Synthesis and luminescence properties of analogues of the green fluorescent protein chromophore

Article abstract of DOI:10.1016/j.dyepig.2020.108267

The green fluorescent protein (GFP) is extensively used as a biomarker for fluorescence biological imaging. The chromophore in GFP is only fluorescent when confined into the β–barrel of the protein. Similarly, synthetic analogues of the fluorophore of GFP

Full text of DOI:10.1016/j.dyepig.2020.108267

Journal Pre-proof
Synthesis and luminescence properties of analogues of the green fluorescent protein
chromophore
Cátia I.C. Esteves, Inês da Silva Fonseca, João Rocha, ArturM.S. Silva, Samuel
Guieu
PII:
S0143-7208(19)32582-3
DOI:
https://doi.org/10.1016/j.dyepig.2020.108267
DYPI 108267
Reference:
To appear in: Dyes and Pigments
Received Date: 5 November 2019
Revised Date: 6 February 2020
Accepted Date: 6 February 2020
Please cite this article as: Esteves CáIC, da Silva Fonseca Inê, Rocha Joã, Silva AS, Guieu S,
Synthesis and luminescence properties of analogues of the green fluorescent protein chromophore,
Dyes and Pigments (2020), doi: https://doi.org/10.1016/j.dyepig.2020.108267.
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Cátia I. C. Esteves: Investigation, Visualization, Writing – original draft,
Inês da Silva Fonseca: Investigation, Visualization,
João Rocha: Funding acquisition, Writing – review & editing,
Artur M. S. Silva: Funding acquisition, Writing – review & editing,
Samuel Guieu: Conceptualization, Funding acquisition, Project administration,
Supervision, Writing – original draft, Writing – review & editing,

Synthesis and Luminescence Properties of Analogues of
the Green Fluorescent Protein Chromophore
a
a
b
a
Cátia I. C. Esteves, Inês da Silva Fonseca, João Rocha, Artur M. S. Silva, Samuel
Guieu
a,b,*
a
LAQV/REQUIMTE, QOPNA, Department of Chemistry, University of Aveiro, 3810-193
Aveiro, Portugal.
b
CICECO-Aveiro Institute of Materials, Department of Chemistry, University of Aveiro,
3
810-193 Aveiro, Portugal.
E-mail: sguieu@ua.pt ; rocha@ua.pt ; artur.silva@ua.pt ; catiaiesteves@ua.pt
Abstract
The green fluorescent protein (GFP) is extensively used as a biomarker for fluorescence
biological imaging. The chromophore in GFP is only fluorescent when confined into the β–
barrel of the protein. Similarly, synthetic analogues of the fluorophore of GFP are usually
non-emissive in solution, due to free rotation around the aryl-alkene bond and (Z/E)-
isomerization of the double bond. Here, the synthesis and characterization of three analogues
of the fluorophore of GFP are reported. The introduction of more electron donating
substituents induces a red-shift in the absorption and emission. The fluorophores are more
emissive in the solid state than in solution, and a study of their crystal structure reveals that
the (Z/E)-isomerization is efficiently blocked in the crystals.
Key words
Green fluorescent protein; Aggregation-Induced Emission Enhancement; Synthesis;
fluorescence
1
. Introduction
The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is extensively used
as a biomarker for fluorescence biological imaging [1] Fusion of GFP with other proteins
.
allows their visualization in living cells, usually without affecting their function [2]. GFP is
therefore used in molecular and cell biology, in cell-labeling and bioimaging applications, as
genetically encoded fluorescent marker in biology, in live cell imaging and in medicine [3].
Other fluorescent proteins emit light with different colors, but all have a similar cylindrical
structure, with the chromophore enclosed in a central α-helix. In the wild type GFP, the
chromophore responsible for the fluorescence is the p-hydroxybenzylideneimidazolidinone
(Figure 1), which emits through excited-state proton transfer from the phenolic oxygen to the
protein β-barrel [4]. An interesting point is that the chromophore is non-emissive when it is
-3)
isolated from the protein (Φ < 10 , due to a non-radiative relaxation channel through the

(Z/E)-isomerization of the benzylidene substituent. The chemical environment ensuring the
autocatalytic formation of the chromophore from three amino acids is also responsible for the
rigidly defined and firmly packed structure of the protein [5]. The fluorescent proteins
efficiently block the (Z/E)-isomerisation of the fluorophore, turning it emissive (Φ = 0.8) [6].
Synthetic analogues of the GFP chromophore have been prepared [2] and applied to
biological imaging, with some success [4,5]. The synthesis usually starts with the
condensation of a hippuric acid derivative with an aldehyde, the so called “Erlenmeyer
azlactone synthesis”, followed by further modifications [4,8]. Various strategies have been
developed in order to turn the analogues emissive in dilute solutions, such as rigidification of
the backbone through chemical modification [8b] or introduction of boron complex [9].
Nevertheless, the fluorophores are always more emissive when their motion is restraint than
they are when in dilute solutions, and crystallization or precipitation are efficient ways of
restricting the molecular motion in small organic molecules.
Some analogues of the GFP fluorophore show Aggregation-Induced Emission Enhancement
(AIEE) properties [10]. Following previous works on AIEE [11], we have now designed
fluorophores with aromatic groups rotating around the imidazolidinone core, in order to
increase the AIEE effect.
Figure 1. Structure of the GFP chromophore and analogues.
2
. Experimental
Melting points were measured with a Büchi Melting Point B-540 apparatus and are
uncorrected. NMR spectra were recorded on a Bruker DRX Avance 300 spectrometer (300.13
1
13
MHz for H, 75.47 MHz for C), in CDCl or DMSO-d as solvents, if not stated otherwise.
3
6
Chemical shifts (δ) are reported in ppm from TMS and coupling constants (J) in Hz; internal
1
3
standard was residual peak of the solvent. Unequivocal C assignments were made with the
aid of 2D gHSQC and gHMBC (delays for one-bond and long-range J C/H couplings were
optimised for 145 and 7 Hz, respectively) experiments. Positive-ion ESI mass spectra were
acquired using a Q-TOF 2 instrument [Nitrogen was used as nebuliser gas and argon as
collision gas. The needle voltage was set at 3000 V, with the ion source at 80°C and
desolvation temperature at 150°C. Cone voltage was 35 V]. Preparative thin-layer
chromatography was performed with Merck silica gel (60 DGF ). Column chromatography
2
54

was performed with Merck silica gel 60 (70−230 mesh). All other chemicals and solvents
used were obtained from commercial sources and were either used as received or dried by
standard procedures.
2
.1. General procedure for the synthesis of Oxazolone derivatives 2a-c
Hippuric acid (5 mmol), sodium acetate (5 mmol), benzaldehyde derivative (5 mmol) and
acetic anhydride (40 mmol) were stirred at 100 ºC for 1-24 h. The reaction was allowed to
cool down to room temperature. It was poured onto ice (50 g) and H O (100 mL) and the
2
precipitate that formed was collected by filtration. Then, the obtained solid was dissolved in
CH Cl and dried over anhydrous Na SO . The solvent was evaporated to dryness and the
2
2
2
4
obtained residues were recrystallized from EtOH. Compounds 2a-c have already been
described in the literature [20].
2
.1.1 (Z)-4-[4-(dimethylamino)benzylidene]-2-phenyl-1,3-oxazol-5(4H)-one (2a) [20]
1
The product was isolated as a red solid (0.598 g, 2.0 mmol, 41 %). M.p. = 218-219 ºC. H
NMR (300 MHz, CDCl ) δ = 3.10 (s, 6H, N(CH ) ), 6.74 (d, J = 9.3 Hz, 2H, H-3’,5’), 7.20 (s,
3
3 2
1
H, CH), 7.50-7.54 (m, 3H, H-3’’, H-4’’ and H-5’’), 8.12-8.16 (m, 4H, H-2’,6’ and H-2’’,6’’)
ppm.
2
.1.2 (Z)-4-(4-methoxybenzylidene-2-phenyl-1,3-oxazol-5(4H)-one (2b)
1
The product was isolated as a yellow solid (0.654 g, 2.3 mmol, 47 %). M.p. = 157-158 ºC. H
NMR (300 MHz, CDCl ) δ = 3.89 (s, 3H, OCH ), 7.00 (dd, J = 9.0 and 2.4 Hz, 2H, H-3’,5’),
3
3
7
.22 (s, 1H, CH), 7.52-7.60 (m, 3H, H-3’’, H-4’’ and H-5’’), 8.15-8.21 (m, 4H, H-2’,6’ and
H-2’’,6’’) ppm.
2
.1.3 (Z)-4-(4-nitrobenzylidene-2-phenyl-1,3-oxazol-5(4H)-one (2c)
1
The product was isolated as a yellow solid (0.496 g, 1.7 mmol, 34%). M.p. = 239-240 ºC. H
NMR (300 MHz, CDCl ) δ = 7.24 (s, 1H, CH), 7.56-7.61 (m, 2H, H-3’’,5’’), 7.66-7.71 (m,
3
1
H, H-4’’), 8.21-8.24 (m, 2H, H-2’’,6’’), 8.31-8.40 (m, 4H, H-2’,6’ and H-3’,5’) ppm.
2
.2. General procedure for the synthesis of Benzamide derivatives 3a-c
Oxazolone derivatives (1 mmol) and p-anisidine (1.5 mmol) were stirred solvent free at ~70ºC
for 4-24 h. Then, the solid was taken up in CH Cl (30 mL) and dried over anhydrous
2
2
Na SO . The solvent was evaporated to dryness and the obtained residues were recrystallized
2
4
from ethyl acetate.
2
.2.1. (E)-N-(1-(4-(dimethylamino)phenyl)-3-((4-methoxyphenyl)amino)-3-oxoprop-1-en-2-
yl)benzamide (3a) [20]
The product was isolated as a yellow solid (0.330 g, 0.79 mmol, 79 %). M.p. = 243-244 ºC.
1
H NMR (300 MHz, DMSO-d ) δ = 2.93 (s, 6H, N(CH ) ), 3.74 (s, 3H, OCH ), 6.69 (d, J 9.0
6
3 2
3
Hz, 2H, H-3’,5’), 6.89 (d, J = 9.0 Hz, 2H, H-3’’,5’’), 7.19 (s, 1H, CH), 7.49-7.63 (m, 7H, H-
’,6’, H-2’’,6’’, H-3’’’, 5’’’ and H-4’’’), 8.07 (d, J = 6.9 Hz, 2H, H-2’’’,6’’’), 9.79 (s, 1H,
2
1
3
NH), 9.90 (s, 1H, NH) ppm. C NMR (75 MHz, DMSO-d ) δ = 41.0 (N(CH ) ), 55.1
6
3 2
(
OCH ), 111.6 (C-3’,5’), 113.6 (C-3’’,5’’), 121.5 (C-1’), 121.7 (C-2’’,6’’), 125.9 (NHCCO),
3
1
27.9 (C-2’’’,6’’’), 128.3 (C-3’’’,5’’’), 130.2 (CH), 131.1 (C-3’,5’), 131.6 (C-4’’’), 132.6 (C-

1
’’), 133.9 (C-1’’’), 150.5 (C-4’), 155.1 (C-4’’), 164.1 (CCONH), 165.9 (CONHC) ppm. MS
+
m/z (ESI %): 416.2 ([M+H] ); HMRS: m/z (ESI) calc. for C H N O 416.1969, found
2
5
26
3
3
4
16.1990.
2
.2.2.
(E)-N-(1-(4-methoxyphenyl)-3-((4-methoxyphenyl)amino)-3-oxoprop-1-en-2-
yl)benzamide (3b)
1
The product was isolated as a white solid (0.319 g, 0.79 mmol, 73 %). M.p. = 233-235 ºC. H
NMR (300 MHz, DMSO-d ) δ = 3.74 (d, J = 1.8 Hz, 3H, OCH ), 3.77 (d, J = 1.6 Hz, 3H,
6
3
OCH ), 6.90 (dd, J = 8.9 and 1.8 Hz, 2H, H-3’’,5’’), 6.96 (dd, J = 8.4 and 1.6 Hz, 2H, H-
3
3
’,5’), 7.17 (s, 1H, CH), 7.51-7.64 (m, 7H, H-2’,6’, H-2’’,6’’, H-3’’’,5’’’ and H-4’’’), 8.05 (d,
1
3
J = 8.1 Hz, 2H, H-2’’’,6’’’), 9.94 (s, 1H, NH), 10.00 (s, 1H, NH) ppm. C NMR (75 MHz,
DMSO-d ) δ = 55.1 (OCH ), 55.2 (OCH ), 113.6 (C-3’’,5’’), 114.0 (C-3’,5’), 121.7 (C-
6
3
3
2
’’,6’’), 126.7 (C-1’), 127.9 (C-2’’’,6’’’), 128.3(C-2’,6’), 128.5 (CH), 128.8 (C-4’’’), 131.1
(
1
C-3’’’,5’’’), 131.7 (NHCCO), 132.4 (C-1’’), 133.6 (C-1’’’), 155.3(C-4’’), 159.5 (C-4’),
64.1 (CCONH), 165.9 (CONHC) ppm. MS m/z (ESI %): 403.2 ([M+H] ); HMRS: m/z (ESI)
+
calc. for C H N O 403.1652, found 403.1669.
2
4
23
2
4
2
.2.3.
(E)-N-(1-(4-nitroxyphenyl)-3-((4-methoxyphenyl)amino)-3-oxoprop-1-en-2-
yl)benzamide (3c)
1
The product was isolated as a white solid (0.143 g, 0.34 mmol, 50%). M.p. = 232-233 ºC. H
NMR (300 MHz, DMSO-d ) δ = 3.74 (br s, 3H, OCH ), 6.92 (d, J = 9.0 Hz, 2H, H-3’’,5’’),
6
3
7
2
.15 (s, 1H, CH), 7.51-7.66 (m, 5H, H-2’’,6’’, H-3’’’,5’’’ and H-4’’’), 7.86 (d, J = 8.6 Hz,
H, H-2’,6’), 8.01 (d, J = 8.5 Hz, 2H, H-2’’’,6’’’), 8.25 (d, J = 8.6 Hz, H-3’,5’), 10.23 (s, 1H,
13
NH), 10.27 (s, 1H, NH) ppm. C NMR (75 MHz, DMSO-d ) δ = 55.2 (OCH ), 113.7 (C-
6
3
3
3
1
’’,5’’), 121.5 (C-2’’,6’’), 123.6 (C-3’,5’), 124.4 (CH), 128.0 (C-2’’’,6’’’), 128.4 (C-
’’’,5’’’), 130.2 (C-2’,6’), 132.0 (C-4’’’), 132.2 (C-1’’), 133.2 (C-1’’’), 134.5 (NHCCO),
41.6 (C-1’ or C-4’), 146.4 (C-1’ or C-4’), 155.5 (C-4’’), 163.5 (CCONH), 165.9 (CONHC)
+
ppm. MS m/z (ESI %): 418.1 ([M+H] ); HMRS: m/z (ESI) calc. for C H N O 418.1397,
2
3
20
3
5
found 418.1418.
2
.3. General procedure for the synthesis of Imidazolone derivatives 4a-c
Anhydrous ZnCl (2.5 mmol) was added to the benzamide derivative (0.5 mmol) in toluene
2
(
10 mL). The reaction mixture was refluxed for 5-18 h followed by addition of H O (25 mL),
2
the pH was adjusted to 3-4 with diluted HCl and the solution extracted with CH Cl (3×25
2
2
mL). The organic layer was collected, dried with anhydrous Na SO , and the solvent was
2
4
evaporated to give a solid residue that was purified by silica gel column chromatography
using dichloromethane as eluent.
2
.3.1. (Z)-5-(4-(dimethylamino)benzylidene)-3-(4-methoxyphenyl)-2-phenyl-3,5-dihydro-4H-
imidazol-4-one (4a)
The product was isolated as an orange solid (0.112 g, 0.28 mmol, 55 %); R 0.46 (CH Cl ).
f
2
2
1
M.p. = 263-264 ºC. H NMR (300 MHz, CDCl ) δ = 3.09 (s, 6H, N(CH ) ), 3.83 (s, 3H,
3
3 2
OCH ), 6.75 (d, J = 8.5 Hz, 2H, H-3’,5’), 6.93 (dd, J = 8.9 and 1.1 Hz, 2H, H-3’’,5’’), 7.11
3
(dd, J = 8.9 and 1.1 Hz, 2H, H-2’’,6’’), 7.27-7.33 (m, 2H, H-3’’’,5’’’), 7.30 (s, 1H, CH), 7.36-
7
.42 (m, 1H, H-4’’’), 7.58 (dd, J = 8.4 and 1.5 Hz, 2H, H-2’’’,6’’’), 8.23 (d, J = 8.5 Hz, 2H,
1
3
H-2’,6’) ppm. C NMR (75 MHz, CDCl ) δ = 40.2 (N(CH ) ), 55.6 (OCH ), 111.9 (C-3’,5’),
3
3 2
3
1
2
14.8 (C-3’’,5’’), 122.7 (C-1’), 128.1 (C-1’’), 128.3 (C-3’’’,5’’’), 128.7 (C-2’’,6’’), 129.1 (C-
’’’,6’’’), 129.6 (C-4), 130.7 (C-4’’’), 131.0 (CH), 134.7 (C-1’’’), 134.9 (C-2’,6’), 151.9 (C-

+
4
’), 157.3 (C-2), 159.3 (C-4’’), 171.0 (CO) ppm. MS m/z (ESI %): 398.4 ([M+H] ); HMRS:
m/z (ESI) calc. for C H N O 398.1863, found 398.1887.
2
5
24
3
2
2
4
.3.2. (Z)-5-(4-methoxybenzylidene)-3-(4-methoxyphenyl)-2-phenyl-3,5-dihydro-4H-imidazol-
-one (4b)
The product was isolated as a yellow solid (0.107 g, 0.27 mmol, 54 %); R 0.64 (CH Cl ).
f
2
2
1
M.p. = 195-197 ºC. H NMR (300 MHz, CDCl ) δ = 3.09 (s, 6H, N(CH ) ), 3.83 (s, 3H,
3
3 2
OCH ), 6.75 (d, J = 8.5 Hz, 2H, H-3’,5’), 6.93 (dd, J = 8.9 and 1.1 Hz, 2H, H-3’’,5’’), 7.11
3
(dd, J = 8.9 and 1.1 Hz, 2H, H-2’’,6’’), 7.27-7.33 (m, 2H, H-3’’’,5’’’), 7.30 (s, 1H, CH), 7.36-
7
.42 (m, 1H, H-4’’’), 7.58 (dd, J = 8.4 and 1.5 Hz, 2H, H-2’’’,6’’’), 8.23 (d, J = 8.5 Hz, 2H,
1
3
H-2’,6’) ppm. C NMR (75 MHz, CDCl ) δ = 40.2 (N(CH ) ), 55.6 (OCH ), 111.9 (C-3’,5’),
3
3 2
3
1
2
4
14.8 (C-3’’,5’’), 122.7 (C-1’), 128.1 (C-1’’), 128.3 (C-3’’’,5’’’), 128.7 (C-2’’,6’’), 129.1 (C-
’’’,6’’’), 129.6 (C-4), 130.7 (C-4’’’), 131.0 (CH), 134.7 (C-1’’’), 134.9 (C-2’,6’), 151.9 (C-
+
’), 157.3 (C-2), 159.3 (C-4’’), 171.0 (CO) ppm. MS m/z (ESI %): 385.3 ([M+H] ); HMRS:
m/z (ESI) calc. for C H N O 385.1547, found 385.1563.
2
4
21
2
3
2
4
.3.3. (Z)-5-(4-nitroxybenzylidene)-3-(4-methoxyphenyl)-2-phenyl-3,5-dihydro-4H-imidazol-
-one (4c)
The product was isolated as a yellow solid (0.092 g, 0.23 mmol, 46 %); R 0.73 (CH Cl ).
f
2
2
1
M.p. = 198-200 ºC. H NMR (300 MHz, CDCl ) δ = 3.85 (s, 3H, OCH ), 6.96 (dd, J = 8.9
3
3
and 2.6 Hz, 2H, H-3’’,5’’), 7.12 (dd, J = 8.9 and 2.6 Hz, 2H, H-2’’,6’’), 7.28 (s, 1H, CH),
.34-7.39 (m, 2H, H-3’’’,5’’’), 7.47-7.52 (m, 1H, H-4’’’), 7.64 (dd, J = 7.2 and 1.5 Hz, 2H,
7
1
3
H-2’’’, H6’’’), 8.30 (d, J = 8.7 Hz, 2H, H-3’,5’), 8.44 (d, J = 8.7 Hz, 2H, H-2’,6’) ppm. C
NMR (75 MHz, CDCl ) δ = 55.5 (OCH ), 114.8 (C-3’’,5’’), 123.8 (C-3’,5’), 124.8 (C=C-H),
3
3
1
4
1
4
26. 8 (C-1’’), 128.2 (C-1’’’), 128.4 (C-2’’,6’’ and C-3’’’,5’’’), 129.4 (C-2’’’,6’’’), 132.0 (C-
’’’), 132.8 (C-2’,6’), 140.5 (C-4’), 141.1 (C-4), 147.8 (C-1’), 159.6 (C-4’’), 163.0 (C-2),
+
70.6 (CO) ppm. MS m/z (ESI %): 400.3 ([M+H] ); HMRS: m/z (ESI) calc. for C H N O
2
3
18
3
4
00.1292, found 400.1314.
3
. Results and discussion
3
.1. Synthesis and characterization
The synthetic route chosen to obtain the fluorophores comprised three steps (Scheme 1). First,
the aldehydes 1a-c were reacted with hippuric acid and sodium acetate in acetic anhydride,
following Erlenmeyer azlactone synthesis, and the oxazolones 2a-c were obtained in 34-47%
yields after recrystallization [12]. They were reacted with p-anisidine at ca. 70 ºC without
solvent, to give the ring-opened benzamides 3a-c in 50-79% yields. Finally, the cyclization of
the benzamides in refluxing toluene with anhydrous ZnCl afforded the fluorophores 4a-c in
2
1
13
4
6-55% yields [13]. All new compounds were fully characterized by H and C-NMR and
HRMS.

Scheme 1: Synthesis of the chromophores 4a-c.
3
.2. Absorption and emission properties
The absorption and emission properties of the fluorophores 4a-c were characterized in THF
solutions, and the results are summarized in Table 1 and Figure 2. In dilute solution, the
fluorophores present a major absorption band at 400-450 nm. As expected [14], the
introduction in 4a of a more electron-donating group, as compared to 4b, red shifts the
absorption and increases the molar extinction coefficient. Likewise, it also red shifts the
maximum of emission to 530 nm. The effect of the nitro substituent is more difficult to assess,
because compound 4c presents two emission bands of very low intensities, around 440 and
4
70 nm. Indeed, fluorophores with nitro substituents tend to present a strong solvatochromism
[
11i] for both absorption and emission, and double bands emission has been described for
fluorophores bearing a nitro substituent [15]. The fluorophores 4a-c are only weakly emissive
in dilute solution, with quantum yields of less than 0.5%. These low quantum yields are
expected in solution, because GFP fluorophore analogues are known to relax through the (Z-
E)-isomerisation of the carbon-carbon double bond, making them non-emissive [8b].
Table 1: UV-visible absorption and fluorescence data of compounds 4a-c in THF.
Solid state
Fluorescence
UV/vis in THF
Fluorescence in THF
Stokes’ shift
Dye
-
1
-
λ
abs
ε (M cm
λ
em (nm)
(c)
(d)
ϕf (%)
ϕf (%)
1
(a)
(b)
-1
(
nm)
)
(cm )
4
a
450
400
410
51 000
25 000
22 000
530
470
440, 470
3354
3723
3982
< 0.5
< 0.5
< 0.1
1
1
6
4
b
4
c
(
(
(
a)
b)
c)
-4
-6
ε was determined by linear regression of 4 measurements in the range 10 M to 10 M.
Excitation at the maximum of absorption.
Quantum yields were determined by comparison with fluorescein (quantum yield 0.90 at excitation 470 nm in a solution of NaOH 0.01 M
in water) [16].
(d)
Quantum yields were determined using an integrating sphere.

0
0
0
0
,3
,2
,1
,0
0,3
0,2
0,1
0,0
Abs
Em
4a
4b
4c
3
00
400
500
600
700
Wavelength (nm)
Figure 2. Absorption and emission spectra of fluorophores 4a-c in THF.
This isomerization can be effectively blocked in the β-barrel of GFP, or when the fluorophore
is in the crystalline state. The latter is a particular case of AIEE, when the formation of
aggregates or crystals limits internal conversion and forces a dye to emit. In order to assess
the AIEE features of the dyes 4a-c, the emission of their solutions and suspensions in THF-
water mixtures was recorded. As 4a and 4b present a similar behavior only 4a is shown here
as an example (see Figure 3 and SI). It is almost non-emissive when fully dissolved in THF
and when the amount of water is increased up to 60%. When the proportion of water reaches
7
0% of the mixture, the dye starts to precipitate, and the emission intensity increases by a
factor of 100. Because the emission wavelength does not vary, the formation of J- or H-
aggregates can be ruled out [17]. The same observation can be made for 4b, but not for 4c
which remains almost non-emissive even in the aggregated form, which was expected because
water can have a quenching effect for fluorophores bearing a nitro group [11i,18]. These
observations were corroborated with the measure of the solid state quantum yields, which are
higher than in dilute solution for all three fluorophores.
Figure 3. Aggregation-Induced Emission Enhancement observed for compound 4a. Emission
spectra of solutions and suspension of 4a in THF-water mixtures (left) and relative emission
intensity as a function of the water percentage (right).

3
.3. Crystal structures
Single crystals suitable for X-ray diffraction were obtained for the fluorophores 4b and 4c. All
angles and bond lengths are in normal range [19], and the crystal structures confirm the Z
configuration of the double bond (Figures 4 and 5).
Compound 4b crystallizes in the triclinic group P-1. One weak intramolecular hydrogen bond
C-H···N can be noticed. In the crystal packing, 4b arranges into columns held together via
weak intermolecular hydrogen bonds and Van der Waals interactions (Figure 4).
Figure 4. Molecular structure of compound 4b (left) and columnar arrangement (right,
hydrogen atoms not shown, for clarity). Thermal ellipsoids are shown at the 50% probability
level, hydrogen atoms are depicted with an arbitrary radius (0.30Å), and the hydrogen bond in
dashed black line. C, grey; O, red; N, blue; H, white.
Compound 4c crystallizes with one molecule of chloroform in the triclinic group P-1. The
chloroform molecule forms a hydrogen bond with the amide oxygen, and a weak
intramolecular hydrogen bond is present between an aromatic C-H and a nitrogen atom. In the
crystal packing, the fluorophores arrange into dimers, held together via weak hydrogen bonds
and Van der Waals interactions, and the dimers further arrange into columns (figure 5).
Figure 5. Molecular structure of compound 4c and chloroform (left) and columnar
arrangement (right, hydrogens atoms not shown, for clarity). Thermal ellipsoids are shown at
the 50% probability level, hydrogen atoms are depicted with an arbitrary radius (0.30Å). C,
grey; O, red; N, blue; H, white; Cl, green.
The crystal structures reveal that no strong intermolecular interactions are present, and the
fluorophores do not form J- or H-aggregates. This is accord with the fact that the emission
wavelengths in the solution and solid state are the same. Moreover, the crystal packing

efficiently blocks the Z/E isomerisation of the double bond, turning the fluorophore more
emissive in the crystalline state than in solution.
4
. Conclusion
Three analogues of the GFP fluorophore were synthesized in moderate yields using a
straightforward strategy. In solution, they display fluorescence quantum yields of less than
0
.5% and moderate Stokes' shifts, between 70 and 80 nm. Their emission intensity increases
dramatically in the solid state, thanks to the restriction of their intramolecular motion. The
crystal structures confirmed the (Z)-conformation of the double bond, which is blocked in the
solid state but not in solution, confirming the (Z/E)-isomerization is the main relaxation
pathway in solution. The introduction of more electron-donating substituents on the aromatic
ring connected to the carbon-carbon double bond red shifts the absorption and emission. In
contrast, the anisole ring is almost perpendicular to the imidazolidinone, meaning that it does
not participate in the conjugation of the aromatic system of the dye, and could be modified in
order to tune its physical-chemical properties without modifying its photophysics.
Acknowledgment
Thanks are due to University of Aveiro, FCT/MEC for the financial support to the QOPNA
research Unit (FCT UID/QUI/00062/2019) and also to the Portuguese NMR Network. This
work was developed within the scope of the project CICECO-Aveiro Institute of Materials,
FCT Ref. UID/CTM/ 50011/2019, financed by national funds through the FCT/MCTES. S.
Guieu acknowledges the fundings from national funds (OE), through FCT – Fundação para a
Ciência e a Tecnologia , I.P., in the scope of the framework contract foreseen in the numbers
4
5
, 5 and 6 of the article 23, of the Decree-Law 57/2016, of August 29, changed by Law
7/2017, of July 19, and from the Integrated Programme of SR&TD “pAGE – Protein
aggregation Across the Lifespan” (reference CENTRO-01-0145-FEDER-000003). C.I.C.
Esteves acknowledges the 016385-PAC NETDIAMOND project (POCI-01-0145-FEDER-
0
16385) for her Post-Doctoral grant (BPD/UI50/7664/2017). Professor Rute AS Ferreira
(phantom-g, CICECO, Department of Physics, University of Aveiro) is acknowledged for the
solid state quantum yield measurements.
Appendix A. Supplementary data.
Experimental procedures, compound characterization data and NMR spectra of the new
compounds. CCDC-1958327-1958328 contain the supplementary crystallographic data for
this
paper.
These
data
can
be
obtained
free
of
charge
at
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data
Centre, 12, Union Road, Cambridge CB2 1EZ, UK. Fax: C44 1223 336 033. E-mail:
deposit@ccdc.cam.ac.uk).

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Highlights
A new synthesis of analogues of the Green Fluorescent Protein fluorophore is presented.
They are almost non emissive in solution.
Their emission intensity increases up to a hundred times upon crystallization.
Changing the substituents tunes their absorption and emission spectra.

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: