PHOXI: A High Quantum Yield, Solvent-Sensitive Blue Fluorescent 5-Hydroxytryptophan Derivative Synthesized within Ten Minutes under Aqueous, Ambient Conditions.

Multiple tryptophan (Trp) proteins are not amenable to fluorescence study because individual residue emission is not resolvable. Biosynthetic incorporation of an indole analogue such as 5-hydroxyindole has not provided sufficient spectroscopic resolution because of low quantum yield and small emission shift. Here, 5-hydroxyindole is used as the starting framework for building a blue emitting fluorophore of high quantum yield, 2-phenyl-6H-oxazolo[4,5-e]indole (PHOXI). This is a three reagent reaction completed in 10 min under ambient conditions in borate buffer at pH 8. Reaction conditions have been optimized using 5-hydroxyindole. Derivatization is demonstrated on tryptophanyl 5-hydroxytryptophan (5-HTP) and a stable ß-hairpin "zipper" peptide with four tryptophan residues, TrpZip2, where Trp 4 has been replaced with 5-HTP, W4 → 5-HTP. Reaction optimization yields a PHOXI fluorophore that is essentially free of byproducts. Reaction specificity is demonstrated by the lack of reaction with N-acetyl-cysteine and amyloid ß-40, a peptide containing all amino acids except tryptophan, proline, and cysteine and lacking 5-HTP. Fluorescence study of PHOXI-derivatized 5-hydroxyindole in different solvents reveals the sensitivity of PHOXI to solvent polarity with a remarkable 87 nm red-shift in water relative to cyclohexane while maintaining high quantum yield. Thus, PHOXI joins the ranks of solvatochromic fluorophores such as PRODAN. Surprisingly, DFT calculations reveal coplanarity of the oxazolo/indole extended ring system and the phenyl substituent for both the HOMO and LUMO orbitals. Despite the crowded environment of three additional Trps in TrpZip2, CD spectroscopy shows that the TrpZip2 ß-hairpin structure is partially retained upon PHOXI incorporation. In an environment of smaller residues, PHOXI incorporation can be less disruptive of protein secondary structure, especially at molecular interfaces and other environments where there is typically less steric hindrance.

The Color of Cation-π Interactions: Subtleties of Amine-Tryptophan Interaction Energetics Allow for Radical-like Visible Absorbance and Fluorescence.

Several peptides and a protein with an inter- or intramolecular cation-π interaction between tryptophan (Trp) and an amine cation are shown to absorb and fluoresce in the visible region of the spectrum. Titration of indole with sodium hydroxide or ammonium hydroxide yields an increasing visible fluorescence as well. Visible absorption and multipeaked fluorescence excitation spectra correlate with experimental absorption spectra and the vibrational modes of calculated absorption spectra for the neutral Trp radical. The radical character of the cation-indole interaction is predicted to stem from the electrostatic dislocation of indole highest occupied molecular orbital (HOMO) charge density toward the cation with a subsequent electronic transition from the HOMO-2 to the HOMO. Because this is a vertical transition, fluorescence is possible. Hydrogen bonding at the indole amine most likely stabilizes the radical-like state. These results provide new spectroscopic tools for the investigation of cation-π interactions in numerous biological systems, among them, proteins and their myriad ligands, and show that one, or at most, two, point mutations with natural amino acids are all that is required to impart visible fluorescence to proteins.

Excited State Electron Distribution and Role of the Terminal Amine in Acidic and Basic Tryptophan Dipeptide Fluorescence.

The results of quantum yield (QY) study of tryptophanyl glutamate (Trp-Glu), tryptophanyl lysine (Trp-Lys) and lysinyl tryptophan (Lys-Trp) dipeptides over the pH range, 1.5 - 13, show that the charge state of the N-terminal amine, and not the nominal molecular charge determines the QY. When the terminal amine is protonated, QY is low (10-2) for all three dipeptides. As the terminal amine cation is found proximal to the indole ring in Trp-Glu and Trp-Lys conformers but not in those for Lys-Trp, its effect may lie only in the partitioning of energy between nonradiative processes, not on QY reduction. QY is also low when both the N-terminal amine and indole amine are deprotonated. These two low QY states can be distinguished by fluorescence lifetime measurement. Molecular dynamics simulation shows that the Chi 1 conformers persist for tens of nanoseconds such that 100 - 101 nanosecond lifetimes may be associated with individual Chi 1 conformers. The ground state electron density or isosurface of high QY (0.30) 3-methyindole has a uniform electron density over the indole ring as do the higher QY Trp dipeptide conformers. This validates the association of ground state isosurfaces with QY. Excited state orbitals from calculated high intensity, low energy absorption transitions are typically centered over the indole ring for higher QY dipeptide species and off the ring in lower QY species. Thus excited state orbitals substantiate the earlier finding that the ground state isosurface charge density pattern on the indole ring can be predictive of QY.

Electronic spectroscopy and computational studies of glutathionylco(III)balamin.

We have studied glutathionylcobalamin (GS-Cbl) by optical spectroscopy and with density functional theory (DFT) and time-dependent DFT (TD-DFT) electronic structure methods of truncated geometric models. We examined the geometric structure of the models by comparison of DFT calculations with recent high-resolution experimental X-ray structure data ( Hannibal, L. et al. Inorg. Chem. 2010, 49, 9921) for GS-Cbl, and we examined the TD-DFT excitation simulations by comparison of the models with measured optical spectra. The calculations employed the B3LYP hybrid functional and the nonhybrid BP86 functional in both vacuum and water (conductor polarized continuum model (cpcm)) with the 6-311G(d,p) basis set. The optimized geometric structure calculations for six truncated models were made by varying the chemical structure, solvent model, and the two DFT functionals. All showed similar geometry. Charge decomposition analysis (CDA) and extended charge decomposition analysis (ECDA), especially with BP86 shows the similar charge transfer nature of the Co-S bond in GS-Cbl and the Co-C bond in CH(3)Cbl. Mayer and Wiberg bond orders illustrate the similar covalent nature of the two bonds. Finally, absolute optical spectral simulation calculations were compared with the experimental UV-visible extinction spectrum and the electronic circular dichroism (ECD) differential extinction spectrum. The BP86 method shows more spectral features, and the best fit was found for a GS-Cbl model with 5,6-dimethylbenzimidazole at the BP86/6-311G(d,p) level with a water cpcm solvent model. The excited state transitions were investigated with Martin's natural transition orbitals (NTOs). The BP86 calculations also showed π bonding interactions between Co and the axial S of the GS- ligand in the molecular orbitals (MOs) and NTOs.

Contributions of the 8-methyl group to the vibrational normal modes of flavin mononucleotide and its 5-methyl semiquinone radical.

Resonance Raman spectroscopy is a powerful tool to investigate flavins and flavoproteins, and a good understanding of the flavin vibrational normal modes is essential for the interpretation of the Raman spectra. Isotopic labeling is the most effective tool for the assignment of vibrational normal modes, but such studies have been limited to labeling of rings II and III of the flavin isoalloxazine ring. In this paper, we report the resonance and pre-resonance Raman spectra of flavin mononucleotide (FMN) and its N5-methyl neutral radical semiquinone (5-CH 3FMN(*)), of which the 8-methyl group of ring I has been deuterated. The experiments indicate that the Raman bands in the low-frequency region are the most sensitive to 8-methyl deuteration. Density functional theory (DFT) calculations have been performed on lumiflavin to predict the isotope shifts, which are used to assign the calculated normal modes to the Raman bands of FMN. A first assignment of the low-frequency Raman bands on the basis of isotope shifts is proposed. Partial deuteration of the 8-methyl group reveals that the changes in the Raman spectra do not always occur gradually. These observations are reproduced by the DFT calculations, which provide detailed insight into the underlying modifications of the normal modes that are responsible for the changes in the Raman spectra. Two types of isotopic shift patterns are observed: either the frequency of the normal mode but not its composition changes or the composition of the normal mode changes, which then appears at a new frequency. The DFT calculations also reveal that the effect of H/D-exchange in the 8-methyl group on the composition of the vibrational normal modes is affected by the position of the exchanged hydrogen, i.e., whether it is in or out of the isoalloxazine plane.