Switching the recognition ability of a photoswitchable receptor towards phosphorylated anions

Article abstract of DOI:10.1039/d0cc00926a

An azobenzene based photoswitchable macrocyclic receptor displays different binding affinities in its E and Z forms towards various phosphorylated coenzymes under physiological conditions with remarkable selectivity for ATP in the E-form and selectivity towards GTP in the photoisomerized Z-form. Linear discriminant analysis clearly separated the analytes using the E-form. An application of this method enabled monitoring the progress of enzymatic phosphorylation using a tyrosine kinase enzyme.

Full text of DOI:10.1039/d0cc00926a

View Article Online
View Journal
ChemComm
Chemical Communications
Accepted Manuscript
This article can be cited before page numbers have been issued, to do this please use: S.
Bandyopadhyay, M. S. Hossain, Sk. A. Rahaman, J. Hatai and M. Saha, Chem. Commun., 2020, DOI:
10.1039/D0CC00926A.
Volume 54
This is an Accepted Manuscript, which has been through the
Royal Society of Chemistry peer review process and has been
accepted for publication.
Number
January 2018
Pages 1-112
1
4
ChemComm
Chemical Communications
rsc.li/chemcomm
Accepted Manuscripts are published online shortly after acceptance,
before technical editing, formatting and proof reading. Using this free
service, authors can make their results available to the community, in
citable form, before we publish the edited article. We will replace this
Accepted Manuscript with the edited and formatted Advance Article as
soon as it is available.
You can find more information about Accepted Manuscripts in the
Information for Authors.
Please note that technical editing may introduce minor changes to the
text and/or graphics, which may alter content. The journal’s standard
Terms & Conditions and the Ethical guidelines still apply. In no event
shall the Royal Society of Chemistry be held responsible for any errors
or omissions in this Accepted Manuscript or any consequences arising
from the use of any information it contains.
ISSN 1359-7345
COMMUNICATION
S. J. Connon, M. O. Senge et al.
Conformational control of nonplanar free base porphyrins:
towards bifunctional catalysts of tunable basicity
rsc.li/chemcomm

Please do not adjust margins
Page 1 of 4
ChemComm
View Article Online
DOI: 10.1039/D0CC00926A
COMMUNICATION
Switching the recognition ability of a photoswitchable receptor
towards phosphorylated anions
Received 00th January 20xx,
Accepted 00th January 20xx
Munshi Sahid Hossain,a Sk. Atiur Rahaman,a Joydev Hatai,b Monochura Sahaa and Subhajit
Bandyopadhyay*a
DOI: 10.1039/x0xx00000x
An azobenzene based photoswitchable macrocyclic receptor reported.20 Selective recognition of ATP has also been
displays different binding affinities in its E and Z forms towards reported.21 A phosphate anion receptor was reported to
various phosphorylated coenzymes under physiological conditions achieve selectivity in affinity for ATP through π‒stacking of
with remarkable selectivity for ATP in the E–form while selectivity pyrene-adenine-pyrene sandwitch.22 An acyclic azobenzene
towards GTP in the photoisomerized Z–form. Linear discriminant based dipicolylamine modified γ-CyD−Cu2+ complexes that can
analysis clearly separated the analytes using the E–form. An recognize ATP has been reported to display a high selectivity
application of this method enabled monitoring the progress of an over other phosphoric acid derivatives.23
Competitive
enzymatic phosphorylation with a tyrosine kinase enzyme.
molecular recognition of GTP and ATP by adenylate kinase has
also been reported.24 Thus, there exists a number of excellent
probes for each of these coenzymes separately. It would be
beneficial if a single probe can detect and discriminate among
ATP, ADP, AMP, Pi and GTP anions.25 This motivated us to
design a switchable receptor that can detect these coenzymes
via a cross-reactive sensor array that can alter the selectivity
upon external inputs like light which could be cost effective
and beneficial in term of practical applications. Recently,
photomodulation of anion recognition through non-covalent
Photoswitchable molecules having efficient switching
behavior can be exploited to regulate molecular functions
reversibly using light.1, 2 Light-driven switches toggle reversibly
between two or more states, which gives rise to species having
different electronic structures, sizes, and shapes.3 Several
photochromic systems are used for switching among which
6
diarylethenes4, spiropyrans5, acyclic as well as macrocyclic
azobenzene7–11 are the most frequently used.
The design and development of receptor for selective
recognition of anions has received considerable attention in
the recent years,12, 13 not only for academic interest but also
interaction using photoswitchable system was reported by
27
Flood and by our group.26,
A spirooxazine based
photochromic sensor, differentiated among various metal ions
via a cross-reactive sensor array.28
because of their applications in diagnostics, imaging, and
15
environmental sciences.14,
Among various anions,
Here we report
a
macrocyclic azobenzene based
recognition of nucleotides, phosphates, pyrophosphates have
fluorescent receptor
1 that can recognize ATP selectively,
17
significant role in a plethora of biological processes.16,
preferentially in the 1E form and discriminate the rest of the
anions as a cross-reactive sensor array. However, upon 366 nm
light excitation, the photoisomerized 1Z form of the receptor
showed altered selectivity towards GTP. Thus, the
photoswitchable cross-reactive receptor stands as a unique
example through photoisomerization that detected various
phosphorylated anions via that linear discriminant analysis
(LDA). 29, 30
Adenosine triphosphate (ATP) is a multi-functional nucleotide
that plays an important role in energy transduction in
organisms and controls several metabolic processes.18–19 It also
turns on and off the activity of a protein by phosphorylation.
Selective recognition and discrimination of ATP and other
phosphate containing coenzymes in aqueous media has
emerged as an important area of research. Recently, a cationic
pillar[6]arene that can selectively bind to ATP has been
a.Department of Chemical Sciences, Indian Institute of Science Education and
Research (IISER) Kolkata, Mohanpur, Nadia, West Bengal 741246, India.
E-mail: sb1@iiserkol.ac.in
b.Institute of Organic Chemistry, University of Duisburg-Essen, Universitatsstraße
745141, Essen, Germany.
E-mail: joydev.hatai@uni-due.de
Scheme 1. Photomodulation of the receptor 1
Electronic Supplementary Information (ESI) available: [details of any
supplementary information available should be included here]. See
DOI: 10.1039/x0xx00000x
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 2 of 4
COMMUNICATION
Journal Name
As a potential application, this system was investigated for
the easy monitoring of a phosphorylation reaction. Post-
translational modifications of proteins or peptides upon
phosphorylation regulate cellular signalling pathways and
protein-protein interactions. Generally progress of the
reaction could be monitored by mass spectrometry in a
biological experiment.31 However, recently a few luminescent
probes having metal ions have also been employed for
monitoring the kinase activity.32 In this work, a metal free
probe, 1E has been used as an ATP-specific sensor to monitor
the phosphorylation of tyrosine. This enabled fast and real-
time in vitro monitoring of a kinase activity.
View Article Online
DOI: 10.1039/D0CC00926A
Initially, the receptor
1 was synthesized and characterized
by spectroscopic and spectrometric methods and also via
single crystal X-ray diffraction (CCDC number-1941103, see SI
for detailed experiments and characterisation). The 1E
⇌ 1Z
photoswitching behavior of the receptor was performed via
UV-Vis as well as 1HNMR spectroscopy, displayed characteristic
of azobenzene photo switching properties (Fig. S1-S3).
The optical sensing of ATP, ADP, AMP, Pi and GTP anions
were demonstrated by the fluorescent response of the
receptor 1E as well as 1Z due to its high sensitivity over UV
Fig. 1 Emission intensity of (A) 1E (10 µM) (B) 1Z (10 µM) with phosphate anions (10
equiv. each) in a Tris buffer of pH 6.8. The same scale have been maintained in all the
figures for the ease of comparison. (C) Systematic 1H NMR titration of 1E (5 mM) with
ATP (0 to 1 equivalent).
absorption (Fig. S4-S5). The influence of the anions (sodium to an aqueous solution of 1Z displayed only a modest
salts) on the fluorescent emission spectrum of receptor in enhancement in the emission intensity (1.5 fold and 1.9 fold
1
both form (10 µM) was investigated in an aqueous solution of respectively). However, addition of ADP, AMP and Pi (10
o
Tris (1 mM, buffered at pH 6.8) at 25 C. Upon addition of ATP equivalents each) did not influence the emission spectra (Fig.
to an aqueous solution of 1E, the emission spectrum shifted 1B and Fig. S7-S8B). Therefore, these fluorescence
(15 nm) towards the longer wavelengths. An increase in the enhancement and spectral shifts showed that the receptor 1E
ATP concentration from 0 to 10 equivalents resulted in a ~7 can easily detect and differentiate among these phosphate
fold fluorescence enhancement. However, the addition of 10 containing anions simultaneously through a combination of ∆λ
equivalents of ADP and GTP to 1E showed only a 3.5 fold and and ∆F values (see Table 1) and at the same time display
3.1 fold enhancement accompanied by a 16 nm and an 8 nm altered selectivity towards GTP upon isomerization. The
red shift respectively. In the case of AMP and Pi receptor, 1E binding of the ATP with 1E was also studied by 1H NMR
showed only a subtle emission intensity enhancement (~2 fold titration in deuterated solvent. Addition of ATP upto 1
and ~1.5 fold respectively) but with large red shifts (32 nm and equivalent displayed significant upfield shift of the aromatic as
30 nm respectively) (Fig. 1A and Fig. S6 and S8A). The degree well as aliphatic protons of the 1E to some extent (Fig. 1C).
of fluorescence enhancement was ascertained to follow the This clearly demonstrated that the 1E form binds strongly to
order ATP> ADP> GTP> AMP> Pi. On the contrary, no the anions. The Job’s plot showed 1:1 binding stoichiometry
appreciable spectral changes were observed upon addition of (χ[ATP] = 0.53) between 1E and the ATP (Fig. S10, binding
pyrophosphate (P2O74–) and a whole host of other common constant for the analytes are given in the Table S1 and Fig.
–
anions Cl–, AcO–, NO3–, NO2–, N3–, F–, HCO3 , CO32–, S2–, SO42– to S11-S15).
the 1E isomer (Fig. S9). Therefore, it was interesting to note
The geometry of the host receptor
1 (in the E isomer as
that the degree of enhancement along with the spectral shift well as in the
Z
isomer), negative charge of the guest anions
of each of these phosphate species served as a unique and the structure of the phosphate derivatives dictate the
fingerprint for their detection (see Table 1). This led us to the mode of interactions of the receptors with the various anions.
multivariate analysis of our data using linear discriminatory Crystal structure of 1E (crystalized from 1:1 v/v CH2Cl2/MeOH,
analysis (see later: multivariate analysis). Interestingly, upon CCDC No.-1941161) showed that it exists as a protonated form
exposure to the 366 nm light, ATP
conversion to the ATP 1Z system. The switching from the
the complex was marked by a significant decrease in the Fig. 2). Furthermore, the coenzymes ATP, ADP, AMP and GTP
emission intensity. Similar, reduction of the fluorescence also exists as ATP4–, ADP3–, AMP2– and GTP4– respectively
intensity was also observed for the ADP 1E, AMP 1E
under neutral pH condition.33 Receptor 1E experiences strong
GTP 1E as well as Pi
1E complexes upon exposure to the UV affinity towards ATP4– most likely via the coulombic interaction
⸦
1E complex underwent through the pyridine nitrogen even at
a neutral pH
⸦
E
to (protonated form captured in presence of ClO4– counter anion,
Z
⸦
⸦
,
⸦
⸦
light, although the extent of the decrement in the intensity between the negatively charged oxygen of the anions and the
was much lesser. Quantitative measurement of the binding of positively charged protonated form of the receptor. In
the anions with 1Z was also checked by systematic titration. It addition, H–bonding interaction of the guest anion with the
was found that the addition of 10 equivalents of ATP and GTP amine group of the adenosine moiety and the –N=N– group of
2 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins

Please do not adjust margins
Page 3 of 4
ChemComm
Journal Name
COMMUNICATION
the receptor is also possible (Fig. S18). The binding mode of the coenzymes. The emission pattern thus obtained subjected
View Article Online
DOI: 10.1039/D0CC00926A
to the chemometric methods (LDA) to discriminate among
these patterns. The LDA mapping clearly indicated that sensor
can detect and discriminate among the analytes. Even the
discrimination between AMP and Pi that is difficult using
univariate analysis, the LDA plot showed a clear discrimination
with 100% cross-validation (Fig. 3D). The LDA plot was used to
identify the 23 of 25 unknown samples of the analytes (ATP,
ADP, AMP, GTP and Pi), indicating 92% accuracy (Table S5).
The two-dimensional score plot for the 1E exhibited a
distinct separation among ATP, ADP and GTP with rest of the
observables (AMP or Pi) but the clustered data of AMP and Pi
in a close proximity. These observations were in conformity
with the previous results for the 1E and hence, reinforced the
capacity of differentiating among the phosphate based
analytes with the azobenzene system (Fig. 3B). The
Fig. 2 (A) Single crystal X-ray diffraction of 1E and (B) protonated 1E at 50% ellipsoid
probability.
the anions with the receptor can be speculated from the introduction of another factor to the score plot enhanced the
crystal structure as well as from the optimized geometry of the separation among the observables in the 3D plot which
receptor. The interactions mentioned earlier are most likely enabled a better separation. This result confirmed spectacular
with the 1E form because of its open structure (Fig. 2A) which ability of the receptors to recognize and differentiate among
the guest anions can access. This can lead to H–bonding the various analytes of biological importance (Fig. 3C).
interactions between the lone pair of the azo –N=N– of the
host and the NH moieties of the adenosine unit. The an ATP as the coenzyme producing a phosphorylated protein
coulombic interaction plays major role is intuitively and the ADP. Since our system can detect and discriminate
The biological phosphate transfer reaction uses kinases and
a
envisaged since the host and the guest bear the opposite among the phosphate derivatives, to explore the real-life use
charges under the working pH of 6.8. The columbic of our system, a biological kinase reaction was carried out to
interactions bring the guests and the host in the close monitor the kinase activity with the receptor 1E. Addition of
proximity and thereby facilitate the H–bonding interactions tyrosine by itself to the receptor 1E (2.5 µM, in Tris–buffered
between them. The weaker interaction of the GTP compared at pH 6.8) did not show any significant fluorescence signal
to ATP with 1E could stem from the lack of the H–bonding prior to the addition of ATP. Upon addition of ATP (25 µM), a
between the adenosinyl-NH2 and the lone pair of the –N=N– of drastic increase in the fluorescence signal with a sevenfold
the host that is present for the later. This was supported by enhancement of the fluorescence intensity at 390 nm was
the DFT studies at the B3LYP level using the polarizable observed. This is consistent with the response of 1E in the
continuum model (PCM) (Fig. S18). Interestingly the 1Z form presence of ATP. For real-time monitoring of the kinase
does not allow both the interactions because of the activity, the enzyme c–Src kinase was subsequently added and
geometrical constrains. Therefore, the molecular recognition the time course of the fluorescence spectra of the mixture
of 1E isomer with the guest anions is stronger than the showed a gradual decrease at 390 nm corresponding to a
corresponding 1Z isomer (Fig. S17).
The unique spectral shift as well as change in fluorescent
intensity in the presence of each of the anions further enabled
the discrimination among the analytes. For a qualitative
discrimination among the coenzymes, the fluorescence
intensities of 1E (10 µM) upon the addition of analytes in
aqueous media were also recorded (λex = 292 nm, Tris-
buffered at pH 6.8 at 25 oC) (Fig. 1). The changes in the
fluorescence intensities at different wavelength led to the
generation of unique optical fingerprints (Fig. 3A) for each of
Table 1. Fluorescence enhancement and wavelength shift of receptor 1 in E form as
well as in the Z form upon addition of the anions.
1E
1Z
Anions
ATP
Fold (F/Fo)
Shift (∆λ)
Fold (F/Fo)
Shift (∆λ)
Fig. 3 (A) Recognition patterns for the analytes (10 equiv. each) based on the variation
of fluorescence intensity of 1E (10 μM) at different wavelengths; (B) 2D and (C) 3D
score plot for an array of 1E discriminating the analytes; (D) The confusion matrix
obtained (using LDA classifier) as a classification accuracy trained on the full data set
for all the analytes in aqueous solution of Tris (buffered at pH 6.8) at 25 oC.
6.5
+15 nm
1.5
-6 nm
ADP
AMP
Pi
3.55
1.9
+16 nm
+32 nm
+30 nm
+8 nm
1.1
-
-3 nm
-
-
1.4
3.1
-
GTP
1.9
+4 nm
This journal is © The Royal Society of Chemistry 20xx
J. Name., 2013, 00, 1-3 | 3
Please do not adjust margins

Please do not adjust margins
ChemComm
Page 4 of 4
COMMUNICATION
Journal Name
138, 13111-13114; (b) J. Trads, K. Hüll, B. S. VMiewaAtsrtuiculerOan,linLe.
Laprell, T. Fehrentz, N. Görldt, K. A. Kozek, C. D. Weaver, N.
Klöcker, D. M. Barber and D. Trauner,DAOnI:g1e0w.1.03C9h/eDm0C.CIn0t0.9E2d6A.,
2019, 58, 15421-15428.
J. M. Mativetsky, G. Pace, M. Elbing, M. A. Rampi, M. Mayor
and P. Samori, J. Am. Chem. Soc., 2008, 130, 9192-9193.
M. Dong, A. Babalhavaeji, C. V. Collins, K. Jarrah, O. Sadovski,
8
9
Q. Dai and G. A. Woolley, J. Am. Chem. Soc. 2017, 139
13483-13486.
,
10 P. K. Hashim and N. Tamaoki, Angew. Chem. Int. Ed., 2011,
50, 11729- 11730.
11 Z. Yu, S. Weidner, T. Risse and S. Hecht, Chem. Sci., 2013, 4,
4156-4167.
12 (a) N. Busschaert, C. Caltagirone, W. V. Rossom and P. A.
Gale, Chem. Rev., 2015, 115, 8038-8155; (b) X. Chi, W. Cen, J.
A. Queenan, L. Long, V. M. Lynch, N. M. Khashab and J. L.
Sessler, J. Am. Chem. Soc., 2019, 141, 6468-6472; (c) T. Saha,
M. S. Hossain, D. Saha, M. Lahiri and P. Talukdar, J. Am.
Chem. Soc., 2016, 138, 7558-7567.
Fig. 4 Scheme of kinase activity with probe 1E (top); Kinase study of 1E (2.5 µM) with
ATP (25 µM) in presence of Tyrosine upon addition of c-Src kinase (1 µM) in Tris
(buffered at pH 6.8) at 25 oC (bottom).
decrease in the concentration of ATP (Fig. 4).
13 J. R. Dobscha, S. Debnath, R. E. Fadler, E. M. Fatila, M. Pink,
K. Raghavachari and A. H. Flood, Chem. Eur. J., 2018, 24
9841-9852.
,
In summary, an azobenzene based macrocyclic probe in
the
E–isomer showed strong binding affinity towards ATP
14 P. A. Gale, R. Pérez-Tomás and R. Quesada, Acc. Chem. Res.,
2013, 46, 2801-2813.
whereas the selectivity towards GTP was displayed by the
Z
–
isomer. All the species showed distinct patterns in terms of the
enhancement of their emission intensity (∆Fλ values) and the
spectral shifts (∆λ). Therefore, taking a combination of these
fluorescence enhancements at various wavelengths and their
spectral shifts with the receptors 1E, these phosphate
containing anions can easily be detected and differentiated
using the linear discrimination analysis method. On the
contrary, the other photoisomerized form 1Z exhibited very
poor discrimination ability towards the anions. Thus, one can
tune the recognition and discrimination ability of the receptor
15 B. S. Khakh and R. A. North, Nature, 2006, 442, 527.
16 A. K. H. Hirsch, F. R. Fischer and F. Diederich, Angew. Chem.
Int. Ed., 2007, 46, 338-352.
17 A. Ojida, Y. Mito-oka, K. Sada and I. Hamachi, J. Am. Chem.
Soc., 2004, 126, 2454-2463.
18 W. N. Lipscomb and N. Strater, Chem. Rev., 1996, 96, 2375-
2434.
19 T. Tabary and L. J. Ju, Immunol. Methods, 1992, 156, 55-60.
20 G. Yu, J. Zhou, J. Shen, G. Tang and F. Huang, Chem. Sci.,
2016, 7, 4073-4078.
21 (a) P. Mahato, A. Ghosh, S. K. Mishra, A. Shrivastav, S. Mishra
and A. Das, Chem. Commun., 2010, 46, 9134-9136; (b) T.
Yamada, S. Fujiwara, K. Fujita, Y. Tsuchido, T. Hashimoto and
T. Hayashita, Molecules, 2018, 23, 635.
1
via light. The practical application of this probe was
demonstrated with 1E as fluorescent probe for the
a
22 Z. Xu, N. J. Singh, J. Lim, J. Pan, H. N. Kim, S. Park, K. S. Kim
and J. Yoon, J. Am. Chem. Soc., 2009, 131, 15528-15533.
23 K. Fujita, S. Fujiwara, T. Yamada, Y. Tsuchido, T. Hashimoto
and T. Hayashita, J. Org. Chem., 2017, 82, 976-981.
24 P. Rognea, M. Rosselina, C. Grundströma, C. Hedberga, U. H.
Sauera and M. Wolf-Watza, Proc. Natl. Acad. Sci. U. S. A.,
2018, 115, 3012-3017.
monitoring of the progress of an enzymatic phosphate`
transfer reaction using a tyrosine kinase enzyme (c–Src kinase)
which enabled a real time monitoring of the in vitro kinase
activity.
This work is supported by DST-SERB (Grant No.
EMR/2017/003720). The authors thank Ms. Piyali Mukherjee
for her help with the DFT analysis.
25 J. L. Kolanowski, F. Liu and E. J. New, Chem. Soc. Rev.,
2018, 47, 195-208.
26 (a) F. C. Parks, Y. Liu, S. Debnath, , S. R. Stutsman, K.
Raghavachari and A. H. Flood, J. Am. Chem. Soc., 2018, 140
,
Conflicts of interest
There are no conflicts to declare.
17711-17723. (b) S. Lee and A. H. Flood, J. Phys. Org. Chem.,
2013, 26, 79-86.
27 A. Rananaware, M. Samanta, R. S. Bhosale, M. A. Kobaisi, B.
Roy, V. Bheemireddy, S. V. Bhosale, S. Bandyopadhyay and S.
Notes and references
V. Bhosale, Sci. Rep., 2016,
28 Y. Huang, F. Li, C. Ye, M. Qin, W. Ran and Y. Song, Sci. Rep.,
2015, , 9724.
6, 22928.
1
A. S. Lubbe, W. Szymanski and B. L. Feringa, Chem. Soc. Rev.,
2017, 46, 1052-1079.
(a) S. Kassem, T. van Leeuwen, A. S. Lubbe, M. R. Wilson, B. L.
Feringa and D. A. Leigh, Chem. Soc. Rev., 2017, 46, 2592-
2621; (b) M. Saha, S. Bandyopadhyay, Chem. Commun. 2019,
55, 3294-3297.
. yma ski, J. M. Beierle, H. A. V. Kistemaker, W. A.
Velema and B. L. Feringa, Chem. Rev., 2013, 113, 6114-6178.
M. Berberich, F. Würthner, Chem. Sci., 2012, 3, 2771-2777.
A. Koçer, M. Walko, W. Meijberg and B. L. Feringa, Science,
2005, 309, 755-758.
R. Klajn, Chem. Soc. Rev., 2014, 43, 148-184.
(a) M. ammerich, C. ch , C. tahler, . entes, F.
hricht, . ppner and R. Herges, J. Am. Chem. Soc., 2016,
5
29 J. Hatai, L. Motiei and D. Margulies, J. Am. Chem. Soc., 2017,
139, 2136-2139.
2
30 J. Han, B. Wang, M. Bender, K. Seehafer and U. H. Bunz, ACS
Appl. Mater. Interfaces, 2016, 8, 20415-20421.
31 S. Morelli, C. Buitrago, G. Vazquez, A. R. D. Boland and R.
Boland J. Biol. Chem., 2000, 275, 36021-36028.
3
32 (a) S. H. Hewitt, J. Parris, R. Mailhot and S. J. Butler, Chem.
Commun., 2017, 53, 12626-12629; (b) E. A. Weitz, J. Y.
Chang, A. H. Rosenfield, E. A. Morrow and V. C. Pierre, Chem.
Sci., 2013, 4, 4052-4060.
33 R. B. Stockbridge and R. Wolfenden, J. Biol. Chem., 2009,
284, 22747-22757.
4
5
6
7
4 | J. Name., 2012, 00, 1-3
This journal is © The Royal Society of Chemistry 20xx
Please do not adjust margins