Optically modulatable blue fluorescent proteins.

Blue fluorescent proteins (BFPs) offer visualization of protein location and behavior, but often suffer from high autofluorescent background and poor signal discrimination. Through dual-laser excitation of bright and photoinduced dark states, mutations to the residues surrounding the BFP chromophore enable long-wavelength optical modulation of BFP emission. Such dark state engineering enables violet-excited blue emission to be increased upon lower energy, green coillumination. Turning this green coillumination on and off at a specific frequency dynamically modulates collected blue fluorescence without generating additional background. Interpreted as transient photoconversion between neutral cis and anionic trans chromophoric forms, mutations tune photoisomerization and ground state tautomerizations to enable long-wavelength depopulation of the millisecond-lived, spectrally shifted dark states. Single mutations to the tyrosine-based blue fluorescent protein T203V/S205V exhibit enhanced modulation depth and varied frequency. Importantly, analogous single point mutations in the nonmodulatable BFP, mKalama1, creates a modulatable variant. Building modulatable BFPs offers opportunities for improved BFP signal discrimination vs background, greatly enhancing their utility.

Collapse and recovery of green fluorescent protein chromophore emission through topological effects.

Housed within the 11-stranded ß-barrel of the green fluorescent protein (GFP) is the arylideneimidazolidinone (AMI) chromophore, the component responsible for fluorescence. This class of small-molecule chromophore has drawn significant attention for its remarkable photophysical and photochemical properties, both within the intact protein and after its denaturation. All of the proteins so far isolated that have visible light fluorescence have been found to contain an AMI chromophore. These proteins comprise an extensive rainbow, ranging from GFP, which contains the simplest chromophore, p-hydroxybenzylideneimidazolidinone (p-HOBDI), to proteins having molecules with longer conjugation lengths and a variety of intraprotein interactions. The fluorescence invariably almost vanishes upon removal of the protective ß-barrel. The role of the barrel in hindering internal conversion has been the subject of numerous studies, especially in our laboratories and those of our collaborators. A better understanding of these chromophores has been facilitated by the development of numerous synthetic protocols. These syntheses, which commonly use the Erlenmeyer azlactone method, have evolved in recent years with the development of a [2 + 3] cycloaddition exploited in our laboratory. The synthetic AMI chromophores have allowed delineation of the complex photophysics of GFP and its derivatives. Upon denaturation, AMI chromophores are marked by 4 orders of magnitude of diminution in emission quantum yield (EQY). This result is attributed to internal conversion resulting from conformational freedom in the released chromophore, which is not allowed within the restrictive ß-barrel. To date, the photophysical properties of the AMI chromophore remain elusive and have been attributed to a variety of mechanisms, including cis-trans isomerization, triplet formation, hula twisting, and proton transfer. Advanced studies involving gas-phase behavior, solvent effects, and protonation states have significantly increased our understanding of the chromophore photophysics, but a comprehensive picture is only slowly emerging. Most importantly, mechanisms in structurally defined chromophores may provide clues as to the origin of the "blinking" behavior of the fluorescent proteins themselves. One approach to examining the effect of conformational freedom on rapid internal conversion of the chromophores is to restrict the molecules, both through structural modifications and through adjustments of the supramolecular systems. We thus include here a discussion of studies involving the crystalline state, inclusion within natural protein-binding pockets, complexation with metal ions, and sequestration within synthetic cavities; all of this research affirms the role of restricting conformational freedom in partially restoring the EQY. Additionally, new photochemistry is observed within these restricted systems. Many of the studies carried out in our laboratories show promise for these molecules to be adapted as molecular probes, wherein inclusion turns on the fluorescence and provides a signaling mechanism. In this Account, we present an overview of the AMI chromophores, including synthesis, overall photophysics, and supramolecular behavior. A significant amount of work remains for researchers to fully understand the properties of these chromophores, but important progress achieved thus far in photophysics and photochemistry is underscored here.

Microcrystals with enhanced emission prepared from hydrophobic analogues of the green fluorescent protein chromophore via reprecipitation.

Certain synthetic analogues of the green fluorescent protein (GFP) chromophore are almost nonfluorescent in dilute solutions but are strongly light-emissive in the solid state, thus exhibiting aggregation-induced emission (AIE) behavior. In the present work, two such hydrophobic derivatives of the GFP chromophore known to be fluorescent in the crystalline state (p-hexyloxy- and p-dodecyloxybenzylideneimidazolinone) were used to prepare aqueous suspensions of particles via a mild solvent-exchange reprecipitation (RP) method. This evolution was monitored at various experimental conditions by UV-vis absorption and fluorescence spectroscopy, fluorescence microscopy, as well as electron transmission and scanning microscopy. Both compounds spontaneously produced platelet-like microcrystals, the size and shape of which were influenced by the experimental conditions. The dodecyl derivative also led to the concomitant formation of nanofibers, a tendency reinforced by addition of poly(acrylic acid) to the RP medium. The photoluminescence properties of the solids produced by RP were compared to pristine microcrystalline powders obtained by crystallization in an organic solvent. Significant differences in the emission properties were found and are discussed. This study illustrates the fact that AIE fluorescence is strongly dependent on the nature of the particles and hence on the preparation methods. Being aware of these variations is important for the preparation and subsequent use of AIE-active compounds as fluorescent materials.

Conformationally locked chromophores as models of excited-state proton transfer in fluorescent proteins.

Members of the green fluorescent protein (GFP) family form chromophores by modifications of three internal amino acid residues. Previously, many key characteristics of chromophores were studied using model compounds. However, no studies of intermolecular excited-state proton transfer (ESPT) with GFP-like synthetic chromophores have been performed because they either are nonfluorescent or lack an ionizable OH group. In this paper we report the synthesis and photochemical study of two highly fluorescent GFP chromophore analogues: p-HOBDI-BF2 and p-HOPyDI:Zn. Among known fluorescent compounds, p-HOBDI-BF(2) is the closest analogue of the native GFP chromophore. These irrreversibly (p-HOBDI-BF(2)) and reversibly (p-HOPyDI:Zn) locked compounds are the first examples of fully planar GFP chromophores, in which photoisomerization-induced deactivation is suppressed and protolytic photodissociation is observed. The photophysical behavior of p-HOBDI-BF2 and p-HOPyDI:Zn (excited state pK(a)'s, solvatochromism, kinetics, and thermodynamics of proton transfer) reveals their high photoacidity, which makes them good models of intermolecular ESPT in fluorescent proteins. Moreover, p-HOPyDI:Zn is a first example of "super" photoacidity in metal-organic complexes.

Excited-state proton transfer in N-methyl-6-hydroxyquinolinium salts: solvent and temperature effects.

The excited-state proton transfer (ESPT) reaction of the "super"photoacid N-methyl-6-hydroxyquinolinium (MHQ) was studied using both fluorescence upconversion and time-correlated single photon counting (TCSPC) techniques. The ultrafast ESPT kinetics were investigated in various alcohols and water and determined to be solvent-controlled. The ESPT temperature dependence of MHQ was also studied in various alcohols and compared to that observed for another "super"photoacid, 5,8-dicyano-2-naphthol (DCN2). A full set of kinetic and thermodynamic parameters describing the ESPT was obtained. The protolytic photodissociation rate constant for MHQ was higher than that for DCN2, while the ESPT activation energies of MHQ were smaller. These findings are attributed to the approximately 3 orders of magnitude differences in excited-state acidities of MHQ and DCN2.

Steric and electronic effects in capsule-confined green fluorescent protein chromophores.

The turn-on of emission in fluorescent protein chromophores sequestered in an "octaacid" capsule is controlled by stereoelectronic effects described by a linear free energy relationship. The stereochemical effects are governed by both the positions and volumes of the aryl substituents, while the electronic effects, including ortho effects, can be treated with Hammett σ parameters. The use of substituent volumes rather than A values reflects packing of the molecule within the confines of the capsule.

Hydroxydialkylamino cruciforms: amphoteric materials with unique photophysical properties.

Two amphoteric cruciforms 6 and 7 (XF; 4,4'-[(1E,1'E)-(2,5-bis{[4-(dibutylamino)phenyl]ethynyl}-1,4-phenylene)bis(ethene-2,1-diyl)]diphenol, 4,4'-[{2,5-bis[(E)-4-(dibutylamino)styryl]-1,4-phenylene}bis(ethyne-2,1-diyl)]diphenol) were prepared by a Horner reaction followed by a Sonogashira coupling and subsequent deprotection. The XFs display significant changes in absorption and emission when exposed to trifluoroacetic acid, tetrabutylammonium hydroxide, and metal triflates. The substitution pattern of 6 and 7 leads to spatial separation of the frontier molecular orbitals, which allows the HOMO or LUMO of the XF to be addressed independently by acidic or basic agents. XF 6, which has hydroxyl groups on the styryl axis, displays changes in emission color upon exposure to ten amines in eight different solvents. The change in fluorescence upon the addition of amines was analyzed by linear discriminant analysis. These XFs may have potential in sensor applications for metal cations and amines.

Water-soluble distyrylbenzenes: one core with two sensory responses--turn-on and ratiometric.

The synthesis of four water-soluble distyrylbenzenes (compounds 1-4) is reported. Their acidochromicity in aqueous media was investigated. Blue shifts and increases in the quantum yields were observed as a general response. The pH-dependent photophysics of 1b-3b in water reveal unexpected protonation sequences upon titration: compound 1b is green-yellow fluorescent at high pH (10) but becomes very weakly fluorescent between pH 5 and pH 3, whereas below pH 2 strong blue fluorescence is observed. This behavior can be explained in terms of the interplay in the protonation of aniline and of the carboxylate groups. In compound 4, a higher basicity of the amino group is observed and ratiometric fluorescence change takes place upon protonation or on reaction with zinc salts in water. Compound 4 can therefore act as a weak ratiometric zinc ligand in water, even though it has only a dimethylamino unit as a binding motif.

Fluorescence turn on by cholate aggregates.

Bile salts, including sodium cholate (NaCh), are amphiphilic molecules with a concave hydrophilic side and a convex hydrophobic side. By forming aggregates in aqueous solution, these natural surfactants fulfill vital biological roles in the solubilization of cholesterol, lipids, and fat-soluble vitamins and thus are involved in the transport and absorption of important biological molecules. Following our success with the encapsulation of fluorescent protein chromophore (FP) analogs by synthetic hydrophobic and hydrophilic hosts, based upon substitution patterns, we now report the binding and turn on of other analogs by bile salt aggregates, observations which may lead to new tools for studying trafficking in these important systems.

Diferrocenyl molecular wires. The role of heteroatom linkers.

Diferrocenyl molecular "wires", in which two ferrocenes are linked by a conjugated chain, allowing the communication of redox information between the ferrocenes, are a versatile platform on which to investigate notions of molecular conductivity. In this paper, we examine the role of heteroatoms--including O, P, S, and Se, as well as C atoms in various oxidation states--separated from the ferrocenes by intervening double bonds which minimize any steric effects. Surprisingly, oxygen is a better electronic mediator than sulfur, a phenomenon we attribute to superior molecular orbital overlap. These fundamental studies on redox coupling will help to guide the design of efficient organic conductors for organic electronics.

Activation of fluorescent protein chromophores by encapsulation.

Chromophores related to fluorescent proteins, when sequestered into the "octaacid" capsule, recover their fluorescence. The fluorescence recovery is related to the inhibition of torsional motions within the cavity, implicating the single-bond torsion as an important contributor to internal conversion within this important class of chromophores.

Topochemistry and photomechanical effects in crystals of green fluorescent protein-like chromophores: effects of hydrogen bonding and crystal packing.

To obtain insight into the effects of the environment on the photophysics and photochemistry of the green fluorescence protein (GFP), eight crystal structures of six synthetic aryl-substituted analogues (2-fluoro, 2-methyl, 3-hydroxy, 3-methoxy, 2,4-dimethyl and 2,5-dimethyl) of the GFP chromophore (4-hydroxy-benzylidenedimethylimidazolinone) were determined and correlated with their two-dimensional steady-state and time-resolved solid-state excitation-emission spectra. The stacking between the molecules greatly affected the emission energy and the lifetime of the emission of the chromophore, implying that pi-pi interactions could be critical for the photophysics of GFP. The reaction pathways were dependent on the excitation energy, resulting either in [2 + 2] photodimerization at the bridging double bond (UV excitation) or flipping of the imidazolone ring (visible excitation). The meta-hydroxy chromophore (3-HOBDI) was the only GFP-chromophore analogue that was obtained as more than one stable polymorph in the pure state thus far. Due to the asymmetric substitution with hydrogen bond donors and acceptors, 3-HOBDI is tetramorphic, the forms showing distinctly different structure and behavior: (1) while one of the polymorphs (3-HOBDI-A), having multilayer structure with alternating stereochemistry of linear hydrogen-bonded motifs, undergoes photodimerization under UV light, (2) another (3-HOBDI-C), which has dimeric head-to-tail structure, shows Z-to-E isomerization via tau-one-bond flip of the imidazolone ring by excitation in the visible region. X-ray diffraction analysis of a partially reacted single crystal of 3-HOBDI-C provided the first direct evidence of tau-one-bond flip occurring in a GFP-like compound. Moreover, the cooperative action of the photodimerization of 3-HOBDI-A appears as a photomechanical effect of unprecedented magnitude for a single crystalline specimen, where photoexcited single crystals bend to more than 90 degrees without breaking.

Unsymmetrical cruciforms.

Five new, unsymmetrical 1,4-distyryl-2,5-bisphenylethynylbenzenes (cruciforms, XF) have been prepared by a sequential Horner reaction of the bisphosphonate of 2,5-diiodo-1,4-xylene with two different aromatic aldehydes. The obtained diiodide was coupled to phenylacetylene under Sonogashira conditions with (Ph(3)P)(2)PdCl(2) as catalyst. The resulting XFs carry dibutylamino, pyridyl, cyano, and diphenylamino residues on their styryl arms to give rise to donor-, acceptor-, and donor-acceptor-substituted XFs. The optical properties of these XFs were investigated. Titration studies using trifluoroacetic acid tracked changes in the electronic structure of the XFs upon protonation. Donor XFs display a blue shift in absorption and emission upon protonation, while the pyridyl-substituted XF displays red shift in absorption and emission upon protonation. In the case of the donor-acceptor XF carrying a pyridyl and an aminostyryl arm, the first protonation occurs either on the pyridine or on the dibutylamino arm; a red shift is seen in absorption (for the former) and a blue shift is observed in emission (for the latter). The titration studies indicate that the protonated XFs do not display kinetic photoacidity when operating either in dichloromethane or acetonitrile solutions. The trends observed for protonation were mirrored when the XFs bind to metal cations. While the binding constants of the metal cations to the XFs were lower than for that for protons, as in some cases full metalation of the XF could not be obtained, the results were qualitatively the same. We did not find dynamic excited-state decomplexation events in the XFs that we have investigated. The XFs, stilbene derivatives, are different from other reported, similarly structured fluorophores as they show significant ratiometric changes in emission upon metal complexation; thus, distyrylbenzene-derived fluorophores may be, in the end, viable choices as platforms for metal ion detection.

Guest/Host Complexes of Octa Acid and Amphiphilic Benzylidene-3-methylimidazolidinones Exchange Hosts within the NMR Time Scale.

Cavitand octa acid (OA) is established to form a stable capsular assembly with one or two hydrophobic guest molecules (1:2 or 2:2 guest/host complex). Examples are known in which the guest molecule tumbles within the capsule without disrupting the structure of the capsuleplex. This process makes the two OA molecules that form the capsule magnetically equivalent. In this study, we have examined the dynamics of capsules that host amphiphilic benzylidene-3-methylimidazolidinone molecules as guests. In these capsuleplexes, although the guest does not tumble, the two OA molecules become magnetically equivalent because the two OA molecules that form the capsule exchange their positions in the NMR time scale. This is equivalent to the content of the capsule remaining stationary while the capsule swirls around it. Benzylidene-3-methylimidazolidinones form both 1:1 and 1:2 supramolecular complexes with cavitand OA. Two-dimensional NMR, ROESY, and NOESY data suggest that in a 300 ms time scale, the two halves of the capsule exchange between themselves and with free OA. The conclusion drawn here provides valuable information concerning the stability of the OA capsuleplex and cavitandplex that is used as the well-defined space to control the excited-state chemistry and dynamics of confined guest molecules.

Activation and tuning of green fluorescent protein chromophore emission by alkyl substituent-mediated crystal packing.

O-Alkyl synthetic analogues of the green fluorescent protein chromophore are nonfluorescent in solution but demonstrate a bright emission in the crystalline state. Three-dimensional steady-state and time-resolved emission spectroscopies in the solid state, as well as single-crystal X-ray diffraction, reveal the nature of complex emission in the crystals. A hypsochromic emission shift with an increase of the alkyl size is determined by the monomer-aggregate emission ratio.

Traversing the "top-down/bottom-up" divide: molecular-scale lithography of self-assembled ribbons.

Use of graphitic (hexabenzocoronene-derived) molecules produces cholesteric ribbons which serve as molecular resists in a fluorine plasma. This procedure allows the shape of the molecular assemblies to be etched into the underlying silicon, validating the concept of "molecular resists".

Bis(4'-dibutylaminostyryl)benzene: spectroscopic behavior upon protonation or methylation.

We have investigated the UV/Vis absorption and emission of 1,4-bis(4'-dibutylaminostyryl)benzene (1) upon protonation with trifluoroacetic acid in dichloromethane and in acetonitrile. We find that 1 does not display significantly dynamic acidity in the excited state, that is, it is not a photoacid. Three protonation states of 1 were investigated, all of which, neutral, singly protonated, and bis-protonated, are fluorescent. As an isolable model for the mono-protonated species, a methylated derivative of 1 was prepared by reaction with methyl triflate. This species displays redshifted emission but similar absorption as the non-quaternized parent, 1. The strong observed emission of all three protonated states of 1 suggests that the kinetic photoacidity of 1* is low or absent. We assume that the lifetime of the excited state is too short to allow solvent reorganization and therefore we cannot make a statement on the thermodynamic photoacidity of the protonated forms of 1.

Excited-State Proton Transfer in Chiral Environments: Photoracemization of BINOLs.

We have studied excited-state proton transfer (ESPT) from chiral proton donors to chiral and achiral acceptors. The key role of the exergonicity of the reaction and the transition-state position along the reaction coordinate for the existence of an enantiomeric effect was established. This effect was observed for "super" photoacids (ΔG ≪ 0) and vanished for endergonic reactions (ΔG > 0) where a "late" transition state similar to planar achiral binaphtholate anion occurs. As a result, photoracemization was observed, as confirmed by circular dichroism spectroscopy. The photoracemization effects were studied for several chiral photoacids (BINOLs and their ethers) and proton acceptors (amines, aminoalcohols, and water) using UV-vis, steady-state fluorescence, and time-resolved fluorescence spectroscopies. The nature of the solvent and the proton acceptor, as well as the chemical structure of the BINOL, played a pivotal role in the photochemical reactivity of the system. Two proposed pathways competed for photoracemization: excited-state inter- and intra-molecular proton transfer, the former being more effective. Irradiation of the dimethoxy BINOL derivative, which lacks an acidic proton and cannot undergo ESPT, produced no appreciable reaction or racemization.

Fluorescence self-quenching of a mannosylated poly(p-phenyleneethynylene) induced by concanavalin A.

We report that static quenching of a mannosylated conjugated polymer (sugar-PPE) by Concanavalin A is positively dependent upon sugar-PPE concentration, that is, the recorded Stern-Volmer constants increase with increasing sugar-PPE concentration. Comparison with data obtained from isothermal titration calorimetry (ITC) display the increased sensitivity of the quenching method when compared to ITC. The proposed mechanism suggests the interaction of two or more chains of PPE with one Con A molecule leading to a quenched sugar-PPE-Con A construct.

Isomerization in fluorescent protein chromophores involves addition/elimination.

The green fluorescent protein (GFP) chromophore undergoes both photochemical and thermal isomerizations. Typically, the Z form is more stable and undergoes photochemical conversion to the E form followed by thermal reversion over a period of seconds or minutes. Although the mechanism of the thermal reversion has been the subject of some investigations, the surprisingly low activation energy for this process has not sparked any controversy. We now show that the chromophore is surprisingly stable in both E and Z forms and that the facile thermal reversion is the result of a novel nucleophilic addition/elimination mechanism. This observation may have implications for the intervention of such processes, as well as blinking and kindling, in fluorescent proteins.