The Role of N-Substituents in Radiationless Deactivation of Aminated Derivatives of a Locked GFP Chromophore

Article abstract of DOI:10.1002/ejoc.201700805

We report the creation of a novel group of the ABDI-BF₂ fluorescent dyes based on the conformationally locked GFP chromophore. We studied the intramolecular mechanism of radiationless deactivation of the ABDI-BF₂ fluorophore by introducing the various substituents at the nitrogen atom. The results of this study and our previous work allowed us to claim that in case of ABDI-BF₂ this deactivation is determined by the formation of the nonfluorescent internal charge transfer exited state with a planar quinoidal structure. The electronic effects have a greater impact on the radiationless deactivation than the conformation. Thus, the introduction of an electron-donating group is more effective than the creation of rigid derivatives. The presented dyes are characterized by high fluorescence quantum yields and pH-independence in the physiological pH range, making them promising candidates for a wide spectrum of fluorescence labeling applications.

Full text of DOI:10.1002/ejoc.201700805

A Journal of
Accepted Article
Title: The role of N-substituents in radiationless deactivation of
aminated derivatives of locked GFP chromophore
Authors: Nadezhda S Baleeva, Snezhana O Zaitseva, Dmitriy A
Gorbachev, Alexander Yu Smirnov, Marina B Zagudaylova,
and Mikhail S. Baranov
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To be cited as: Eur. J. Org. Chem. 10.1002/ejoc.201700805
Link to VoR: http://dx.doi.org/10.1002/ejoc.201700805
Supported by

European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
The role of N-substituents in radiationless deactivation of
aminated derivatives of locked GFP chromophore
[
a]
[a]
[a]
[a]
Nadezhda S. Baleeva, Snezhana O. Zaitseva, Dmitriy A. Gorbachev, Alexander Yu. Smirnov,
[a],
Marina B. Zagudaylova, Mikhail S. Baranov *
Abstract: We report the creation of novel group of the ABDI-BF
2
quinoidal structure. The electronic effects have a greater impact on
the radiationless deactivation than conformational. Thus, the
electron-donating group introduction is more effective then the rigid-
derivatives creation. The presented dyes are characterized by high
fluorescence quantum yields and pH-independence in the
physiological pH range making them promising candidates for a wide
spectrum of fluorescent labeling applications.
fluorescent dyes based on the conformationally locked GFP
chromophore. We studied the intramolecular mechanism of
radiationless deactivation of ABDI-BF
various substituents at the nitrogen atom. The results of this study
and our previous work allowed us to claim that in case of ABDI-BF
2
fluorophore by introducing the
2
this deactivation is determined by the formation of the non-
fluorescent internal charge transfer exited state with a planar
Introduction
dyes substituted with differently sized nitrogen-containing rings
as well as non-rigid derivative with one flexible substituent.
Study of the optical properties of these compounds and
comparison with our previous results made it possible to better
understand the influence of conformational and electronic effects
of the substituents on formation of non-fluorescent ICT state.
Synthetic analogues of the chromophores of fluorescent
proteins have become a subject of extensive research for the
last three decades. The syntheses of various substituted
benzylidene imidazolones (BDI) allowed to reveal real
chromophores structures and led to better understanding of the
fluorescent proteins spectral behavior and their maturation
[1]
mechanism.
The other investigations revealed
a
broad
Results and Discussion
diversity of spectral and photochemical properties of synthetic
BDI derivatives and provoked several attempts of their practical
Synthesis
[2]
applications. Thus, various BDI derivatives have been used as
[3]
[4]
[5]
Conformationally locked aminated GFP chromophore
two-photon
and polymeric dyes,
ion sensors
and
[6]
analogues (ABDI-BF
imidazolones 1 by coordination-assisted borylation reaction
which was previously used in the synthesis of confomationally
2
, 2) were synthesized from corresponding
aggregation-induced emitters. Also, unique properties of BDI
[13]
[
7]
allowed to use them as fluorogenic labels for RNA and
[8]
proteins.
Recently we have proposed
[
9-12]
locked derivatives of the fluorescent proteins chromophores
and other dyes
a
strategy for dramatic
[14]
(Scheme 1). The detailed synthetic
improvement of fluorescence quantum yields (FQY) of various
BDI derivatives based on introducing of difluoroboryl group,
which provides the conformational lock at the benzylidene part
of the molecule responsible of non-radiative relaxation of the
excited state. This technique allowed us to synthesize several
procedures and characterization of compounds are presented in
Supporting Information.
[9]
[10]
fluorescent analogues of GFP , CFP
and Kaede
chromophores. We also demonstrated that the introducing of
amino group (giving ABDI-BF core) provides an improvement of
spectral properties of the BDI derivatives by making them red-
[11]
2
[
11,12]
shifted and pH-independent in the physiological range.
However, the possible formation of non-fluorescent excited
state with internal charge transfer (ICT) of the ABDI-BF dyes led
2
to the solvent-dependence of FQY and impedes their usage as
the common fluorescent markers.
Here, we present the creation of a novel group of ABDI-BF
2
[a]
Institute of Bioorganic Chemistry, Russian Academy of
Sciences,
Miklukho-Maklaya 16/10, 117997 Moscow, Russia
E-mail: baranovmikes@gmail.com
http://www.ibch.ru/en/structure/groups/TotalSynthesis/955
2
Scheme 1. Various ABDI-BF 2 and their synthesis
The yield of compounds 2a-c,e varied within the range of 50-
0%. At the same time, compound 2d was obtained with a yield
Supporting information for this article is given via a link at the end of
the document.
7
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European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
of only 10%, since the borylation of the azetidine derivative 1d
was accompanied by opening of the four-membered ring.
approach successfully achieved in the design of wide range of
[23,24]
the fluorescent dyes.
Probably, the introduction of such
small cycles impedes the changes of the angle between the C-
N-C atoms in the aminogroup, which affects the formation of the
ICT state. In this regard, in the present study we have created
2
Quantum yields of ABDI-BF dyes 2
[12]
[23]
Previously,
various ABDI-BF
we had already synthesized a number of
dyes and found that the derivatives with two
2
a series of ABDI-BF derivatives with differently sized nitrogen-
2
flexible alkyl substituents at the nitrogen atom (e.g. compound 2f, containing rings (ranging from azepane to aziridine, compounds
Scheme 1) showed noticeable dependence of FQYs on the
solvents nature (Fig 1).
2a-d, Fig 1).
As expected, borylation of the corresponding ABDI 1 led to
significant increase of FQY (Table 1, Table S2). Yet, noticeable
dependence of FQY on the solvents nature of the compounds
2a-d has been revealed (Table 1, Fig 1, Table S2). It was found,
that the most prominent FQY variation was observed for
compounds 2a and 2b with more than tenfold FQY difference
between water and dioxane solutions (Table 1, Table S1), that
was not greatly diverse them from the previously obtained
derivative 2f (Table 1, Fig 1). However, for derivatives with
smaller cycles (2c and 2d) this effect was much less
pronounced. The FQY variations of these compounds were
2
close to that of the rigid ABDI-BF 2g and 2h synthesized
previously. Thus, for more polar and protic solvents, a decrease
in the cycle size led to a noticeable increase in the quantum
yield (Fig. 1). Thus, according to the data obtained in this and
Fig. 1. Fluorescence quantum yields of compounds 2 in different solvents
The similar solvent dependence of FQY has been also
described for other fluorescent dyes, comprising dialkylamino
previous study we can guess that in case of ABDI-BF
2
[15]
[16]
substituents, such as Nile red and Nile blue,
other dyes.
Laurdan
This type of behavior is determined by formation
of an additional excited state, characterized by high charge
and
derivatives ICT state has planar and quinoidal character and its
formation cannot be completely suppressed by various kind of
amino group substituents fixation, as it was previously reported
[17]
[
18]
[25]
separation – an ICT state.
Formation of ICT state usually
for N-phenylpyrroles.
[19]
leads to a decrease of overall FQY, and may in several cases
In our previous work we showed that the least variation was
observed for rigid ABDI-BF containing hydrogen atoms in the
2
[20,16e,17a]
produce additional ICT emission band.
It is generally
considered, that in case of amino-derivatives, the ICT state can
be additionally accompanied by the conformational change to
form twisted geometry (TICT – Twisted Intramolecular Charge
amino group (2i, Scheme 1, Table 1).
In the present study we have created a non-rigid analogue of
this compound (2e, Scheme 1) and showed that FQY of this
[21]
Transfer, Scheme 2)
or alternatively, forming a quinoidal
compound was the least solvent-dependent among ABDI-BF
2
structure with planar geometry (PICT – Planar Intramolecular
Charge Transfer, Scheme 2).
derivatives synthesized in this work (Table 1, Fig 1, Table S2).
Obviously, that formation of the ICT state with the high degree of
charge delocalization is governed by the electronic effects of the
substituent in amino group. The hydrogen atom is the weaker
electron donors than alkyl groups and it less stabilizes the
positive charge on the nitrogen atom (Scheme 2) which
suppresses the formation of ICT state. Thus, we can guess that
[22]
2
in case of ABDI-BF derivatives electronic effects have a greater
impact on the formation of ICT state than conformational.
Solvatochromism of ABDI-BF dyes 2
2
Scheme 2. Possible structure of planar and twisted forms of ABDI-BF
excited ICT state
2
[12]
As reported previously for the other ABDI-BF
2
dyes
One of the possible approaches to reduce the formation of
ICT state is fixation of substituents at the nitrogen atom in the
ortho position of the aromatic system. Thus, we previously
created the group of the rigid analogues (Scheme 1, 2g and
substantial dependence of the absorption and emission maxima
on the nature of the solvent was observed for 2a-d (see Fig 2 for
2d and Table 1 and Fig S1-5 for other compounds).
[12]
2
h).
However, these compounds also demonstrate the FQY
variation, albeit to a lesser degree, than the derivatives with free
alkyls. Thus, the ICT state is most likely not twisted since the
formation of the twisted geometry is not possible for the
compounds 2g and 2h.
Another typical approach to avoid the ICT state formation is
[23]
introduction of cyclic substituents at the nitrogen atom.
This
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European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
shifе) upon excitation was also revealed that indicate a dramatic
shift in their photoacidity. Herewith the values of all the
parameters were not very different for the whole group of
compounds, however, the difference between the ground state
and the excited one decreased with decreasing cycle size.
3
Table 2. Solvatochromic spectral parameters (in 10 /cm) of 2a,c,d
compared to 2e
Cmpd
a
b
p
ν
o
R
[
12]
2
e Abs
-0.5
-0.6
-0.5
-0.6
-0.3
-0.3
-0.3
-0.3
0.6
0.6
0.6
0.3
-0.5
-0.4
-0.5
-0.4
-0.4
-0.3
-0.4
-0.6
-1.2
-1.1
-1.0
-1.0
20.2
20.0
20.1
20.9
19.4
19.1
19.2
19.3
0.84
0.87
0.86
0.90
0.95
0.97
0.96
0.95
Fig. 2. Fluorescence and absorbance spectra of 2d in different solvents
A pronounced bathochromic shift was observed in polar and
protic solvents, thus the difference between water and hexane
reached 30-50 nm. This phenomenon was shown previously for
2
a Abs
c Abs
d Abs
2
[26]
the other ABDIs
dyes.
and a group of dialkykamino-substituted
2
[
15-17]
[
12]
2
f Em
The nature of N-alkyl substituent also influenced on the
maxima positions (Fig 3). The derivatives with large cyclic
substituents 2a,b had the positions of the maxima almost
identical to the diethylamine derivative 2f, whereas the maxima
of the derivatives with smaller cycles of 2c,d were slightly (10-20
nm) blue-shifted, which correlates well with the previously
2
a Em
2
c Em
2
d Em
[
23]
reported data.
even larger blue-shift of absorption and emission spectra (Table
), which is explained by the less donor nature of the substituent.
This effect was previously observed for other fluorescent dyes
The compound 2e was characterized by an
2
Acid-base behavior of ABDI-BF dyes 2
The study of aqueous solutions of compounds 2 has shown
that an acidification leads to a noticeable bathochromic shift in
absorption maxima associated with protonation of the aromatic
amino group (Fig 4, S10-13). As it was shown earlier, all
compounds 2 in protonated form possessed merely identical
spectral characteristics, since conjugation of the amino group
lone pair is lost upon protonation leading to the disappearance
of the nitrogen atom substituents' influence.
1
[15,16]
[26a,e]
containing amino groups,
relates to the influence of the substituents nature on the position
of the Frontier Orbitals (HOMO and LUMO).
as well as other BDI.
and
Fig. 3. Fluorescence and sbsorbance spectra of compounds 2b-d,i in
acetonitrile
Analysis of the emission spectra of compounds 2a-d in
different solvents (Table S3) revealed no unusual emission
maxima, as it was shown previously for 2f and others, which
indicates a nonradiative decay of ICT state. This claim is
additionally confirmed by the fact that the positions of emission
Fig. 4. pH-titration of compound 2c
2
maxima of the ABDI-BF 2 dyes was is in s good agreement with
[27]
[12]
All the new compounds 2 obtained in this study were
characterized by a fairly low pKa value (2.5-1.5). At the same
time we surprisingly observed no significant differences between
pKa of the cyclic derivatives 2a-d. All these compounds were
characterized by pKa value close to 2, which is noticeably lower
than that of the dialkyl derivative 2f and its analogues (Table S4,
Fig S6-9).
a Kamlet-Taft model as it was shown previously for 2f. This
model correlates the spectral shift ν of the solute with the solvent
parameters that are responsible for the acidic (α), basic (β), and
polar solvating (π*) properties of the latter:

(1/cm) =  + p* + a + b
0
Multivariable regression of absorption and emission data for
compounds 2a,c,d,f data are summarized in Table 2.
As it was shown previously for 2f both the dipole moment
Stability of ABDI-BF dyes 2
2
(
parameter p) and acidity (parameter b) increase upon excitation
of other ABDI-BF derivatives 2. An unusual switch of b sign
from positive (hypsochromic shift) to negative (bathochromic
[
10]
Earlier,
we found out that the borylated derivatives of
2
chromophores are easily destructed in basic conditions. In this
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European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
regard, we also have studied the chemical and photo-stability of
the derivatives 2, which were synthesized in this work, as well as
and studied the impact of the ring size on the formation of non-
fluorescent ICT state. The results of this study and our previous
a
rigid and non-rigid compounds 2g and 2f obtained
2
work allowed us to claim that in case of ABDI-BF ICT exited
[12]
previously.
state is non-fluorescent and has quinoidal character with non-
twisted planar structure (PICT). Additional synthesis of the non-
rigid compound 2e bearing hydrogen atom in amino group
demonstrated that electronic effects have a greater impact on
the formation of ICT state than conformational. Thus, the
electron-donating group introduction is more effective then the
rigid-derivatives creation.
Highly-fluorescent cycloalkylamino (2c,d) and non-rigid (2e)
derivatives, demonstrated significantly greater chemical and
photo-stability than the rigid-derivatives which we proposed
earlier. They are also more chemically accessible compounds
and seem to be promising fluorescent dyes for various
applications.
We have established that the action of an aqueous solution
of methylamine (this reagent was chosen because it does not
lead to a decrease in solubility and is also used in the synthesis
of fluorescently labeled oligonucleotides) leads to the destruction
of all compounds 2. In this case, the disappearance of
fluorescence is also accompanied by loss of color, which is
evidence of the previously proposed mechanism of the
decomposition associated with the opening of the imidazolone
[10]
ring. The rigid derivative 2g which was obtained by us earlier
was the least stable, whereas the cyclic derivatives 2c,d
synthesized in the present study showed much greater stability
(
Fig 5A, Fig S14-15).
Experimental Section
Synthetic procedures, additional spectral data and pH titration curves are
presented in Supporting information.
Acknowledgements
The authors gratefully acknowledge support from the Russian
Foundation for Basic Research Grant 15-03-02856-a. This
research was carried out using equipment provided by the IBCH
core facility (CKP IBCH).
Keywords: GFP • chromophore • fluorescence • borylation •
ICT-state
Fig 5. Time-dependent decrease of compounds 2 fluorescence in
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in the present study a pronounced dependence of photostability
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European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
Table 1. Optical properties of compounds 2 in different solvents
Water
EtOH
CH
3
CN
EtOAc
Dioxane
pK(water)
Abs
Em
QY
Abs
Em
QY
Abs
Em
QY
Abs
Em
QY
Abs
Em
QY
Abs
Em
QY
Abs
519(30000)
567
509(33000)
558
495(31000)
553
492(34000)
541
497(34500)
538
2
a
2.0
0.04
~505*
0.26
500(33000)
564
0.34
484(37000)
558
0.82
481(39000)
546
0.76
487(42000)
540
2b
~560*
-*
~0.05*
516(33000)
562
0.33
508(51500)
562
0.17
508(35500)
560
0.65
493(55500)
556
0.30
493(35500)
552
0.65
482(59000)
551
0.78
491(38500)
541
0.77
480(62500)
542
0.78
496(39500)
537
0.76
484(62000)
535
2c
1.6
2.0
2.5
3.9
2d
0.53
495(31500)
548
0.76
500(35500)
544
0.70
475(32500)
536
0.80
477(36000)
527
0.84
481(36000)
521
2e
0.65
520(53500)
563
0.03
515(45000)
0.82
505(48000)
557
0.22
512(45000)
0.77
494(45500)
554
0.31
487(44000)
0.93
492(47000)
538
0.63
492(43500)
0.89
497(51000)
538
0.72
494(45000)
2
f
2i
Em
QY
560
556
547
540
533
2.2
0.63
0.64
0.67
0.64
0.65
(
* - non soluble enough)
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European Journal of Organic Chemistry
10.1002/ejoc.201700805
FULL PAPER
FULL PAPER
Nadezhda S. Baleeva,[a] Snezhana O.
Zaitseva, [a] Dmitriy A. Gorbachev, [a]
Alexander Yu. Smirnov,[a] Marina B.
Zagudaylova, Mikhail S. Baranov, [a],*
The role of N-substituents in
radiationless deactivation of
aminated derivatives of locked GFP
chromophore
We report the creation of a group of the fluorescent dyes based on the locked GFP
chromophores aminated derivatives. Their properties study allowed us to claim that
their fluorescence quantum yield variation is determined by the formation of the
non-fluorescent internal charge transfer exited state with a planar quinoidal
structure. Thus, the electronic effects have a greater impact on the radiationless
deactivation than conformational.
Key Topic: fluorescent dyes
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