Rational design of latent fluorophores from water-soluble hydroxyphenyltriazine dyes suitable for lipase sensing

Article abstract of DOI:10.1002/ejoc.201500047

Derivatives of 2-(2′-hydroxy-5′-dimethylaminobenzyl)-4,6-dimethylamino-1,3,5-triazine were synthesized by an original multistep protocol that afforded, after quaternization of the N,N-dimethylaminobenzyl moiety, water-soluble fluorescent dyes. These fluor

Full text of DOI:10.1002/ejoc.201500047

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SHORT COMMUNICATION
DOI: 10.1002/ejoc.201500047
Rational Design of Latent Fluorophores from Water-Soluble
Hydroxyphenyltriazine Dyes Suitable for Lipase Sensing
Alicia Jacquemet,[a] Sandra Rihn,[a] Gilles Ulrich,[a] Pierre-Yves Renard,[b]
Anthony Romieu,[c,d] and Raymond Ziessel*[a]
Keywords: Dyes/pigments / Proton transfer / Fluorophores / Enzymes / Protecting groups
Derivatives of 2-(2Ј-hydroxy-5Ј-dimethylaminobenzyl)-4,6-
dimethylamino-1,3,5-triazine were synthesized by an origi-
nal multistep protocol that afforded, after quaternization of
the N,N-dimethylaminobenzyl moiety, water-soluble fluores-
cent dyes. These fluorophores exhibited excited-state intra-
10000 cm–1). The phenol residue was masked by an
enzyme-labile protecting group based on self-immolative
para-hydroxybenzyl alcohol. Lipases were used to unveil the
fluorescence under physiological conditions (phosphate-
buffered saline, pH 7.2) without aggregation or precipitation
of the fluorescent dyes.
molecular proton transfer and large Stokes shifts (ΔSS
Ͼ
examples of reversible switching have been constructed
from photochromic systems.[10] For enzyme-substrate inter-
actions in living cells, Förster resonance energy transfer
(FRET) is frequently employed.[11]
Introduction
Switchable (or masked) fluorophores are invaluable
molecules with spectroscopic properties that can be tuned
in response to external stimuli such as photons, chemical
reagents, and enzymes.[1,2] A specific functionality (typically
an aniline or a phenol) of these fluorescent organic dyes is
masked by linking a blocking subunit that quenches
fluorescence or inhibits, for instance, proton transfer in the
excited state.[3] Irreversible unmasking (decaging) by a
specific chemical or physical event elicits recovery of the
fluorescence and can be used as a reporter for enzyme ac-
tivity,[4] as an imaging tool,[5] or for sensing specific ana-
lytes.[6] Generally, such fluorophores can be accurately lo-
calized, which allows visualization of biochemical activities
through sophisticated imaging experiments including super-
resolution microscopy.[7]
Intramolecular hydrogen bonding in synthetic fluoro-
phores based on keto–enol tautomers provides an elegant
approach to fluorescence control through an excited-state
intramolecular proton-transfer (ESIPT) process.[12] This is
dependent on the fact that the planar enol form, which is
the dominant isomer in the ground state, can rearrange into
the excited keto form, which is strongly emissive. The sig-
nificant structural reorganization involved gives rise to large
Stokes shifts (ΔSS Ͼ 10000 cm–1 in the visible region).[13] If
the proton transfer is inhibited by a twist of the neighboring
phenyl ring, steric crowding, or chemical functionalization,
the resulting compounds are not emissive. This situation
has been shown to arise in the case of 2-(2Ј-hydroxy-5Ј-
chlorophenyl)-6-chloro-4(3H)-quinazolinone[14] and 2-(2Ј-
hydroxyphenyl)benzothiazole,[15] the phenol groups of
which are masked by O-phosphorylation. A specific enzyme
yields a fluorescent precipitate at the site of the phosphatase
activity that is detectable by fluorescence monitoring. The
released phenol enables proton transfer upon excitation and
an increase in brightness, likely due to formation of stable
aggregates. Aggregation-induced emission is often evoked
to explain the intense signal.[16] Furthermore, this concept
has been scrutinized to detect hydrolase,[17] monoamine
oxidase,[18] esterase,[19] and galactosidase/peptidase activi-
ties.[20]
Activable fluorophores often have
a photoreactive
moiety [azido[8] or ortho-(dimethoxy)nitrobenzyl][9] that
quenches the fluorescence by electron transfer and that can
be cleaved irreversibly by photoirradiation. Representative
[a] Institut de Chimie et Procédés pour l’Energie, l’Environnement
et la Santé (ICPEES), Laboratoire de Chimie Moléculaire et
Spectroscopies Avancées (ICPEES-LCOSA), UMR CNRS
7515, Ecole Européenne de Chimie, Polymères et Matériaux,
25 Rue Becquerel, 67087 Strasbourg Cedex 02, France
E-mail: ziessel@unistra.fr
http://icpees.unistra.fr/lcosa/
[b] Normandie Université, COBRA UMR 6014 & FR 3038, UNIV
Rouen; INSA Rouen; CNRS, IRCOF,
1, Rue Tesnières, 76821 Mont-Saint-Aignan Cedex, France
[c] Institut de Chimie Moléculaire de l’Université de Bourgogne,
UMR CNRS 6302, Université de Bourgogne,
9 Avenue Alain Savary, 21078 Dijon, France
[d] Institut Universitaire de France,
Herein, we describe water-soluble masked-ESIPT fluoro-
phores based on a 2-(2Ј-hydroxy-5Ј-dimethylaminobenzyl)-
4,6-dimethylamino-1,3,5-triazine scaffold with a phenol
moiety that is protected with an enzyme-labile self-immol-
ative protecting group, which allows the “off–on” detection
103 Boulevard Saint-Michel, 75005 Paris, France
Supporting information for this article is available on the
WWW under http://dx.doi.org/10.1002/ejoc.201500047.
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R. Ziessel et al.
SHORT COMMUNICATION
of esterase/lipase activity by steady-state fluorescence over reagent, and this led to compound 2.[24] This compound
the spectral range of 400 to 550 nm in purely aqueous was treated with nBuLi at low temperature to generate 2-
solution (Scheme 1). The choice of the para-acetoxybenzyl lithioaromatic derivatives. Reaction with cyanuric chloride
alcohol moiety was inspired by prodrug medicinal chemis- (also known as 2,4,6-trichloro-1,3,5-triazine) provided the
try[21] and was chosen as an ideal esterase/lipase-labile self- monoaryl derivative, and the remaining chloro substituents
immolative spacer arm because of its easy linkage to the were displaced in situ by reaction with dimethylamine (40%
water-soluble phenol-based dye through a fully stable carb- in water) to give intermediate 3, which was deprotected with
onate linkage by using diphosgene as the reactant. Despite acid to provide compound 4 in fair yield. Alkylation of 4
the presence of the side arm, the dye remains completely was regioselective and very efficient, and it provided cat-
soluble in water. The construction of the target activable ionic compound 5 by using methyl iodide or zwitterionic
fluorogenic dye is derived from our earlier work on the use compound 7 by using 1,3-propanesultone. Counteranion
of 1,3,5-triazine derivatives for optical and FRET purposes exchange by using a Dowex resin (preloaded with nitrate
in organic solutions.[22]
ions) provided water-soluble dye 6 in excellent yield. Note
that dye 5 is only partially soluble in water.
To interrupt the intramolecular proton transfer in the
excited state, O-alkylation seemed to be an adequate
approach. To this end, compound 7 was treated with an
excess amount of diphosgene to generate the activated
chloroformate intermediate, the subsequent reaction of
which with para-acetoxybenzyl alcohol (Ac-PABA) led to
masked fluorophore 8 (Scheme 3). Dye 8 appears to be
totally soluble in water without any organic additives. Simi-
larly, water-insoluble analogue 9[20] was also converted into
corresponding esterase/lipase-reactive latent fluorophore
10. As expected, both probes 8 and 10 were found to be
nonemissive owing to the lack of intramolecular hydrogen
bonding. The structures of these novel ESIPT fluorophores
were unambiguously confirmed by NMR spectroscopy,
ESI-MS, and elemental analyses (see the Supporting Infor-
mation).
Scheme 1. Target masked fluorophores with a self-immolative pro-
tecting module; PHBA = para-hydroxybenzyl alcohol.
Results and Discussion
1
The synthetic protocol is outlined in Scheme 2. The
Analysis of dyes 7 and 9 by H NMR spectroscopy con-
hydroxy group of commercially available 4-hydroxybenz- firmed that the OH proton had an unusual chemical shift
aldehyde was protected by using methoxymethyl chloride (δ Ͼ 14 ppm), and this reflected its involvement in a strong
(MOMCl) and K2CO3 as the base, which afforded 1 in intramolecular hydrogen bond. The presence of a large ex-
quantitative yield.[23] Reductive amination of the aldehyde cess amount of water or phosphate-buffered saline (PBS)
function was performed by using Ti[OCH(CH3)2]4 as the did not change the shape of the emission and absorption
Scheme 2. Step-by-step construction of key phenol-based dyes 5–7.
2
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Fluorophores from Water-Soluble Hydroxyphenyltriazine Dyes
Scheme 3. Synthesis of masked hydroxyphenyltriazine; DIEA = N,N-diisopropylethylamine.
band profiles. Fluorescence quantum yields were very sim- cence of dyes 8 and 10 was totally quenched as a result of
ilar to those determined in purely organic solvents, and ex- the absence of the phenol residue. However, the use of
citation spectra matched the absorption, excluding forma- aqueous conditions (PBS at pH 7.2) revealed the presence
tion of aggregates. Interestingly, the emission profiles were of very weak emission at 460 nm, as highlighted in Fig-
similar for all dyes and did not depend on the solvent dipole ures 2 and 3. This residual emission is likely formed by
moment or the nature of the side arm (neutral, cationic, or slight hydrolysis (Ͻ1 mol-%) of carbonate derivatives 8 and
zwitterionic).
10. At that stage, we noticed that acetylation of the phenol
Examination of the optical properties of dyes 4, 6, and 7 groups in dyes 7 and 9 was feasible, but the resulting non-
in a polar solvent (EtOH or PBS at pH 7.2) revealed an fluorescent dyes were hydrolytically not stable under
unstructured absorption at 310–319 nm, typical of 1,3,5-tri- aqueous PBS conditions.
azine derivatives, and a more intense π–π* absorption
Table 1. Selected spectroscopic data for dyes 4, 6, and 7.
around 250 nm assigned to the phenyl group (Figure 1).[25]
[a]
[b]
[c]
[d]
Dye Solvent
λabs
ε
λem
ΔSS
Φem
By irradiation in the range of the less energetic absorption
band, a strong fluorescence was observed around 470 nm
with a large Stokes shift of about 10000 cm–1 (Table 1),
which was assumed to be due to emission from the excited
keto form produced by intramolecular proton transfer to
the triazine in the excited state (Figure 1).[26] This transfer
is expected to be facilitated by the existence of a related H-
bond with the ground-state enol. As expected, the fluores-
(nm) (m–1 cm–1)
(nm) (cm–1)
(%)
4
6
7
7
8
EtOH
EtOH
PBS
PBS/DMSO (9:1) 312
PBS
PBS/hog pan-
creas
CH2Cl2
319
320
312
6800
6950
5500
5900
6300
470
472
460
460
–
10100[b]
10200
10400
10400
–
10
12
7 (10)[e]
11
315
312
327
–
Ͻ0.1
8
9
9
–
460
497
480
10400
10500
–
10
7550
–
30[f]
PBS/DMSO
63
(9:1)[g]
PBS/DMSO
(9:1)/Amano
10
–
–
480
–
60
[a] Concentration about 10–4 m. [b] Concentration about 10–5 m.
[c] Stokes shift. [d] Determined by using rhodamine 6G as a stan-
dard (ΦF = 0.76 in water).[27] [e] Addition of bovine serum albumin
(BSA) protein (1 mg) as surfactant in solution (2 mL). [f] Measured
in an integrating sphere. [g] Suspension of dye.
To assess the possibilities for decaging probe 8 by remov-
ing its acetyl ester and subsequent 1,6-elimination of self-
immolative spacer arm, several lipases were screened by
fluorescence-based bioassays. The most active lipases were
those from hog pancreas and Rhizopus oryzae from about
10 different lipases (Table S1, Supporting Information).
Two different reaction media, namely, PBS/DMSO (9:1 v/v,
Figure 2) or pure PBS (pH 7.2, Figure 3) were used. Under
the first conditions, the fluorescence emission at 460 nm
Figure 1. Absorption, emission (λex = 330 nm), and excitation (λem
= 500 nm) spectra of phenolic dye 7 in PBS at pH 7.2 (1ϫ10–4
for absorption and 1ϫ10–5 m for emission and excitation).
m
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SHORT COMMUNICATION
ure S17). For the blank experiment, bovine serum albumin
(BSA) was used to mimic the enzyme environment.
Decaging undoubtedly occurs through a cascade reaction
(also known as a domino process[28]) to provide para-
hydroxybenzyl alcohol (PHBA), acetic acid, carbon dioxide,
and a free-phenol-based ESIPT fluorophore.[29] In the ab-
sence of DMSO, similar results were obtained (Figure 3)
with an increase in the fluorescence by a factor 8, and the
maximum was reached after around 10 min. Under these
conditions, the absolute quantum yield measured in an inte-
grating sphere was 10%, similar to the one found for free-
phenol dye 7 in PBS (Table 1), which confirmed the comple-
tion of the lipase-induced fluorophore release process.
Again, for the blank experiment, the increase in fluores-
cence was only 1.1-fold over that on the same time scale,
temperature, and buffer conditions by using BSA to mimic
the enzyme environment.
Finally, with water-insoluble dye 10, the use of DMSO as
the solvent was required, and self-immolation of the PHBA
spacer was induced by a lipase working in organic media
(i.e., Amano lipase from Burkholderia cepacia) in the same
timeframe of about 15 min and with high quantum yield
(Table 1). In this case, the insoluble fluorophore probably
inserted within a hydrophobic pocket of the enzyme and
the solution remained transparent for weeks. In the absence
of the enzyme and with the addition of an increasing
amount of water into a DMSO solution of dye 9, it precipi-
tated in the form of highly fluorescent nanoobjects. Further
photophysical characterization of this system is currently in
progress and aggregation-induced fluorescence enhance-
ment by conformation restriction is likely to be at the origin
of this observation.[16]
Figure 2. Fluorescence emission spectra (λex = 330 nm) measured
as a function of time of latent fluorophore 8 [2 mL of solution at
1ϫ10–5 m in PBS/DMSO (9:1 v/v)] in the presence of 7.2 units of
lipase from hog pancreas, incubation at 40 °C. Spectrum at t = 0
was corrected for dilution. The scale on the right-hand side is given
in minutes.
Conclusions
For the first time, we succeeded in solubilizing substi-
tuted hydroxyphenyl-1,3,5-triazine dyes in water through
effective postsynthetic quaternization of a dimethylamino
moiety preintroduced onto the 2-hydroxyphenyl substitu-
ent. Caging their intrinsic fluorescence through the masking
of the hydroxy group with an enzyme-labile self-immolative
spacer derived from PHBA was readily achieved under mild
conditions. Stable, water-soluble, and nonfluorescent dyes
were successfully produced. Incubation of the resulting
Figure 3. Fluorescence emission spectra (λex = 330 nm) measured
as a function of time of latent fluorophore 8 (2 mL of solution at
1ϫ10–5 m in PBS, pH 7.2) in the presence of 7.2 units of lipase
from hog pancreas (500 μL solution in PBS, pH 7.2), incubation at
40 °C. Spectrum at t = 0 was corrected for dilution. The scale on
the right-hand side is given in minutes.
gradually increased in the presence of lipase from hog pan- latent fluorophores with lipases (working either in aqueous
creas at 40 °C. A maximum fluorescence was reached after buffers or in organic solvents) led to quantitative release of
about 35 min of incubation, which provided an absolute the phenol, restoration of the intramolecular proton trans-
quantum yield of 10% (measured with an integrating fer, and, thus, the fluorescence. These molecules extend the
sphere, Table 1). This value is in keeping with the quantum scope of fluorogenic dyes useful for tracking enzyme activi-
yield of unmasked phenolic dye 7 measured under similar ties.[30] Herein, the window of signal analysis was larger
conditions but in the absence of the hydrolytic enzyme than 120 nm in the visible region owing to the fact that
(Table 1). In the presence of the lipase, the fluorescence in- these excited-state intramolecular proton-transfer-based
creased by a factor 15, whereas in its absence the increase fluorophores exhibit very large Stokes shifts. Further mo-
in fluorescence was only 1.2-fold over the same time scale, lecular engineering will be regarded to redshift their fluores-
temperature, and buffer conditions, which thus clearly cence, particularly by increasing delocalization pathways
demonstrated the overall stability of latent fluorophore 8 and/or by grafting Förster resonance energy transfer mod-
under these simulated physiological conditions (Fig- ules to the aryl moiety of “para-hydroxybenzyl alcohol like”
4
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Fluorophores from Water-Soluble Hydroxyphenyltriazine Dyes
tored by TLC). The solution was extracted with CH2Cl2, washed
with water, dried with MgSO4, and concentrated. The residue was
purified by column chromatography (silica gel; petroleum ether/
CH2Cl2, 4:6 to 0:1) to give a white solid (80.3 mg, 94%). 1H NMR
self-immolative spacers. The rationale design, construction,
and studies of similar phenolic dyes that are completely
soluble in water and aimed at sensitive biosensing applica-
tions are currently in progress.
4
3
(300 MHz, CDCl3): δ = 7.95 (d, J = 1.8 Hz, 1 H), 7.23 (dd, J =
8.2 Hz, 4J = 1.8 Hz, 1 H), 7.23 (ABsys, JAB = 8.4 Hz, υoδ =
4
92.0 Hz, 4 H), 7.06 (d, J = 1.8 Hz, 1 H), 5.17 (s, 2 H), 3.13 (s, 12
H), 2.97 (s, 3 H), 2.32 ppm (s, 3 H). 13C NMR (75 MHz, CDCl3):
δ = 169.7, 169.5, 165.4, 153.7, 150.9, 147.7, 136.0, 132.8, 132.0,
131.8, 130.8, 129.8, 122.8, 121.9, 69.4, 36.1, 21.3, 21.1 ppm. MS
(ESI): m/z (%) = 466.1 (100) [M + H]+. C24H27N5O5 (465.20):
calcd. C 61.92, H 5.85, N 15.04; found C 61.72, H 5.49, N 14.82.
Experimental Section
For general procedures and description of the intermediates see the
Supporting Information.
Compound 7: 2-[4,6-Bis(dimethylamino)-1,3,5-triazin-2-yl]-4-[(di-
methylamino)methyl]phenol (88 mg, 0.28 mmol) was solubilized in
toluene (2.5 mL). 1,3-Propanesultone (109 mg, 0.83 mmol,
3 equiv.) was added, and the mixture was stirred for 5 days at room
temperature. Then, EtOAc was added, and the mixture was centri-
fuged. The white solid was washed several times with EtOAc to
give the sulfobetain compound (112 mg, 92%). 1H NMR
(400 MHz, [D6]DMSO): δ = 2.13 (m, 2 H, CH2), 2.50 (m, 2 H,
CH2), 2.93 (s, 6 H, +NCH3), 3.16 [s, 6 H, NMe2(triazine)], 3.19 [s,
6 H, NMe2(triazine)], 3.33 (m, 2 H, CH2), 4.50 (s, 2 H, +NCH2),
6.99 (d, J = 8.7 Hz, 1 H, HPh6), 7.56 (dd, J = 2.1, 8.3 Hz, 1 H,
HPh5), 8.43 (d, J = 2.6 Hz, 1 H, HPh3), 14.37 ppm (s, 1 H, OH).
13C NMR (100 MHz, [D6]DMSO): δ = 19.0 (CH2), 35.9 [NCH3(tri-
azine)], 36.1 [NCH3(triazine)], 47.8 (CH2), 48.7 (+NCH3), 48.9
(+NCH3), 62.6 (CH2), 65.8 (+NCH2), 117.8 (CPh6), 117.9 (CPh2),
118.0 (CPh4), 133.7 (CPh3), 137.7 (CPh5), 162.6 (CPh1), 162.7
(Ctriazine), 168.4 ppm (Ctriazine). MS (ESI): m/z (%) = 439.2 (100)
[M + H]+. C19H30N6O4S (438.54): calcd. C 52.04, H 6.90, N 19.16;
found C 51.78, H 6.69, N 18.87.
Acknowledgments
The authors thank the Centre National de la Recherche Sci-
entifique (CNRS) and Agence Nationale de la Recherche (ANR)
(Programme Blanc 2009, ANR-09-Blan-0081-01, ANR “Fluo-
Mag”) for financial support of this work, especially for a Ph.D.
grant to S. R. and a postdoctoral fellowship to A. J. P.-Y. R. thanks
Labex SynOrg (ANR-11-LABX-0029) and Région Haute-Norm-
andie (CRUNCh network) for financial support. A. R. thanks the
Institut Universitaire de France (IUF) and the Burgundy Region
(FABER programme, PARI Action 6, SSTIC 6 “Imagerie, instru-
mentation, chimie et applications biomédicales”) for financial sup-
port. Prof. Jack Harrowfield (ISIS in Strasbourg) is greatly ac-
knowledged for critical reading of this manuscript prior to publica-
tion.
Compound 8: A mixture of phenol (31.4 mg, 0.07 mmol) and di-
phosgene (3 mL) was stirred at room temperature. After 24 h, the
residual diphosgene was removed under vacuum and a solution of
para-acetoxybenzyl alcohol (17 mg, 0.11 mmol, 1.5 equiv.; pre-
viously dried with a Dean Stark system) in anhydrous chloroben-
zene (7 mL) was added. The resulting mixture was stirred for 24 h,
and then, anhydrous triethylamine (33 μL, 0.23 mmol, 3.3 equiv.)
was added. After evaporation under reduced pressure, the crude
product was purified by reversed-phase column chromatography
(CH3CN/H2O, 98:2 to 96:4) to give a white solid (18 mg, 40%). 1H
NMR (300 MHz, [D6]DMSO): δ = 2.14 (m, 2 H, CH2), 2.28 (s, 3
H, CH3), 2.50 (m, 2 H, CH2), 2.96 (s, 6 H, +NCH3), 3.08 [s, 12 H,
NMe2(triazine)], 3.45 (m, 2 H, CH2), 4.61 (s, 2 H, NCH2), 5.17 (s,
2 H, CH2O), 7.12 (d, J = 8.7 Hz, 2 H, HPhPABA), 7.42 (m, 3 H,
HPhPABA and HPh6), 7.74 (dd, J = 2.1, 8.3 Hz, 1 H, HPh5), 8.26 ppm
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(d,
J = 2.6 Hz, 1
H, HPh3). 13C NMR (100 MHz, [D6]-
DMSO): δ = 19.1 (CH2), 20.8 (CH3 acetate), 35.5 [NCH3(triazine)],
47.8 (CH2), 48.9 (+NCH3), 63.3 (CH2), 65.1 (NCH2), 69.2 (CH2O),
121.9, 123.8, 126.2, 129.7, 131.0, 132.3, 135.7, 135.9, 150.4, 150.6,
152.3 (C=O), 164.6, 168.2, 169.1 ppm (C=O). MS (ESI): m/z (%)
= 631.1 (100) [M + H]+. C29H38N6O8S (630.71): calcd. C 55.22, H
6.07, N 13.22; found C 54.85, H 5.87, N 13.07.
Compound 10: A Schlenk flask was charged with 9 (50.0 mg,
0.183 mmol) in dry CH2Cl2 (2 mL). The solution was cooled to
0 °C, and then trichloromethyl chloroformate or diphosgene
(0.031 mL, 0.256 mmol, 50.7 mg) and freshly distilled DIEA
(0.031 mL, 0.183 mmol, 1 equiv.) were added. The solution was
stirred 30 min at 0 °C and then for an additional 30 min at room
temperature. The solvent was evaporated. The crude product was
dissolved in dry CH2Cl2 (2 mL), 4-(hydroxymethyl)phenyl acetate
(0.026 mL, 0.183 mmol, 30.4 mg) and freshly distilled triethylamine
(0.025 mL, 0.183 mmol) were added. The solution was stirred at
40 °C until complete consumption of the starting material (moni-
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R. Ziessel et al.
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Received: January 13, 2015
Published Online: ᭿
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Eur. J. Org. Chem. 0000, 0–0

Job/Unit: O50047
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Date: 02-02-15 13:55:17
Pages: 7
Fluorophores from Water-Soluble Hydroxyphenyltriazine Dyes
Latent Fluorophores
The phenol residue of water-soluble 2-(2Ј-
hydroxy-5Ј-dimethylaminobenzyl)-4,6-di-
methylamino-1,3,5-triazine is masked by an
enzyme-labile protecting group constructed
with a self-immolative para-hydroxybenzyl
alcohol. The fluorescence of the dyes is re-
stored with lipases under physiological
conditions without aggregation or precipi-
tation of the fluorescent dyes.
A. Jacquemet, S. Rihn, G. Ulrich, P.-
Y. Renard, A. Romieu, R. Ziessel* .... 1–7
Rational Design of Latent Fluorophores
from Water-Soluble Hydroxyphenyltriazine
Dyes Suitable for Lipase Sensing
Keywords: Dyes/pigments / Proton trans-
fer / Fluorophores / Enzymes / Protecting
groups
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