Synthesis, spectral properties and evaluation of carboxy-functionalized 3-thiazolylcoumarins as blue-emitting fluorescent labeling reagents

Article abstract of DOI:10.1016/j.tetlet.2020.152227

A series of UV-light excitable 7-hydroxy-, methoxy- and acetoxy-substituted [3-(thiazol-2-yl)]coumarin derivatives containing a carboxyalkyl functional group on the thiazole fragment were synthesized for the fluorescent labeling of biomolecules. The spectral properties of these compounds in methanol and aqueous buffers (pH 6.3 and 10.5) were studied. The dyes exhibit a bright blue fluorescence with maxima in the range of 438–457 nm and large Stokes shifts of 72–83 nm (in methanol). Emission maxima of the ionized forms of the 7-hydroxyl derivatives in basic medium are located at 482–484 nm. Thiazolylcoumarins are characterized by high fluorescence quantum yields (up to 0.85). Dye conjugates with L-trileucine and (2′-5′)-triadenylate were obtained via the active ester approach.

Full text of DOI:10.1016/j.tetlet.2020.152227

Journal Pre-proofs
Synthesis, spectral properties and evaluation of carboxy-functionalized 3-thia‐
zolylcoumarins as blue-emitting fluorescent labeling reagents
Iaroslav Kuziv, Larysa Dubey, Igor Dubey
PII:
S0040-4039(20)30694-8
DOI:
Reference:
https://doi.org/10.1016/j.tetlet.2020.152227
TETL 152227
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Tetrahedron Letters
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3 April 2020
1 July 2020
6 July 2020
Please cite this article as: Kuziv, I., Dubey, L., Dubey, I., Synthesis, spectral properties and evaluation of
carboxy-functionalized 3-thiazolylcoumarins as blue-emitting fluorescent labeling reagents, Tetrahedron Letters
(2020), doi: https://doi.org/10.1016/j.tetlet.2020.152227
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Synthesis, spectral properties and evaluation of carboxy-functionalized 3-
thiazolylcoumarins as blue-emitting fluorescent labeling reagents
Iaroslav Kuziv, Larysa Dubey, Igor Dubey*
Institute of Molecular Biology and Genetics, NAS of Ukraine
150 Zabolotnogo str., 03680 Kyiv, Ukraine
* Corresponding author: Tel. +(380)442000377, Fax +(380)445260759,
e-mail: dubey@imbg.org.ua
Abstract
A series of UV-light excitable 7-hydroxy-, methoxy- and acetoxy-substituted [3-(thiazol-2-
yl)]coumarin derivatives containing a carboxyalkyl functional group on the thiazole fragment were
synthesized for the fluorescent labeling of biomolecules. The spectral properties of these
compounds in methanol and aqueous buffers (pH 6.3 and 10.5) were studied. The dyes exhibit a
bright blue fluorescence with maxima in the range of 438-457 nm and large Stokes shifts of 72-83
nm (in methanol). Emission maxima of the ionized forms of the 7-hydroxyl derivatives in basic
medium are located at 482-484 nm. Thiazolylcoumarins are characterized by high fluorescence
quantum yields (up to 0.85). Dye conjugates with L-trileucine and (2'-5')-triadenylate were obtained
via the active ester approach.
Keywords: coumarins, fluorescence, labeling, conjugates
1. Introduction
Fluorescence-based techniques employing appropriate fluorescent probes are some of the
most informative approaches to detect biomolecules and study their cellular transport or interactions
with other biological and synthetic molecules. Fluorescent labeling is a key element of modern
biomedical research and diagnostics allowing the optical detection and quantification of labeled
proteins, nucleic acids, oligonucleotides and other biomolecules has been extensively used to study
biomolecular interactions, cell and tissue imaging, immunofluorescent analysis, microarray
technologies and medical diagnostics [1-3].
There are a wide variety of organic dyes with intense green or red fluorescence. Many of them
have been developed for the excitation with commercially available argon (488 nm) and He-Ne
(633 nm) lasers. However, the range of fluorescent labels emitting in the blue spectral region is
quite limited, and there are relatively few bright fluorophores available for UV-light excitation, e.g.

with a standard violet laser (405 nm). At the same time, blue-emitting dyes are needed for numerous
applications, especially those requiring multi-color sets of fluorophores (nucleic acid sequencing,
microarrays, in situ hybridization, bioimaging) where a blue probe provides a contrasting color that
is easily distinguished from the green, yellow or red fluorescence of the longer-wavelength probes
[2, 4].
Coumarins (2-oxo-2H-benzopyrans) represent one of the most important classes of
fluorescent dyes. In particular, a large number of intense blue fluorophores belong to the coumarin
family [2-7]. A variety of substituents have been introduced into the dye structure, most often
amino, alkylamino, hydroxyl or methoxy groups at C-7, COOH at C-3 or C-4, fluorine, CF3 and
other groups at various positions of the dye, as well as annulated aromatic rings. Recent advances in
the field of coumarin fluorophores include coumarin-based boron complexes [8] and coumarin-
fused heterocycles [9]. Coumarin derivatives are used as laser dyes [10], chemosensors for metal
ions [11-13], anions [12-14] amino acids [13], biothiols [15-17] and other compounds [13, 15, 18],
as well as environment- and pH-sensitive probes [19, 20]. At the same time, coumarin dyes are
among the most important non-radioactive labels in biomedical research [2-4, 6, 21-24].
A broad range of methods is available for the covalent attachment of fluorescent dyes to
biomolecules [2, 21-25]. The labels can be introduced via the reaction of appropriately
functionalized dyes with amino, carboxyl or thiol functional groups present (or introduced) in
biological molecules. The most common approach is the reaction between amines and activated
carboxylic derivatives resulting in the formation of an amide bond. Thus, carboxy-modified dyes
are extensively used for coupling with amine-containing biomolecules (as well as natural and
synthetic polymers or low-molecular compounds). “Non-amide’ approaches to the labeling of
proteins and aminoalkyl-functionalized oligonucleotides are based on the reaction of their amino
groups with pyrylium [26] or dioxaborine [27] dyes which do not require preliminary activation.
Some alternative conjugation approaches were also proposed for coumarins. Azidocoumarin
derivatives and, less frequently, alkyne-functionalized dyes have been used to prepare fluorescent
conjugates via Huisgen 1,3-dipolar cycloaddition [28, 29]. Staudinger ligation of
diphenylphosphino coumarin derivatives with compounds containing an azide group can also be
used in bioconjugation [30]. The thiol group is an important nucleophilic center for selective
labeling, and SH alkylation with reactive dyes such as chloro(bromo)methylcoumarins [29],
maleimide [31] or acrylamide derivatives [32] has been reported. Nevertheless, amide bond
formation remains the main strategy for fluorescent labeling, and carboxy-modified dyes are
perhaps the most commonly used reagents for bioconjugation.
A number of carboxylated blue-emitting coumarins and their active esters (as well as thiol-
specific probes), are commercially available, including a range of reactive dyes based on simple

amino-, hydroxy- and alkoxycoumarins, such as AMCA (7-amino-4-methylcoumarin-3-acetic acid),
and more advanced sulfonylated Alexa Fluor® 350 or fluorinated Pacific BlueTM dyes (Fig. 1).
Figure 1. Commercially available carboxy-functionalized blue-emitting coumarin dyes and their
excitation/emission maxima [2].
A general approach to the modification of coumarins is the introduction of a heteroaromatic
fragment at various positions of the dye. Fluorescent derivatives with heteroaryl (e.g. thienyl,
thiazolyl, benzothiazolyl, oxazolyl) substituents at the C-3 position and amino or hydroxyl groups at
the C-7 position of the coumarin nucleus have attracted attention due to numerous advantages,
including intense fluorescence in the blue spectral region, high chemical and photostability and
broad possibilities for further modification. The presence of the heteroaromatic fragment at the C-3
position of coumarins, and thus extension of the conjugated chromophore system, strongly affects
the optical properties of the dyes [10, 33-35]. This modification causes a bathochromic shift of the
absorption and fluorescence bands and an increase in the absorption and emission intensity.
However, the literature on covalent attachment of such dyes to biomolecules is limited [35-37].
We have previously prepared a number of carboxy-modified furyl, thiazolyl and
phenylthiazolyl coumarin derivatives suitable for introduction into oligonucleotides and other
biomolecules [38, 39]. In order to extend the range of such reagents, herein we report the synthesis
of a series of functionalized 3-thiazolylcoumarins bearing carboxyalkyl groups of various lengths,
designed for dye attachment to amine-containing natural and synthetic molecules. In addition to 3-
heteroaryl fragments, the coumarin core of the reagents contains fluorescence-enhancing
substituents at the C-7 position.

2. Results and Discussion
2.1. Synthesis of the functionalized dyes
Numerous synthetic approaches have been reported for the construction of the coumarin
skeleton containing aliphatic, aromatic or heteroaromatic substituents at the C-3 position [40]. The
most popular methods are based on Knoevenagel [41] and Pechmann [42] condensation using
various solvents and catalysts, and the Suzuki cross-coupling reaction [43]. In particular, 3-(2-
thiazolyl)coumarins with alkyl and aryl substituents at the C-4 position of the thiazole ring were
synthesized by Knoevenagel reaction between o-hydroxybenzaldehydes and 2-
cyanomethylthiazoles under basic catalysis conditions followed by hydrolysis of the obtained 2-
iminocoumarins [34, 44].
To prepare carboxyl functionalized 3-thiazolylcoumarins, we have used the classical
Knoevenagel condensation between salicylic aldehyde and readily available cyanomethyl thiazoles
containing carboxymethyl or carboxypropyl groups. This convenient approach allows the direct
synthesis of COOH-functionalized thiazolylcoumarins, instead of post-synthetic modification of the
thiazolyl fragment of the dye, such as N-acylation by succinic anhydride reported for isomeric 2-
amino-4-(coumarin-3-yl)thiazoles [45]. Two series of reactive dyes were thus synthesized; the
structures are marked with a and b indexes corresponding to compounds containing methylene or
propylene linkers between the dye and a carboxylic functional group, respectively.
As outlined in Scheme 1, the preparation of core dyes 3a and 3b began with the synthesis of
appropriate COOH-functionalized thiazole derivatives. 2-Cyanomethyl-4-carboxyalkyl thiazoles
2a,b were obtained by the condensation of 2-cyanothioacetamide with α-halogenoketones 1a or 1b
in methanol at reflux. It should be noted that the preparation of compound 1b (bromination of
acetylglutarate followed by hydrolysis and decarboxylation with HCl and subsequent esterification)
produces the mixture of chloro- and bromomethylketones; both of these compounds are converted
into 2b during the cyclization reaction.
The construction of the 3-thiazolylcoumarin core was carried out by heating 2a or 2b and 2,4-
dihydroxybenzaldehide in methanol at reflux in the presence of piperidine as a catalyst. The imino
group of the formed 2-iminocoumarin intermediate was hydrolyzed upon addition of concentrated
HCl to form the corresponding coumarin. Then the reaction mixture was basified to pH>10 to
achieve full hydrolysis of the ester group resulting in the formation of carboxyl-functionalized
compounds 3a,b.

Scheme 1. Synthesis of coumarin derivatives. Reagents and conditions: i. NCCH2C(S)NH2, MeOH,
reflux; ii. (a) 2,4-dihydroxybenzaldehyde, piperidine, MeOH, reflux, (b) HCl, reflux, (c) aq. NaOH,
rt; iii. MeOH, H2SO4, reflux; iv. Ac2O, Py, rt; v. Me2SO4, K2CO3, acetone, reflux; vi. (a) Me2SO4,
K2CO3, acetone, reflux, (b) aq. NaOH, rt; vii. NaOH, 1,4-dioxane-water (3:2 v/v), rt.
Core carboxyalkyl-modified thiazolyl coumarins 3a,b containing the hydroxyl group were
used as starting compounds to obtain a series of dye derivatives with other substituents at the C-7
position and free or esterified COOH groups (Scheme 1). Methyl ester derivatives were obtained in
order to examine the effect of ionizable COOH groups on the photophysical characteristics of the
dyes. Thus, the esterification of 3a and 3b in methanol catalyzed by sulfuric acid afforded methyl
esters 4a and 4b. Fully methylated analogs 7a,b were obtained by the reaction of core compounds
3a,b with dimethyl sulfate in acetone in the presence of potassium carbonate. Compound 8a was
prepared by the base-catalyzed methylation of 3a with dimethyl sulfate followed by in situ
hydrolysis of the ester using sodium hydroxide. Coumarin derivative 8b was synthesized via the
sodium hydroxide hydrolysis of ester 7b. Finally, acetylated dyes 5a,b and 6a,b were obtained by
reacting the corresponding 7-hydroxycoumarins (3a,b or 4a,b, respectively) with acetic anhydride
in pyridine. The dyes 3-8 were purified by preparative silica gel column chromatography and/or
recrystallization. The purity of all compounds was over 97% by LC-MS.
2.2. Synthesis of peptide-dye conjugate

The obtained carboxy-functionalized dyes can be used for the introduction of fluorescent
labels with bright blue emission to a variety of amine-containing low- and high-molecular
compounds, including biomolecules and natural and synthetic polymers. To evaluate the efficiency
of the new thiazolylcoumarin derivatives as reagents for biomolecular fluorescent labeling, we have
performed a model conjugation of L-trileucine with hydroxycoumarin derivative 3b using a
commonly utilized active ester approach.
This hydrophobic tripeptide has been covalently attached to transport polymers and cell-
penetrating peptides as a pH-dependent endosomal escape moiety for enhancing the delivery of
therapeutic agents such as antisense oligonucleotides [46], small interfering RNA (siRNA) [47] and
low-molecular drugs [48] to the cell. Dye-labeled peptides could be used to study its mechanism of
biological activity.
Peptide-dye conjugate 9b was synthesized in a high yield via the activation of the carboxylic
functional group of 3b with N,N'-dicyclohexyl carbodiimide (DCC) and N-hydroxysuccinimide
(NHS). Dye activation was performed in anhydrous, amine-free DMF, however, the subsequent
coupling of NHS-activated dye with a terminal amino group of trileucine was complicated due to
the low solubility of this free hydrophobic peptide in common solvents.
The solubility problem of trileucine has been previously solved by including 1.25 eq. of
trifluoroacetic acid and an excess of pyridine in DMF [49]. We have found DMF–acetonitrile–water
mixture (4:1.5:1) containing N,N-diisopropylethylamine (DIPEA, 2 eq.) to be a suitable solvent for
trileucine, and the peptide smoothly reacted with the active ester of the dye in this aqueous-organic
system. The coupling reaction was carried out at room temperature using 1.2 eq. of the activated
dye over the peptide amino component (Scheme 2).
Scheme 2. Fluorescent labeling of L-trileucine. Reagents and conditions: i. DCC, NHS, DMF, rt, 4
h; ii. Leu-Leu-Leu, DIPEA (2 eq.), DMF-CH3CN-H2O 4:1.5:1 v/v, rt, 1 h, 89%.
Coumarin-labeled tripeptide 9b was isolated in 89% yield and the purity of the conjugate was
1
>97% by LC-MS (see Fig. S31). The H NMR spectrum of this compound contains signals of the

dye and trileucine fragment. In particular, the resonances of three amide NH protons in DMSO-d6
are observed at 7.71, 7.96 and 8.03 ppm.
2.3. Preparation of the coumarin-labeled oligonucleotide
To test the efficiency of the new labeling reagents for use with another class of biomolecules,
we have also prepared a coumarin-labeled oligonucleotide, namely, (2'-5')-triadenylate. Short 5'-
phosphorylated 2'-5'-linked oligoadenylates (2-5A) play an essential role in the innate immunity to
viral infections [50]. In contrast, “core” (dephosphorylated) triadenylates demonstrate other types of
activity. In particular, previous studies revealed their ability to bind to a number of biologically
relevant proteins [51, 52] and to modulate protein kinase activity [53]. Fluorescently labeled 2-5As
would be valuable tools in the studies of their cellular functions.
Coumarin-oligonucleotide conjugate 11a was obtained via the active ester approach. Dye 3a
was activated by the reaction with DCC in the presence of 1-hydroxybenzotriazole (HOBT) in DMF
and then coupled to the 5'-amino group of 5'-aminohexyl-functionalized (2'-5')-triadenylate 10
(Scheme 3). The latter was prepared by solution-phase phosphotriester synthesis [54] followed by
functionalization via the classical carbonyl diimidazole approach [55]. The conjugation reaction
was performed in a slightly basic aqueous-organic medium (2:1 mixture of DMF and sodium
bicarbonate buffer, pH 8).
Scheme 3. Synthesis of fluorescently labeled triadenylate: i. DCC, HOBT, DMF, rt, 3 h; ii. 10,
DMF–sodium bicarbonate (100 mM, pH 8) 2:1 v/v, rt, overnight, 42%.
Product 11a was isolated by anion-exchange column chromatography in 42% yield. The
oxybenzotriazole active ester of the carboxy-modified dye was more efficient than the NHS ester; in
the latter case the coupling reaction in the same medium was slower, and the yield of the conjugate
was 15% lower.

The structure and purity of the oligonucleotide-dye conjugate were confirmed by UV-Vis
spectroscopy and LC-MS analysis with electrospray (ES) ionization in both positive and negative
modes. Mass spectra of the conjugate contain peaks corresponding to the protonated or
deprotonated species with one, two or three positive or negative charges (see Fig. S32). To the best
of our knowledge, this is a first example of the synthesis of fluorescently labeled “core” (2'-5')-
oligoadenylate.
2.4. Spectral properties of 3-thiazolylcoumarin derivatives
The spectral-fluorescent characteristics of the obtained heteroarylcoumarins were investigated
in methanol and, for 7-hydroxy derivatives, also in basic and acidic aqueous buffers (pH 10.5 and
6.3) to evaluate the effect of OH ionization on spectral properties (the pKa of 7-hydroxycoumarin is
7.7).
Both the absorption and fluorescence spectra of the new compounds are quite typical for dyes
of this class [10, 33, 34]. Most of them have two absorption maxima in methanol, except for 7-
acetoxy derivatives which have three. In methanol, the short-wavelength band is located at 246-261
nm (ε = 6000-7800 M-1 cm-1), whereas the highly intense long-wavelength band for 7-hydroxy- and
7-methoxy-substituted dyes is observed at 378-382 nm (ε = 24500-30500 M-1 cm-1). An additional
band present in the spectra of 7-acylated dyes (5a,b, 6a,b) is located at 290-292 nm (ε = 4000-4600
M-1 cm-1); their main absorption bands are less intense (ε = 22300-24700 M-1 cm-1) in comparison
with other dyes and blue-shifted to 359-364 nm.
The dyes exhibit bright blue fluorescence, with emission maxima in the range 438-458 nm (in
MeOH). The dyes are excitable in the UV region, and their excitation spectra are close to the
corresponding absorption spectra in both organic and aqueous medium. The dyes demonstrate quite
large Stokes shifts of 72-83 nm in methanol; for 7-hydroxy derivatives 3a,b in acidic aqueous
buffer the shift is smaller (59 nm), but it is as high as 100 nm at pH 6.3.
Bright coumarin dyes are characterized by high fluorescence quantum yields: Φ = 0.67-0.85
in methanol (except for compound 5a with Φ = 0.19), while the yield is somewhat lower (0.55-
0.68) for hydroxy-derivatives 3a,b in both acidic and basic aqueous buffers. Quantum yields were
determined using coumarin-1 and coumarin-314 dyes as standards with Φ = 0.50 and 0.68,
respectively [56]. Optical properties of compounds are presented in Supplementary Table S1.
The effect of the 7-substituent on spectral parameters generally correlates with its electron
donor/acceptor properties. Thus, the presence of 7-acetoxy group leads to hypsochromic shift and
decrease of molar extinction coefficients of absorption bands in methanol in comparison with the 7-
methoxy substituent. A small bathochromic shift (~2 nm) with little hypochromic effect is observed
for 7-hydroxyl derivatives as compared to 7-methoxy analogs, along with the appearance of a small

shoulder in 430-480 nm region. While the spectra of methoxy- and acetoxycoumarins are not
sensitive to pH, both the absorption and fluorescence spectra of compounds 3a,b and 4a,b are pH-
dependent which is typical for 7-hydroxycoumarins [57, 58]. The presence of the above mentioned
long-wavelength shoulder in their UV-Vis spectra in MeOH may be due to some degree of
ionization of the OH-group. With increasing pH the intensity of this shoulder increases, and the
absorption maximum shifts to longer wavelengths.
Deprotonation of the 7-hydroxyl group in alkaline buffer (pH 10.5) causes a large red shift
(41-42 nm for the main absorption band and 21-22 nm for the short-wavelength band for 3a,b) in
comparison with the spectra of the corresponding dyes in methanol, associated with a significant
increase of absorption intensity. In contrast, the absorption spectra of the hydroxyl derivatives in
slightly acidic aqueous solution (pH 6.3) are very close to those in methanol, with ε decrease. The
absorption spectra of representative dyes are shown in Figure 2.
1.00
3a (MeOH)
3a (pH10.5)
5a (MeOH)
8a (MeOH)
0.75
0.50
0.25
0.00
250
300
350
400
450
500
Wavelength (nm)
Figure 2. Normalized UV-Vis absorption spectra of coumarin derivatives in methanol and
phosphate buffer (pH 10.5).
Emission spectra of thiazolylcoumarins also demonstrate the dependence of band position and
intensity on the nature of the functional group at the C-7 position. In normalized fluorescence
spectra, the emission bands of compounds containing 7-acetoxy substituent are shifted in the blue
region by 6-12 nm, whereas those with OH group are red-shifted by ~4 nm in comparison with
methoxy analogs. Methoxy and hydroxyl derivatives have similar fluorescence intensities in
methanol, whereas the intensity is considerably lower for 7-acetoxy-substituted dyes.
Similar to the UV-Vis spectra, the emission maxima of deprotonated 7-hydroxycoumarins in
basic phosphate buffer are significantly red-shifted as compared to the spectra of these dyes in

methanol or in acidic aqueous medium (pH 6.3), and the emission intensity strongly increases
(Table S1); these compounds exhibit greenish fluorescence in alkaline solution. The emission
spectra of selected dyes are presented in Figure 3.
Dyes with a pH-dependent emission and fluorescent biomolecules can be applied in life
sciences as fluorescent indicators of intracellular pH to control the pH changes in various
compartments of the cell using fluorescence microscopy [59].
Figure 3. Fluorescence spectra of compounds in methanol and aqueous phosphate buffers (dye
concentration 10-6 M).
The excitation of 7-hydroxy derivatives in MeOH at the longwave shoulder (437 nm) also
allows the emission spectra of the ionized dyes to be obtained. The ionized forms of 7-
hydroxycoumarins 3a,b and 4a,b demonstrate almost identical normalized fluorescence spectra in
methanol (excitation at 437 nm) and basic aqueous medium (pH 10.5, excitation at the main
absorption band). In both cases, the emission maxima are located at 482-484 nm (Table S1, Fig. S1;
see also Fig. 4).
Chemical or enzymatic (esterase) hydrolysis of 7-acetoxycoumarins leads to the formation of
hydroxy derivatives. In basic medium where the phenolic OH group is ionized this transformation is
associated with a significant increase in fluorescence intensity and a large red shift of the emission
band from 438-447 nm to 482-484 nm. Relative fluorescence intensity increases more than 6-fold
upon the transformation of 5a into 3a at pH 10.5 (Fig. 3). Such spectral changes may be useful in
chemo- and biosensing applications of acetylated dyes (see [36, 60]), although we have not studied
this in detail.

In most cases, the length (and thus the hydrophobicity) of the linker between the dye and a
carboxylic function has little effect on optical properties. The pairs of 7-hydroxyl derivatives 3a and
3b, 4a and 4b have identical normalized absorption spectra, as well as the pair of 7-methoxy
derivatives with a carboxylic group (8a, 8b). Coumarin 7a exhibits a small hypsochromic shift of 1-
2 nm and a slightly lower absorption in the 250-315 nm region in comparison with its analog 7b
containing a longer linker.
Excitation spectra of “methylene” and “propylene” series of the dyes are identical in most
cases. In the case of methyl esters, the emission spectra of “methylene” dyes are blue-shifted as
compared to “propylene” analogs by 1-6 nm. However, the linker has limited effect on the
normalized emission spectra of carboxyl derivatives 3a,b, 5a,b, 8a,b; all of these spectra are
identical to the normalized emission spectra of “propylene” methyl esters 4b, 6b, 7b. At the same
time, acetylated dyes 5a,b with a free COOH group have identical normalized emission spectra, but
the fluorescence intensity of 5a is significantly lower than that for 5b (Table S1). An additional
effect of carboxyl esterification was observed for methyl esters 4a,b demonstrating approximately
two times higher absorption in the long-wavelength shoulder region (430-480 nm) than COOH-
containing analogs 3a,b (Fig. S2). This band corresponds to the absorbance of molecules with an
ionized 7-OH-group, and the effect could be due to the ability of the acidic COOH group to
suppress hydroxyl ionization.
2.5. Spectral properties of coumarin conjugates with biomolecules
Conjugation has not significantly affected the spectral properties of the dye, although it is
associated with some decrease in absorption and fluorescence intensity. Peptide-dye conjugate 9b
and parent dye 3b have almost identical normalized UV-Vis spectra in methanol or in phosphate
buffer (pH 10.5); however, the extinction coefficients for both short- and long-wavelength bands of
the labeled trileucine are about 20% lower than those for the free 3b. Both excitation and emission
spectra of the conjugate are quite similar to those for 3b; the band positions remain the same, but
the relative fluorescence intensity of the conjugated dye decreases by 15-20% (Table S1).
Just as in the case of free parent dye 3b, excitation of the conjugate in MeOH at the long-
wavelength shoulder at 437 nm results in the shift of emission band from 457 to 482 nm, and the
spectrum becomes similar to that in basic aqueous buffer (Fig. 4).
The UV-Vis spectrum of oligonucleotide conjugate 11a clearly confirms the presence of
oligonucleotide and dye fragments. The spectrum contains two intense bands, with λmax = 259 and
432 nm in basic phosphate buffer (Fig. 5). The first one corresponds mainly to the strong absorption
of the trinucleotide moiety, with some contribution (about 10%) of the short-wavelength absorption
of the attached dye; for (2'-5')A3, λmax = 259 nm, ε259 = 37.6×103 M-1cm-1 [54], and for 3a ε259 = 4.2×103 at

pH=10.5. The long-wavelength absorption band belongs exclusively to the dye fragment of 11a.
After the conjugation, the long-wavelength absorption band of the dye is red-shifted by 8 nm. The
observed UV-to-visible absorption ratio A259/A432 = 1.19 is in good agreement with the theoretical
value of 1.10 indicating the presence of one dye residue in the labeled product and confirming the
efficient removal of the unreacted dye. The fluorescence spectrum of the conjugate (Emmax = 485
nm) is similar (in both shape and position of the emission band) to that of the parent dye 3a (Fig. 5),
although the fluorescence quantum yield of the conjugate decreases by 7%, in comparison to the
free dye (Table S1).
Figure 4. Normalized absorption, excitation and emission spectra of coumarin-peptide conjugate 9b
in methanol (A) and phosphate buffer, pH 10.5 (B). Em – emission spectrum obtained with
excitation at maximum of the main absorption band, Ex* – excitation spectrum obtained for λem
483 nm, Em* – emission spectrum with excitation at 437 nm.
=

Figure 5. Normalized absorption and emission spectra of the dye 3a and dye-labeled
oligonucleotide 11a in phosphate buffer (pH 10.5).
3. Conclusion
We have synthesized and characterized a series of new fluorescent labeling reagents with
intense blue emission based on a UV-excitable 3-thiazolylcoumarin core modified with
carboxyalkyl groups of various lengths. The dyes demonstrate excellent photophysical properties,
such as relatively high molar extinction coefficients, high fluorescence quantum yields and large
Stokes shifts. The fluorescence spectra of the dyes contain intense bands of 7-substituted
heteroarylcoumarin chromophores with emission maxima observed in the range of 438-484 nm,
depending on the dye structure, solvent and excitation wavelength. These reactive dyes are versatile
reagents for the conjugation with amine-containing biomolecules, polymers and low-molecular
compounds via the formation of amide bonds. The efficiency of newly developed reagents was
confirmed by the synthesis of a fluorescently labeled peptide and oligonucleotide.
Supplementary data
Supplementary data associated with this article include synthetic protocols, photophysical
data, 1H and 13C NMR spectra of compounds and LC-MS of the conjugates.
References
1. Lavis LD, Raines RT. ACS Chem. Biol. 2014; 9: 855-866.
2. The Molecular Probes® Handbook: A Guide to Fluorescent Probes and Labeling
Technologies. 11th ed.; Spence MTZ, Johnson ID, Eds.; Life Technologies: Carlsbad, CA,
2010.

3. Stockert JC, Blázquez-Castro A. Fluorescence Microscopy in Life Sciences. Bentham Science,
2017
4. Gonçalves MST. In Advanced Fluorescence Reporters in Chemistry and Biology I;
Demchenko AP, Ed.; Springer: Berlin-Heidelberg, 2010; pp 27-64.
5. Sun W-C, Gee KR, Haugland RP. Bioorg Med Chem Lett. 1998; 8: 3107-3110.
6. Panchuk-Voloshina N, Haugland RP, Bishopn-Stewart J, Bhalgat MK, Millard PJ, Mao F,
Leung W-Y, Haugland RP. J Histochem Cytochem. 1999; 47: 1179-1188.
7. Matta A, Bahadur V, Taniike T, Van der Eycken J, Singh BK. Dyes Pigments. 2017; 140: 250-
260.
8. 8 Zhang P, Liu W, Niu G, Xiao H, Wang M, Ge J, Wu J, Zhang H, Li Y, Wang P. J Org Chem.
2017; 82: 3456-3462.
9. Soares AMS, Hungerford G, Costa SPG, Gonçalves MST. Dyes Pigments. 2017; 137: 91-100.
10. Anufrik SS, Tarkovskii VV, Nikitchenko VM. J Appl Spectr. 1999; 66: 772-779.
11. Song Y, Chen Z, Li H. Curr Org Chem. 2012; 16: 2690-2707.
12. Roy D, Chakraborty A, Ghosh R. Spectrochim Acta A. 2018; 191: 69-78.
13. Li H, Cai L, Chen Z. In Advances in Chemical Sensors; Wang W, Ed.; InTech: Rijeka, Croatia,
2012; pp 121-150.
14. Yanar U, Babür B, Pekyılmaz D, Yahaya I, Aydıner B, Dede Y, Seferoğlu Z. J Mol Struct.
2016; 1108: 269-277.
15. Babür B, Seferoğlu N, Öcal M, Sonugur G, Akbulut H, Seferoğlu Z. Tetrahedron. 2016; 72:
4498-4502.
16. Renault K, Renard P-Y, Sabot C. Eur J Org Chem. 2018: 6494-6498.
17. Zhang H, Wang B, Ye Y, Chen W, Song X. Org Biomol Chem. 2019; 17: 9631-9635.
18. Liu Y, Ren D, Zhang J, Li H, Yang X-F. Dyes Pigments. 2019; 162: 112-119.
19. Pajk S. Tetrahedron Lett. 2014; 55: 6044-6047.
20. Shiraishi T, Saito T, Kagechika H, Hirano T. Tetrahedron Lett. 2014; 55: 6784-6786.
21. Hermanson GT. Bioconjugate Techniques. 2nd ed.; Academic Press: Amsterdam-Boston-
Heidelberg, 2008.
22. Gonçalves MST. Chem. Rev. 2009; 109: 190-212.
23. Gong Y, Pan L. Tetrahedron Lett. 2015; 56: 2123-2132.
24. Cui L, Rao J. In The Chemistry of Molecular Imaging; Long N, Wong W-T, Eds.; John Wiley
& Sons: Hoboken, NJ, 2015; pp. 25-53.
25. Lavis LD. Biochemistry. 2017; 56: 5165-5170.
26. Yarmoluk SM, Kostenko AM, Dubey IY. Bioorg Med Chem Lett. 2000; 10: 2201-2204.

27. Gerasov A, Shandura M, Kovtun Y, Losytskyy M, Negrutska V, Dubey I. Anal Biochem. 2012;
420: 115-120.
28. Sivakumar K, Xie F, Cash BM, Long S, Barnhill HN, Wang Q. Org. Lett. 2004; 6: 4603-4606.
29. Schmid K, Adobes-Vidal M, Helm M. Bioconjug Chem. 2017; 28: 1123-1134.
30. Wodtke R, König J, Pigorsch A, Köckerling M, Mamat C. Dyes Pigments. 2015; 113: 263-273.
31. Song HY, Ngai MH, Song ZY, MacAry PA, Hobley J, Lear MJ. Org Biomol Chem. 2009; 7:
3400-3406.
32. Liao Y-C, Venkatesan P, Wei L-F, Wu S-P. Sensors Actuators B. 2016; 232: 732-737.
33. Takechi H, Oda Y, Nishizono N, Oda K, Mashida M. Chem Pharm Bull. 2000; 48: 1702-1710.
34. Deligeorgiev T, Tsvetkova T, Ivanova D, Timtcheva I. Color Technol. 2008; 124: 195-203.
35. Roubinet B, Chevalier A, Renard PY, Romieu A. Eur J Org Chem. 2015: 166-182.
36. Bentsen JG, Mickelson CA, Knudson OB, Lewandowski KM. U.S. Patent 6566508, 2003.
37. Jansen TP, Rodeghiero G, Foglietta D, Perciaccante R, Della Ciana L. Eur. Patent Application
3219712, 2017.
38. Kuziv IB, Ishchenko VV, Khilya VP, Dubey IY. Ukr Bioorg Acta. 2008; 6(1): 3-12.
39. Kuziv IB, Ishchenko VV, Khilya VP, Dubey IY. Ukr Bioorg Acta. 2009; 7(2): 47-54.
40. Medina FG, Marrero JG, Macías-Alonso M, González MC, Córdova-Guerrero I, Teissier
García AG, Osegueda-Robles S. Nat Prod Rep. 2015; 32: 1472-1507.
41. Vekariya RH, Patel HD. Synth Commun. 2014; 44: 2756-2788.
42. Zambare AS, Kalam Khan FA, Zambare SP, Shinde SD, Sangshetti JN. Curr Org Chem. 2016;
20: 798-828.
43. Zen AA, Aylott JW, Chan WC. Tetrahedron Lett. 2014; 55: 5521-5524.
44. Khilya OV, Shablykina OV, Frasinyuk MS, Turov AV, Ishchenko VV, Khilya VP. Chem
Heterocycl Compds. 2004; 40: 1408-1420.
45. Zhuravel IO, Kovalenko SM, Vlasov SV, Chernykh VP. Molecules. 2005; 10: 444-456.
46. Ding H, Inoue S, Ljubimov AV, Patil R, Portilla-Arias J, Hu J, Konda B, Wawrowsky KA,
Fujita M, Karabalin N, Sasaki T, Black KL, Holler E, Ljubimova JY. Proc Natl Acad Sci USA.
2010; 107: 18143-18148.
47. Ullah I, Chung K, Beloor J, Kim J, Cho M, Kim N, Lee KY, Kumar P, Lee SK. J Drug Target.
2017; 25: 320-329.
48. Patil R, Portilla-Arias J, Ding H, Inoue S, Konda B, Hu J, Wawrowsky KA, Shin PK, Black
KL, Holler E, Ljubimova JY. Pharm. Res. 2010; 27: 2317-2329.
49. Mitin YV. Int J Pept Protein Res. 1996; 48: 374-376.
50. Gusho E, Baskar D, Banerjee S. Cytokine. 2016. DOI: 10.1016/j.cyto.2016.08.009.

51. Levchenko SM, Rebriev AV, Tkachuk VV, Dubey LV, Dubey IY, Tkachuk ZY. Biopolym
Cell. 2013; 29: 42-48.
52. Levchenko SM, Rebriev AV, Tkachuk VV, Tkachuk LV, Dubey LV, Dubey IY, Yashchuk
VM, Tkachuk ZY. Mol Cryst Liq Cryst. 2014; 590: 213-220.
53. Skorobogatov OY, Kukharenko AP, Kozlov OV, Dubey IY, Tkachuk ZY. J Proteomics
Bioinform. 2017; 10: 320-323.
54. Dubey IY, Dubey LV. Biopolym Cell. 2007; 23: 538-544.
55. Wachter L, Jablonski J, Ramachandran KL. Nucleic Acids Res. 1986; 14: 7985-7994.
56. Reynolds GA, Drexhage KH. Opt Commun. 1975; 13: 222-225.
57. Fink DW, Koehler WR. Anal Chem. 1970; 42: 990-993.
58. Nizomov N, Kholov AU, Ishchenko AA, Ishchenko VV, Khilya VP. J Appl Spectr. 2007; 74:
626-634.
59. Han J, Burgess K. Chem. Rev. 2010; 110: 2709-2728.
60. Zhu R, Wang S, Su X, Liu J. Tetrahedron Lett. 2018; 59: 4376-4379.

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A series of new reagents for biomolecular fluorescent labeling were synthesized
The reagents are based on carboxyalkyl-modified 3-(thiazol-2-yl)coumarin
The dyes are UV-light excitable and exhibit a bright blue fluorescence
Coumarin-labeled L-trileucine and (2'-5')-triadenylate were obtained

Declaration of interests
☐ The authors declare that they have no known competing financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered as
potential competing interests:
Declaration of interests: none