory, University of Bayreuth, Universitaetsstr. 30, 95440, Bayꢀ
reuth, Germany
ABSTRACT: Dark fluorescence quenchers are nonꢀfluorescent dyes that can modulate the fluorescence signal of an appropriate
fluorophore donor in a distanceꢀdependent manner. Dark quenchers are extensively used in many biomolecular analytical applicaꢀ
tions, such as studies with fluorogenic protease substrates or nucleic acids probes. A very popular dark fluorescence quencher is
dabcyl, which is a hydrophobic azobenzene derivative. However, its insolubility in water may constitute a major drawback, espeꢀ
cially during the investigation of biochemical systems whose natural solvent is water. We designed and synthesized a new azobenꢀ
zeneꢀbased dark quencher with excellent solubility in aqueous media, which represents a superior alternative to the muchꢀused
dabcyl. The advantage of hydrodabcyl over dabcyl is exemplarily demonstrated for the cleavage of the fluorogenic substrate hyꢀ
drodabcylꢀSerꢀPheꢀEDANS by the proteases thermolysin and papain.
Biomolecular processes are extensively studied by employꢀ
ing fluorescence dyes1. There is a broad range of fluorescent
dyes the spectroscopic characteristics of which depend on
several physicoꢀchemical parameters such as their hydrophoꢀ
bicity and oxidation state, or the pH value and ionic strength
of the medium2. The strategy of using such singleꢀlabeled
probes was refined by the development of dualꢀlabeled probes
which combine a reporter dye with a quencher moiety3. Typiꢀ
cally, these probes may exist in two conformations differing in
their fluorescence properties: a ‘closed’ form in which the
reporter and the quencher are in close proximity and an ‘open’
form in which these groups are spatially separated. If the
quencher is a fluorescence dye itself, both the increase in the
fluorescence of the quencher and the decrease in the fluoresꢀ
cence of the reporter can be followed. However, an overlap of
quencher and reporter fluorescence spectra may cause backꢀ
ground noise necessitating a meticulous instrumental setꢀup
and data analysis. Dark quenchers (e.g., nonꢀfluorescent dyes)
offer a solution to this problem because they do not occupy
any crucial emission bandwith. Dualꢀlabeled probes comprisꢀ
ing a reporter and a dark quencher are also called fluorogenic
probes since the biochemical event to be observed causes their
transition from a nonꢀfluorescent to a (typically strongly)
fluorescent form.
Dabcyl (4ꢀ(4'ꢀdimethylaminophenylazo)benzoic acid (1),
Figure 1) is a dark quencher in dualꢀlabeled probes widely
used for a variety of biomolecular applications such as enzyꢀ
matic catalysis and nucleic acid probes4–7. The absorption
band of dabcyl (1) in the range of 350ꢀ550 nm overlaps with
the emission band of many common fluorescent dyes such as
5ꢀ((2'ꢀaminoethyl)amino)naphthaleneꢀ1ꢀsulfonic
acid
(EDANS) (λem, Max = 490 nm), bimane (λem, Max = 480 nm), and
many fluorescein, coumarin and rhodamine derivatives.
Dabcyl (1) is an azobenzene pushꢀpull functionalized at the
paraꢀpositions of the phenyl rings. Its conjugated πꢀsystem and
its lack of polar substituents render it a hydrophobic comꢀ
pound, virtually insoluble in aqueous media. In fact, stock
solutions of dabcyl (1) need to be prepared in DMSO. Altꢀ
hough dabcyl (1) is one of the most popular acceptors for
developing FRETꢀbased probes, its insolubility in water seꢀ
verely limits its use in biological systems where the natural
solvent is water. Even when this hydrophobicity can be partly
compensated for by the hydrophilicity of the substrate to
which dabcyl (1) is linked (e.g., long DNA segments or pepꢀ
tide chains), it poses a real problem in the case of comparaꢀ
tively small substrates. Such solubility problems were reported
for various dabcylꢀlabeled substrates5,8. In particular, any
incomplete dissolution leads to inaccurate estimations of the
concentrations and consequently to erroneous calculations of
stability and rate constants. Most attempts to overcome the
problems stemming from the insolubility of dabcyl (1) such as
performing the enzymatic assays in mixtures of water and
DMSO5 can be source of new problems due to the modificaꢀ
tion of the polar environment of the reaction. Instead of adjustꢀ
ing the polarity of the solvent we designed and synthesized a
new azobenzene derivative, hydrodabcyl (2), that is spectroꢀ
scopically dabcylꢀlike, yet far more hydrophilic when comꢀ
pared to dabcyl (1).
Figure
1.
Chemical
structures
of
4ꢀ(4'ꢀ
dimethylaminophenylazo)benzoic acid (dabcyl (1)) and 4ꢀ(2',6'ꢀ
dihydroxyꢀ4'ꢀdimethylaminophenylazo)ꢀ2ꢀhydroxybenzoic acid
(hydrodabcyl (2)).
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All procedural information can be found in the Supporting
Information.
ionic solubilizers such as sulfonate groups12. Consequently,
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hydrodabcyl (2) will not significantly modify the electrostatic
profile of the molecule to which it is linked and thus the bindꢀ
ing properties of the labeled molecule are expected not to be
altered much. This aspect has important implications for inꢀ
vestigations of biological systems in which molecular interacꢀ
tions are frequently driven by electrostatics (e.g. enzymatic
reactions). Additionally, the carboxyl group of hydrodabcyl
(2) remains available for the coupling to an amino group in the
substrate through a standard amide bond formation. Practical
advantages of the good solubility of hydrodabcyl (2) are the
ease of preparing solutions and of cleansing used glassware.
RESULT AND DISCUSSION
Technically, hydrodabcyl (2) (4ꢀ(2',6'ꢀdihydroxyꢀ4'ꢀ
dimethylaminophenylazo)ꢀ2ꢀhydroxybenzoic acid, Figure 1) is
a trishydroxy derivative of the parent dabcyl (1).Since phenolꢀ
ic OH groups were known to affect the emitting properties of a
molecule, which is apparent for instance from the intensity
increase and redꢀshift of the fluorescence when going from
phenylalanine to tyrosine, it took some deliberation to ensure
the darkness of the new chromophore. We found that the addiꢀ
tion of three hydroxy groups to the azobenzene of 1 led to a
nonꢀfluorescent molecule. This finding is in line with a previꢀ
ous work on the coumarin chromophore that showed that the
addition of an OH group at Cꢀ7 of coumarin gives rise to a
distinctly fluorescent molecule while attachment of three OH
groups affords nonꢀfluorescent coumarin derivatives9. Accordꢀ
ingly, trihydroxydabcyl derivatives were not fluorescent eiꢀ
ther. Moreover, the three hydroxy groups had to be positioned
in a way that precludes the formation of catechol chelate comꢀ
plexes with biologically relevant metal ions (e.g. Fe(III)10)
which would possibly interfere with the system under investiꢀ
gation. This aspect is particularly important for the investigaꢀ
tion of enzymatic reactions in which metals are essential coꢀ
factors and for possible applications in vivo.
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Saturated solutions of hydrodabcyl (2) in water show a pH
of 4.5, which is significantly lower than the pH of pure water.
In fact, both dabcyl (1) and hydrodabcyl (2) bear a carboxyl
group and have therefore an acidic character. The acidic enviꢀ
ronment favors the protonation of the carboxyl group, thus
increasing the hydrophobic character of the molecule. While
the hydroxy groups of hydrodabcyl (2) compensate for this
effect, dabcyl (1) turns even less soluble in water as the pH
drops. Nevertheless, experiments are normally carried out at
controlled pH, therefore, we performed the same test in a
buffered aqueous solution at pH 8.0 where the carboxyl group
is supposed to be deprotonated and thus charged. Expectedly,
in an aqueous buffer of pH 8.0 the solubility of both dabcyl (1)
and hydrodacyl (2) is increased, distinctly so in the case of
hydrodabcyl (2) (25 mM) but still unsufficiently so in the case
of dabcyl (1) (cf. Table 1 in SI), confirming the superiority of
hydrodabcyl.
Scheme 1. Synthesis of hydrodabcyl (2).
Hydrodabcyl (2) was synthesized in two steps (Scheme 1,
for details see SI†). Phloroglucinol (3) was converted with
dimethylamine to 5ꢀdimethylaminoꢀresorcinol (4), which in
turn was reacted with 4ꢀdiazosalicylic acid (5) to give hyꢀ
Figure 2. (a) Solubility in water: on the left, evident precipitation
of dabcyl (1) (intended concentration ca. 7 µM); on the right, a
clear solution of hydrodabcyl (2) (7 µM). (b) Concentration deꢀ
pendence of the absorbance at 445 nm of hydrodabcyl (2) in a
buffered solution at pH 7.0 (black) and at pH 4.3 (red). The soluꢀ
tion at pH 4.3 was obtained by gradual acidification of the soluꢀ
tion at pH 7.0 (SI for details). The temperature was set at 20 °C.
drodabcyl (2). The intermediate 4 was prepared according to a
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protocol by Petrzilka and Lusuardi
and obtained as pink
crystals as described (Figure S1 in SI). However, when used as
such for the azo coupling reaction with 5, it afforded a fluoresꢀ
cent product that proved unsuitable as a dark quencher. A
careful examination of every single step of the procedure
pointed to an impurity as the cause of the pink coloration and
possibly of the fluorescence of the final product (cf. SI). In
fact, when the unspecified impurity was removed by a precedꢀ
ing column chromatography, 4 was obtained as colorless crysꢀ
tals (Figure S1 in SI). Its reaction with 5 afforded 2 as a nonꢀ
fluorescent compound in 58 % yield over both steps. Though
seemingly marginal, this modification to the original protocol
is crucial for obtaining 2 in a quality that allows its use as a
dark quencher.
In the majority of their applications, quenchers such a
dabcyl (1) or hydrodabcyl (2) are linked to the actual moleꢀ
cules of interest, e.g. to peptide substrates in order to monitor
protease activities. Both dabcyl (1) and hydrodabcyl (2) can be
readily coupled to an amino group of a peptide via a standard
amide bond formation. However, this linkage eliminates the
free carboxylic acid on the quenchers and so the option to
improve their solubility, or that of their peptide conjugates, by
elevating the pH. To investigate this aspect, we linked the two
dark quenchers to the amino group of a lysine to obtain the
compounds Lysꢀdabcyl and Lysꢀhydrodabcyl. Interestingly, in
a buffered aqueous solution at pH 8.0, the concentration of
Lysꢀdabcyl was only 7.6 × 10ꢀ6 M whereas we could prepare a
6.6 mM solution of Lysꢀhydrodabcyl without reaching saturaꢀ
Hydrodabcyl (2) is soluble in water, up to a concentration of
5.7 × 10ꢀ4 M at 20 °C. In contrast, it was not possible to preꢀ
pare an aqueous solution of dabcyl (1) due to precipitation
even at low concentration (Figure 2a). The attachment of
hydroxy groups to the dabcyl core imparts water solubility
without adding electric charges as would have the addition of
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tion. Accordingly, the solubility of Lysꢀhydradabcyl is preꢀ hydrodabcyl (2) as quencherꢀacceptor and phenylalanine laꢀ
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sumably much higher than 6.6 mM and therefore close to that
of LꢀLys which is about 40 mM or 5.8 g kgꢀ1 of water13. In
other words, amide conjugation of hydrodabcyl (2) to peptides
does not significantly alter the solubility of the latter, whereas
conjugation to dabcyl (1) is expected to lower their solubility
in aqueous media. The modification of the solubility of a
reactant in a biochemical process may hamper and distort its
experimental study considerably. For instance, a distinctly
reduced solubility of the product of an enzymatic reaction may
hinder its release from the active site, resulting in an artifactuꢀ
al inhibiting effect. The solubility of hydrodabcyl was accuꢀ
rately measured spectroscopically at different pH by certifying
the linear dependence between absorbance and concentration
(Figure 2b and SI for more details). From basic pH values
down to pH 6.0, solutions with concentrations in the millimoꢀ
lar range can be prepared directly at the desired pH. At pH <
6.0 the solubility is lower, however, solutions with millimolar
concentrations can still be prepared by gradual acidification of
an alkaline solution down to a pH value of 4.3. The excellent
solubility of hydrodabcyl (2) and its conjugates with biomoleꢀ
cules makes it the quencher of choice in most biochemical
applications.
beled with EDANS as fluorophore donor (Figure 4 and SI for
details of the synthesis procedure). A dipeptide is the minimal
substrate for protease reactions, ideal to prove the applicability
or even superiority of hydrodabcyl (2) in labeling small moleꢀ
cules. The dipeptide SerꢀPhe was chosen because it is a prefꢀ
erential cleavage site of the P1’ protease thermolysin, an enꢀ
zyme that is commercially available. Furthermore, the same
dipeptide labeled with EDANS and dabcyl (1) had already
been tested with several proteases14, therefore allowing a comꢀ
parison of dabcyl (1) and hydrodabcyl (2) during a typical
biochemical application. It should be noted that in this case
hydrodabcyl (2), due to its high polarity, needs to have its
hydroxy groups protected by acetylation prior to the amide
bond formation in order to increase yields and reactivity. Subꢀ
sequent deprotection under mild conditions then restores the
advantageous polarity and solubility of the conjugate (cf. SI).
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The absorption spectra of dabcyl (1) and hydrodabcyl (2) in
DMSO are quite similar with the latter showing a slight bathoꢀ
chromic shift of the main absorption band and a greater molar
absorbance (Figure 3). Both effects enhance the quenching
power of hydrodabcyl (2) at larger wavelengths and advocate
its preferential use, even in applications where dabcyl (1) has
been customarily employed, so far. On the other hand, in
aqueous solution, the absorption maximum of hydrodabcyl (2)
(λMax = 445 nm, ε445 = 43000 Mꢀ1 cmꢀ1) is shifted toward
smaller wavelength (hypsochromic shift) in comparison to its
spectrum in DMSO (λMax = 470 nm, ε470 = 37000 Mꢀ1 cmꢀ1) as
shown in Figure 3. However, this hypsochromic shift is partly
compensated by its greater molar absorbance in aqueous soluꢀ
tion, thus rendering hydrodabcyl (2) an effective quencher at
wavelengths up to 500 nm also in aqueous solution.
Figure 4. a) Fluorescence spectra of 20 ꢁM hydrodabcylꢀSerꢀPheꢀ
EDANS (7) alone (black) and after 5 h incubation with thermolyꢀ
sin (blue). For comparison, the fluorescence spectra of 20 ꢁM
PheꢀEDANS alone (green) and an equimolar amount of hydrodaꢀ
bcyl (2) + PheꢀEDANS (red). The spectra were recorded in
100 mM TrisꢀHCl (pH 7.5) containing 2 mM CaCl2 at 37 °C; λEx=
336 nm. b) Chemical structure of hydrodabcylꢀSerꢀPheꢀEDANS
(7) The quencher hydrodabcyl (2) and the fluorophore EDANS
are marked in gray and green, respectively. The protease cleavage
site is indicated by a blue bar.
In the doubleꢀlabeled substrate, the fluorescence of EDANS
linked to the Phe moiety is largely quenched by hydrodabcyl
(2) linked to the Ser moiety (black curve in Figure 4). The
fluorescence of EDANS decreases also when an equimolar
amount of hydrodabcyl (2) is added to PheꢀEDANS (green
and red curves in Figure 4). However, when the fluorophore
and the quencher are not kept close together, as in the double
labeled dipeptide, the quenching efficiency is lower in line
with the distanceꢀdependence of energyꢀtransferꢀbased proꢀ
cesses. Interestingly, the fluorescence intensity curve detected
when the labeled dipeptide was incubated with the enzyme
thermolysine (blue curve in Figure 4) coincided with the curve
of PheꢀEDANS in presence of an equimolar amount of hyꢀ
drodabcyl (2) (red curve in Figure 4) and not with the fluoresꢀ
cence intensity curve of PheꢀEDANS alone (green curve in
Figure 4). This finding indicates that the hydrodabcyl moiety
has a certain quenching ability even when not covalently
linked to the fluorophore and that this ability is roughly the
same for hydrodabcyl (2) and its amide with serine. This fact
has to be taken into account during the procedure to determine
the reference points for calibrating the signal accurately. Sevꢀ
eral type of physical quantities can be estimated provided the
appropriate calibration of the signal is made.
Figure 3. Comparison of molar absorbances of dabcyl (1) in
DMSO (red curve, λMax = 451 nm, ε451 = 32000 Mꢀ1 cmꢀ1), hyꢀ
drodabcyl (2) in DMSO (black curve, λMax = 470 nm, ε470 = 37000
Mꢀ1 cmꢀ1) and 2 in buffered aqueous solution pH 8.0 (blue curve
λMax = 445 nm, ε445 = 43000 Mꢀ1 cmꢀ1); all at 20 °C.
Hydrodabcyl (2) has the requisite properties for serving as a
useful dark quencher in fluorogenic probes. In order to obꢀ
serve hydrodabcyl (2) in action we synthesized a peptidic
substrate featuring only two amino acids: serine labeled with
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Figure 5a shows the restoration of the fluorescence of The mixture resulting from the reaction of thermolysin with an
1
EDANS as a function of time upon adding different concentraꢀ
tions of thermolysin to a solution of the labeled dipeptide. As
expected, the larger the excess of the substrate with respect to
the enzyme, the slower the reaction. Interestingly, despite the
different concentration ratio between labeled substrate and
enzyme, the same fluorescence level is eventually reached,
indicating that the same amount of substrate molecules has
been cleaved. When the enzyme is added in stoichiometric
amount (1:1) to the labeled substrate the reaction is so fast that
it cannot be observed on the chosen time scale. Under these
conditions, the initial amount of substrate is expected to be
fully cleaved, defining the maximum fluorescence level correꢀ
sponding to a complete cleavage of the substrate. Consequentꢀ
ly, since all the time traces reached the same fluorescence
level, we conclude that the cleavage reaction proceeded to
completion, independently of the amount of enzyme added. In
order to confirm the exhaustive cleavage of the substrate, fresh
enzyme was added to the mixture of substrate and enzyme
after reaching signal saturation. Since no appreciable fluoresꢀ
cence increase was observed, we excluded the presence of
nonꢀcleaved substrate and concluded that the fluorescence
level truly corresponds to the fluorescence signal of the fully
processed substrate.
excess of doubleꢀlabeled substrate (ratio 1:200) was applied in
lane 4. The presence of a bright spot with the same Rf as Pheꢀ
EDANS together with the absence of a dark spot with the
same Rf as the intact doubleꢀlabeled substrate indicates that
thermolysin cleaved the SerꢀPhe peptide bond in the doubleꢀ
labeled substrate. The dark spot with an Rf of 0.33 overlapping
with the bright spot of PheꢀEDANS most likely corresponds to
hydrodabcylꢀSer, the other product of the cleavage. Interestꢀ
ingly, the Rf of the putative spot of hydrodabcylꢀSer is lower
than 0.53, the Rf of the putative spot of dabcylꢀSer, as reported
in the literature14 . The same trend can be recognized when
observing that the doubleꢀlabeled substrate including hydrodaꢀ
bcyl (2) has a lower retention factor (Rf = 0.5) than the one
including dabcyl (1) (Rf = 0.72) 14. As a lower retention factor
Rf is an indication of higher polarity, this result is in keeping
with the higher polarity conferred by hydrodabcyl (2) as comꢀ
pared to dabcyl (1). Taken together our results demonstrate the
retention of the activity of thermolysin in cleaving the peptide
bond between serine and phenylalanine in the hydrodabcylꢀ
SerꢀPheꢀEDANS (7) despite the presence of the chromoꢀ
phores. They also corroborate the usefulness and the adꢀ
vantages of hydrodabcyl (2) in fluorogenic probes.
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Furthermore, we tested the proteolysis of hydrodabcylꢀSerꢀ
PheꢀEDANS (7) by another enzyme, the cysteine protease
papain (Figure 5). In the work of Weimar et al.14, where the
fluorogenic substrate was dabcylꢀSerꢀPheꢀEDANS, only a
small portion of the dipeptide was processed after an incubaꢀ
tion of several hours with papain. In our experiment, we incuꢀ
bated the same amount of hydrodabcylꢀSerꢀPheꢀEDANS (7)
(20 ꢀM) with 6 ꢀM of papain. This relatively high concentraꢀ
tion of papain was necessary to allow the detection of the
reaction in a comparable time range. This shows that papain is
much slower than thermolysin in cleaving the dipeptide, in
agreement with the findings of Weimar et al.14. However, in
contrast to them, papain was able to cleave the entire amount
of the fluorogenic substrate, when dabcyl (1) was substituted
with hydrodabcyl (2), as evidenced both by the evolution of
the fluorescence signal and the analysis of the degradation
products by TLC (Figure 5). This result is noteworthy, as it
shows that the hydrophobicity of dabcyl (1) may indeed repreꢀ
sent a handicap for fluorogenic probes when used in biochemꢀ
ical system (e.g. interfering with proteolysis) and points out
the higher applicability of hydrodabcyl (2).
Figure 5: a) Time traces of the hydrolysis of 20 ꢁM of hydrodaꢀ
bcylꢀSerꢀPheꢀEDANS (7) at 37 °C, (λEx= 336 nm, λEm=515 nm)
by thermolysin (different concentrations) and papain (6 ꢁM).
Thermolysin was added in different enzyme to substrate concenꢀ
tration ratios: 1:2000 (green), 1:200 (blue), 1:1 (red). The magenta
curve describes the hydrolytic reaction by papain. The black line
corresponds to the fluorescence level of the substrate alone, beꢀ
fore adding the enzyme. b) Products of hydrodabcylꢀSerꢀPheꢀ
EDANS after (7) proteolytic cleavage indicated by TLC: hyꢀ
drodabcylꢀSerꢀPheꢀEDANS (7) (lane 1, Rf = 0.5), PheꢀEDANS
(lane 2, Rf = 0.3), EDANS (lane 3, Rf = 0 ꢀ 0.1), reaction mixture
after proteolytic cleavage with thermolysin (lane 4, bright spot
Rf = 0.3 and dark spot Rf = 0.33) and with papain (lane 5, bright
spot Rf = 0.3 and dark spot Rf = 0.33).
CONCLUSION
Hydrodabcyl (2) is a new dark fluorescence quencher with
an optimal solubilityꢀstabilityꢀabsorption profile. Its small
dimension, the absence of charged groups, and its absorption
range make hydrodabcyl (2) the dark quencher of choice in
tandem with many commercially available fluorescence doꢀ
nors. Hydrodabcyl (2) overcomes the problem of insolubility
in aqueous media, doing away with the need of organic coꢀ
solvents, and it lends itself ideally to highꢀthroughput enzyꢀ
matic tests. Thus, hydrodabcyl (2) represents a vastly imꢀ
proved and superior alternative to the very popular dabcyl (1)
in the design of fluorogenic probes.
Additional evidence for the cleavage of the substrate was
provided by the determination of the degradation product with
thinꢀlayer chromatography (TLC) (Figure 5b). Hydrodabcyl
(2) confers an orangeꢀred color to the substrate to which it is
attached making it visible under UV light as a dark spot. Acꢀ
cordingly, the doubleꢀlabeled substrate hydrodabcylꢀSerꢀPheꢀ
EDANS (7) appeared as a dark spot with a retention factor (Rf)
of 0.5 (Figure 5b, lane 1). The references of the potential
products EDANS and PheꢀEDANS are shown as white spots
in lanes 2 and 3 of Figure 5b. In agreement with the work of
Weimer et al.14, EDANS remained close to the starting line
(Rf = 0 ꢀ 0.1), whereas PheꢀEDANS had an Rf value of 0.3.
ASSOCIATED CONTENT
Supporting Information
The Supporting Information is available free of charge on the
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Johansson, M. K.; Cook, R. M. Chem. - Eur. J. 2003, 9,
Bernacchi, S.; Mely, Y. Nucleic Acids Res. 2001, 29, E62ꢀ
Matayoshi, E. D.; Wang, G. T.; Krafft, G. A.; Erickson, J.
3466–3471.
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Supporting data, chemical synthesis and characterization (PDF)
(4)
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AUTHOR INFORMATION
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Corresponding Author
Science 1990, 247, 954–958.
(6)
(7)
Tyagi, S. Nat. Biotechnol. 1996, 14, 947–948.
Tyagi, S.; Marras, S. A.; Kramer, F. R. Nat. Biotechnol.
* elisa.bombarda@uniꢀbayreuth.de.
2000, 18, 1191–1196.
Notes
(8)
Holskin, B. P.; Bukhtiyarova, M.; Dunn, B. M.; Baur, P.;
The authors declare no competing financial interests. A patent for
hydrodabcyl is pending (WO 2016/083611 A1, issued
02.06.2016).
de Chastonay, J.; Pennington, M. W. Anal. Biochem. 1995, 227, 148–
155.
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(9)
Balaiah, V.; Seshadri, T. R.; Venkateswarlu, V. Proc. -
Indian. Acad. Sci. 1942, 16, 68–82.
ACKNOWLEDGMENT
(10) Avdeef, A.; Sofen, S. R.; Bregante, T. L.; Raymond, K. N.
J. Am. Chem. Soc. 1978, 100, 5362–5370.
(11) Petrzilka, T.; Lusuardi, W. G. Helv. Chim. Acta 1973, 56,
510–518.
(12) Loudwig, S.; Bayley, H. J. Am. Chem. Soc. 2006, 128,
12404–12405.
(13) Lide, D. R. CRC Handbook of Chemistry and Physics, 85th
Edition.
(14) Weimer, S.; Oertel, K.; Fuchsbauer, H. L. Anal. Biochem.
2006, 352, 110–119.
The present work was supported by the DFG grant BO 3578/1 and
by the RTG 1640 (Photophysics of Synthetic and Biological
Multichromophoric Systems). We thank Prof. Dr. G. Matthias
Ullmann for fruitful discussions and Markus Petermichl for helpꢀ
ing in acquiring NMR spectra.
REFERENCES
(1)
ed.; Springer: New York, 2006.
Lakowicz, J. R. Principles of fluorescence spectroscopy, 3rd
(2)
Hauglang, R. P. Handbook of fluorescence probes and reꢀ
search products, 9th ed.; Molecular Probes, Inc.: Eugene, OR (USA),
2002.
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