A synthetic approach to GFP chromophore analogs from 3-azidocinnamates. Role of methyl rotors in chromophore photophysics

Article abstract of DOI:10.1039/c3cc41948g

We have suggested a novel combinatorial approach for synthesis of otherwise inaccessible GFP chromophore analogs, and studied the influence of aliphatic substituents on their pH-dependent spectral properties. We found that the demethylation at C or N posi

Full text of DOI:10.1039/c3cc41948g

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A synthetic approach to GFP chromophore analogs
from 3-azidocinnamates. Role of methyl rotors in
chromophore photophysics†
Mikhail S. Baranov,a Kyril M. Solntsev,*b Konstantin A. Lukyanova and
Ilia V. Yampolsky*a
Cite this: DOI: 10.1039/c3cc41948g
Received 15th March 2013,
Accepted 3rd May 2013
DOI: 10.1039/c3cc41948g
www.rsc.org/chemcomm
We have suggested a novel combinatorial approach for synthesis with aldehydes,11 and copper-catalyzed condensation of amidines
of otherwise inaccessible GFP chromophore analogs, and studied with 2-bromocinnamic acid.12
the influence of aliphatic substituents on their pH-dependent
In the course of our systematic synthetic studies on the
spectral properties. We found that the demethylation at C or N range of GFP chromophore analogs, we were interested in
positions of the imidazolone ring leads to a decrease in the excited obtaining GFP chromophores bearing H and CF3 substituents
state lifetime.
at position 2 of the imidazolone core (2f and 2g). Utilization of
the abovementioned methods was unsuccessful, so we looked
Recently, the chromophore of Green Fluorescent Protein (GFP) for some alternative combinatorial synthetic approaches to
and its synthetic analogs have attracted much interest due to develop a library of C-modified GFP chromophores.
the unique combination of their spectral and photochemical
One example of the construction of 4-arylideneimidazolone,
properties. Synthetic GFP chromophore (p-hydroxybenzylidene which has a structure identical to that of the GFP core, based on the
imidazolone, p-HOBDI, 2 in Scheme 1) derivatives were used to reaction of SEM-protected 2-azido-3-(indol-3-yl)acrylamide with
gain a more detailed understanding of spectral behavior,1 methyldiphenylphosphine and 2-(indol-3-yl)-2-oxoacetyl chloride,
chromophore maturation,2 enhanced state proton transfer in is known in the literature. This method was applied in the synthesis
fluorescent proteins,3 and as a platform for novel fluorescent4–6 of marine alkaloid rhopaladin D.13 We have utilized this approach
and fluorogenic7 dyes. Several synthetic approaches to different to the synthesis of the GFP chromophore analogs.
functionalized analogs of the GFP chromophore were developed,8
We have studied the reaction of (Z)-2-azido-3-(4-methoxy-
including classical Erlenmeyer azlactone synthesis,9 cyclization phenyl)-N-methylacrylamide (obtained by aminolysis of ester 1,
with aromatic aldimines,10 condensation of saturated imidazolones see ESI†) with triphenylphosphine and carboxylic anhydrides or
acyl chlorides (Scheme 1).‡
We have found that among acyl chlorides, only pivaloyl
chloride gave moderate yield (34%) in this reaction. Further-
more, we found that isobutyryl chloride gave poor yield (17%),
while propionyl and acetyl chloride gave no desired product.
This difference can be attributed to side reactions associated
with deprotonation at the alpha-position of the acyl chloride.14
At the same time, carboxylic anhydrides demonstrated reverse
applicability: the best yields were obtained with sterically non-
hindered radicals, Me, Et, CF3 and H, while the secondary
radical (iPr) gave poor yield and the tertiary radical (tBu) did not
Scheme 1 Synthesis of GFP chromophore analogs 2a–g.
react. At the final stage of the synthesis, standard phenol
demethylation conditions (BBr3, CH2Cl2) were used to obtain
a Institute of Bioorganic Chemistry, Russian Academy of Sciences,
Miklukho-Maklaya 16/10, 117997 Moscow, Russia. E-mail: ivyamp@ibch.ru;
Fax: +7 499-7428122; Tel: +7 499-7428122
b School of Chemistry and Biochemistry, Georgia Institute of Technology,
901 Atlantic Drive, Atlanta, GA 30332-0400, USA. E-mail: solntsev@gatech.edu;
Fax: +1 404-894-7452; Tel: +1 404-385-3117
the desired GFP chromophore analogs (Table 1).
The reported combinatorial synthetic approach appears to
be useful for the synthesis of otherwise inaccessible GFP
chromophore analogs. This approach allows introduction of a
CF3 substituent into an imidazolone core as well as providing
the ability to obtain a parent heterocycle in this range, which
bears a hydrogen at position 2.
† Electronic supplementary information (ESI) available: Synthetic procedures,
NMR and MS spectral data and titration curves. See DOI: 10.1039/c3cc41948g
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Table 1 Reactivity of carboxylic anhydrides and acyl chlorides in synthesis of
C-derivatized GFP chromophores 2a–g
Table 2 pKa’s and absorption maxima of novel GFP chromophore analogs
compared with the known chromophore 2a (measured in 0.2 M phosphate
buffer)
Yield at stage iii/%
Overall yield
pKa
Abs/nm
Product
R1
Anhydride
Acyl chloride
(4 stages)/%
Neutral Anionic Cationic
2a
2b
2c
2d
2e
2f
Me
Et
61
35
26
15
0
42
26
16
12
28
27
22
Chromophore R1 R2 Nitrogen Phenol form
form
form
Traces
—
17
34
—
Pr
2a
2e
2f
2g
2h
Me Me 2.95
tBu Me 2.2
CF3 Me dcmp
7.7
7.7
7.15
7.80
7.7
368
373
388
374
365
424
430
470
434
422
390
402
dcmp
399
386
iPr
tBu
CF3
H
Traces
45
H
Me
Me 1.9
2.2
2g
30a
—
H
a
Mixed anhydride with acetic acid CH3COOCHO was used.
strong electronic withdrawing trifluoromethyl group. The latter
chromophore is also characterized by an B50 nm bathochromic
shift in anionic form compared to 2a.
The series of compounds obtained by the above method
made it possible to study the influence of aliphatic substituents in
the GFP chromophore on its pH-dependent spectral properties.
We have also included the N-unsubstituted chromophore 2h
(ESI†) in this comparative study.
Previously, one of us studied the subtle dependence of the
p-HOBDI photophysics on the nature and position of alkyl
substituents.16 The availability of a wide variety of aliphatic
derivatives of p-HOBDI prepared in this work made possible the
testing of intriguing hypothesis of involvement of methyl rotors
in the p-HOBDI deactivation mechanism proposed by Agmon
et al.17 They suggested that free barrierless rotation of C- and
N-methyl groups on the imidazolone ring supplies ‘‘a dense
thermal bath of rotational states that couple with the internal
conversion. These modes can enhance the nonradiative transi-
tion by accepting the excess energy’’. In other words, demethy-
lated derivatives of p-HOBDI were proposed to have slower rates
of internal conversion and longer excited-state lifetimes.
We have compared the photoinduced behavior of the C- and
N-demethylated derivatives of 2a (2g and 2h, correspondingly)
and compared it with the behavior of 2a and 2b. To exclude
possible selective hydrogen-bonding solute–solvent interac-
tions, room-temperature acetonitrile was chosen as a solvent
for these studies.
Similarly to 2a, studied by one of us earlier,15 all homologs
2b–h have three pH-dependent spectral forms: anionic, neutral
and cationic (Fig. 1 and Table 2). Chromophores 2e, 2g and 2h
have lowered pKa associated with protonation of the imidazole
nitrogen, compared to a reference chromophore 2a. This can be
attributed to steric effects in the case of the tBu substituent (2e)
and to electronic effects in the case of dealkylated chromo-
phores 2g and 2h. A significantly lowered phenolic pKa (7.15 vs.
7.7 for 2a) was observed in the case of 2f, which carries the
Transient absorption spectra of the GFP-synthetic chromo-
phores are shown in Fig. 2. The spectra are very close to those of
the parent p-HOBDI studied by Vengris et al.18 and one of us19
earlier. All spectra consisted of overlapped photoinduced
absorption (PA) bands at 460, 570, and 770 nm, their ratio
varied among the compounds. The analysis of the transient
data of p-HOBDI and its derivatives was presented earlier.18,19 It
was demonstrated that upon photoexcitation the relaxation path-
way is characterized by a sum of exponentials and involves a
transition through a succession of states along a reaction coordi-
nate that includes contributions from intramolecular twisting
motion, solvation dynamics, and vibrational cooling. Therefore,
the experimental time constants determined from the analysis of
PA data can be considered as average values for a distribution of
the population in both the excited and the ground state. In this
work we will not analyse in detail the complex behavior of the
subpicosecond photoinduced kinetics of the novel chromophores,
but will perform a comparative analysis of the kinetics of a series
of dealkylated derivatives. Based on the similarity of the ground-
and excited-state absorption spectra of all homologs, we assumed
that the excited-state deactivation mechanism is the same. Then
we fitted the decay of the common 570 nm PA band using a
polyexponential function.
Fig. 1 pH-titration of chromophores 2a–h.
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Foundation for Basic Research Grant 11-04-01293-a, and
the Program of the Government of the Russian Federation
‘Measures to attract leading scientists to Russian educational
institutions’ (Grant no. 11.G34.31.058). M.S.B. was supported
by a stipend from a program of the President of the Russian
Federation. K.M.S. acknowledges generous support from the
National Science Foundation (CHE-1213047). The authors
thank Joseph Perry (Georgia Tech) and the members of his
group for the help with transient absorption measurements.
Notes and references
‡ Typical synthetic procedure: (Z)-2-azido-3-(4-methoxyphenyl)-N-
methylacrylamide, obtained by the action of MeNH2 on azidoester 1
(ESI†) (5.2 g, 22.4 mmol), and triphenylphosphine (7.0 g, 26.7 mmol)
were heated in dry toluene (100 mL) under argon at 65 1C for 30 min.
A yellow precipitate separated and effervescence was observed. The
reaction mixture was cooled to room temperature and trifluoroacetic
anhydride (4.2 g, 20 mmol) and DIPEA (1.3 g, 10 mmol) were added. The
mixture was heated to 40 1C and stirred for 2 h, then cooled, diluted
with 200 mL CHCl3, washed with aqueous NaHCO3 (5%, 100 mL), water
(2 Â 100 mL), brine (2 Â 100 mL) and dried over Na2SO4. The solvent
was evaporated and the product ((Z)-4-(4-methoxybenzylidene)-1-
methyl-2-(trifluoromethyl)-1H-imidazol-5(4H)-one) was purified using
column chromatography (CHCl3 : EtOH
= 50 : 1). Yellowish solid,
1.28 g (45%), mp 99–101 1C.
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Fig. 2 Left column: transient absorption spectra of GFP chromophores in MeCN
after excitation at 370 nm. The À0.4 ps trace corresponds to the spectral baseline
in the absence of pump excitation. Right column: normalized kinetics of the TA
signals. Color coding is the same for each column.
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The PA kinetics of the GFP-synthetic chromophores are
shown in Fig. 2. At all wavelengths the decays of the C- and
N-dealkylated derivatives (2g and 2h, correspondingly) were
faster than those of 2a. The characteristic decay times were
0.35 ps and 0.37 ps for 2g and 2h vs. 0.52 ps for 2a and 0.57 ps
for C-ethyl derivative 2b.
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The authors gratefully acknowledge support from the MCB
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