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Dual Fluorescence in GFP Chromophore Analogues: Chemical Modulation of Charge Transfer and Proton Transfer Bands Tanmay Chatterjee, Mrinal Mandal, Ananya Das, Kalishankar Bhattacharyya, Ayan Datta, and Prasun K. Mandal J. Phys. Chem. B, Just Accepted Manuscript • DOI: 10.1021/acs.jpcb.6b01993 • Publication Date (Web): 21 Mar 2016 Downloaded from http://pubs.acs.org on March 22, 2016
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The Journal of Physical Chemistry
Dual Fluorescence in GFP Chromophore Analogues: Chemical Modulation of Charge Transfer and Proton Transfer Bands Tanmay Chatterjeeα, Mrinal Mandalα, Ananya Dasα, Kalishankar Bhattacharyyaβ, Ayan Dattaβ, and Prasun K. Mandalα* α
Department of Chemical Sciences, Indian Institute of Science Education and Research (IISER) - Kolkata, Mohanpur, WestBengal, 741246, India. e-mail:
[email protected] β
Department of Spectroscopy, Indian Association for the Cultivation of Science, 2A and 2B Raja S. C. Mullick Road, Kolkata, West Bengal, 700032, India Supporting Information
ABSTRACT: Dual fluorescence of GFP chromophore analogues has been observed for the first time. OHIM (o-Hydroxy imidazolidinone) shows only Charge transfer (CT) band, CHBDI (p-Cyclicamino o-hydroxy benzimidazolidinone) shows comparable intensity CT and PT (proton transfer) band, and MHBDI (p-methoxy o-hydroxy benzimidazolidinone) shows higher intensity PT band. It could be shown that differential optical behaviour is not due to conformational variation in solid or in solution phase. Rather control of excited state electronic energy level and excited state acidity constant by functional group modification could be shown to be responsible for differential optical behaviour. Chemical modification induced electronic control over relative intensity of charge transfer and proton transfer bands could thus be evidenced. Support from single crystal X-ray structure, NMR, Femtosecond to nanosecond fluorescence decay analysis, TDDFT based calculation provided important information and thus helped us understand the photophysics better. Dual fluorescence has been an interesting phenomenon in electronic spectroscopy as one ground state species creates two excited state species and thus exhibits two emission bands.1 Several researchers have been working on charge transfer and/or proton transfer fluorescence behaviour of GFP chromophore analogues,2-11however, dual fluorescence of GFP chromophore analogues has not been reported so far. In case of GFP chromophore (p-HBDI, see Chart 1) only normal emission band is observed,6,7 whereas in case of GFP chromophore analogue o-HBDI only proton transfer band is observed.2-5,10 oHBDI has been chemically modified by introducing CF3, OCH3 etc. groups, however, in these cases although emission maximum gets shifted, only proton transfer emission band is observed.4 Other researchers have replaced -OH group of pHBDI with NR2 group and only charge transfer emission band has been observed.9-12 Later, a few derivatives have been synthesized where both NR2 group at the para position (4position) as well as -OH group at the ortho position (2position) have been introduced but even in these cases only normal charge transfer emission band has been observed.13 Thus, strong quest to observe dual fluorescence for GFP chromophore analogues is quite active. To observe dual fluorescence of GFP chromophore analogues, researchers have introduced charge transfer (CT) donor moiety as well as -OH group for proton transfer (PT).13 Moreover, it is reported that rotation around exocyclic single and double bond (Φ & τ twist) has significant effect on the photophysics of GFP chromophore analogues.5,14-26 In an effort to
observe dual fluorescence we have synthesized OHIM, CHBDI, MHBDI (see Chart 1). Rationale behind the choice of these molecules are as follows: OHIM has both CT donor moiety (NEt2 group) and o-OH group for PT, CHBDI has a nitrogen containing bicyclic ring for CT and an o-OH group for PT, MHBDI has -OMe group (in place of NEt2 group) for CT and o-OH group for PT. -OMe group has lesser CT donor ability (σp = -0.27) than NEt2 group (σp = -0.72).27 In CHBDI the rotation of CT donor NR2 group (present in OHIM) will be fully hindered and Φ twist will be partly hindered. Thus, structural as well as electronic control over optical behaviour could be probed into. Three hydroxy (OHIM, CHBDI, MHBDI) and corresponding methoxy derivatives (OMIM, CMBDI, MMBDI, see Chart 1) will provide important insight about (chemical) structure- (optical) property relationship in GFP chromophore analogues. Dual fluorescence and significant structural and/or electronic control over it have been observed. Support from single crystal X-ray structure, NMR, femtosecond fluorescence decay, excited state electronic energy level diagram have been provided. Chart 1: Chemical structure of the compounds (non IUPAC number).
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Figure 1: Absorption and emission spectra of OHIM (a), CHBDI (b), and MHBDI (c) in four solvents of different polarity.
OHIM and its methoxy analogue (OMIM) have been synthesized following a literature procedure.13 CHBDI and its methoxy analogue (CMBDI) have been synthesized from 9-Formyl8-hydroxyjulolidine. MHBDI and its methoxy analogue (MMBDI) have been synthesized starting from 2-hydroxy-4methoxybenzaldehyde. Details of syntheses and characterisation have been provided in supp. info. S5. Absorption and emission spectra of OHIM, CHBDI, and MHBDI have been depicted in Fig. 1. In all the cases there exists only one absorption band. OHIM exhibits only one prominent emission band, whereas, CHBDI exhibits dual emission bands with equal intensity (in hexane), and MHBDI exhibits higher intensity longer wavelength emission band and lower intensity lower wavelength emission band (in MeOH). In case of CHBDI the relative intensity of longer wavelength emission band decreases as the polarity of the solvent increases. In case of MHBDI the relative intensity of lower wavelength emission band increases with increase in polarity of the solvent. Thus, interesting dual fluorescence, and chemical structural as well as solvent polarity control over the relative intensity of dual fluorescence bands could be observed. Let us now investigate the reason behind these interesting observations. We have explored several aspects in order to understand the underlying factors playing significant roles. The first possible reason behind differential emission behaviour could be different molecules adopting different conformations. In order to have an insight in this direction, we have obtained single crystal X-ray structure of all three derivatives and have been depicted in Fig. 2. As can be seen from Fig. 2, all three compounds adopt similar conformation in the solid state. For all three compounds strong H-bonding exists (shown as dotted line) between -OH group and imidazolidinone sp2 nitrogen (see supp. info. S6 for detail). O-N distances are ~2.60 Å (for OHIM), ~2.64 Å (for CHBDI) and ~2.61 Å (for MHBDI) and ° ° ∠O-H-N are 175.5 (for OHIM), ∠173.2 (for CHBDI) and ° ∠175.6 (for MHBDI).
Similar solid state conformation, however, does not necessarily guarantee the same in solution phase. In order to have an insight regarding solution phase conformation, 1H NMR study has been carried out. The 1H NMR peak correspond to hydroxyl proton appears at a δ value of 14.28 ppm for OHIM, 14.29 ppm for CHBDI, and 14.23 ppm for MHBDI resulting in Hbond strength of ~10.39 ± 0.2 KCal/mole for OHIM, ~10.40 ± 0.2 KCal/mole for CHBDI, and ~10.34 ± 0.2 KCal/mole for MHBDI (see supp. info. S3 for detail). From these data it can be concluded that H-bonding is quite strong even in solution phase and the solution phase conformation is similar to that in solid phase. However, this becomes quite intriguing that when ground state conformation is similar both in solid and solution phase then how is it possible that the emission behaviour are so different. What immediately comes to mind is that perhaps photoinduced excited state conformations are different for different compounds. In order to shed light in this direction, we have investigated photoinduced twisting around exocyclic double bond connecting the phenolic and imidazolidinone moieties (see supp. info. S12 for detail). We could not observe any photoinduced twisting leading to Z-E isomerisation in these three hydroxylic compounds which further strengthens the fact that H-bonding is quite strong.3,28,29 The photoinduced twisting around double bond leading to Z-E isomerisation could however be observed for the corresponding methoxy derivatives (OMIM, CMBDI, and MMBDI, see chart 1) (see supp. info. S12). Thus, excited state twisting leading to different conformations in the excited state and hence different photophysical behaviour for these three molecules is also ruled out. Thus, structural flexibility (in OHIM and MHBDI) or partial physical rigidity (in CHBDI) can't be the reason for the observed differential optical behaviour.
Figure 2: Single crystal X-ray structure OHIM (a), CHBDI (b), and MHBDI (c). Thermal ellipsoids are drawn at 50 % probability level.
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Table 1: Photophysical data of hydroxylic derivatives in different solvents. Compound
Solvent
Hex
OHIM
λem
ε
(nm)
(nm)
(M-1cm-1)
442
467
Ψ
λmon
τ1 (B1)
τ2 (B2)
τ3 (B3)
(nm)
(ps)
(ps)
(ps)
0.76 (0.25)
32.8 (0.75)
6.7x10-3 4
480
448
472
2.9x10
DCM
449
494
3.0x104
4.6x10-3
500
ACN
442
497
3.1x104
3.5x10-3
500
1.20 (0.20)
15.4 (0.80)
MeOH
452
505
3.1x104
1.4x10-3
500
0.70 (0.30)
14.8 (0.70)
Hex
459
484, 605
Ψ
1.4x10-3
Tol
468
498, 596
5.1x104
3.3X10-3
ACN
MeOH
Hex
473
463
475
509,606
511, 605
518, 603
5.0x10
4
4.9x104
5.1x104
6.5x10
Ψ
-3
Tol
DCM
CHBDI
ɸfl
λabs
2.1X10
-3
1.6x10-3
7.4x10-4
395
567
Ψ
1.0x10-3
397
445, 566
2.5x104
1.2x10-3
Tol
23.0
Ψ 500
