Synthesis of methylsulfanyl analogs of Kaede protein chromophore

Article abstract of DOI:10.1007/s10593-020-02673-w

[Figure not available: see fulltext.] 5-Arylidene-1-methyl-2-[2-(methylsulfanyl)-2-phenylethenyl]-1H-imidazol-5(4H)-ones were synthesized as a result of the reaction of 5-arylidene-1-methyl-2-(methylsulfanyl)-1H-imidazol-5(4H)-ones with terminal acetylene

Full text of DOI:10.1007/s10593-020-02673-w

DOI 10.1007/s10593-020-02673-w
Chemistry of Heterocyclic Compounds 2020, 56(3), 399–402
SHORT COMMUNICATION
Synthesis of methylsulfanyl analogs of Kaede protein chromophore
Elvira R. Zaitseva1,2, Alexander Yu. Smirnov2*, Sofya I. Shcerbinina2,
Vlada V. Zasedateleva2, Konstantin S. Mineev2, Mikhail S. Baranov2,3
1 D. Mendeleyev University of Chemical Technology of Russia,
9 Miusskaya Sq., Moscow 125047, Russia
2 Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences,
16/10 Miklukho-Maklaya St., Moscow 117997, Russia; е-mail: alexmsu@yandex.ru
3 Pirogov Russian National Research Medical University,
1 Ostrovitianova St., Moscow 117997, Russia
Translated from Khimiya Geterotsiklicheskikh Soedinenii,
2020, 56(3), 399–402
Submitted January 2, 2020
Accepted February 18, 2020
5-Arylidene-1-methyl-2-[2-(methylsulfanyl)-2-phenylethenyl]-1H-imidazol-5(4H)-ones were synthesized as a result of the reaction of
5-arylidene-1-methyl-2-(methylsulfanyl)-1H-imidazol-5(4H)-ones with terminal acetylenes in the presence of a palladium catalyst and
copper iodide. This reaction presents a rare example of the formation of a compound with a methylsulfanylethenyl group. A study of the
optical properties of the obtained compounds showed that they are characterized by a significant bathochromic shift of spectral maxima
in comparison with similar derivatives of the GFP chromophore.
Keywords: acetylenes, imidazolones, Kaede protein, chromophores, fluorogens, GFP chromophore.
One of the most important modern methods for studying
intracellular processes is fluorescence microscopy based on
the use of fluorescent dyes. Among them, benzylidene-
imidazolones, analogs of the chromophore of green
fluorescent protein (GFP), occupy an important place.1
These compounds are widely used due to the great
variability of their optical properties, low toxicity, and
synthetic availability.2,3 One of the subclasses of this group
of dyes is the Kaede protein chromophore analogs, which
are characterized by a significant bathochromic shift of
spectral maxima compared to the original derivatives of the
GFP chromophore.4,5
It is known that aryl alkyl sulfides can undergo various
cross-coupling reactions catalyzed by transition metal
complexes.6 However, very few examples of such reactions
with terminal acetylenes are described in the literature.
Moreover, these transformations lead to the formation of
both substituted acetylenes as a result of the Sonogashira
reaction7,8 and products containing a carbon–sulfur bond.9,10
5-Arylidene-2-alkylsulfanylimidazolones 2 are readily
available stable reagents.11 They are structurally similar to
the GFP chromophore and were previously used in the
synthesis of its derivatives by arylation12 using metal
complex catalysis. The aim of this work was to study the
reactions of 5-arylidene-2-methylsulfanylimidazolones 2
with terminal acetylenes.
As a result of our experiments, it turned out that the
reaction of 5-arylidene-2-methylsulfanylimidazolones 2,
obtained from 5-arylidene-2-thiooxoimidazolidinones 1,
with acetylenes resulted in the formation of methylsulfanyl
compounds 3 (Scheme 1). Moreover, in all cases, no
appreciable amounts of acetylene derivative were found in
the reaction mixture. Thus, the reaction we have proposed
is not only a novel example of the synthesis of the Kaede
protein chromophore derivatives, but also a rare example of
the creation of a methylsulfanylethenyl group in a
molecule. Moreover, we have shown that not only terminal
arylacetylenes but also alkylacetylenes can be used in this
0009-3122/20/56(3)-0399©2020 Springer Science+Business Media, LLC
399

Chemistry of Heterocyclic Compounds 2020, 56(3), 399–402
Scheme 1
Scheme 2
Ar
reaction. We found that the transformation proceeds best in
the presence of the Pd(dppf)Cl2 catalyst, as well as CuI. We
used DIPEA as the base, and the reaction was carried out
without solvent. In total, we synthesized 5 new compounds.
The structures of the obtained compounds 3, as well as
the configuration of double bonds, were additionally
confirmed by the 1H–13C HSQC, 1H–13C HMBC, and
1H–15N HMBC two-dimensional NMR spectroscopy
experiments on compound 3c as an example (Fig. 1). In
some cases, the products contained a small inseparable
amount of the E-isomer (relative to the multiple bond at the
sulfur atom) impurity, but its amount rarely exceeded 10%.
In the case of compound 3c, this impurity was also
characterized by NMR.
Ar
Cu
R
Ln
Pd
SMe
N
N
SMe
N
O
N
O
Me
Me
HI
H
LnPd0
Ar
CuI
R
Ln
Pd
N
Ar
MeS
R
R
N
N
O
Me
SMe
N
O
Me
Our studies showed that the revealed transformation
does not take place either in the absence of a palladium
catalyst or in the absence of CuI. This suggests that the
mechanism of this transformation is similar to the classical
Sonogashira reaction. However, we did not find the desired
arylacetylenes and methanethiol in the reaction products
(the reaction was carried out both in a sealed vessel and in
a stream of argon, which was supposed to remove this
gaseous product). This fact suggests that the methyl-
sulfanyl group does not add to the prepared acetylene
according to the nucleophilic mechanism, but is transferred
in the catalytic cycle (Scheme 2), as was previously stated.9
We studied the optical properties of the new compounds
3a–e, and also performed a comparison with similar
derivatives of the GFP chromophore 4a–e (Table 1). The
quantum fluorescence yields of all the studied compounds
turned out to be small (less than 0.2%), which is typical of
emission bands in the spectra of the new compounds 3a–e,
compared with compounds 4a–e, as well as an increase in
their Stokes shift was noted. The positions of the
absorption and emission maxima also depended on the
nature of the introduced substituents: the presence of a
more donating group in the benzylidene fragment shifted
the spectral bands to the long-wavelength region. And in
the emission and absorption spectra of compound 3d (as
compared with compound 3c), a distinct shift of the bands
to the shorter wavelength region, as well as a decrease in
the Stokes shift could be observed.
To conclude, we have developed a new method for the
synthesis of methylsulfanyl analogs of the Kaede protein
chromophore as a result of the study. Five new compounds
Table 1. Optical properties of compounds 3a–e, 4a–c,e in MeCN
such derivatives and facilitates their use as fluorogens.4,5
A
significant bathochromic shift of both the absorption and
Compound Absorption maximum, nm
Emission maximum, nm
368
486
425
418
425
368
354
425
368
368
461
591
541
466
540
461
428
488
435
438
3a
3b
3c
3d
3e
3a
4a
4b
4c
4e
Figure 1. Major correlations and chemical shifts (shifts of 1H are
indicated in red, shifts of 13C – in blue, shifts of 15N – in green;
1
1
1
δ, ppm) in H–13C HSQC, H–13C HMBC, and H–15N HMBC
spectra of compound 3c.
400

Chemistry of Heterocyclic Compounds 2020, 56(3), 399–402
were obtained, the optical properties of which suggest the
3CH(CH3)2); 1.27 (3H, sept, J = 7.3, 3CH(CH3)2); 2.72
(3H, s, NCH3); 3.08 (3H, s, SCH3); 6.83 (1H, s, CH=); 6.94
(2H, d, J = 8.7, H Ar); 8.16 (2H, d, J = 8.8, H Ar).
13C NMR spectrum, δ, ppm: 12.1; 12.5; 17.7; 26.3; 120.0;
122.1; 126.5; 133.8; 136.7; 157.1; 161.7; 170.9. Found,
m/z: 405.2022 [M+H]+. C21H33N2O2SSi. Calculated, m/z:
405.2027.
promise of their use as novel dyes. The presence in the
composition of all new compounds of the methylsulfanyl
group opens up possibilities for even further modification.
Experimental
IR spectra were registered on a Thermo Scientific
Nicolet iS10 spectrometer with the Smart iTR attachment.
1H and 13C NMR spectra were acquired on a Bruker
Avance III spectrometer (700 and 176 MHz, respectively)
in DMSO-d6, with TMS or the residual solvent signal
Synthesis of compounds 3a–e (General method).
Compound 1a–c,e (1 mmol), alkyne (2 mmol), CuI (190 mg,
1 mmol), Pd(dppf)Cl2·CH2Cl2 (41 mg, 0.05 mmol), and
DIPEA (2.58 g, 20 mmol) were mixed under an argon
atmosphere. The mixture was heated at 100°C in an oil
bath for 12 h, cooled, and filtered. EtOAc (200 ml) was
added to the filtrate, and the mixture was washed with
saturated aqueous NaCl (3×50 ml). The organic layer was
evaporated under reduced pressure. In the case of
compound 2e, the intermediate silylated product without
additional purification was dissolved in THF (10 ml),
Bu4NF·3H2O (630 mg, 2 mmol) was added, and the mixture
was stirred for 3 h. EtOAc (100 ml) was then added, and the
mixture was washed with saturated aqueous NaCl (3×30 ml).
The organic layer was dried over Na2SO4 and evaporated
under reduced pressure. The product was in all cases
isolated by column chromatography, eluent CHCl3 (for
compounds 2a–d) or CHCl3–EtOH, 20:1 (for compound 2e).
(5Z)-5-Benzylidene-3-methyl-2-[(Z)-2-(methylsulfanyl)-
2-phenylethenyl]-3,5-dihydro-4H-imidazol-4-one (3a).
Content of E-isomer less than 5%. Yield 184 mg (55%),
yellow powder, mp 158–160°C. IR spectrum, ν, cm–1:
3388, 3065, 2997, 2913, 2432, 2161, 2027, 1978, 1698,
1594, 1495, 1429, 1385, 1314, 1269, 1166, 1032, 961, 753,
1
(2.50 ppm for Н nuclei and 39.5 ppm for 13С nuclei) as
internal standard. High-resolution mass spectra were recorded
on a Bruker micrOTOF II, electrospray ionization. Melting
points were determined on an SMP 30 apparatus and are
uncorrected. All operations with moisture-sensitive
substances were carried out in an atmosphere of dry argon
using the standard Schlenk technique.
Acros Organics reagents were used without additional
purification; freshly distilled solvents were used for the
reactions. Compounds 1b,13 1e,14 1, 2 a,c,15 4a,b,16 4c,e17
were synthesized according to literature methods.
(5Z)-5-[4-(Diethylamino)benzylidene]-3-methyl-
2-(methylsulfanyl)-3,5-dihydro-4H-imidazol-4-one (2b).
Compound 1b (825 mg, 3.0 mmol) was dissolved in MeCN
(30 ml), MeI (1.70 g, 12.0 mmol) and K2CO3 (1.66 g,
12.0 mmol) were added, and the resulting mixture was
heated under reflux for 8 h. After cooled, EtOAc (200 ml)
was added, and the mixture was washed with saturated
aqueous NaCl (3×50 ml). The organic layer was evaporated
under reduced pressure, and the product was isolated by
column chromatography, eluent CHCl3. Yield 645 mg
1
688, 584. H NMR spectrum, δ, ppm (J, Hz): 2.45 (3H, s,
1
(71%), red powder, mp 163–165°C. H NMR spectrum,
SCH3); 2.76 (3H, s, NCH3); 7.02 (1H, s, SC=CH); 7.22
(2H, d, J = 7.4, H Ar); 7.25 (1H, s, ArCH=); 7.26 (1H, t,
J = 7.2, H Ar); 7.32 (2H, t, J = 7.5, H Ar); 7.42–7.52 (3H,
m, H Ar); 8.22 (2H, d, J = 6.9, H Ar). 13C NMR spectrum,
δ, ppm: 14.9; 26.5; 125.8; 127.4; 128.1; 128.3; 128.6; 128.7;
130.5; 130.6; 132.3; 133.6; 134.9; 138.1; 160.7; 169.3. Found,
m/z: 335.1208 [M+H]+. C20H19N2OS. Calculated, m/z:
335.1213.
δ, ppm (J, Hz): 1.12 (6H, t, J = 7.1, 2CH2CH3); 2.69 (3H,
s, NCH3); 3.06 (3H, s, SCH3); 3.42 (4H, q, J = 7.1,
2CH2CH3); 6.72 (2H, d, J = 9.2, H Ar); 6.76 (1H, s, CH=);
8.05 (2H, d, J = 8.4, H Ar). 13C NMR spectrum, δ, ppm:
12.4; 12.5; 26.2; 43.8; 111.1; 120.9; 124.3; 133.9; 134.0;
148.7; 160.9; 168.9. Found, m/z: 304.1474 [M+H]+.
C16H22N3OS. Calculated, m/z: 304.1478.
(Z)-3-Methyl-2-(methylsulfanyl)-5-{4-[(triisopropyl-
silyl)oxy]benzylidene}-3,5-dihydro-4H-imidazol-4-one
(2e). Compound 1e (3.0 mmol) was dissolved in THF (50 ml),
then triisopropylsilyl chloride (640 mg, 3.3 mmol), DIPEA
(490 mg, 3.6 mmol), and imidazole (10 mg) were added.
The formed solution was stirred for 24 h and evaporated
under reduced pressure. EtOAc (200 ml) was added, and
the solution was washed with saturated aqueous NaCl
(3×50 ml). The organic layer was dried over Na2SO4 and
evaporated under reduced pressure. Without purification,
the residue was dissolved in MeCN (30 ml), MeI (1.70 g,
12.0 mmol) and K2CO3 (1.66 g, 12.0 mmol) were added,
and the resulting mixture was heated under reflux for 8 h.
After cooled, EtOAc (200 ml) was added, and the mixture
was washed with saturated aqueous NaCl (3×50 ml). The
organic layer was evaporated under reduced pressure, and the
product was isolated by column chromatography, eluent
CHCl3. Yield 820 mg (69%), yellow powder, mp 160–163°C.
1H NMR spectrum, δ, ppm (J, Hz): 1.07 (18H, d, J = 7.3,
(5Z)-5-[4-(Diethylamino)benzylidene]-3-methyl-2-[(Z)-
2-(methylsulfanyl)-2-phenylethenyl]-3,5-dihydro-4H-imid-
azol-4-one (3b). Content of E-isomer less than 3%. Yield
278 mg (69%), red powder, mp 174–176°C. IR spectrum,
ν, cm–1: 3066, 2966, 2949, 2433, 2161, 2028, 1977, 1636,
1575, 1428, 1318, 1266, 1189, 1079, 1015, 985, 768, 687,
1
586. H NMR spectrum, δ, ppm (J, Hz): 1.14 (6H, t,
J = 7.0, 2CH2CH3); 2.01 (3H, s, SCH3); 3.14 (3H, s,
NCH3); 3.44 (4H, q, J = 7.0, 2CH2CH3); 6.39 (1H, s,
SC=CH); 6.74 (2H, d, J = 9.0, H Ar); 6.95 (1H, s, ArCH=);
7.41–7.48 (3H, m, H Ar); 7.48–7.53 (2H, m, H Ar); 8.21
(2H, br. s, H Ar). 13C NMR spectrum, δ, ppm: 12.4; 16.7;
25.9; 43.6; 110.6; 111.2; 121.3; 126.4; 128.2; 128.4; 128.5;
134.2; 134.8; 138.8; 148.8; 154.7; 155.2; 169.0. Found,
m/z: 406.1943 [M+H]+. C24H28N3OS. Calculated, m/z:
406.1948.
(5Z)-5-(4-Methoxybenzylidene)-3-methyl-2-[2-(methyl-
sulfanyl)-2-phenylethenyl]-3,5-dihydro-4H-imidazol-4-one
(3c). Yield 164 mg (45%), yellow powder, mp 158–160°C.
401

Chemistry of Heterocyclic Compounds 2020, 56(3), 399–402
IR spectrum, ν, cm–1: 3057, 2997, 2932, 2834, 2161, 2028,
134.2; 136.8; 138.7; 155.9; 156.5; 157.4; 159.6; 169.3.
Found, m/z: 351.1154 [M+H]+. C20H19N2O2S. Calculated,
m/z: 351.1162.
1698, 1595, 1506, 1430, 1362, 1110, 1074, 990, 809, 707,
663. Found, m/z: 365.1318 [M+H]+. C21H21N2O2S.
Calculated, m/z: 365.1318.
1
Z-Isomer (34%). H NMR spectrum, δ, ppm (J, Hz):
The study was carried out with the financial support of
the Russian Foundation for Basic Research within the
framework of the scientific project No. 20-33-70266.
2.02 (3H, s, SCH3); 3.15 (3H, s, NCH3); 3.84 (3H, s,
OCH3); 6.41 (1H, s, SC=CH); 7.04 (1H, s, ArCH=); 7.05
(2H, d, J = 8.8, H Ar); 7.44 (2H, d, J = 7.1, H Ar); 7.45–
7.52 (3H, m, H Ar); 8.37 (2H, d, J = 8.6, H Ar). 13C NMR
spectrum, δ, ppm: 17.0; 26.1; 55.4; 110.2; 113.7; 114.4;
124.7; 127.3; 128.3; 128.7; 133.9; 137.6; 138.7; 157.3;
158.1; 160.8; 169.4.
References
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1
E-Isomer (11%). H NMR spectrum, δ, ppm (J, Hz):
2.57 (3H, s, SCH3); 3.19 (3H, s, NCH3); 3.80 (3H, s,
OCH3); 6.11 (1H, s, SC=CH); 6.74 (1H, s, ArCH=); 6.76
(2H, d, J = 8.6, H Ar); 7.35 (2H, d, J = 7.2, H Ar); 7.35–
7.54 (5H, m, H Ar). 13C NMR spectrum, δ, ppm: 15.8;
26.1; 55.2; 105.2; 113.7; 123.8; 127.2; 128.0; 128.1; 128.5;
133.8; 137.5; 138.4; 156.5; 157.1; 160.6; 169.3.
(5Z)-5-(4-Methoxybenzylidene)-3-methyl-2-[(Z)-3-
methyl-2-(methylsulfanyl)but-1-en-1-yl]-3,5-dihydro-4H-
imidazol-4-one (3d). Content of E-isomer 6%. Yield 207 mg
(63%), yellow powder, mp 141–143°C. IR spectrum, ν, cm–1:
2917, 2849, 2433, 2160, 2089, 1978, 1698, 1594, 1511,
1446, 1384, 1308, 1253, 1168, 1032, 959, 850, 731, 687,
1
586. H NMR spectrum, δ, ppm (J, Hz): 1.01 (3H, t,
J = 7.3, CH2CH2CH3); 1.61–1.66 (2H, m, CH2CH2CH3);
2.48 (3H, s, SCH3); 2.63–2.67 (2H, m, CH2CH2CH3); 3.12
(3H, s, NCH3); 3.83 (3H, s, OCH3); 6.34 (1H, s, SC=CH);
6.93 (1H, s, ArCH=); 7.02 (2H, d, J = 9.0, H Ar); 8.30 (2H,
d, J = 8.6, H Ar). 13C NMR spectrum, δ, ppm: 13.2; 14.2;
22.6; 25.9; 38.2; 55.2; 114.1; 123.2; 127.3; 133.3; 133.5;
137.7; 157.4; 160.1; 160.4; 169.4. Found, m/z: 331.1475
[M+H]+. C18H23N2O2S. Calculated, m/z: 331.1475.
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(5Z)-5-(4-Hydroxybenzylidene)-3-methyl-2-[(Z)-
2-(methylsulfanyl)-2-phenylethenyl]-3,5-dihydro-4H-
imidazol-4-one (3e). Content of E-isomers less than 3%.
Yield 168 mg (48%), orange powder, mp 160–162°C.
IR spectrum, ν, cm–1: 3152, 2919, 2849, 2601, 2160, 2027,
1674, 1594, 1429, 1362, 1276, 1141, 1028, 934, 762, 695,
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1
596. H NMR spectrum, δ, ppm (J, Hz): 2.01 (3H, s,
SCH3); 3.14 (3H, s, NCH3); 6.41 (1H, s, SC=CH); 6.86
(2H, d, J = 9.0, H Ar); 6.99 (1H, s, ArCH=); 7.42–7.48
(3H, m, H Ar); 7.51 (2H, d, J = 7.4, H Ar); 8.25 (2H, d,
J = 8.2, H Ar); 10.11 (1H, s, OH). 13C NMR spectrum, δ, ppm:
16.9; 26.0; 110.3; 115.8; 125.3; 125.8; 128.1; 128.2; 128.6;
16. Zaitseva, S. O.; Golodukhina, S. V; Baleeva, N. S.; Levina, E. A.;
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402