Synthesis of the New Green Fluorescent Protein Chromophore Analogue Starting from a Cinnamic Aldehyde Derivative

Article abstract of DOI:10.1134/S1068162019030075

Abstract: A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphenyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was prepared by the synthetic approach based on the use of car

Full text of DOI:10.1134/S1068162019030075

ISSN 1068-1620, Russian Journal of Bioorganic Chemistry, 2019, Vol. 45, No. 3, pp. 214–216. © Pleiades Publishing, Ltd., 2019.
Russian Text © The Author(s), 2019, published in Bioorganicheskaya Khimiya, 2019, Vol. 45, No. 3, pp. 333–336.
LETTER TO EDITOR
Synthesis of the New Green Fluorescent Protein Chromophore
Analogue Starting from a Cinnamic Aldehyde Derivative
S. O. Zaitsevaa, 1 and M. S. Baranova
aShemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, Moscow, 117997 Russia
Received November 30, 2018; revised December 4, 2018; accepted December 19, 2018
Abstract—A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphe-
nyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was
prepared by the synthetic approach based on the use of carboxyimidate. Comparison of optical properties of
the obtained compound and of the classical GFP chromophore analogue (Z)-5-(4-hydroxybenzylidene)-
2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one showed the shift of absorption and emission maxima to the
long-wavelength region induced by the increase of the conjugated π-system in the benzylidene fragment.
Keywords: imidazolones, chromophores, fluorescent dyes, GFP, optical properties
DOI: 10.1134/S1068162019030075
The shift is often known to result from the exten-
sion of the conjugated π-system in the dye molecule.
As for various derivatives of the GFP chromophore
(5-(4-hydroxybenzylidene)-3,5-dihydro-4H-imidazol-
4-one) (Scheme 1), such a modification is most often
introduced at position 2 of the imidazolone cycle. In
particular, introduction of a styrene group at position 2
leads to Kaede protein derivatives (Scheme 1) exhibit-
ing notable bathochromic shifts [6]. These com-
pounds find application as dyes and can be used as
sensitizing dyes in solar cells [7].
Synthetic analogues of fluorescent protein chro-
mophores—that is, various benzylidene imidazolo-
nes—have been a subject of careful researchers’ attention
for almost four decades. Intense coloration, small size,
high solubility, and easily tuned spectral characteristics
allow successful application of these compounds in
chemical and biological studies [1]. As fluorescent [2]
and fluorogenic [3] dyes, such molecules can be used to
visualize cell structures, label various objects, or
develop ion- and pH-responsive sensors [4, 5].
The main characteristic determining the possibility
of application of a chromophore analogue as a dye is
the position of its absorption and emission maxima.
Since the absorption region of most biological objects
resides in the short-wavelength region of the spec-
trum, design of compounds demonstrating a signifi-
cant bathochromic shift is a relevant task.
In the work, we propose an alternative way to
increase the system of conjugated bonds in a GFP
chromophore derivative to shift the positions of
absorption and emission maxima, i.e., through modi-
fication of the benzylidene fragment (Scheme 1).
OH
OH
OH
4''
OTBDMS
3''
2''
5''
6''
OTBDMS
OTBDMS
1''
1) MeNH2, CHCl3
DIBALH
PDC
CHCl3
3'
2'
Ar
toluene
−80°C
N
N
NCH2COOEt
2)
N
N
N1
5
OMe
1'
O
4
O
2
CH2OH
(II)
O
3N
(IV)
COOEt
(I)
TBAF
O
Kaede
GFP
(III)
Scheme 1. Synthesis of compound (IV).
Abbreviations: DIBALH, diisobutylaluminum hydride; GFP, green fluorescent protein; PDC, pyridinium dichromate; TBAF, tetra-
butylammonium fluoride; TBDMS, tert-butyldimethylsilyl.
1 Corresponding author: phone: +7 (499) 724-81-22; fax: +7 (495) 135-14-05; e-mail: snezhana_zaitseva@mail.ru.
214

SYNTHESIS OF THE NEW GREEN FLUORESCENT PROTEIN CHROMOPHORE
215
300 350 400 450 500 550
500
550
600
650
700
Wavelength, nm
Fig. 1. Normalized absorption (left) and emission (right) spectra of the GFP chromophore (black) and compound (IV) (grey)
solutions in water at pH 10.
Synthesis of compound (IV) was carried out using a bathochromic shift of the absorption and emission
a cinnamic aldehyde derivative (Scheme 1), since one maxima.
of the most universal methods to synthesize ben-
Therefore, we obtained a new analogue of the GFP
zylidene imidazolones is interaction of aldehyde-
chromophore from a cinnamic aldehyde derivative.
derived Schiff-bases with carboximidate [8]. The
Maxima of absorption and emission of the compound
required aldehyde (III) was obtained with fair yield
lie in the longer-wavelength region of the spectrum,
from a commercially available silylated ethylphenylac-
compared to the GFP chromophore analogue, due to
rylate (I) by reduction and further oxidation (Scheme 1).
extended conjugated system of π-bonds in the ben-
Surprisingly, formation of the Schiff base was not
zylidene fragment.
hampered by side reactions of the α,β-unsaturated
aldehyde. Due to the presence of conjugated double
bonds, the final compound (IV) was obtained as a
mixture of E- and Z-isomers of each of the two double
bonds. However, it could be easily transformed into an
individual isomer—E-isomer at the 3' position and
Z-isomer at the 1' position of the allylidene moiety—
in the course of slow recrystallization from methanol,
which is supported by the NMR data: the spin–spin
interaction constant between two hydrogen atoms at
the 2'–3' bond is 11.4 Hz, which corresponds to the
trans configuration, while position of the signal at the
1' atom corresponds to the typical position of Z-iso-
mers of other GFP chromophore derivatives, in which
this configuration is the most stable [9].
EXPERIMENTAL
NMR spectra (δ, ppm, J, Hz) were registered on a
Bruker Avance III (700 MHz, United States) equip-
ment in DMSO-d6 and CDCl3 (Me4Si was used as
internal standard). UV-visible absorption spectra were
recorded on a Varian Cary 100 Bio (United States)
spectrophotometer. Fluorescence spectra were regis-
tered on a Varian Cary Eclipse (United States) fluo-
rescence spectrophotometer. Melting points were
determined on an SMP-30 (Great Britain) instrument
and are reported uncorrected.
(E)-3-(4-((tert-Butyldimethylsilyl)oxy)phenyl)prop-
2-en-1-ol (II). A solution of 2.97 g (10 mmol) of ethyl-
(E)-3-(4-(((tert-butyldimethylsilyl)oxy)phenyl)acry-
late) (I) [10] in 60 mL of toluene was cooled to –80°C.
Diisobutylaluminum hydride (DIBALH) (10.6 mL,
Study of optical properties of compound (IV) (Fig. 1;
Table 1), as well as comparison with properties of
GFP, showed that introduction of a larger conjugated
π-system in the benzylidene part of the molecule led to 60 mmol) was added, and the reaction mixture was
Table 1. Positions of absorption and emission maxima of the GFP chromophore and compound (IV)
GFP
(IV)
Solvent
λ
abs, nm
λ
em, nm
λ
abs, nm
λ
em, nm
Dioxane
369
367
368
370
367
425
435
420
438
436
453
491
388
386
385
397
395
458
482
491
508
513
458
600
Ethyl acetate
Acetonitrile
Methanol
Water (neutral form)
Water (deprotonated form)
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY Vol. 45 No. 3 2019

216
ZAITSEVA, BARANOV
kept at –80°C for 20 h. Then, 400 mL of ethyl acetate
and 150 mL potassium tartrate solution were added
and the mixture was stirred for 1 h. Then, the aqueous
phase was extracted with ethyl acetate (2 × 150 mL),
and organic fractions were combined and washed with
saturated NaCl solution (2 × 100 mL). The solution
was dried over anhydrous Na2SO4 and evaporated, and
the product was purified by column chromatography
(EtOAc–hexane, 1 : 3). Product (II) yield was 2.14 g
(81%). 1H NMR: 7.28 (d, J2 8.7, 2Н, H2'', H6''), 6.80
(d, J2 8.7, 2Н, H3'', H5''), 6.56 (d, J2 16.0, 1H, H3'),
6.25 (dt, J2 15.8, 6.0, 1Н, Н2'), 4.30 (t, J2 5.2, 2H,
СН2), 1.00 (s, 9Н, t-Bu), 0.21 (s, 6Н, 2 СН3). Agrees
with the literature data [11].
FUNDING
The study was supported by the Russian Foundation
for Basic Research (project no. 17-00-00401_KOMFI).
The work was performed using the equipment of the
Center for Collective Use (Shemyakin–Ovchinnikov
Institute of Bioorganic Chemistry) supported by the
Ministry of Education and Science of the Russian
Federation (agreement ID RFMEFI62117X0018).
COMPLIANCE WITH ETHICAL STANDARDS
Conflict of Interest
The authors declare that they have no conflict of
interest.
(E)-3-(4-((tert-Butyldimethylsilyl)oxy)phenyl)-
acrylaldehyde (III). To a solution of alcohol (II) in
50 mL chloroform, 20 eq. of pyridinium dichromate
were added. The solution was stirred for 1.5 h at room
temperature; the reaction was followed by TLC in the
EtOAc–hexane, 1 : 3, system. The reaction mixture
was filtered through 1 cm of silica gel and evaporated.
Statement of the Welfare of Animals
This article does not contain any studies involving
animals or human participants performed by any of
the authors.
1
Yield: 823 mg (39%). H NMR: 9.67 (d, J2 7.7, 1Н,
REFERENCES
СНО), 7.48 (d, J2 8.5, 2Н, H2'', H6''), 7.42 (d, J2 15.8,
1Н, H3'), 6.89 (d, J2 8.5, 2Н, H3'', H5''), 6.62 (dd, J2
15.8, 7.7, 1Н, Н2'), 1.00 (s, 9Н, t-Bu), 0.25 (s, 6Н, 2
СН3). Agrees with the literature data [12].
1. Walker, C.L., Lukyanov, K.A., Yampolsky, I.V.,
Mishin, A.S., Duraj-Thatte, A.M., Bahareh, A., Tol-
bert, L.M., and Solntsev, K.M., Curr. Opin. Chem.
Biol., 2015, vol. 27, pp. 64–74.
2. Yuan, L., Lin, W., Chen, H., Zhu, S., and He, L.,
Angew. Chem., 2013, vol. 52, pp. 10018–10022.
3. Povarova, N.V., Bozhanova, N.G., Sarkisyan, K.S.,
Gritcenko, R., Baranov, M.S., Yampolsky, I.V., Luk-
yanov, K.A., and Mishin, A.S., J. Mater. Chem. C,
2016, vol. 4, pp. 3036–3040.
5-([1Z,3E]-4-(4-Hydroxyphenyl)allylidene)-2,3-
dimethyl-3,5-dihydro-4H-imidazol-4-one (IV). (1) To
a solution of 262 mg (1 mmol) aldehyde (III) in 20 mL
chloroform, 0.3 mL 40% aqueous methylamine and
anhydrous sodium sulfate (2 g) were added. The mix-
ture was stirred for 48 h at room temperature, then the
organic phase was separated, dried over anhydrous
Na2SO4, and evaporated.
4. Baldridge, A., Solntsev, K.M., Song, C., Tanioka, T.,
Kowalik, J., and Tolbert, L.M., Chem. Commun., 2010,
vol. 46, pp. 5686–5688.
5. Olsen, S., Baranov, M.S., Baleeva, N.S., Antonova, M.M.,
Johnsond, K.A., and Solntsev, K.M., Phys. Chem.
Chem. Phys., 2016, vol. 18, pp. 26703–26711.
6. Yampolsky, I.V., Kislukhin, A.A., Amatov, T.T.,
Shcherbo, D., Potapov, V.K., Lukyanov, S., and
Lukyanov, K.A., Bioorg. Chem., 2008, vol. 36, pp. 96–
104.
7. Chuang, W.T., Chen, B.S., Chen, K.Y., and Cheng-
Chih, H., Chem. Commun., 2009, vol. 45, pp. 6982–6984.
8. Baldridge, A., Kowalik, J., and Tolbert, L.M., Synthe-
sis, 2010, no. 14, pp. 2424–2436.
9. Bozhanova, N.G., Baranov, M.S., Sarkisyan, K.S.,
Gritcenko, R., Mineev, K.S., Golodukhina, S.V., Bale-
eva, N.S., Lukyanov, K.A., and Mishin, A.S., ACS
Chem. Biol., 2017, vol. 12, pp. 1867–1873.
10. Boschi, D., Tron, G.C., Lazzarato, L., Chegaev, K.,
Cena, C., Di Stilo, A., Giorgis, M., Bertinaria, M.,
Fruttero, R., and Gasco, A., J. Med. Chem., 2006,
vol. 49, pp. 2886–2897.
11. Hiroyuki, A., Kawahara, E., Kishida, M., and Kato, K., J.
Mol. Catal. B: Enzym., 2006, vol. 40, pp. 8–15.
12. Satoshi, I., Rika, O., Chihiro, M., Masahiro, K., Kazu-
toshi, H., Mitsuhiro, N., Yoshiharu, A., Satoshi, K.,
Takashi, H., Shojiro, M., and Haruki, N., Tetrahedron,
2013, vol. 69, pp. 3847–3856.
(2) The imine (1 mmol) was mixed with ethyl-2-
((1-methoxyethylidene)amino)acetate (175 mg, 1.1 mmol)
and stirred at room temperature for 36 h. Then, 50 mL
of ethyl acetate was added to the reaction mixture,
then it was washed with water (2 × 50 mL) and satu-
rated NaCl solution (2 × 5 mL). To the organic phase,
10 eq. tetrabutylammonium fluoride was added; the
mixture was stirred for 10 min and 2 mL glacial acetic
acid was added. The reaction mixture was washed with
water (5 × 5 mL) and saturated NaCl solution (3 × 5 mL).
The solution was dried over anhydrous Na2SO4, evap-
orated, and purified with column chromatography
(EtOAc–EtOAc : i-PrOH, 1 : 4 gradient). Individual
isomer (E-isomer at position 3' and Z-isomer at posi-
tion 1') was obtained by slow recrystallization from
methanol. Yield: 88 mg (36%); yellow crystals;
1
mp ~300°C with decomposition. H NMR: 9.88 (s,
1Н, ОН), 7.44 (d, J2 8.2, 2Н, H2'', H6''), 7.29 (dd, J2
15.4, 11.6, 1Н, H2'), 7.11 (d, J2 15.6, 1Н, H1'), 6.84 (d,
J2 11.4, 1Н, H3'), 6.79 (d, J2 8.2, 2Н, H3'', H5''), 3.06
(s, 3Н, 3-CH3), 2.29 (s, 3Н, 2-CH3). 13C NMR: 168.7
(C4), 161.0 (C2), 158.9 (C4''), 141.6 (C3'), 138.3 (C1'),
129.1 (C2'', C6''), 127.4 (C1''), 127.3 (C5), 120.0 (C2'),
115.9 (C3'', C5''), 26.1 (3-CH3), 15.0 (2-CH3).
Translated by N. Onishchenko
RUSSIAN JOURNAL OF BIOORGANIC CHEMISTRY
Vol. 45
No. 3
2019