Synthesis and Optical Properties of the New Acetylene Kaede Chromophore Analog

Article abstract of DOI:10.1134/S1068162020030231

Abstract: A novel derivative of acetylene Kaede protein chromophore (Z)-2-((4-(diethylamino) phenyl) ethynyl)-5-(4-hydroxybenzylidene)-3-methyl-3,5-dihydro-4H-imidazol-4-one was synthesized by a modified Sonogashira reaction. In comparison with the classi

Full text of DOI:10.1134/S1068162020030231

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2020, Vol. 46, No. 3, pp. 458–461. © Pleiades Publishing, Ltd., 2020.
Russian Text © The Author(s), 2020, published in Bioorganicheskaya Khimiya, 2020, Vol. 46, No. 3, pp. 332–336.
LETTER TO THE EDITOR
Synthesis and Optical Properties of the New Acetylene Kaede
Chromophore Analog
a, b
a
a, 1
a, c
E. R. Zaitseva , A. Yu. Smirnov , A. S. Mishin , and M. S. Baranov
a
Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
b
Mendeleev University of Chemical Technology of Russia, Moscow, 125047 Russia
Pirogov Russian National Research Medical University, Moscow 117997 Russia
c
Received November 22, 2019; revised November 29, 2019; accepted December 26, 2019
Abstract—A novel derivative of acetylene Kaede protein chromophore (Z)-2-((4-(diethylamino) phenyl)
ethynyl)-5-(4-hydroxybenzylidene)-3-methyl-3,5-dihydro-4H-imidazol-4-one was synthesized by a modi-
fied Sonogashira reaction. In comparison with the classical GFP chromophore analog, the compound exhib-
its a prominent shift of absorption and emission maxima to the long-wavelength region. The value of Stock’s
shift of the newly synthesized compound reached almost 150 nm.
Keywords: imidazolones, chromophores, fluorescent dyes, GFP, Kaede, Sonogashira reaction, optical prop-
erties
DOI: 10.1134/S1068162020030231
9
] in biomedical studies. However, for the entire time
The key to modern approaches to the study of pro-
cesses occurring in living cells is visualization methods
associated with the emission of light. The most
important component of these methods is the intro-
duction of a luminescent or fluorescent label into the
object under study. Among the latter, it is worth noting
separately the numerous derivatives of the chromo-
phore of green fluorescent proteins (GFP) containing
that the Kaede protein chromophore analogs have
been studied, their acetylene derivatives have not been
obtained. In the present work, we first developed an
approach to the synthesis of this class of compounds
and, using one example, examined their availability
and optical properties.
As the target compound, we selected the substance
benzylideneimidazolone in their composition [1]. This (V) (Fig. 1). One of the most common methods for the
class of compounds has great variability in optical prop- synthesis of acetylenes is the Sonogashira reaction, its
erties, significant Stokes shift, good solubility in water, use involves the terminal alkyne and aryl or hetaryl
high permeability through the cytoplasmic membrane, halide in the reaction. 2-Halogenimidazolones are not
low toxicity, and synthetic availability [2–4].
described in the literature, however, there are exam-
ples of the Sonogashira reaction involving alkylthio
derivatives in which palladium complexes catalyzed by
the replacement of the sulfur-containing group with
acetylene [10, 11]. There are also several examples of
the modification of thiohydantoins [12] and 2-alkylth-
ioimidazolones [13] as a result of the Libskind-Srogl
reaction. Therefore, for the synthesis of the target
compound, we chose a modified method for the
preparation of acetylenes by the Sonogashira reaction
with desulfurization of the corresponding thiohydan-
toin derivative (Scheme 1).
Biological objects are characterized by high
absorption of shortwave radiation (which often dis-
rupts the ongoing biological processes) and low
absorption of longwave radiation. Therefore, when
conducting biomedical research, it is preferable to use
labels with maximum absorption and emission of flu-
orescence in the long wavelength part of the visible
spectrum. One of the main synthetic strategies for
achieving a bathochromic shift is to increase the size of
the conjugate π-systems of the chromophore mole-
cule. An example of this approach in the case of ben-
zimidazolones is the Kaede protein chromophore
analogs containing a styrene substituent in the second
position of the imidazolone cycle [5]. These com-
pounds are already used as fluorescent [6, 7] or fluo-
rogenic (substances that increase the quantum yield of
fluorescence upon binding to the target object) tags [8,
Compound (IV) was synthesized using a rather rare
modification of the Sonogashira reaction using a
derivative containing an SCH group [14]. The catalyst
3
used was copper (I) iodide and Pd (dppf) Cl CH Cl .
2
2
2
In the beginning, as a result of the condensation of
4
4
-hydroxybenzaldehyde with 2-thiooxoimidazolin-
-one the compound was obtained (I). Since the
1
Corresponding author: phone: +7 (495) 995-55-57 (2074); alkylation of thiooxoimidazoline could also occur with
e-mail: mishin@ibch.ru.
the participation of the hydroxy group of the ben-
58
4

SYNTHESIS AND OPTICAL PROPERTIES
459
400
450
500
500
600
700
800
Wavelength, nm
Fig. 1. The absorption and emission spectra of the compound (V) in acetonitrile.
zylidene moiety, the next step was its protection with a trofluorometer (United States). Melting points were
triisopropylsilyl group. The resulting compound (II) was determined on an SMP 30 device (Great Britain) and
methylated with methyl iodide to give the product (III). were not corrected. High-resolution mass spectra were
After this, the Sonogashira reaction was carried out, as recorded on a Bruker micrOTOF II instrument, elec-
a result of which the compound (IV) with an acetylene trospray ionization.
fragment in the structure. To remove the triisopropyl-
silyl protection compound (IV) was treated with tetra-
butylammonium fluoride. As a result, we obtained a
new compound (V) with a good exit.
(
Z)-5-(4-Hydroxybenzylidene)-2-thiooxoimidazolin-
4
-one (I). 4-Hydroxybenzaldehyde (793 mg, 6.5 mmol),
2
-thiooxoimidazolin-4-one (580 mg, 5.0 mmol) and
sodium acetate (1.76 g, 21.5 mmol) were dissolved in
Next, the optical properties of the new compound (V) 15 mL of glacial acetic acid, the resulting mixture was
in acetonitrile (Fig. 1). It was found that the emission boiled for 3 h and cooled to room temperature. With
of fluorescence of compound (V) is small, which is vigorous stirring, 50 mL of water was added, the pre-
consistent with previously obtained data for other cipitate formed filtered out and washed with water
Kaede protein chromophore analogs [6]. The maxi- (20 mL) and diethyl ether (10 mL), and then dried in
mum absorption (457 nm) and emission (approxi- vacuum. Yellow powder (935 mg, 85%), mp 280°C
1
mately 605 nm) of the compound (V) are shifted to a
much longer wavelength region compared with the
classical chromophore GFP (Z)-5-(4-hydroxyben-
zylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazole-
with decomposition; H NMR: 6.43 (s, 1 H), 6.81 (d,
J 8.6, 2 H), 7.63 (d, J 8.6, 2 H), 10.02 (broad s., 1 H),
2
1
2
1.95 (broad s., 1 H), 12.22 (broad s., 1 H).
(
Z)-2-Thioxo-5-(4-((triisopropylsilyl)oxy)benzylidene)
4
4
-one (absorption and emission maxima 368 nm and
38 nm, respectively) [8]. Also noteworthy is the fact
imidazolidin-4-one (II). (Z)-5-(4-Hydroxyben-
zylidene)-2-thiooxoimidazolin-4-one (I, 1.10 g,
that the Stokes shift of the new acetylene derivative is
almost 150 nm, which makes the dye obtained prom-
ising for multicolor labeling of biological samples.
5
.0 mmol), triisopropylsilyl chloride (1.06 g,
.5 mmol), DIPEA (0.78 g, 6.0 mmol) and imidazole
5
(
20 mg) were dissolved in THF (50 mL), and then
Thus, we were the first to develop an approach to
the synthesis of acetylene analogs of the Kaede protein
chromophore using compound (V). The absorption
and emission maxima of this compound lie in the lon-
ger wavelength region than that of the analogous GFP
chromophore due to the presence of a conjugated
acetylene system. This fact, as well as the low fluores-
cence of this compound, suggests that the new deriva-
tive (V) or its analogs may turn out to be promising flu-
orogenic dyes.
stirred overnight. The resulting solution was evapo-
rated, the residue was dissolved in ethyl acetate
(
200 mL), washed with saturated sodium chloride
solution (3 × 50 mL), dried over anhydrous Na SO
2
4
and evaporated. The product was purified by flash chro-
matography (eluent—chloroform). Yellow powder
1
(
1.48 g, 79%), mp 275°C with decomposition; H NMR:
1
.08 (d, J 7.4, 18 H), 1.25–1.31 (sept., J 7.4, 3 H),
2
2
6
.45 (s, 1 H), 6.89 (d, J 8.6, 2 H), 7.68 (d, J 8.6, 2 H),
2
2
1
3
1
2.03 (broad s., 1 H), 12.28 (broad s., 1 H); C NMR:
1.9, 17.5, 111.6, 119.8, 125.4, 126.1, 132.0, 156.5,
165.6, 178.6; HRMS (ESI), m/z: found M, 377.1711;
1
EXPERIMENTAL
+
+
calculated for C H N O SSi , [M + H] 377.1714.
NMR spectra (δ ppm, J Hz) are registered on the
19 29th
2
2
Bruker Avance III device (700 MHz, United States) in
(Z)-3-Methyl-2-(methylthio)-5-(4-((triisopropylsi-
DMSO-d (internal standard—Me Si). UV and visible lyl)oxy)benzylidene)-3,5-dihydro-4H-imidazol-4-one
6
4
absorption spectra were recorded on a Varian Cary 100 (III). (Z)-2-Thiooxo-5-(4-((triisopropylsilyl)oxy)-ben-
spectrophotometer Bio (United States). Fluorescence zylidene)imidazolidin-4-one (II, 1.13 g, 3.0 mmol),
spectra were recorded on a Varian Cary Eclipse spec- iodomethane (1.70 g, 12.0 mmol) and potash (1.66 g,
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 46 No. 3 2020

4
60
ZAITSEVA et al.
1
2.0 mmol) were mixed in acetonitrile (30 mL) and inated (V) and used in the next stage without complete
1
boiled for 8 h. The reaction mixture was cooled, fil- purification. H NMR: 1.08 (d, J 7.4, 18 H), 1.13
2
tered, the solution was evaporated three quarters and
ethyl acetate (150 mL) was added. The resulting solu-
tion was washed, saturated sodium chloride solution
(
sept., J 7.1, 6 H), 1.30 (t, J 7.6, 1 H), 3.22 (s, 3 H),
2 2
.42 (q, J 7.1, 4 H), 6.75 (d, J 9.0, 2 H), 6.97 (d, J
3
8
8
2 2
2
2
.8, 2 H), 7.02 (s, 1 H), 7.52 (d, J 9.0, 2 H), 8.16 (d, J
2
(
3 × 30 mL), dried over anhydrous Na SO and evap-
2 4
.6, 2 H); HRMS (ESI), m/z: found M, 530.3198; cal-
orated. The product was purified by flash chromatog-
+
+
culated for C H N O Si , [M + H] 530.3197.
3
2
44
3
2
raphy (eluent—chloroform). Yellow powder (820 mg,
1
6
1
9%), mp 160–163°C; H NMR: 1.07 (d, J 7.3,
2
(Z)-2-((4-(Diethylamino)phenyl)ethynyl)-5-(4-
hydroxybenzylidene)-3-methyl-3,5-dihydro-4H-imid-
8 H), 1.27 (sept., J 7.3, 3 H), 2.72 (s, 3 H), 3.08 (s, 3
2
H), 6.83 (s, 1 H), 6.94 (d, J 8.7, 2 H), 8.16 (d, J 8.8, 2 azol-4-one (V). (Z)-2-((4-(Diethylamino)phenyl)-
2
13
H); C NMR: 12.1, 12.5, 17.7, 26.3, 120.0, 122.1, ethynyl)-3-methyl-5-(4-((triisopropylsilyl)oxy)ben-
26.5, 133.8, 136.7, 157.1, 161.7, 170.9; HRMS (ESI), zylidene)-3,5-dihydro-4H-imidazol-4-one (IV, 159 mg,
1
m/z: found M, 405.2022; calculated for 0.3 mmol) was dissolved in THF (4 mL), tetrabutylam-
+
+
C H N O SSi , [M + H] 405.2027.
monium trihydrate fluoride (474 mg, 1.5 mmol) was
added, after which the resulting solution was stirred for
2
1
33
2
2
(
Z)-2-((4-(Diethylamino)phenyl)ethynyl)-3-methyl-
1
h. Ethyl acetate (100 mL) was added and washed
5
4
-(4-((triisopropylsilyl)oxy) benzylidene)-3,5-dihydro-
with saturated sodium chloride solution (10 × 15 mL).
The organic phase was dried over anhydrous Na SO
H-imidazol-4-one (IV). (Z)-3-Methyl-2-(meth-
2
4
ylthio)-5-(4-((triisopropylsilyl)oxy)benzylidene)-3,5-
dihydro-4H-imidazol-4-one (III, 406 mg, 1 mmol),
copper(I)iodide (190 mg, 1 mmol) and a complex
and evaporated. The product was purified by flash
chromatography (eluent—a mixture of chloroform
and ethanol, 50 : 1). Dark red powder (64 mg, 57%),
(
1,1'-bis(diphenylphosphino)ferrocene of palla-
1
dium(II)chloride with dichloromethane (41 mg, mp 188–190°C; H NMR: 1.12 (t, J
7.0, 6 H,
2
0
.05 mmol). The flask was evacuated, filled with NCH CH ), 3.21 (s, 3 H, NCH ), 3.41 (sq. J 6.9, 4 H,
2 3 3 2
argon, DIPEA (2.58 g, 20 mmol) and N,N-diethyl-4- NCH CH ), 6.73 (d, J 9.0, 2 H, H5', H7'), 6.87 (d, J
2
3
2
2
ethynylaniline (340 mg, 2 mmol) were added in a
stream of argon. The reaction mixture was kept at a
temperature of 100°C for 12 hours, then ethyl acetate
8
.8, 2 H, H3'', H5''), 6.99 (s, 1 H, H7''), 7.51 (d, J 8.8,
2
2
H, H4', H8'), 8.10 (d, J 8.6, 2 H, H2'', H6 ''), 10.24
2
13
(
(
br s, 1 H, OH); C-NMR: 12.3 (NCH CH ), 27.0
2 3
(
100 mL) was added and washed with saturated
NCH ), 43.8 (NCH CH ), 78.4 (C1'), 102.6 (C2'),
3
2
3
sodium chloride solution (3 × 30 mL). The organic
phase was dried over anhydrous Na SO and evapo-
1
03.6 (C3'), 111.3 (C5', C7'), 115.9 (C3'', C5''), 125.4
C7''), 127.4 (C1''), 134.2 (C2'', C6''), 134.6 (C4', C8'),
36.4 (C2), 146.2 (C5), 149.1 (C6'), 160.1 (C4''), 168.5
C4); HRMS (ESI), m/z: found M, 374.1864; calcu-
2
4
(
rated. The product was purified by flash chromatogra-
phy (eluent was a 1 : 5 mixture of ethyl acetate and
hexane). Dark powder (227 mg, 43%), mp 166–
1
(
+
2
+
1
68°C; the product was partially (up to 20%) contam- lated for C H N O , [M + H] 374.1863.
2
3
24
3
OH
OH
OTIPS
OTIPS
H
N
MeI,
TIPSCI,
DIPEA
NaOAc
K2CO3
S
+
AcOH
Δ
Imidazole,
THF
CH CN,
NH
3
H
N
H
N
O
Δ
N
O
S
S
SMe
NH
I)
NH
(II)
N
O
O
O
(
(III)
OTIPS
OH
4''
5''
3''
2''
NEt2
TBAF · 3H2O
THF
6''
PdCl2 · dppf · CH2Cl2,
1''
7''
8'
7'
1
5
CuI, DIPEA, Δ
N
N
NEt2
1'
2'
NEt2
6'
2
3'
4
4'
5'
N
N3
O
O
(IV)
(V)
Scheme 1. Compound synthesis scheme (V).
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 46
No. 3
2020

SYNTHESIS AND OPTICAL PROPERTIES
461
FUNDING
5. Yampolsky, I.V., Kislukhin, A.A., Amatov, T.T.,
Shcherbo, D., Potapov, V.K., Lukyanov, S., and Luk-
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pp. 96–104.
The study was supported by the Russian Science Foun-
dation as part of research project No. 16-14-10364.
6
. Baleeva, N.S., Myannik, K.A., Yampolsky, I.V., and
Baranov, M.S., Eur. J. Org. Chem., 2015, vol. 2015,
pp. 5716–5721.
COMPLIANCE WITH ETHICAL STANDARDS
This article does not contain any research involving
humans and animals as research objects.
7. Ermakova, Y.G., Sen, T., Bogdanova, Y.A., Smirnov, A.Y.,
Baleeva, N.S., Krylov, A.I., and Baranov, M.S., J.
Phys. Chem. Lett., 2018, vol. 9, pp. 1958–1963.
8
. Povarova, N.V., Zaitseva, S.O., Baleeva, N.S., Smir-
nov, A.Y., Myasnyanko, I.N., Zagudaylova, M.B.,
Bozhanova, N.G., Gorbachev, D.A., Malyshevskaya, K.K.,
Gavrikov, A.S., Mishin, A.S., and Baranov, M.S.,
Chemistry, 2019, vol. 25, pp. 9592–9596.
Conflict of Interests
The authors declare they have no conflict of interest.
9
. Jung, K.H., Fares, M., Grainger, L.S., Wolstenholme,
C.H., Hou, A., Liu, Y., and Zhang, X.A., Org. Biomol.
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