Green Fluorescent Protein Chromophore Analogues Research Articles

Self-assembly solid-state enhanced fluorescence emission of GFP chromophore analogues: Formation of microsheets and microtubes oriented by molecular skeleton

The p-, m- and o-N,N-dimethylamino analogs of the green fluorescent protein (GFP) chromophore (denoted as p-DBHI, m-DBHI and o-DBHI) were synthesized by 2,3-cycloaddition. These three compounds were structurally characterized by NMR, HRMS and single crystal X-ray diffraction and were shown to be in the Z-form in both the solid phase and solution. Their fluorescence properties and self-assembly behaviors were investigated by UV–Vis, photoluminescence spectroscopy, fluorescence microscopy and scanning electron microscopy. They exhibited low fluorescence quantum yields in both protic and aprotic solvents, which was consistent with the reported results, and strong emissions in the solid state, thus exhibiting aggregation-induced emission (AIE) behaviors. By a solvent exchange method, the p-DBHI and o-DBHI were assembled into microsheets, while the m-DBHI was assembled into microtubule-like structures. The photoluminescence properties of the assemblies were compared with those of the pristine microcrystalline powders obtained by evaporation from organic solvents. The fluorescence quantum yields of the microcrystals obtained by self-assembly were recorded to 9.86 %, 3.37 % and 31.65 %, respectively, which were much higher than those of the corresponding pristine powders (4.71 %, 2.51 % and 17.03 %). This indicated that the fluorescence properties in the solid state depended on the morphologies of the particles.

Cocrystal Approach to Modulate the Photoluminescent Properties of a GFP Chromophore Analogue: Role of Halogen/Hydrogen Bonding in Achieving a Wide Range of Solid-State Fluorescence Emissions

Fine-tuning the photophysical properties of fluorescent organic solids is essential to attain multicolor displays and meet the demand for futuristic light-emitting materials. Here, we report the tunable luminescence of a green fluorescent protein (GFP) chromophore analogue, 3,4,5-TIA (A), based on the formation of two-component molecular cocrystals with six different coformers. Coformers selected to synthesize the binary cocrystals include 1,4-diiodotetrafluorobenzene (B), perfluoronaphthalene (C), 1,4-dibromotetrafluorobenzene (D), 2,3,5,6-tetrafluoroterephthalic acid (E), benzene-1,2,4,5-tetracarbonitrile (F), and benzene-1,2,4,5-tetracarboxylic acid (G). Interestingly, the cocrystals A•C and A•F showed molecular crystal polymorphism with a slight variation in fluorescence, revealing an aggregation-induced emission (AIE). A crystal structure analysis showed the interplay of hydrogen bonding, halogen bonding, and aromatic π-stacking interactions in associating neutral solid components in the cocrystal. All of the novel cocrystals displayed a wide range of photoluminescence ranging from blue to dark orange. The time-dependent density functional theory (TD-DFT) calculations indicate the changes in the energy level structures (HOMO to LUMO) in cocrystals that resulted in variations in fluorescence emission. The study aims to further understand the structure–property relationship between molecular arrangement and photoluminescence.

Effects of hydrogen bonds on two-photon absorption of Green fluorescent protein chromophore analogue

Effects of hydrogen bonds on two-photon absorption (TPA) of a new donor-acceptor type green fluorescent protein chromophore analogue are investigated by employing a combined molecular dynamics and quantum chemistry method. The probable configurations of the chromophore in water are extracted from molecular dynamics simulation and the TP A properties of more than twenty hydrogen bond complexes are computed by quadratic response theory. Thereby, the structure and property relations are established. Three types of hydrogen bonds including O⋯H–O, N–H⋯O and N⋯H–O can be formed between the chromophore and water molecules. The O⋯H–O induces a little decrease of TPA cross section with a red-shifted wavelength. The N–H⋯O gives rise to a great enhancement of TP A at a longer wavelength, while the N⋯H–O decreases TP A significantly and makes the wavelength blue-shifted. The reasons for these effects are rationalized well by using a two-state model analysis. The related molecular orbitals are also plotted to visualize the charge transfer characters. In addition, the averaged TP A spectrum is obtained by calculating the probabilities of various hydrogen bond complexes. Our research could provide a good insight into the design of two-photon materials by making use of hydrogen bond networks.

Ultrafast excited state relaxation of a model green fluorescent protein chromophore: Femtosecond fluorescence and transient absorption study

The intrinsic excited state properties of green fluorescent protein (GFP) chromophore have inspired syntheses of its structural analogues with a motivation to replicate or improve its excited state properties for biological imaging. In this work, we report torsional-motion mediated multi-coordinate relaxation pathway to explain the excited state dynamics of a GFP chromophore analogue, (4Z)-4-(4-N,N-Dimethylaminobenzylidene)-1,2-diphenyl-1,4-dihydro-5H-imidazolin-5-one (DPI), using femtosecond fluorescence up-conversion, femtosecond transient absorption and quantum chemical calculations. The excited state dynamics of this chromophore involves the formation of an N,N-disubstituted amine twisting mediated intramolecular charge transfer state on a sub-picosecond timescale. This intramolecular charge transfer event prepares an intermediate state that undergoes a “non-volume conserving” twist motion along an exocyclic double bond. The quantum chemical computations further predict that the twist motion along the exocyclic double bond is activated and leads to the formation of a conical intersection between S1 and S0 states of DPI, which is the main nonradiative channel of deactivation. This study confirms that for a structural analogue of GFP chromophore to exhibit excited state properties like the GFP, would require preventing such nonradiative pathways, which can be achieved by restricting the twisting degree of freedom about the exocyclic double bond.

A Biomimetic Emitter Inspired from Green Fluorescent Protein.

The unique tripeptide structure of green fluorescent protein (GFP), a Ser-Tyr-Gly motif, generates the mature chromophore in situ to define the emission profiles of GFP. Here, we describe the rational design and discovery of a biomimetic fluorescent emitter, MBP, by mimicking the key structure of the Ser-Tyr-Gly motif. Through systematically tailoring the tripeptide, a family of four chromophores were engineered, while only MBP exhibited bright fluorescence in different fluid solvents with highly enhanced quantum yields. Distinct to previous hydrogen-bonding-induced fluorescence quenching of GFP chromophore analogues, the emission of MBP was only slightly decreased in protic solvents. Heteronuclear multiple bond correlation techniques demonstrated the fundamental mechanism for enhanced fluorescence emission owing to the synergy of the formation of the intramolecular hydrogen-bonding-ring structure and the self-restricted effect, which was further illustrated via theoretical calculations. This work puts forward an extraordinary approach toward highly emissive biomimicking fluorophores, which gives new insights into the emission mechanisms and photophysics of GFP-like chromophores.

Mechanical-bending-induced fluorescence enhancement in plastically flexible crystals of a GFP chromophore analogue

Mechanical-bending-induced fluorescence enhancement in plastically flexible crystals of a GFP chromophore analogue

A green fluorescent protein chromophore analogue to promote self-assembly of diphenylalanine into fluorescent microtubes

It is known that misfolding and aggregation of amyloid-beta (Aβ) peptide was thought to be the causing of Alzheimer's disease (AD). Until now, there are no effective drugs for this disease. Therefore, it is necessary to develop excellent probes for early diagnosis and assisting drug research and development of AD. Diphenylalanine peptide (FF), as a key and the smallest core recognition sequence of amyloid-beta (Aβ) peptide, has attracted much attention. In this paper, we designed and synthesized meta- and para-amino green fluorescent protein chromophore analogues m-DBI and p-DBI. Comparing their fluorescence properties, we found that m-DBI displayed relatively strong fluorescence in aprotic organic solvents due to a large barrier for the Z→E photoisomerization, while it showed weak fluorescence in protic solvents as a result of solvent-solute H-bonding interactions. Importantly, it could drive and assemble with FF into microtubes with “turn-on” fluorescence. In contrast, p-DBI exhibited very weak fluorescence in both protic and aprotic solvents, which is due to the ultrafast Z→E photoisomerization. Furthermore, it could not co-assemble with FF and even displayed weak fluorescence in solid state. In addition, FF-m-DBI co-assembly microtubes could be partially disassembled through incubating with a potent inhibitor of Aβ aggregation EGCG, accompanied by a decrease in fluorescence intensity, shows the potential application of screening possible inhibitor molecules of AD diseases.

Five concomitant polymorphs of a green fluorescent protein chromophore (GFPc) analogue: understanding variations in photoluminescence with π-stacking interactions.

The synthetically modified green fluorescent protein chromophore analogue 3,4,5-trimethoxybenzylidene imidazolinone (1) yielded five polymorphs (I, II, III, IV, V) concomitantly irrespective of the solvent used for crystallization. The pentamorphic modification of 1 is solely due to the interplay of iso-energetic weak intermolecular interactions in molecular associations as well as the conformational flexibility offered by a C-C single bond, which connects the electron-deficient moiety imidazolinone with the electron-rich trimethoxybenzylidene group. A common structural feature observed in all the polymorphs is the formation of a `zero-dimensional' centrosymmetric dimeric unit through a short and linear C-H...O hydrogen bond engaging phenyl C-H and imidazolinone carbonyl oxygen. However, the networking of these dimeric units showed a subtle difference in all the polymorphs. The 2D isostructurality was observed between polymorphs I, II and III, while the other two polymorphs IV and V revealed only `zero-dimensional' isostructurality. The different fluorescence emissions of Form I (blue) and Forms II to V (yellow) were attributed to the differences in π-stacking interactions. It shows that one can modulate the photophysical properties of these smart materials by slightly altering their crystal structure. Such an approach will aid in developing new multi-colour organic fluorescent materials of varying crystal structures for live-cell imaging and fluorescent sensing applications.

Mechanical‐Bending‐Induced Fluorescence Enhancement in Plastically Flexible Crystals of a GFP Chromophore Analogue

AbstractSingle crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.

Mechanical-Bending-Induced Fluorescence Enhancement in Plastically Flexible Crystals of a GFP Chromophore Analogue.

Single crystals of optoelectronic materials that respond to external stimuli, such as mechanical, light, or heat, are immensely attractive for next generation smart materials. Here we report single crystals of a green fluorescent protein (GFP) chromophore analogue with irreversible mechanical bending and associated unusual enhancement of the fluorescence, which is attributed to the strained molecular packing in the perturbed region. Soft crystalline materials with such fluorescence intensity modulations occurring in response to mechanical stimuli under ambient pressure conditions will have potential implications for the design of technologically relevant tunable fluorescent materials.

Unraveling the Effect on Luminescent Properties by Postsynthetic Covalent and Noncovalent Grafting of gfp Chromophore Analogues in Nanoscale MOF-808.

Here, we demonstrate mimicking of photophysical properties of native green fluorescent protein (gfp) by immobilizing the gfp chromophore analogues in nanoscale MOF-808 and further exploring the bioimaging applications. The two virtually nonfluorescent gfp chromophore analogues carrying different functionalities, BDI-AE (COOH/COOMe) and BDI-EE (COOMe/COOMe) were immobilized in nanosized MOF-808 via postsynthetic modification. An 1H NMR and IR study confirms that BDI-AE was coordinated in NMOF-808, whereas BDI-EE was just noncovalently encapsulated. Interestingly, the extremely weakly fluorescent monomers BDI-AE and BDI-EE (QY = 0.01-0.03%, lifetime = 0.01-0.03 ns) showed a 102-fold increase in quantum efficiency with a significantly longer excited-state lifetime (QY = 1.8-5.6%, lifetime 0.89-1.49 ns) after immobilization in the NMOF-808 scaffold. Moreover, BDI-AE@MOF-808 has 4 times higher quantum efficiency as well as longer excited-state lifetime in comparison to BDI-EE@NMOF-808 due to the rigidity imposed in the chromophore upon coordination with Zr4+ in the former case. Further, a cell viability test performed for BDI-AE@NMOF-808 in HeLa cells confirmed the nontoxic nature of the material and, more importantly, bioimaging applications have also been explored successfully.

Photochromic Conjugated Microporous Polymer Manifesting Bio-Inspired pcFRET and Logic Gate Functioning.

Design and synthesis of solid-state photochromic materials remain a challenge because of high structural constrain. However, this can be mitigated in attaining structural flexibility by introducing permanent porosity into the system. Here, we report for the first time the design and synthesis of a photochromic conjugated microporous polymer (pcCMP) by assembling photochromic dithienylethene aldehyde and benzene-1,3,5-tricarbohydrazide. The yellow photo-isomer pcCMP-O gets converted to a deep-green photo-isomer pcCMP-C by UV-light irradiation, which can be reverted to pcCMP-O by visible light or thermal treatment. Owing to the thermo-irreversible nature, the pcCMP is found to be suitable for designing an INH functioning logic gate. pcCMP-C shows highly enhanced conductivity (92 times) because of enhanced conjugation compared to pcCMP-O. Furthermore, we demonstrate the bio-inspired photo-switchable pcFRET process by encapsulation of a red-emissive green fluorescent protein (gfp) chromophore analogue into the pcCMP. This material shows high processibility and has been exploited further for secret writing.

Short Duplex Module Coupled to G-Quadruplexes Increases Fluorescence of Synthetic GFP Chromophore Analogues

Aptasensors became popular instruments in bioanalytical chemistry and molecular biology. To increase specificity, perspective signaling elements in aptasensors can be separated into a G-quadruplex (G4) part and a free fluorescent dye that lights up upon binding to the G4 part. However, current systems are limited by relatively low enhancement of fluorescence upon dye binding. Here, we added duplex modules to G4 structures, which supposedly cause the formation of a dye-binding cavity between two modules. Screening of multiple synthetic GFP chromophore analogues and variation of the duplex module resulted in the selection of dyes that light up after complex formation with two-module structures and their RNA analogues by up to 20 times compared to parent G4s. We demonstrated that the short duplex part in TBA25 is preferable for fluorescence light up in comparison to parent TBA15 molecule as well as TBA31 and TBA63 stabilized by longer duplexes. Duplex part of TBA25 may be partially unfolded and has reduced rigidity, which might facilitate optimal dye positioning in the joint between G4 and the duplex. We demonstrated dye enhancement after binding to modified TBA, LTR-III, and Tel23a G4 structures and propose that such architecture of short duplex-G4 signaling elements will enforce the development of improved aptasensors.

Dual-Self-Restricted GFP Chromophore Analogues with Significantly Enhanced Emission.

The tremendous gap of fluorescence emission of synthetic green fluorescent protein (GFP) chromophore to the protein itself makes it impossible to use for applications in molecular and cellular imaging. Here, we developed an efficient methodology to enhance the photoluminescence response of synthetic GFP chromophore analogues by constructing dual-self-restricted chromophores. Single self-restricted chromophores were first generated with 2,5-dimethoxy substitution on the aromatic ring, which were further modified with phenyl or 2,5-dimethoxy phenyl to form dual-self-restricted chromophores. These two chromophores showed an obvious solvatofluorochromic color palette across blue to yellow with a maximum emission Stokes shift of 95 nm and dramatically enhanced fluorescence emission in various aprotic solvents, especially in hexane, where the QY reached around 0.6. Importantly, in acetonitrile and dimethyl sulfoxide, the fluorescence QYs of both chromophores were over 0.22, which were the highest reported so far in high polar organic solvents. Meanwhile, the fluorescence lifetimes also improved obviously with the maximum of around 4.5 ns. Theoretical calculations revealed a more favorable Mülliken atomic charge translocation over the double-bond bridge and illustrated much higher energy barriers for the Z/E photoisomerization together with larger bond orders compared with the GFP core chromophore, p-HBDI. Our work significantly improved the fluorescence emission of synthetic GFP chromophore analogues in polar solvents while reserved the multicolor emitting function, providing a solid molecular motif for engineering high-performance fluorescent probes.

A novel carbazolyl GFP chromophore analogue: synthesis strategy and acidic pH-activatable lysosomal probe for tracing endogenous viscosity changes

A novel, acidic pH-activatable carbazolyl GFP chromophore analogue was designed for tracing lysosomal viscosity changes.

S1/S0 Potential Energy Surfaces Experience Different Types of Restricted Rotation: Restricted Z/ E Photoisomerization and E/ Z Thermoisomerization by an Out-of-Plane Benzyl Group or In-Plane m-Pyridinium Group?

Any method that can enhance the fluorescence of fluorophores is highly desirable. Fluorescence enhancement accomplished by restricted Z/ E photoisomerization through intramolecular steric hindrance or relatively high bond order of a C═C double bond in a S1 excited state has rarely been studied. In this article, we used green fluorescent protein (GFP) chromophore analogues as a model to get new physical insights into the restricted Z/ E photoisomerization and E/ Z thermoisomerization phenomena. We found that the S1 and S0 potential energy surfaces (PESs) of the GFP chromophore analogues experience two dramatically different types of restricted rotation, and 2b can be a representative example. In its S1 PES, it is not the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group but the relatively high bond order of the I-bond in the S1 excited state of 2b that makes it have a higher barrier for the Z/ E photoisomerization, a smaller Z/ E photoisomerization quantum yield, and a higher fluorescence quantum yield. In its S0 PES, it is not the reduced bond order of the I-bond in the S0 ground state of 2b but the intramolecular steric hindrance between the out-of-plane benzyl group and the in-plane m-pyridinium group that makes it have an extra higher barrier for E/ Z thermoisomerization and a much smaller E/ Z thermoisomerization rate constant.

Synthesis of the New Green Fluorescent Protein Chromophore Analogue Starting from a Cinnamic Aldehyde Derivative

A novel derivative of green fluorescent protein chromophore (Z)-5-((E)-3-(4-hydroxyphenyl)allylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one was synthesised. The desired compound was prepared by the synthetic approach based on the use of carboxyimidate. Comparison of optical properties of the obtained compound and of the classical GFP chromophore analogue (Z)-5-(4-hydroxybenzylidene)-2,3-dimethyl-3,5-dihydro-4H-imidazol-4-one showed the shift of absorption and emission maxima to the long-wavelength region induced by the increase of the conjugated π-system in the benzylidene fragment.

Synthesis and spectroscopic characterization of novel GFP chromophore analogues based on aminoimidazolone derivatives.

In order to improve the fluorescence properties of the green fluorescent protein chromophore, p‑HOBDI ((5‑(4‑hydroxybenzylidene)‑2,3‑dimethyl‑3,5‑dihydro‑4H‑imidazol‑4‑one), sixteen dihydroimidazolone derivates were synthesized from thiohydantoin and arylaldehydes. The synthesis developed is an efficient, novel, one-pot procedure. The study provides a detailed description of the spectroscopic characteristics of the newly synthesized compounds, using p‑HOBDI as a reference. The new compounds all exhibited significantly stronger fluorescence than p‑HOBDI, up to 28 times higher quantum yields. An experimental and theoretical investigation of the relationship of the fluorescence properties with the molecular structure was also carried out. A good correlation was found between the emission wavenumber and the Hammett constant of the functional group, which suggests the intermolecular charge transfer (ICT) mechanism between the aromatic groups.

Synthesis of Green Fluorescent Protein Chromophore Analogues for Interdisciplinary Learning for High School Students

Interdisciplinary outreach activities with easy-to-implement experiments have been developed to enhance high school students’ interest in chemistry and their inquisitive minds with active learning strategies. The synthesis of a model chromophore of the green fluorescent protein was employed as a key motif. Students simply enjoyed observing changes in the substances as well as witnessing a variety of fluorescent emissions. They gradually realized the importance of molecular design in the understanding and development of chemistry, biology, physics, and medicine.

Protonation of the imino nitrogen deactivates the excited state of imidazolin-5-one in the solid state

The imino nitrogen of p-methoxybenzylideneimidazolinone (pMBDI) was protonated using aqueous $$\hbox {HClO}_{4}$$ and HCl acid and crystallized in monoclinic crystal system with $$\textit{P2}_{\textit{1}}/n$$ space group. Interestingly, the protonated compounds are fluorescent in polar solvents but non-fluorescent in non-polar solvent as well as in the solid state. These results point to the existence of a non-radiative pathway involving the imidazole nitrogen in the quenching of excited states in these compounds. SYNOPSIS The imino nitrogen of p-methoxybenzylideneimidazolinone (pMBDI) was protonated and crystallized in monoclinic crystal system with $$\textit{P2}_{\textit{1}}/n$$ space group. Interestingly, the protonation at imino nitrogen of imidazolinone ring results in the loss of the fluorescence property of gfp chromophore analogue (p-methoxybenzylideneimidazolinone).