A new family of bioorthogonally applicable fluorogenic labels

Article abstract of DOI:10.1039/c3ob40296g

Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical properties were elaborated by experimental and theoretical investigations. Of the newly synthesized fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic performance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while their "clicked" triazole congeners showed intense fluorescence. One of these labels showed a very large Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluorogenic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen of possible fluorogenic tags will enable the generation of libraries of such tags in the future.

Full text of DOI:10.1039/c3ob40296g

Organic &
Biomolecular Chemistry
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A new family of bioorthogonally applicable
fluorogenic labels†‡
Cite this: Org. Biomol. Chem., 2013, 11,
3297
András Herner,a Ivana Nikić,b Mihály Kállay,c Edward A. Lemkeb and Péter Kele*a
Synthetic procedures for the construction of fluorogenic azido-labels were developed. Photophysical
properties were elaborated by experimental and theoretical investigations. Of the newly synthesized
fluorogenic and bioorthogonally applicable dyes two were selected on the basis of their fluorogenic per-
formance and further subjected to in vitro and in vivo studies. Both tags exhibited excellent fluorogenic
properties as in aqueous medium, the azide form of the selected dyes is virtually non-fluorescent, while
their “clicked” triazole congeners showed intense fluorescence. One of these labels showed a very large
Stokes shift. To the best of our knowledge this is the first reported case of mega-Stokes type of fluoro-
genic labels. These studies have justified that these two fluorogenic tags are remarkably suitable for
bioorthogonal tagging schemes. The developed synthetic approach together with the theoretical screen
of possible fluorogenic tags will enable the generation of libraries of such tags in the future.
Received 7th November 2012,
Accepted 5th March 2013
DOI: 10.1039/c3ob40296g
www.rsc.org/obc
of the fluorescent signal with remarkable spatial and temporal
resolution and their potential for multichannel imaging.
Introduction
Small molecular modulation of biomatter is of great impor- Indeed, labeling of amino acids, proteins, lipids, nucleic acids,
tance in modern chemical biological research. The develop- sugars, or even cells and viruses with synthetic fluorescent
ment of bioorthogonal chemistry schemes has greatly dyes has become an indispensible tool in bioanalytical
facilitated the exploration of biochemical processes. Continu- sciences.3
ous efforts have been made lately for the development of new
One advantage of applying synthetic fluorophores is the
bioorthogonal reagents that offer selective, often site-specific, ability to employ chemical approaches to control the photo-
and high-yielding reactions with biological targets. Up to now, physical properties and chemical reactivity of fluorescent tags.
these efforts have resulted in the development of several Applying modern synthetic organic chemistry strategies enables
bioorthogonal reagents e.g. (cyclo)alkynes, azides or tetr- efficient tailoring of the chemical structure to obtain probes for
azines.1 Non-natural biomolecular building blocks containing specific needs. Fluorescence tagging schemes often follow a two
one or more of these bioorthogonal functions offer easy and (or more)-step procedure, where excessive amounts of the
specific incorporation of these chemical reporters into target fluorophore are administered to the sample to be labeled. After
structures by taking advantage of gene technology or the meta- the incubation time the unreacted reagent should be removed.
bolic machinery of living organisms.2 Of the biomolecular However, this task is not easy in many instances and the
labeling techniques fluorescent modification of biological remaining, non-specifically bound fluorescent labels contribute
matter is by far the most popular one. Its popularity can be to very high levels of background fluorescence therefore com-
owed to the highly sensitive and relatively cheap detectability promising the sensitivity of detection (low signal-to-noise ratio).
Solution to this problem is offered by the use of so-called fluoro-
genic (switchable, “turn-on”) probes where the reagents are
weakly or non-fluorescent at the wavelength of detection.4 The
fact that these labels are introduced as virtually non-fluorescent
derivatives eliminates the need for the separation of excessive
reagents without compromising sensitivity. Many fluorogenic
dyes have been developed to sense for example differences in
microenvironments, however, very few fluorogenic reagents are
available for covalent modification of biomatter.4,5
aInstitute of Chemistry, Eötvös Loránd University, H-1117, Pázmány Péter sétány 1a,
Budapest, Hungary. E-mail: kelep@elte.hu; Fax: (+36)1 372 2909;
Tel: (+36)1 209 0555 ext. 1610
bStructural and Computational Biology Unit, EMBL, Meyerhofstrasse 1, D-69117
Heidelberg, Germany
cDepartment of Physical Chemistry and Materials Science, Budapest University of
Technology and Economics, P. O. Box 91, H-1521, Budapest, Hungary
†This work is dedicated to Roger M. Leblanc on the occasion of his birthday.
‡Electronic supplementary information (ESI) available: 1H- and 13C-NMR spectra
Bioorthogonal tagging schemes have benefited greatly from
the use of the azide group. The significance of this functional
group lies behind its high energetic feature together with its
for small molecules and fluorescence spectra for 1a–c and 9a–c, further images
of live cell labeling experiments and optimized geometries are provided. See
DOI: 10.1039/c3ob40296g
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spectral parameters by incorporating electron donating
(methoxy) or withdrawing (nitro) groups. Based on our earlier
work with polymethine-fluorophores we also hoped that some
of these dyes will exhibit large Stokes shifts in their fluorescent
form.9
Fig. 1 Modular setup of fluorogenic tags.
Synthesis
stability under a wide variety of reaction conditions.6 Note-
worthy is its selective reactivity that enables reactions solely
with particular counterparts in the presence of several other
functional groups present in biological media. Its participation
in conventional bioorthogonal modulation schemes e.g.
copper-catalyzed or strain promoted cycloaddition with
alkynes or the Staudinger ligation technique with phosphanes
as reaction partners has just raised the azide group to the
most popular one.7 Another less emphasized feature of the
azide group is the fact that its incorporation into fluorescent
scaffolds often results in dramatic drop in fluorescence inten-
sity. These fluorogenic compounds remain non-fluorescent as
long as the azide function is intact. They become intensely flu-
orescent, however, when the azide group is converted into a
different functionality. Examples show that conversion of
azides into amines or triazoles results in changes in the elec-
tronic system and the originally quenched fluorescence
reinstates.4,5 Quite simply, the azide moiety offers a bioortho-
gonally applicable, switchable two-in-one combination under
certain circumstances. Despite their obviously advantageous
features azido quenched fluorescent tags are rarely reported.
Design of fluorogenic labels mostly relies on empirical trial
and error methods and very few examples use theoretical pre-
diction of such features. Moreover, the so-far presented fluoro-
genic azido-tags lack the possibility of fine-tuning e.g.
emission wavelengths, as simple fluorogenic scaffolds bear the
azide moiety. Herein we introduce an approach that could be
used in the design of more complex fluorogenic units with
theoretically predictable photophysical features (Fig. 1).
Commercially available benzothiazole scaffolds 2a and 2b were
condensed with 4-nitrobenzaldehyde in molten p-TsOH in a
minimum amount of refluxing toluene providing exclusively
the trans-styryl products (4a–b) in moderate to good yields
(Scheme 1, Method A). Reduction of the nitro-group with tin(II)
chloride under acidic conditions and subsequent diazotization
based azide group incorporation resulted in fluorogenic dyes
1a–b in medium yields. In the case of 2-methyl-6-nitro-1,3-
benzothiazole this reaction route was not applicable for 2c
bearing two nitro groups. 2c was therefore reacted with
4-azidobenzaldehyde (6) in DMSO at r.t. in the presence of
NaOMe giving rise to 7c in medium yield. This intermediate
was then subjected to acid catalyzed elimination effected by
TFA to obtain fluorogenic dye 1c in moderate yield (Scheme 1,
Method B). Method B was found to be less efficient for the syn-
thesis of 1a as it gave a much lower overall yield (6%). This
method was also not suitable for the production of 1b as it
resulted in a mixture of products in low yields.
The conditions of the elimination step in Method B were
found to be strongly dependent on the nature of the substitu-
ents. For example, 1a and 1c were not formed when the basic
medium was applied (NaOMe-DMSO at r.t.) to 7a and 7c. Pro-
ducts in these cases were only formed under acid catalyzed
conditions. On the other hand, the electron-donating methoxy
substituent facilitated the base catalyzed elimination step but
resulted in an inseparable mixture of products. It is note-
worthy to mention that in the case of Method B when the
addition–elimination steps were carried out in a sequential
manner, separation and the purification of the desired pro-
ducts were greatly facilitated by the intermediate primer
adduct (7).
Results and discussion
Design
We have chosen the 1,3-benzothiazole (Fig. 1) scaffold as a
starting module for its appealing synthetic and fluorescent fea-
tures. 1,3-Benzothiazoles are easily modified at their 2nd and
6th positions; therefore we have also intended to use these
sites for further modifications. On the one hand we planned
to shift the emission wavelength of the benzothiazole core
towards the red regime by means of extending the conjugated
system. On the other hand we intended to modulate the emis-
sion intensity of the fluorescent scaffold by incorporating an
azide moiety into the framework. Based on former studies and
theoretical investigations,5,8 we have anticipated that incorpor-
ation of a 4-azidostyryl moiety that can either be incorporated
directly or in the form of its nitro-precursor will satisfy both
Scheme 1 Synthesis of fluorogenic dyes. (a) 1.1 eq. p-TsOH × H2O, toluene,
110 °C, 18 h; (b) 5 eq. Sn(II)Cl2 × 2H2O, ccHCl, r.t./45 °C, 5–20 h; (c) i. NaNO2,
ccHCl, 0 °C, ii. NaN3, 0 °C→r.t., 3 h; (d) 1.2 eq. NaOMe, DMSO, r.t., 6–20 h;
needs. We will show that position 6 is suitable for tuning the (e) catalytic TFA, 3 Å MS, toluene, 60–70 °C, 6–16 h.
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hydrogen bond formation with water decreased the fluore-
scence intensities (ESI‡).
In fact, in the case of 1b and 1c practically no fluorescence
could be detected in aqueous solutions therefore the fluore-
scence of the corresponding triazoles (9b, 9c) implies remark-
able increase in the fluorescence signal (Fig. 2 and 3). These
results suggest that 1b and 1c are particularly suitable to be
applied in aqueous solutions. Excitation wavelengths were
found to be very similar for all fluorogenic dyes but emission
maxima were shifted towards the red regime in the order of 9a
< 9b ≪ 9c. Noteworthy is to mention the spectral properties of
9c. It exhibits a large Stokes-shift (called “mega-Stokes”10) of
190 nm in water providing us with the possibility of more sen-
sitive detection by eliminating the interfering scattered light
and self-absorption. Moreover, to the best of our knowledge,
such a combination of fluorogenic and mega-Stokes properties
is unprecedented in the literature. Dyes of this kind are excep-
tionally useful in Förster resonance energy transfer (FRET)
applications as the dye emission does not interfere with the
spectral bands of the second fluorophore, which is a common
problem in FRET technology.
Scheme 2 (a) cat. [Cu(PPh3)2]NO3, cat. TEA, CH2Cl2, r.t., 10 h.
Method A can easily be generalized and extended for a large
variability of substrates for modular approaches. For example,
several heterocyclic derivatives with relatively acidic methyl
groups (picolynium, lepidinium, (benz)oxazoles, etc.) could be
applied and even libraries of fluorogenic dyes become accessi-
ble in a reasonable number of synthetic steps. Work elaborat-
ing this option is currently underway in our lab and results
will be reported in due course.
To our delight, all fluorogenic dyes showed very low emis-
sion intensities as a result of the quenching effect of the azide
moiety. Next, we were curious, whether it is possible to turn on
the fluorescence of the fluorogenic cores as a result of chemi-
cal reaction with appropriate reaction partners. Therefore we
have selected N-(prop-2-ynyl)pivalamide probe (8) as a reaction
partner for 1a–c in a copper catalyzed azide–alkyne click reac-
tion (Scheme 2).
Regarding the emission wavelengths, we observed a small
but persistent blue shift in all cases for the triazoles compared
to their corresponding azides. This is most likely in connection
Compound 8 was selected on the basis of not having any
aromatic moieties that could affect fluorescence of the pro-
ducts by any means. The click reactions were performed in a
dichloromethane solvent with a drop of triethylamine and a
catalytic amount of Cu(I)-catalyst10 at room temperature. Pro-
ducts (9a–c) were then collected by filtration and analyzed for
their fluorescent properties.
Spectroscopic measurements
We have studied the fluorescent properties of the click-pro-
ducts, 9a–c, in comparison to their parent azides, 1a–c, in
DMSO, CH2Cl2 and water (Table 1). In DMSO very low intensity
of fluorescence was observed for all azides. Triazoles on the
other hand showed orders of magnitudes stronger fluore-
scence, with 9b showing the best performance. In all cases
Fig. 2 Enhancement of fluorescence intensity at λmax (em) of the triazoles in
DMSO and water solvent. The concentration of azides and the corresponding
triazoles were the same.
Table 1 Photophysical properties of azides and the corresponding triazoles
Fluorophore
1a
9a
1b
9b
1c
9c
λmax (abs)/nma
ε/104 M−1 cm−1a,b
λmax (exc)/nma
λmax (em)/nma
Stokes-shift/nma
360
4.00
362
455
93
368
5.30
346
417
71
371
3.60
357
446
89
368
4.22
361
445
84
388
3.21
392
586
194
355
2.27
385
530
145
a,d
ΦF
0.001 0.045 0.002 0.439 0.047 0.068
45 220 1.5
ΦF (triazole)/ΦF
(azide)a
λmax (exc)/nmc
λmax (em)/nmc
λmax (exc)/nme
λmax (em)/nme
Stokes-shift/nme
350
410
343
414
352
427
357
455
98
352
390
522
381
489
437
355
453
98
n.d. f 335
n.d. f 421
n.d. f 86
n.d. f 350
n.d. f 540
n.d. f 190
a In DMSO. b Measured at λmax (abs). c In CH2Cl2. d Relative to quinine
sulfate in 0.5
background.
M
H2SO4.11 e In water. f Not detectable, in the
Fig. 3 Normalized emission spectra of 1b–9b and 1c–9c.
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with the change in molecular dipole moments associated with
the evolution of the triazole motif.
Table 2 Calculated transition wavelengths (λ) and oscillator strengths for the
fluorophoresa
Absorption
Emission
Quantum chemical calculations
Substituent
λ/nm
Osc. strength
λ/nm
Osc. strength
To interpret the experimentally observed behavior of the fluoro-
phores we performed quantum chemical calculations using
the Gaussian 09 package.12 To accelerate the calculations we
employed propyne as a model system instead of the click
reagent N-(prop-2-ynyl)pivalamide used in the experiments.
The geometries for all the compounds were optimized at the
density functional theory (DFT) level choosing the PBE0 func-
tional13,14 and the 6-311++G** basis set. All the DFT calcu-
lations were performed with dichloromethane as a solvent
invoking the polarized continuum model.15 Though the experi-
ments were carried out in different solvents, we chose dichloro-
methane since the polarity of this solvent and the probability
of the formation of specific solute–solvent interactions are low
enabling the simplified treatment of the solvent effects.
Excited-state properties were calculated using the time-depen-
dent DFT method16 with the same functional, basis set, and
solvation model. To simulate the experimental absorption
(excitation) spectra vertical excitation energies as well as oscil-
lator strengths were calculated at the optimized ground-state
geometries. For the calculation of fluorescence wavelengths
and intensities the geometries of the first excited singlet state
were optimized and vertical excitation energies and oscillator
strengths were calculated at the minima of the excited-state
surfaces.
We note that several isomers (conformers) exist for the
investigated molecules. First, stereoisomerism about the
central double bond can be observed. Since NMR experiments
revealed that only the trans isomers were formed in the syn-
thesis, we considered the latter in the calculations. Second,
because of the restricted rotation about other bonds further cis
and trans isomers can be identified. We calculated the ener-
gies for all the possible isomers and only retained the most
stable one for each molecule for the excited-state calculations.
As we tested for the H-substituted compound the excited-state
properties are largely independent of which isomer is con-
sidered, thus the above restriction does not influence our
conclusions.
Azides
–H
–OMe
–NO2
Triazoles
–H
393 (350)
407 (352)
445 (390)
1.76
1.78
1.51
451 (410)
467 (427)
512 (522)
1.78
1.83
1.60
388 (343)
402 (352)
422 (381)
1.78
1.71
1.58
442 (414)
458 (437)
497 (489)
1.78
1.81
1.66
–OMe
–NO2
a Numbers in parentheses show the experimental results in CH2Cl2.
Provided that the excited-state geometry optimization is
started from a geometry close to the optimized ground-state
structure, the relaxation of the geometry is moderate for all
molecules, and the optimized S1-state geometries are close to
the ground-state ones. Remarkable conformational change
takes place only at the triazole moiety of the triazole deriva-
tives, which is perpendicular to the phenyl ring in the S1 state
of the unsubstituted molecule, while the excited-state confor-
mation is planar for the other compounds. The calculated
transition wavelengths and oscillator strengths are collected in
Table 2.
The agreement of the experimental and theoretical absorp-
tion and emission wavelengths is satisfactory and justifies the
selection of the applied theoretical model. However, the oscil-
lator strengths for the emissions are practically identical for
the azide and the triazole derivatives suggesting that for the
azides the fluorescence from the minima found in the optimi-
zations is not the dominant decay channel.
We found that upon the rotation of the azide group the first
excited state interchanges with a non-emissive state. In the
case the excited-state geometry optimization is started from
the distorted structure, we obtain a minimum on the potential
energy surface of the latter state, which is very close to the
ground-state potential surface (ca. 0.5 eV above the ground
state energy) and the transition probability of the excited state
→ ground state transition is zero. The corresponding geome-
tries are displayed (see ESI‡). Consequently, these results
suggest the existence of an alternative, radiationless decay
channel explaining the low fluorescence intensities observed
for the azide derivatives. Due to the conformation change of
the azide group, a state-switch takes place which opens up the
possibility of the internal conversion of the two lowest excited
states followed by another internal conversion process to the
ground state.
The optimized ground-state geometries for the most stable
isomers are presented (see ESI‡). The geometries are planar
for the azide derivatives, while for the triazoles the triazole
ring forms an angle of about 30 degrees with the plane of the
phenyl ring.
We found that the rotational barrier of the rotation around
the bond connecting the azide/triazole moiety to the phenyl
ring is low, consequently the rotation is allowed at room temp-
erature. Since the molecules are expected to have several low-
lying excited states, and upon the rotation around a bond the
In vitro peptide labeling
excited-state potential energy surfaces (PESs) may cross each Following model reactions and photophysical characterization
other and an internal conversion may take place, we mapped we were curious to test the performance of our new fluorogenic
the excited-state PESs in more detail and started the geometry labels in action. For this we have chosen a cyclooctyne (CyO)
optimization from several initial geometries differing in the bearing peptide sequence (CyO-Gly-Pro-Leu-Gly-Val-Arg-DL-Pra-
torsion angle between the azide/triazole and phenyl groups.
NH2; DL-Pra = racemic propargyl-glycine) from our previous
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Fig. 5 Fluorescent and corresponding Coomassie stained images of
GFPY39TAG+SCO and GFPwt labeling reactions with compounds 1b, 1c (lanes 1 and
2, respectively) inside live E. coli.
work we have genetically encoded cyclooctyne (SCO) bearing
unnatural amino acid 10 using a rationally engineered tRNApyl
/
PylRSAF pair.2a,b An Amber (TAG) mutated Green Fluorescent
Protein (GFP) reporter construct was expressed in E. coli cells
together with a pEvol PylRSAF plasmid in the presence of 10,
leading to site-specific incorporation of SCO into GFP. Living
cells were treated with 1b, 1c and lysed subsequently. The
lysates were loaded onto an SDS PAGE gel for whole cell lysate
analysis. The gel was analysed for fluorescence by exciting the
samples in the UV-range (365 nm) and detecting the emission
intensities. Afterwards the gels were stained with Coomassie
(Fig. 5).
Fig. 4 Top: coupling reaction of 1b and CyO-pep. The dots show the data-
points of the labelled peptide, the triangles belong to the control sample
(without peptide). Bottom: coupling reaction of 1c and CyO-pep. The dots show
the datapoints of the labelled peptide, the triangles belong to the control
sample (without peptide).
Analysis of the gel under UV excitation revealed that both
fluorogenic labels efficiently labelled the SCO-tagged GFP. In
the control experiment where wild type (WT) GFP was
expressed in the cells no labeling of the protein took place.
Furthermore, it was also obvious that 1b and 1c can stand the
harsh conditions applied during lysis and no fluorescent
decomposition products were formed (for full details and full
gel images see ESI‡). These results served as evidence that the
two new fluorogenic labels can be used in bioorthogonal
fluorescent tagging schemes resulting in background fluore-
scence free images.
studies.17 The cyclooctyne moiety enables rapid in situ copper-
free tagging of the peptide e.g. with fluorogenic azides under
physiological conditions (pH = 7.20 phosphate buffer). Due to
their performance in the model reactions we have selected 1b
and 1c for in vitro labeling reactions. The tagging reaction was
monitored in time by following fluorescence intensity changes
at 455 and 540 nm, respectively, while exciting at 355 nm. In
both cases rapid increase in fluorescence intensity could be
observed (Fig. 4).
Reaction with 1b was somewhat faster than in the case of 1c.
The final fluorescence intensity was much higher for the former
fluorogenic tag as well. Control reactions of both fluorogenic
tags in the absence of the peptide showed no change in fluore-
scence intensities, implying that there are no e.g. nitrene
involved decomposition processes that would result in fluore-
scent species. The fact that the non-fluorescent azides of 1b and
1c show very low fluorescence intensities means that the evol-
ving signal can be detected with excellent s/n ratios, a feature
greatly desired in biological tagging schemes.
Experimental
General
Unless otherwise indicated, all starting materials were
obtained from commercial suppliers (Sigma-Aldrich, Fluka,
Merck, Alfa Aesar, Reanal, Molar Chemicals, Romil) and used
without further purification. Dichloromethane stabilized with
ethanol was used, unless otherwise mentioned. SCO was pur-
chased from Sichem (Bremen, Germany). Tamra-azide was
ordered from www.baseclick.eu. Analytical thin-layer chromato-
In vivo protein labeling
We were also curious to explore the performance of selected graphy (TLC) was performed on silica gel 60 F254 precoated
fluorogenic azido-tags under in vivo conditions where the dyes aluminium TLC plates from Merck. Column chromatography
have to stand more challenging conditions. In our previous was carried out with flash silica gel (0.040–0.063 mm) from
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Molar Chemicals. Visualisation of TLC samples was performed
(E)-2-(4-Aminostyryl)-1,3-benzothiazole (5a). SnCl2 × 2H2O
with a 254/365 nm UV lamp or using aqueous KMnO4 solution (639 mg, 2.832 mmol, 4 eq.) in cc. HCl(aq.) (6 ml) was stirred
(1.5 g KMnO4, 10 g K2CO3 and 1.25 ml 10 m% NaOH in 200 ml for minutes. (E)-2-(4-Nitrostyryl)-1,3-benzothiazole (4a)
5
of water). The aromatic azides (1a–c, 6, 7a, 7c) could be visual- (200 mg, 0.7084 mmol, 1 eq.) was added to it in portions and
ised as a yellow spot after 0.5–1 minute irradiation with a the resulting mixture was stirred overnight at r.t. After all nitro
254 nm UV lamp on a TLC plate. NMR spectra were recorded compounds were consumed (followed by TLC) the reaction
on a Bruker DRX-250 or a Varian VNMRS 600 MHz spectro- mixture was poured onto ice-water (50 ml). The pH was made
meter. Chemical shifts (δ) are given in parts per million (ppm) basic with saturated NaOH solution and the resulting mixture
using solvent signals as the reference. Coupling constants (J) was extracted with CH2Cl2 (3 × 20 ml). The combined organic
are reported in Hertz (Hz). Splitting patterns are designated as phases were washed with brine (1 × 30 ml) and dried over
s (singlet), d (doublet), t (triplet), qr (quadruplet), qv (quintu- MgSO4. After filtering and evaporating the solvent a pale
plet), m (multiplet), dd (doublet of a doublet), td (triplet yellow solid was obtained (131 mg, 73%). Rf = 0.11 (hexanes–
doublet), dt (doublet triplet), br s (broad singlet). The exact EtOAc 5 : 1); mp >178 °C (decompose); IR (neat) νmax/cm−1
masses were determined with an Agilent 6230 time-of-flight 3316 br and 1598 (NH2); δH(250 MHz, CDCl3) 7.96 (1 H, d, J =
mass spectrometer. Samples were introduced by the Agilent 8.1 Hz, Ar-H), 7.83 (1 H, d, J = 7.9 Hz, Ar-H), 7.44–7.38 (4 H, m,
1260 Infinity LC system and the mass spectrometer was oper- 3 × Ar-H, CHvCH), 7.33 (1 H, t, J = 7.5 Hz, Ar-H), 7.21 (1 H, d,
ated in conjunction with a JetStream source in positive/nega- J = 16.3 Hz, CHvCH), 6.68 (2 H, d, J = 8.0 Hz, Ar-H), 3.92
tive ion mode. All melting points were measured on a Bibby (1.6 H, brs, NH2); δC(62.5 MHz, CDCl3) 167.9, 153.9, 147.9,
Scientific SMP10 Melting Point Apparatus and are uncorrected. 138.0, 134.1, 129.0, 126.1, 125.8, 124.9, 122.5, 121.4, 118.2,
IR spectra were obtained on a Bruker IFS55 spectrometer on a 115.0; HRMS (ESI) [M + H]+ calcd for [C15H12N2S + H]+:
single-reflection diamond ATR unit. The fluorescence 253.0794, found: 253.0795.
measurements were carried out on a Varian Cary Eclipse
spectrofluorimeter, the UV-VIS measurements on a Jasco V-660
spectrophotometer.
All reactions involving aromatic azides were carried out in
the dark. CAUTION! Working with azide salts in the presence
of halogenated solvents could lead to the formation of hazar-
dous diazomethane.
After 1 month we have observed a slow decomposition of
1a–c fluorogenic dyes even storing it under an inert atmos-
phere at −20 °C in the dark. Although the amount of the
decomposition product is not significant it is advisable to
purify it before usage by passing through a short plug of silica
using CH2Cl2 as an eluent.
(E)-2-(4-Azidostyryl)-1,3-benzothiazole (1a)
Method A (from 5a). (E)-2-(4-Aminostyryl)-1,3-benzothiazole
(5a) (100 mg, 0.3963 mmol, 1 eq.) was dissolved in a mixture
of 2 ml cc. HCl(aq.), 2 ml water and 1 ml MeOH. To this solu-
tion was added an NaNO2 solution (33 mg, 0.4783 mmol, 1.2
eq. in 200 μl water, +100 μl washing water) at 0 °C, dropwise.
Covering the reaction flask with aluminium foil is necessary from
this point! The temperature was kept at 0 °C, and after
10 minutes an NaN3 solution (31 mg, 0.4768 mmol, 1.2 eq. in
200 μl water, +100 μl washing water) was added dropwise,
slowly. After the addition, the mixture was allowed to warm to
r.t. and stirred for a further 3 h. Next, the reaction mixture was
poured onto ice-water (20–30 ml), extracted with CH2Cl2 (4 ×
15 ml) and washed with brine (1 × 15 ml). After drying over
MgSO4 the drying agent was removed by filtration and the fil-
trate was concentrated in vacuo. The product was purified by
column chromatography (SiO2, hexanes–EtOAc = 10 : 1 → 7 : 1)
to result in a pale yellow solid that was further washed with
hexanes to obtain a 65 mg (59%) product. Rf = 0.62 (hexanes–
EtOAc 5 : 1); mp >170 °C (decompose); IR (neat) νmax/cm−1
2115 (N3); δH(600 MHz, CDCl3) 7.99 (1 H, d, J = 7.7 Hz, Ar-H),
7.86 (1 H, d, J = 8.5 Hz, Ar-H), 7.56 (2 H, d, J = 8.5 Hz, Ar-H),
7.48 (1 H, d, J = 16.3 Hz, CHvCH), 7.47 (1 H, t, J = 8.5 Hz,
Ar-H), 7.37 (1 H, t, J = 7.0 Hz, Ar-H), 7.34 (1 H, d, J = 16.3 Hz,
CHvCH), 7.06 (2 H, d, J = 8.5 Hz, Ar-H); δC(62.5 MHz, CDCl3)
166.7, 153.9, 141.0, 136.3, 134.4, 132.3, 128.8, 126.3, 125.3,
123.0, 121.8, 121.5, 119.5; HRMS (ESI) [M + H]+ calcd for
[C15H10N4S + H]+: 279.0699, found: 279.0695.
Synthesis of dyes
(E)-2-(4-Nitrostyryl)-1,3-benzothiazole
(4a). 2-Methyl-1,3-
benzothiazole (2a) (250 mg, 1.675 mmol, 1 eq.), 4-nitro-benz-
aldehyde (253 mg, 1.674 mmol, 1 eq.) and p-toluenesulfonic
acid monohydrate (350 mg, 1.840 mmol, 1.1 eq.) were mixed
in toluene (2 ml) in a rubber septum sealed vial. The reaction
mixture was stirred at 110 °C for 18 h. Toluene was removed by
evaporation in vacuo from the dark red suspension. Ethanol
(6–8 ml) was added to the mixture and boiled for a few
minutes. The suspension was then allowed to cool to r.t. and
filtered. The product was washed few times with ethanol to
give a yellow powder (408 mg, 86%). Rf = 0.47 (hexanes–EtOAc
5 : 1); mp >218 °C (decompose); IR (neat): νmax/cm−1 1517 and
1340 (NO2); δH(600 MHz, DMSO-d6) 8.28 (2 H, d, J = 8.5 Hz,
Ar-H), 8.14 (1 H, d, J = 7.9 Hz, Ar-H), 8.07 (2 H, d, J = 8.5 Hz,
Method B (from 7a). 1-(4-Azidophenyl)-2-(1,3-benzothiazol-
Ar-H), 8.03 (1 H, d, J = 7.9 Hz, Ar-H), 7.88 (1 H, d, J = 16.1 Hz, 2-yl)ethanol (7a) (263 mg, 0.8875 mmol) was stirred in dry
CHvCH), 7.82 (1 H, d, J = 16.1 Hz, CHvCH), 7.55 (1 H, t, J = toluene (7 ml) in the presence of 3 Å molecular sieves. After
7.6 Hz, Ar-H), 7.48 (1 H, t, J = 7.2 Hz, Ar-H); δC(150 MHz, wrapping the flask with an aluminium foil the reaction
DMSO-d6) 165.5, 153.3, 147.2, 141.8, 134.7, 134.3, 128.6, 126.7, mixture was heated to 70 °C. During warming the reactant was
125.9, 125.8, 123.9, 122.8, 122.3; HRMS (ESI) [M + H]+ calcd for dissolved completely. Trifluoroacetic acid was added (2 drops)
[C15H10N2O2S + H]+: 283.0536, found: 283.0542.
and the reaction mixture was stirred for 16 h. After cooling to
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r.t. it was diluted with CH2Cl2 (8 ml) and the organic phase MeOH. To this solution was added NaNO2 solution (23 mg,
was washed with water (2 ml) and dried over MgSO4. The 0.3333 mmol, 1.2 eq. in 300 μl water, +100 μl washing water) at
mixture was then filtered and the solvent was evaporated. The 0 °C, dropwise. Covering the reaction flask with aluminium foil is
residue was purified by column chromatography (SiO2, necessary from this point! The temperature was kept at 0 °C,
hexanes–EtOAc = 10 : 1 → 5 : 1). The pale yellow residue and after 10 minutes an NaN3 solution (21 mg, 0.3230 mmol,
(44 mg) was transferred onto a filter funnel and washed with 1.2 eq. in 300 μl water, +100 μl washing water) was added drop-
hot hexanes (3–5 ml). A pale yellow powder was obtained wise. After the addition the mixture was allowed to warm to r.t.
(30 mg, 12%).
and stirred for a further 3 h. Next, the reaction mixture was
(E)-6-Methoxy-2-(4-nitrostyryl)-1,3-benzothiazole (4b). 2-Methyl- poured onto ice-water (15–20 ml), extracted with CH2Cl2 (3 ×
6-methoxy-1,3-benzothiazole (2b) (300 mg, 1.674 mmol, 1 eq.), 10 ml) and washed with brine (1 × 10 ml). The organic phase
4-nitro-benzaldehyde (253 mg, 1.674 mmol, 1 eq.) and p-tolue- was dried over MgSO4 and filtered. The solvent was evaporated
nesulfonic acid monohydrate (350 mg, 1.840 mmol, 1.1 eq.) on a rotary evaporator and the residue purified by column
were mixed in toluene (2 ml). After closing with a rubber chromatography (SiO2, CH2Cl2) to give a pale yellow solid that
septum the reaction mixture was heated to 110 °C and was transferred onto a filter funnel and washed with a little
stirred for 18 h. Toluene was evaporated from the dark amount of hexanes (3–5 ml), and dried (54 mg, 64%). Rf = 0.50
yellow suspension on a rotary evaporator. Ethanol (10 ml) was (hexanes–EtOAc 5 : 1); mp >145 °C (melt and decompose); IR
added and boiled for a few minutes, then cooled to r.t. and (neat) νmax/cm−1 2114 (N3), 1266 (Caryl–O–Calkyl); δH(250 MHz,
filtered. The filtered solid was washed with a little amount of CDCl3) 7.86 (1 H, d, J = 8.2 Hz, Ar-H), 7.54 (2 H, d, J = 8.2 Hz,
ethanol to give an orange-yellow powder (129 mg, 25%). Rf = Ar-H), 7.44–7.27 (3 H, m, CHvCH and 2 × Ar-H), 7.12–6.98
0.36 (hexanes–EtOAc 5 : 1); mp >256 °C (decompose); IR (neat) (3 H, m, Ar-H and CHvCH), 3.89 (3 H, s, CH3); δC(62.5 MHz,
νmax/cm−1 1506 and 1334 (NO2), 1218 (Caryl–O–Calkyl); CDCl3) 164.3, 158.0, 148.4, 140.7, 135.8, 135.3, 132.5, 128.6,
δH(600 MHz, DMSO-d6) 8.27 (2 H, d, J = 8.9 Hz, Ar-H), 8.04 123.5, 121.9, 119.5, 115.6, 104.1, 55.8; HRMS (ESI) [M + H]+
(2 H, d, J = 8.5 Hz, Ar-H), 7.91 (1 H, d, J = 8.9 Hz, Ar-H), 7.82 calcd for [C16H12N4OS + H]+: 309.0805, found: 309.0807.
(1 H, d, J = 16.2 Hz, CHvCH), 7.72 (1 H, d, J = 2.7 Hz, Ar-H),
1-(4-Azidophenyl)-2-(1,3-benzothiazol-2-yl)ethanol
(7a).
7.70 (1 H, d, J = 16.2 Hz, CHvCH), 7.14 (1 H, dd, J1 = 8.9 Hz, 2-Methyl-1,3-benzothiazole (2a) (298 mg, 2 mmol, 1 eq.) and
J2 = 2.5 Hz, Ar-H), 3.86 (3 H, s, CH3); δC(150 MHz, DMSO-d6) 4-azido-benzaldehyde (6) (294 mg, 2 mmol, 1 eq.) were dis-
165.8, 157.8, 147.1, 142.0, 136.0, 133.5, 128.4, 126.1, 123.4, solved in anhydrous dimethyl sulfoxide (6 ml). NaOMe
124.0, 119.6, 116.2, 104.7, 55.7; HRMS (ESI) [M + H]+ calcd for (119 mg, 2.2 mmol, 1.1 eq.) was added and through septa the
[C16H12N2O3S + H]+: 313.0641, found: 313.0643.
atmosphere was changed to nitrogen in the reaction flask and
(E)-6-Methoxy-2-(4-aminostyryl)-1,3-benzothiazole
(5b). it was covered with aluminium foil. The stirring was continued
SnCl2 × 2H2O (295 mg, 1.308 mmol, 4 eq.) in cc. HCl(aq.) for 20 h at r.t. (The reactants did not react completely). The
(6 ml) was stirred for 5 minutes. (E)-6-Methoxy-2-(4-nitro- dark reaction mixture was diluted with cold water (40 ml) and
styryl)-1,3-benzothiazole (4b) (103 mg, 0.3298 mmol) was extracted with CH2Cl2 (3 × 50 ml) and the combined organic
added to it in portions and the resulting mixture was stirred layers were washed with water (1 × 20 ml) and dried over
for 1 h at 45 °C. After cooling to r.t. the reaction mixture was MgSO4. After filtration and evaporation the residue was puri-
poured onto ice-water (20 ml) and the pH was made basic fied by column chromatography (SiO2, hexanes–EtOAc = 5 : 1
using saturated NaOH solution. The mixture was extracted → 3 : 1). An orange solid was obtained (273 mg, 46%). The
with CH2Cl2 (4 × 15 ml), and the combined organic phases product was sufficiently pure and was used directly in the next
were washed with brine (1 × 20 ml) and dried over MgSO4. step without further purification. Rf = 0.33 (hexanes–EtOAc
After filtration and removal of the solvent the residue was puri- 3 : 1); δH(250 MHz, CDCl3) 8.01 (1 H, d, J = 8.2 Hz, Ar-H), 7.86
fied on a short column (SiO2, CH2Cl2–MeOH = 10 : 0 → 10 : 1) (1 H, d, J = 8.8 Hz, Ar-H), 7.45 (2 H, d, J = 8.8 Hz, Ar-H), 7.49
to obtain the product as a pale yellow solid (89 mg, 96%). Rf = (1 H, t, J = 7.6 Hz, Ar-H), 7.39 (1 H, t, J = 7.5 Hz, Ar-H), 7.03
0.07 (hexanes–EtOAc 5 : 1); mp >183 °C (decompose); IR (neat) (2 H, d, J = 8.8 Hz, Ar-H), 5.29 (1 H, t, J = 6.0 Hz, CH-OH), 4.44
νmax/cm−1 3316 br and 1622 (NH2), 1218 (Caryl–O–Calkyl); (0.9 H, br s, OH), 3.43 (2 H, d, J = 6.3 Hz, CH2); δC(62.5 MHz,
δH(250 MHz, CDCl3) 7.83 (1 H, d, J = 8.8 Hz, Ar-H), 7.38 (2 H, CDCl3) 168.5, 152.7, 139.5, 134.6, 128.8, 127.3, 126.2, 125.2,
d, J = 8.4 Hz, Ar-H), 7.31–7.25 (2 H, m, CHvCH and Ar-H), 122.7, 121.5, 119.1, 72.1, 42.8.
7.17 (1 H, d, J = 16.1 Hz, CHvCH), 7.04 (1 H, dd, J1 = 9.0 Hz,
J2 = 2.5 Hz, Ar-H), 6.68 (2 H, d, J = 8.5 Hz, Ar-H), 3.89 (1.6 H,
brs, NH2), 3.88 (3 H, s, CH3); δC(62.5 MHz, CDCl3) 165.5, 157.6,
148.5, 147.7, 137.0, 135.5, 128.8, 126.0, 123.0, 118.4, 115.2,
115.1, 104.2, 55.8; HRMS (ESI) [M + H]+ calcd for [C16H14N2OS
+ H]+: 283.0900, found: 283.0906.
(E)-6-Nitro-2-(4-azidostyryl)-1,3-benzothiazole (1c)
Method B (from 7c). 1-(4-Azidophenyl)-2-(6-nitro-1,3-benzo-
thiazol-2-yl)ethanol (7c) (231 mg, 0.6767 mmol) was stirred in
dry toluene (5 ml) in the presence of 3 Å molecular sieves.
After covering with aluminium foil the reaction vessel was
warmed to 70 °C. Meanwhile, the reactant was dissolved com-
pletely. Trifluoroacetic acid was added (3 drops) and the reac-
(E)-6-Methoxy-2-(4-azidostyryl)-1,3-benzothiazole (1b)
Method
A (from 5b). (E)-6-Methoxy-2-(4-aminostyryl)-1,3- tion mixture was stirred for 7 h. During the reaction the
benzothiazole (5b) (77 mg, 0.2727 mmol, 1 eq.) was dissolved product precipitated as an orange powder. After cooling to
in a mixture of 1.5 ml cc. HCl(aq.), 1.5 ml water and 2 ml 3–5 °C (in fridge) the precipitate was filtered and washed with
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hot hexanes (2–3 ml). A yellow powder was obtained (82 mg, 16.4 Hz, CHvCH), 7.54 (1 H, t, J = 8.0 Hz, Ar-H), 7.46 (1 H, t,
37%). Rf = 0.48 (hexanes–EtOAc 3 : 1); mp >178 °C (decom- J = 7.6 Hz, Ar-H), 4.39 (2 H, d, J = 5.9 Hz, CH2), 1.13 (9 H, s,
pose); IR (neat) νmax/cm−1 2133 (N3), 1510 and 1340 (NO2); CH3); δC(150 MHz, DMSO-d6) 177.4, 166.1, 153.4, 146.8, 136.8,
δH(250 MHz, CDCl3) 8.79 (1 H, d, J = 1.9 Hz, Ar-H), 8.35 (1 H, 135.9, 135.2, 134.1, 129.0, 126.5, 125.5, 122.7, 122.5, 122.2,
dd, J1 = 8.8 Hz, J2 = 2.5 Hz, Ar-H), 8.05 (1 H, d, J = 8.8 Hz, 120.5, 120.0, 37.9, 34.5, 27.3; HRMS (ESI) [M + H]+ calcd for
Ar-H), 7.61 (1 H, d, J = 15.8 Hz, CHvCH), 7.60 (2 H, d, J = [C23H23N5OS + H]+: 418.1696, found: 418.1701.
9.5 Hz, Ar-H), 7.35 (1 H, d, J = 16.4 Hz, CHvCH), 7.09 (2 H, d,
Click compound 9b. It was obtained from (E)-6-methoxy-2-
J = 8.8 Hz, Ar-H); δC(62.5 MHz, CDCl3) 172.3, 157.7, 144.9, (4-azidostyryl)-1,3-benzothiazole (1b) (15.0 mg, 0.04864 mmol)
141.9, 139.1, 134.7, 131.5, 129.3, 122.9, 122.0, 120.7, 119.7, and N-(prop-2-ynyl)pivalamide (8) (10.2 mg, 0.0733 mmol).
118.1; HRMS (ESI) [M + H]+ calcd for [C15H9N5O2S + H]+: White-pale yellow fibrous solid (13 mg, 60%). Rf = 0.68
324.0555; found: 324.0557.
(CH2Cl2–MeOH 9 : 1); mp >214 °C (decompose); IR (neat) νmax/
1-(4-Azidophenyl)-2-(6-nitro-1,3-benzothiazol-2-yl)ethanol (7c). cm−1 3331 br (aromatic ring), 1624 and 1536 (CONHR).1224
6-Nitro-2-methyl-1,3-benzothiazole (2c) (300 mg, 1.545 mmol, (Caryl–O–Calkyl); δH(600 MHz, DMSO-d6) 8.58 (1 H, s, triazole-
1 eq.) and 4-azido-benzaldehyde (6) (250 mg, 1.799 mmol, H), 8.07 (1 H, t, J = 5.5 Hz, NH), 7.98 (4 H, s, Ar-H), 7.88 (1 H,
1.2 eq.) were dissolved in anhydrous dimethyl sulfoxide (9 ml). d, J = 9.0 Hz, Ar-H), 7.71–7.65 (2 H, m, CHvCH and Ar-H),
NaOMe (100 mg, 1.851 mmol, 1.2 eq.) was added under a 7.64 (1 H, d, J = 16.2 Hz, CHvCH), 7.12 (1 H, dd, J1 = 9.0 Hz,
nitrogen atmosphere and the flask was covered with alu- J2 = 1.7 Hz, Ar-H), 4.39 (2 H, d, J = 5.5 Hz, CH2), 3.86 (3 H, s,
minium foil. The stirring was continued for 17 h at r.t. The O–CH3), 1.13 (9 H, s, CH3); δC(150 MHz, DMSO-d6) 177.4,
dark reaction mixture was diluted with cold water (90 ml) and 163.5, 157.6, 147.8, 146.7, 136.6, 135.6, 135.4, 134.7, 128.8,
extracted with CH2Cl2 (3 × 60 ml). The combined organic 123.1, 122.9, 120.5, 120.0, 115.8, 104.7, 55.7, 37.9, 34.5, 27.3;
phases were washed with water (1 × 70 ml), brine (1 × 70 ml) HRMS (ESI) [M + H]+ calcd for [C24H25N5O2S + H]+: 448.1802,
and dried over MgSO4. After filtration the solvent was removed found: 448.1802.
in vacuo and the residue was purified by column chromato-
Click compound 9c. It was obtained from (E)-6-nitro-2-(4-
graphy (SiO2, hexanes–EtOAc = 5 : 1 → 2 : 1). An orange-yellow azidostyryl)-1,3-benzothiazole (1c) (15 mg, 0.04639 mmol) and
viscous compound was obtained (231 mg, 44%). The product N-(prop-2-ynyl)pivalamide (8) (9.7 mg, 0.0697 mmol). Yellow
was sufficiently pure and was used directly in the next step powder (14 mg, 65%). Rf = 0.70 (CH2Cl2–MeOH 9 : 1); mp
without further purification. Rf = 0.15 (hexanes–EtOAc 3 : 1); >240 °C (decompose); IR (neat) νmax/cm−1 3293 (aromatic
δH(250 MHz, CDCl3) 8.75 (1 H, d, J = 2.2 Hz, Ar-H), 8.32 (1 H, ring), 1516 and 1336 (NO2), 1628 (CONHR); δH(600 MHz,
dd, J1 = 8.9 Hz, J2 = 2.3 Hz, Ar-H), 8.04 (1 H, d, J = 9.0 Hz, Ar- DMSO-d6) 9.20 (1 H, d, J = 2.3 Hz, Ar-H), 8.60 (1 H, s, triazole-
H), 7.40 (2 H, d, J = 8.4 Hz, Ar-H), 7.00 (2 H, d, J = 8.5 Hz, Ar- H), 8.34 (1 H, dd, J1 = 8.8 Hz, J2 = 2.3 Hz, Ar-H), 8.16 (1 H, d,
H), 5.28 (1 H, t, J = 6.2 Hz, CH-OH), 4.01 (0.7 H, br s, OH), 3.49 J = 9.4 Hz, Ar-H), 8.08 (1 H, t, J = 5.6 Hz, NH), 8.05 (2 H, d, J =
(2 H, d, J = 6.0 Hz, CH2); δC(62.5 MHz, CDCl3) 174.7 and 173.3, 8.8 Hz, Ar-H), 8.02 (2 H, d, J = 8.8 Hz, Ar-H), 7.92 (1 H, d, J =
156.9 and 156.2, 144.8 and 144.6, 139.6 and 139.1, 135.8 and 15.8 Hz, CHvCH), 7.82 (1 H, d, J = 16.4 Hz, CHvCH), 4.39
135.2, 127.15 and 127.13 (CH), 122.7 and 122.4 (CH), 121.5 (2 H, d, J = 5.9 Hz, CH2), 1.13 (9 H, s, CH3); δC(150 MHz,
and 121.4 (CH), 119.1 (CH), 118.0 and 117.8 (CH), 71.8 (CH), DMSO-d6) 177.4, 172.5, 157.2, 146.8, 144.2, 138.2, 137.2, 134.8,
43.5 ppm (CH2). Duplication of peaks is most probably due to 129.5, 122.7, 122.2, 121.9, 120.6, 120.0, 119.3, 37.9, 34.4, 27.3;
the chelate effect between OH and Nv. The orders of the HRMS (ESI) [M + H]+ calcd for [C23H22N6O3S + H]+: 463.1547,
carbons were determined by DEPT135 measurement.
found: 463.1553.
Synthesis of click conjugates
Model reactions with CyO-peptide
General method. The azides (1a–c) (1 eq.) and N-(prop-2-
ynyl)pivalamide18 (8) (1.5 eq.) were stirred in CH2Cl2 (1 ml, The reaction mixture contained 1 × 10−4 M azido-dye (1b, 1c)
stabilized with amylene) at r.t. in the presence of triethylamine and 2 × 10−4 M CyO-peptide in a 2 ml phosphate buffered solu-
(4 μl) and a pinch of Cu(PPh3)2NO3 catalyst10 for 8 h. The tion. For fluorescence measurements aliquots were drawn
resulting suspension was diluted with hexanes (4 ml), filtered from the reaction mixture: a 25 μl reaction mixture and a
and the products were washed with a little amount of CH2Cl2 2475 μl phosphate buffer were mixed (final concentration 1 ×
(1–1.5 ml) and hexanes (5 ml).
10−6 M for the starting amount of dye) and emission was
Click compound 9a. It was obtained from (E)-2-(4-azidos- monitored at emission maxima using 355 nm excitation.
tyryl)-1,3-benzothiazole (1a) (15.0 mg, 0.0538 mmol) and
1b + CyO-pep. 650 μl 3.24 × 10−4 M 1b (DMSO solution),
N-(prop-2-ynyl)pivalamide (8) (11.3 mg, 0.0812 mmol). White- 400 μl 1.00 × 10−3 M CyO-pep and 950 μl phosphate buffer (pH
pale yellow fibrous solid (17 mg, 76%). Rf = 0.73 (CH2Cl2– 7.20) incubated at 37 °C, covered with aluminium foil.
MeOH 9 : 1); mp >258 °C (decompose); IR (neat) νmax/cm−1
The control experiment contained 650 μl 3.24 × 10−4 M 1b
3311 br (aromatic ring) 1627 and 1541 (CONHR); δH(600 MHz, (DMSO solution) and 1350 μl phosphate buffer (pH 7.20) and
DMSO-d6) 8.59 (1 H, s, triazole-H), 8.12 (1 H, d, J = 8.2 Hz, incubated at the same temperature.
Ar-H), 8.07 (1 H, t, J = 5.6 Hz, NH), 8.03–7.98 (5 H, m, 5 ×
Ar-H), 7.76 (1 H, d, J = 16.4 Hz, CHvCH), 7.73 (1 H, d, J = 400 μl 1.00 × 10−3 M CyO-pep and 1103 μl phosphate buffer
1c + CyO-pep. 497 μl 4.02 × 10−4 M 1b (DMSO solution),
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(pH 7.20) were incubated at 37 °C, covered with aluminium such tags. Fast screening of these libraries greatly facilitates
foil.
the selection of fluorogenic tags for certain needs (e.g. exci-
The control experiment contained 497 μl 4.021 × 10−4 M 1b tation and emission wavelengths). Work aiming at the syn-
(DMSO solution) and 1503 μl phosphate buffer (pH 7.20) and thesis of such designed libraries is underway in our laboratory
incubated at the same temperature.
and results will be reported in due course. Further aims are to
fine tune the photophysical properties of such tags and gener-
ate fluorogenic azides that are excitable in the red-far-red, near
infrared region as these regimes are more suitable for biologi-
cal tagging and detection schemes.
Live cell labeling and lysate analysis of cyclooctyne expressing
E. coli
Top 10 E. coli cells containing pEvol PylRSAF and pBAD plas-
mids for expressing GFPY39TAG or GFPWT were grown in the
presence of ampicillin and chloramphenicol in a Terrific broth
medium. GFPY39TAG was grown in the presence (GFPY39TAG+SCO
)
Acknowledgements
and absence of SCO (GFPY39TAG−SCO) and GFPWT was grown
also in the presence of SCO.2a,b Cells were induced with arabi-
nose at OD 0.4 and allowed to grow overnight. After harvesting,
the cells were resuspended in 1× phosphate buffered saline
(PBS, pH 7.4) and the OD was adjusted to ∼1.7. A total volume
of 1.5 ml of this suspension was used in further steps. The
cells were centrifuged and washed 3 times with PBS. Pellets
were afterwards resuspended in 1 ml PBS and incubated with
500 μM compounds 1b, 1c (both 50 mM stock in DMSO) or
50 μM of TAMRA-azide (40 mM stock in DMSO) at 37 °C in the
dark shaking for 4.5 h. After labeling, the cells were centri-
fuged and pellets were washed rigorously with PBS containing
5% DMSO. Subsequently samples were lysed and loaded onto
an SDS PAGE gel for whole cell lysate analysis. The gel was ana-
lysed for fluorescence by exciting the samples in the UV-range
(365 nm) and detecting the emission with an ethidium
bromide filter setting on a commercially available gel docu-
mentation system (Alpha Innotech, CA). Afterwards the gel was
stained with Coomassie.
P. K. acknowledges the support of the Hungarian Academy of
Sciences for the János Bolyai Fellowship (BO/00148/11/7).
Financial support of the Hungarian Scientific Research Fund
(OTKA), Grant No. K100134 is also greatly appreciated. M. K.
acknowledges the financial support by the European Research
Council (ERC) under the European Community’s Seventh
Framework Programme (FP7/2007–2013), ERC Grant Agree-
ment No. 200639. I. N. acknowledges support by an EMBO
Long-Term Fellowship and E. A. L. funding of the DFG Emmy
Noether and SPP1623 program.
Notes and references
1 (a) J. C. Jewett and C. R. Bertozzi, Chem. Soc. Rev., 2010, 39,
1272; (b) C. R. Becer, R. Hoogenboom and U. Schubert,
Angew. Chem., Int. Ed., 2009, 48, 4900; (c) K. V. Reyna and
Q. Lin, Chem. Commun., 2010, 46, 1589; (d) K. E. Beatty,
Mol. BioSyst., 2011, 7, 2360; (e) M. F. Debets, S. S. van
Berkel, J. Dommerholt, A. T. J. Dirks, F. P. J. T. Rutjes and
F. L. van Delft, Acc. Chem. Res., 2011, 44, 805;
(f) B. R. Varga, M. Kállay, K. Hegyi, Sz. Béni and P. Kele,
Chem.–Eur. J., 2012, 18, 822.
Conclusion
In conclusion, we have developed synthetic procedures for the
construction of fluorogenic tags. In addition, by using theoreti-
cal chemical calculations we are able to predict and screen for
potential fluorogenic azides that are worth to be synthesized.
We have selected and synthesized three new fluorogenic and
bioorthogonally applicable dyes, 1a–c. In aqueous media the
azides 1a–c are virtually non-fluorescent. Although “clicked”
triazole derivative 9a exhibits moderately increased emission
intensity, 9b and 9c show intense fluorescence. Photophysical
studies have justified 1b and 1c as remarkably suitable for
bioorthogonal tagging schemes. The performance of these two
fluorogenic tags was tested in the fluorescence tagging of a
peptide sequence by means of a strain-promoted azide–alkyne
cycloaddition reaction. Both tags exhibited excellent perform-
ance. Furthermore, the applicability of these fluorogenic tags
was further demonstrated in in vivo cell labeling experiments.
Analysis of the gel-electrophoresis chromatograms of the cell
lysates revealed that both tags can be used in background fluo-
rescence free bioorthogonal tagging schemes.
2 (a) T. Plass, S. Milles, C. Koehler, C. Schultz and
E. A. Lemke, Angew. Chem., Int. Ed., 2011, 50, 3878;
(b) T. Plass, S. Milles, C. Koehler, J. Szymański, R. Mueller,
M. Wießler, C. Schultz and E. A. Lemke, Angew. Chem., Int.
Ed., 2012, 51, 4166; (c) S. J. Luchansky and C. R. Bertozzi,
ChemBioChem, 2004, 5, 1706; (d) N. J. Agard, J. M. Baskin,
J. A. Prescher, A. Lo and C. R. Bertozzi, ACS Chem. Biol.,
2006, 1, 644; (e) K. E. Beatty, F. Xie, Q. Wang and
D. A. Tirrell, J. Am. Chem. Soc., 2005, 127, 14150;
(f) A. Borrmann, S. Milles, T. Plass, J. Dommerholt,
J. M. M. Verkade, M. Wießler, C. Schultz, J. C. M. van Hest,
F. L. van Delft and E. A. Lemke, ChemBioChem, 2012, 13,
2094; (g) G. Charbon, E. Brustad, K. A. Scott, J. Wang,
A. Løebner-Olesen, P. G. Schultz, C. Jacobs-Wagner and
E.
Chapman,
ChemBioChem,
2011,
12,
1818;
(h) A. R. Katritzky and T. Narindoshvili, Org. Biomol. Chem.,
2009, 7, 627.
3 (a) M. S. T. Gonçalves, Chem. Rev., 2009, 109, 190;
(b) S. Chen, X. Li and H. Ma, ChemBioChem, 2009, 10, 1200;
(c) J. H. Lee, J.-H. So, J. H. Jeon, E. B. Choi, Y.-R. Lee,
The synthetic approach and the theoretical screen of poss-
ible fluorogenic tags will allow the generation of libraries of
This journal is © The Royal Society of Chemistry 2013
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Organic & Biomolecular Chemistry
Y.-t. Chang, C.-H. Kim, M. A. Bae and J. H. Ahn, Chem. 10 (a) C. G. Bates, P. Saejueng, J. M. Murphy and
Commun., 2011, 47, 7500; (d) T. Ehrenschwender,
B. R. Varga, P. Kele and H.-A. Wagenknecht, Chem.–Asian J.,
D. Venkataraman, Org. Lett., 2002, 4, 4727; (b) Zs. Gonda
and Z. Novák, Dalton Trans., 2010, 39, 726.
2010, 5, 1761; (e) M. Sato, Y. Ito, N. Arima, M. Baba, 11 D. F. Eaton, Pure Appl. Chem., 1988, 60, 1107.
M. Sobel, M. Wakao and Y. Suda, J. Biochem., 2009, 146, 33; 12 M. J. Frisch, G. W. Trucks, H. B. Schlegel, G. E. Scuseria,
(f) P. Kele, X. Li, M. Link, K. Nagy, A. Herner, K. Lőrincz,
Sz. Béni and O. S. Wolfbeis, Org. Biomol. Chem., 2009, 7,
3486.
M. A. Robb, J. R. Cheeseman, G. Scalmani, V. Barone,
B. Mennucci, G. A. Petersson, H. Nakatsuji, M. Caricato,
X. Li, H. P. Hratchian, A. F. Izmaylov, J. Bloino, G. Zheng,
J. L. Sonnenberg, M. Hada, M. Ehara, K. Toyota, R. Fukuda,
J. Hasegawa, M. Ishida, T. Nakajima, Y. Honda, O. Kitao,
H. Nakai, T. Vreven, J. A. Montgomery Jr., J. E. Peralta,
F. Ogliaro, M. Bearpark, J. J. Heyd, E. Brothers,
K. N. Kudin, V. N. Staroverov, R. Kobayashi, J. Normand,
K. Raghavachari, A. Rendell, J. C. Burant, S. S. Iyengar,
J. Tomasi, M. Cossi, N. Rega, J. M. Millam, M. Klene,
J. E. Knox, J. B. Cross, V. Bakken, C. Adamo, J. Jaramillo,
R. Gomperts, R. E. Stratmann, O. Yazyev, A. J. Austin,
R. Cammi, C. Pomelli, J. W. Ochterski, R. L. Martin,
K. Morokuma, V. G. Zakrzewski, G. A. Voth, P. Salvador,
J. J. Dannenberg, S. Dapprich, A. D. Daniels, Ö. Farkas,
J. B. Foresman, J. V. Ortiz, J. Cioslowski and D. J. Fox,
GAUSSIAN 09 (Revision B.01), Gaussian, Inc., Wallingford
CT, 2009.
4 (a) A. Nadler and C. Schultz, Angew. Chem., Int. Ed., 2013,
52, 2408–2410; (b) C. Le Droumaguet, C. Wang and
Q. Wang, Chem. Soc. Rev., 2010, 39, 1233; (c) K. Sivakumar,
F. Xie, B. M. Cash, S. Long, H. N. Barnhill and Q. Wang,
Org. Lett., 2004, 6, 4603; (d) J. Qi, M.-S. Han, Y.-C. Chang
and C.-H. Tung, Bioconjugate Chem., 2011, 22, 1758;
(e) G. Lukinavičius and K. Johnsson, Curr. Opin. Chem.
Biol., 2011, 15, 768; (f) J. C. Jewett and C. R. Bertozzi, Org.
Lett., 2011, 13, 5937.
5 P. Shieh, M. J. Hangauer and C. R. Bertozzi, J. Am. Chem.
Soc., 2012, 134, 17428.
6 (a) M. F. Debets, Ch. W. J. van der Doelen, F. P. J. T. Rutjes
and F. L. van Delft, ChemBioChem, 2010, 11, 1168;
(b) J. M. Baskin and C. R. Bertozzi, Aldrichimica. Acta, 2010,
43, 15–23.
7 (a) M. Meldal and C. W. Tornøe, Chem. Rev., 2008, 108, 13 J. P. Perdew, K. Burke and M. Ernzerhof, Phys. Rev. Lett.,
2952; (b) P. V. Chang, J. A. Prescher, E. M. Sletten, 1996, 77, 3865; Erratum: 1997, 78, 1396.
J. M. Baskin, I. A. Miller, N. J. Agard, A. Lo and 14 C. Adamo and V. Barone, J. Chem. Phys., 1999, 110, 6158.
C. R. Bertozzi, Proc. Natl. Acad. Sci. U. S. A., 2010, 107, 1821; 15 S. Miertuš, E. Scrocco and J. Tomasi, Chem. Phys., 1981, 55,
(c) C. I. Schilling, N. Jung, M. Biskup, U. Schepers and
S. Bräse, Chem. Soc. Rev., 2011, 40, 4840.
8 (a) Z. Zhou and C. J. Fahrni, J. Am. Chem. Soc., 2004, 126,
117.
16 R. E. Stratmann, G. E. Scuseria and M. J. Frisch, J. Chem.
Phys., 1998, 109, 8218.
8862; (b) L. Yongjun, L. Ying, K. Xianghe, X. Min and 17 P. Kele, G. Mező, D. Achatz and O. S. Wolfbeis, Angew.
L. Chengbu, Theor. Exp. Chem., 2000, 36, 303. Chem., Int. Ed., 2009, 48, 344.
9 (a) G. B. Cserép, K. N. Enyedi, A. Demeter, G. Mező and 18 N. Willand, M. Desroses, P. Toto, B. Dirie, Z. Lens,
P. Kele, Chem.–Asian J., 2013, 8, 494; (b) K. Nagy, E. Orbán,
Sz. Bősze and P. Kele, Chem.–Asian J., 2010, 5, 773.
V. Villeret, P. Rucktooa, C. Locht, A. Baulard and B. Deprez,
ACS Chem. Biol., 2010, 5, 1007.
3306 | Org. Biomol. Chem., 2013, 11, 3297–3306
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