7-membered-ring effect on fluorescence quantum yield: does metal-complexation-induced twisting-inhibition of an amino GFP chromophore derivative enhance fluorescence?

To investigate two aspects, namely, (1) the 7-membered-ring effect on fluorescence quantum yield and (2) whether metal-complexation-induced twisting-inhibition of an amino green fluorescent protein (GFP) chromophore derivative is bound to enhance fluorescence, a novel GFP-chromophore-based triamine ligand, (Z)-o-PABDI, is designed and synthesized. Before complexation with metal ions, the S1 excited state of (Z)-o-PABDI undergoes τ-torsion relaxation (Z/E photoisomerization) with a Z/E photoisomerization quantum yield of 0.28, forming both ground-state (Z)- and (E)-o-PABDI isomers. Since (E)-o-PABDI is less stable than (Z)-o-PABDI, it is thermo-isomerized back to (Z)-o-PABDI at room temperature in acetonitrile with a first-order rate constant of (1.366 ± 0.082) × 10-6 s-1. After complexation with a Zn2+ ion, (Z)-o-PABDI as a tridentate ligand forms a 1 : 1 complex with the Zn2+ ion in acetonitrile and in the solid state, resulting in complete inhibition of the φ-torsion and τ-torsion relaxations, which does not enhance fluorescence but causes fluorescence quenching. (Z)-o-PABDI also forms complexes with other first-row transition metal ions Mn2+, Fe3+, Co2+, Ni2+ and Cu2+, generating almost the same fluorescence quenching effect. By comparison with the 2/Zn2+ complex, in which a 6-membered ring of Zn2+-complexation enhances fluorescence significantly (a positive 6-membered-ring effect on fluorescence quantum yield), we find that the flexible 7-membered rings of the (Z)-o-PABDI/Mn+ complexes trigger their S1 excited states to relax through internal conversion at a rate much faster than fluorescence (a negative 7-membered-ring effect on fluorescence quantum yield), leading to fluorescence quenching regardless of the type of transition metal that complexes with (Z)-o-PABDI.

Against the NEER principle: the third type of photochromism for GFP chromophore derivatives.

Photochromism is the heart of photochromic fluorescent proteins. Excited-state proton transfer (ESPT) is the major cause of photochromism for the green fluorescent protein (GFP) and Z-E photoisomerization through τ-torsion is the major cause of photochromism for Dronpa (a GFP mutant). In this article, s-E-1 opens a third type of photochromism for GFP chromophore derivatives, which involves light-driven φ-torsion with no τ-torsion, followed by excited-state intramolecular proton transfer (ESIPT), and is gated by environmental polarity. Since s-E-1 does not follow Z-E photoisomerization through τ-torsion but undergoes light-driven φ-torsion, which involves equilibration of the excited-state rotamers, it is clearly against the NEER (Non-Equilibration of Excited-state Rotamers) principle.

E-Z Isomerization Mechanism of the Green Fluorescent Protein Chromophore: Remote Regulation by Proton Dissociation of the Phenol Group.

Dronpa, a GFP (green fluorescent protein)-like fluorescent protein, allows its fluorescent and nonfluorescent states to be switched to each other reversibly by light or heat through E-Z isomerization of the GFP chromophore. In this article, a GFP chromophore (p-HBDI) in water is used as a model to explore this E-Z isomerization mechanism. Based on the experimental solvent isotope effect (kH2O/kD2O = 2.30), the E-Z isomerization of p-HBDI in water is suggested to go through the remote-proton-dissociation-regulated direct mechanism with a proton transfer in the rate-determining step. The fractionation factor (ϕ) of the water-associated phenol proton of p-HBDI in the transition state is found to be 0.43, which is exactly in the range of 0.1-0.6 for the fractionation factor (ϕ) of the transferring proton in the transition state of R2O···H···O+H2 in water. This means that the phenol proton of E-p-HBDI in the transition state is on the way to the associated water oxygen during the E-Z isomerization. The proton dissociation from the phenol group of p-HBDI remotely regulates its E-Z isomerization. Less proton dissociation from the phenol group (pKa = 8.0) at pH = 1-4 results in a modest reduction in the E-Z isomerization rate of p-HBDI, while complete proton dissociation from the phenol group at pH = 11-12 also reduces its E-Z isomerization rate by one order of magnitude because of the larger charge separation in the transition state of the p-HBDI anion. All of these results are consistent with the remote-proton-dissociation-regulated direct mechanism but against the water-assisted addition/elimination mechanism.

Aza-ortho-Quinone Methide and Its Conjugated Acid: Reactivity, Stability and Acidity.

Aza-o-quinone methides and their conjugated acids are reactive drug metabolites that might react with nucleophilic sites of DNAs and proteins to cause cancer or immune responses, and their reactivity with water is the key information to judge if these metabolites are harmful in living systems. For the first time, aza-o-quinone methide (1) and its conjugated acid (2) are observed by laser flash photolysis, and their reactivity, stability and acidity in water are determined.

Dramatic changes in the excited-state behaviour of the green fluorescent protein chromophore by a strong π-donating group through significantly lowering the excited-state potential energy surface with photoinduced intramolecular charge transfer.

A strong π-donating group like p-NMe2 dramatically changes the excited-state behavior of the green fluorescent protein (GFP) chromophore, such as realizing charge-transfer absorption and executing significant photoinduced intramolecular charge transfer (ICT), which makes a planar first singlet (S1) excited-state minimum disappear and significantly lowers the S1 excited-state potential energy surface (PES), leading to barrierless τ-torsion and φ-torsion excited-state relaxation and eliciting the φ-torsion S1 excited-state minimum. This finding is critical since a strong π-donating group like p-NMe2 may do the same things to other fluorophores.

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.

Synthetic green fluorescent protein chromophore analogues with a positive charge at the phenyl-like group.

Synthetic green fluorescent protein (GFP) chromophore analogues with a positive charge at the phenyl-like group have the highly electrophilic amidine carbon, smaller LUMO-HOMO energy gap, red-shifted electronic absorptions and fluorescent emissions, and accelerated E-Z thermoisomerization rates. They are water-labile and their hydrolysis results in ring-opening of the imidazolinone moiety with a half life around 25-37 h in D2O at 25 °C.

Insights into Excited State Intramolecular Proton Transfer: An Alternative Model for Excited State Proton Transfer of Green Fluorescence Protein.

The excited state intramolecular proton transfer (ESIPT) that occurs in the o-sulfonamide analogue ( o-TsABDI) of the green fluorescent protein (GFP) chromophore provides an alternative model to get insights into the excited state proton transfer (ESPT) related photophysics of GFP. In this article, we explored the ESIPT-related photophysics of o-TsABDI by electronic absorption and fluorescence emission spectra in a wide polarity range of solvents, cis- trans photoisomerization experiment, and Coulomb-attenuating method (CAM)/time-dependent (TD) density functional theory (DFT) calculations. We found that the whole ESIPT process involves four steps. The first step is photoexcitation of o-TsABDI, which does not involve charge transfer (CT). The second step is ESIPT and accompanying electron transfer from the n orbital of the sulfonamide nitrogen to the half-filled π orbital of the 4-benzylideneimidazolone moiety. The third step is fluorescence emission of the zwitterionic o-TsABDI and accompanying CT from the π* orbital of the 4-benzylideneimidazolone moiety to the half-filled n orbital of the sulfonamide nitrogen. The last step involves irreversible and barrierless proton recombination. In contrast to the isolated GFP chromophore and its p- and m-amino analogues, the S1 excited state of o-TsABDI does not relax by way of cis- trans photoisomerization through the S1/S0 conical intersection CI(I) by rotating around the I-bond, but follows the ESIPT pathway. The low fluorescence quantum yield of the zwitterionic o-TsABDI might be due to (1) the fluorescence that involves the low-probability π* → n charge transfer and (2) nonradiative relaxation through the S1/S0 conical intersection CI(P″) by rotating around the P-bond.

Synthesis and properties of the para-trimethylammonium analogues of green fluorescence protein (GFP) chromophore: The mimic of protonated GFP chromophore.

At low pH, protons from the external, bulk solution can protonate the phenoxide group of the p-HBDI chromophore in wild-type green fluorescent protein (wtGFP) and its mutants, and likely continue to tentatively protonate the phenol hydroxyl group of the same chromophores. Because the protonated GFP chromophore is a transient, we prepare the stable p-trimethylammonium analogues (2a and 2b) of the GFP chromophore to mimic it and explore their properties. What we found is that the p-trimethylammonium analogues of the GFP chromophore have the highly electrophilic amidine carbon, blue-shifted electronic absorption, smaller molar absorptivity, smaller fluorescent quantum yield, and faster E-Z thermoisomerization rate. The amidine carbon of the p-trimethylammonium analogue (2b) of the GFP chromophore is the only site that is attacked by very weak nucleophile of water, resulting in ring-opening of the imidazolinone moiety. The half-life of its decay rate in D2O is around 33 days. Actually, acid-catalyzed hydrolysis of p-HBDI also results in ring-opening of the imidazolinone moiety. The ratio of the acid-catalyzed hydrolysis rate constants [kobs(p-HBDI)/kobs(1b)] between p-HBDI and 1b (p-dimethylammonium analogue of the GFP chromophore) is dramatically increased from 0.30 at pH = 2 to 0.63 at pH = 0. This is the evidence that more and more phenol hydroxyl groups of p-HBDI are tentatively protonated in a low-pH aqueous solution and that accelerates hydrolysis of p-HBDI in the way similar to the quaternary ammonium derivatives 2a and 2b in water. With this view point, 2a and 2b still can partially mimic the cationic p-HBDI with the protonated phenol hydroxyl group. Implication of the experiment is that the amidine carbon of the chromophore in wtGFP and its mutants at very low pH should be highly electrophilic. Whether ring-opening of the imidazolinone moiety of the GFP chromophore would occur or not depends on if water molecules can reach the amidine carbon of the chromophore inside wtGFP and its mutants.

Electron-Withdrawing ß-Substituent, Ring-Strain, and Ortho Effects on Reactivity, Selectivity, and Stability of o-Alkoxybenzyl Carbocations.

o-Alkoxybenzyl carbocations 1 and 2 were generated by laser flash photolysis of the corresponding o-alkoxybenzyl alcohols 3 and 4 to understand how the electron-withdrawing ß-substituent, the ring-strain, and the ortho effects affect the reactivity (electrophilicity), selectivity, and stability of 1 and 2, and to fit the electrophilicity of 1 and 2 into the current carbocation electrophilicity scale (E). Our finding is that both the electron-withdrawing ß-substituent and the ring-strain effects make 1 less stable than 2 by 3.0 kcal/mol. These effects plus the ortho effect of 2 make 1 more reactive than 2, but the selectivity of 1 and 2 toward amine nucleophiles is almost the same within experimental errors. The electrophilicity of 1 and 2 has been fit into the current carbocation electrophilicity scale (E) quite well.

Synthesis, photophysical properties, and application of o- and p-amino green fluorescence protein synthetic chromophores.

The o- and p-amino green-fluorescence-protein synthetic chromophores (GFPSCs) were synthesized from the corresponding o- and p-nitro protecting group. Among the four protecting groups of the o-amino group, the o-nitro protecting group is the only choice to synthesize the o-amino GFPSCs. The first singlet excited states of o- and p-amino GFPSCs carry significant charge-transfer character through the mechanism of photoinduced charge transfer (PCT). The o-amino GFPSCs can serve as wavelength-ratiometric fluorescence sensors that selectively recognize Cr(3+) in aqueous medium through a PCT mechanism.

Twisting-based spectroscopic measure of solvent polarity: the P(T) scale.

The P(T) scale, which is correlated well with the E(T)(30), Y, and Z scales, is the first twisting-based spectroscopic measure of solvent polarity. It is based on two combined mechanisms: (1) the solvent-dependent intramolecular charge-transfer absorption that displays a regular hypsochromic band shift in polar solvents and (2) overcoming the intramolecular hydrogen-bond of o-1 by its differentiated solvation in polar solvents, causing a C-C bond to twist that leads to a regular hypsochromic shift of its lowest energy electronic absorption band.

Synthesis of proline-derived dipeptides and their catalytic enantioselective direct aldol reactions: catalyst, solvent, additive and temperature effects.

A series of dipeptides of L-proline-L-amino acid and L-proline-D-amino acid were synthesized to evaluate the catalytic effect for asymmetric direct aldol reactions. In the direct aldol reaction, a catalyst of L-proline-L-amino acid achieves better enantioselectivity than the corresponding L-proline-D-amino acid catalyst. Solubility of the dipeptide catalysts in the solvents is a key point for achieving a better yield of the direct aldol reaction, while hydrogen bonding of solvent does not play an important role in attaining better enantioselectivity and yield. Yield and enantioselectivity of the direct aldol reaction in water were improved by NMM and SDS additives, but the results that were done in plain DMSO were even better.

Why does a para-amino group make the green fluorescent protein chromophore non-fluorescent: coherent intramolecular charge transfer reduces the Z/E-photoisomerization barrier.

A para-N-phenyl-amino group significantly increases the fluorescence quantum yield of N-phenyl-4-aminostilbene by the "amino conjugation effect", but, in contrast, a para-amino group in the para-amino analogue (p-ABDI) of the green fluorescent protein (GFP) chromophore makes p-ABDI non-fluorescent because the coherent photo-induced intramolecular charge transfer reduces the barrier of the Z/E-photoisomerization.

Synthesis and Properties of the p-Sulfonamide Analogue of the Green Fluorescent Protein (GFP) Chromophore: The Mimic of GFP Chromophore with Very Strong N-H Photoacid Strength.

The para-sulfonamide analogue ( p-TsABDI) of a green fluorescent protein (GFP) chromophore was synthesized to mimic the GFP chromophore. Its S1 excited-state p Ka* value in dimethylsulfoxide (DMSO) is -1.5, which is strong enough to partially protonate dipolar aprotic solvents and causes excited-state proton transfer (ESPT), so it can partially mimic the GFP chromophore to further study the ESPT-related photophysics and the blinking phenomenon of GFP. In comparison with 8-hydroxypyrene-1,3,6-trisulfonate (HPTS) (p Ka = 7.4, p Ka* = 1.3 in water), p-TsABDI (p Ka = 6.7, p Ka* = -1.5 in DMSO) is a better photoacid for pH-jump studies.

Establishment of steric substituent constants in the adamantane system by ab initio calculations.

[reaction: see text] Steric substituent constants, S(A), of both alkyl and aryl substituents were calculated in the adamantane system by both isodesmic reactions and ab initio calculations. The method provides an easy and reliable way to quantify the substituent steric effects.

Addition of Bromine to Ketenes and Bisketenes: Electrophilic Attack at Carbonyl Carbon and Neighboring Group Participation.

Addition of bromine to bisketene (Me(3)SiC=C=O)(2) (1) gave the fumaryl dibromide E-7, whose stereochemistry was proven by X-ray structure determination. Upon warming, E-7 rearranged to the furanone 8, and this process was faster in the more polar CD(3)CN compared to CDCl(3), consistent with an ionization pathway for the rearrangement. The bromination of 1 in CH(2)ClCH(2)Cl followed second-order kinetics with a rate constant (2.1 +/- 0.1) x 10(4) M(-)(1) s(-)(1) at 25 degrees C. The first-order dependence of bromine addition to 1 on [Br(2)] is attributed to intramolecular nucleophilic assistance by the second ketenyl moiety in an initial complex of 1 and Br(2) to give E-7. A transition structure for this process has been calculated by ab initio methods. By contrast PhMe(2)SiCH=C=O 11 and gamma-oxoketene 16 underwent bromination by third-order kinetics, second order in [Br(2)], indicating the absence of neighboring group participation in the rate-limiting step. The bisketene Me(2)Si(CH=C=O)(2) (13) underwent bromination by mixed kinetics with both first- and second-order terms in [Br(2)].

Direct UV observation and kinetic studies of a alpha-alkoxy benzyloxy radical.

A alpha-alkoxy benzyloxy radical 3 was generated by laser flash photolysis of peroxyacetal 2, and its UV spectrum, decomposition pattern, reactivity with PPh(3), O(2), and i-PrOH, and quantum yield were explored. It was found that the radical 3 is very unstable and highly reactive and performs beta-C-H scission much faster than beta-C-O scission and H-abstraction.

Kinetic and mechanistic studies of NEt3-catalyzed intramolecular aminolysis of carbamate.

The mechanism of the NEt 3-catalyzed intramolecular aminolysis of Z- 1 in acetonitrile or aqueous acetonitrile solutions is suggested to involve rate-determining collapse of T (+/-) through simultaneous H-abstraction and ethoxide expulsion of E2 process. The evidence for that includes the primary isotope effect of k H/ k D = 1.66 in acetonitrile, general-base catalysis in aqueous buffer solutions, salt effect, and the proposed water-stabilized rate-determining transition states (TS1 and TS2) in the water-titration experiments.

Hydroquinone-benzonitrile system: intramolecular charge-transfer and computational studies.

A novel intramolecular donor-acceptor system of hydroquinone-benzonitrile was synthesized. Its photo-induced intramolecular charge-transfer (ICT) transition was confirmed by (1) shift of its emission maximum with increasing solvent polarity, (2) high dipole moment for the ICT excited state calculated from the Lippert equation, and (3) its HOMO and LUMO. According to the extent of separation between HOMO and LUMO, it is suggested that substituent position (ortho, meta, or para) in the donor-acceptor biphenyls is not a key point for the photo-induced intramolecular charge transfer and the donor with two alkoxy or hydroxy groups has more photo-induced charge transfer transitions than the one with one alkoxy or hydroxy group. In other words, the hydroquinone-benzonitrile system displays more photo-induced charge transfer transitions than 4COB (4-cyano-4'-butyloxybiphenyl).