skip to main content


Title: A Novel Dialkylamino GFP Chromophore as an Environment-Polarity Sensor Reveals the Role of Twisted Intramolecular Charge Transfer
We discovered a novel fluorophore by incorporating a dimethylamino group (–NMe2) into the conformationally locked green fluorescent protein (GFP) scaffold. It exhibited a marked solvent-polarity-dependent fluorogenic behavior and can potentially find broad applications as an environment-polarity sensor in vitro and in vivo. The ultrafast femtosecond transient absorption (fs-TA) spectroscopy in combination with quantum calculations revealed the presence of a twisted intramolecular charge transfer (TICT) state, which is formed by rotation of the –NMe2 group in the electronic excited state. In contrast to the bright fluorescent state (FS), the TICT state is dark and effectively quenches fluorescence upon formation. We employed a newly developed multivariable analysis approach to the FS lifetime in various solvents and showed that the FS → TICT reaction barrier is mainly modulated by H-bonding capability instead of viscosity of the solvent, accounting for the observed polarity dependence. These deep mechanistic insights are further corroborated by the dramatic loss of fluorogenicity for two similar GFP-derived chromophores in which the rotation of the –NMe2 group is inhibited by structural locking.  more » « less
Award ID(s):
2003550
NSF-PAR ID:
10289260
Author(s) / Creator(s):
; ; ; ;
Date Published:
Journal Name:
Chemosensors
Volume:
9
Issue:
8
ISSN:
2227-9040
Page Range / eLocation ID:
234
Format(s):
Medium: X
Sponsoring Org:
National Science Foundation
More Like this
  1. Twisting intramolecular charge transfer (TICT) is a common nonradiative relaxation pathway for a molecule with a flexible substituent, effectively reducing the fluorescence quantum yield (FQY) by swift twisting motions. In this work, we investigate coumarin 481 (C481) that contains a diethylamino group in solution by femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and theoretical calculations, aided by coumarin 153 with conformational locking of the alkyl arms as a control sample. In different solvents with decreasing polarity, the transition energy barrier between the fluorescent state and TICT state increases, leading to an increase of the FQY. Correlating the fluorescence decay time constant with solvent polarity and viscosity parameters, the multivariable linear regression analysis indicates that the chromophore’s nonradiative relaxation pathway is affected by both hydrogen (H)-bond donating and accepting capabilities as well as dipolarity of the solvent. Results from the ground- and excited-state FSRS shed important light on structural dynamics of C481 undergoing prompt light-induced intramolecular charge transfer from the diethylamino group toward –C=O and –CF3 groups, while the excited-state C=O stretch marker band tracks initial solvation and vibrational cooling dynamics in aprotic and protic solvents (regardless of polarity) as well as H-bonding dynamics in the fluorescent state for C481 in high-polarity protic solvents like methanol. The uncovered mechanistic insights into the molecular origin for the fluorogenicity of C481 as an environment-polarity sensor substantiate the generality of ultrafast TICT state formation of flexible molecules in solution, and the site-dependent substituent(s) as an effective route to modulate the fluorescence properties for such compact, engineerable, and versatile chemosensors. 
    more » « less
  2. Abstract

    Strategic incorporation of ameta‐dimethylamino (–NMe2) group on the conformationally locked green fluorescent protein (GFP) model chromophore (m‐NMe2‐LpHBDI) has drastically altered molecular electronic properties, counterintuitively enhancing fluorescence of only the neutral and cationic chromophores in aqueous solution. A ~200‐fold decrease in fluorescence quantum yield ofm‐NMe2‐LpHBDI in alcohols (e.g., MeOH, EtOH and 2‐PrOH) supports this GFP‐derived compound as a fluorescence turn‐on water sensor, with large fluorescence intensity differences between H2O and ROH emissions in various H2O/ROH binary mixtures. A combination of steady‐state electronic spectroscopy, femtosecond transient absorption, ground‐state femtosecond stimulated Raman spectroscopy (FSRS) and quantum calculations elucidates an intermolecular hydrogen‐bonding chain between a solvent –OH group and the chromophore phenolic ring –NMe2and –OH functional groups, wherein fluorescence differences arise from an extended hydrogen‐bonding network beyond the first solvation shell, as opposed to fluorescence quenching via a dark twisted intramolecular charge‐transfer state. The absence of ameta‐NMe2group twisting coordinate upon electronic excitation was corroborated by experiments on control samples without themeta‐NMe2group or with bothmeta‐NMe2andpara‐OH groups locked in a six‐membered ring. These deep mechanistic insights stemming from GFP chromophore scaffold will enable rational design of organic, compact and environmentally friendly water sensors.

     
    more » « less
  3. null (Ed.)
    Since green fluorescent protein (GFP) has revolutionized molecular and cellular biology for about three decades, there has been a keen interest in understanding, designing, and controlling the fluorescence properties of GFP chromophore ( i.e. , HBDI) derivatives from the protein matrix to solution. Amongst these cross-disciplinary efforts, the elucidation of excited-state dynamics of HBDI derivatives holds the key to correlating the light-induced processes and fluorescence quantum yield (FQY). Herein, we implement steady-state electronic spectroscopy, femtosecond transient absorption (fs-TA), femtosecond stimulated Raman spectroscopy (FSRS), and quantum calculations to study a series of mono- and dihalogenated HBDI derivatives (X = F, Cl, Br, 2F, 2Cl, and 2Br) in basic aqueous solution, gaining new insights into the photophysical reaction coordinates. In the excited state, the halogenated “floppy” chromophores exhibit an anti-heavy atom effect, reflected by strong correlations between FQY vs. Franck–Condon energy ( E FC ) or Stokes shift, and k nr vs. E FC , as well as a swift bifurcation into the I-ring (major) and P-ring (minor) twisting motions. In the ground state, both ring-twisting motions become more susceptible to sterics and exhibit spectral signatures from the halogen-dependent hot ground-state absorption band decay in TA data. We envision this type of systematic analysis of the halogenated HBDI derivatives to provide guiding principles for the site-specific modification of GFP chromophores, and expand design space for brighter and potentially photoswitchable organic chemical probes in aqueous solution with discernible spectral signatures throughout the photocycle. 
    more » « less
  4. null (Ed.)
    Biliverdin is a bile pigment that has a very low fluorescence quantum yield in solution, but serves as a chromophore in far-red fluorescent proteins being developed for bio-imaging. In this work, excited-state dynamics of biliverdin dimethyl ether (BVE) in solvents were investigated using femtosecond (fs) and picosecond (ps) time-resolved absorption and fluorescence spectroscopy. This study is the first fs timescale investigation of BVE in solvents, and therefore revealed numerous dynamics that were not resolved in previous, 200 ps time resolution measurements. Viscosity- and isotope-dependent experiments were performed to identify the contributions of isomerization and proton transfer to the excited-state dynamics. In aprotic solvents, a ∼2 ps non-radiative decay accounts for 95% of the excited-state population loss. In addition, a minor ∼30 ps emissive decay pathway is likely associated with an incomplete isomerization process around the C15C16 double bond that results in a flip of the D-ring. In protic solvents, the dynamics are more complex due to hydrogen bond interactions between solute and solvent. In this case, the ∼2 ps decay pathway is a minor channel (15%), whereas ∼70% of the excited-state population decays through an 800 fs emissive pathway. The ∼30 ps timescale associated with isomerization is also observed in protic solvents. The most significant difference in protic solvents is the presence of a >300 ps timescale in which BVE can decay through an emissive state, in parallel with excited-state proton transfer to the solvent. Interestingly, a small fraction of a luminous species, which we designate lumin-BVE (LBVE), is present in protic solvents. 
    more » « less
  5. The electronically excited singlet states of nitroaromatic compounds are often presumed to be essentially non-fluorescent. Nonetheless, a growing number of reports in the literature have demonstrated that certain structural types of nitroaromatics can indeed fluoresce, and often quite efficiently. Consideration of the mechanisms responsible for the typical fast or ultrafast non-radiative deactivation of the excited singlet states of nitroaromatics points to several general principles for their design that combine the strong electron-withdrawing properties of the nitro group with reasonable fluorescence quantum yields. An overview of published examples of fluorescent nitroaromatics emphasizes these concepts in the context of the importance of chromophore architecture and conformation and the defining roles of excited state charge transfer and solvent polarity in modulating the non-radiative decay channels that compete with fluorescence. Overcoming the stigma that nitroaromatics are intrinsically destined to be non-fluorescent thus paves the way for incorporating the strongly electron-withdrawing nitro group into the existing toolbox for the development of new nitro-substituted fluorophores and chromophores tuned to specific applications. 
    more » « less