Alkylated green fluorescent protein chromophores: dynamics in the gas phase and in aqueous solution.

Fluorescent labelling of macromolecular samples, including using the green fluorescent protein (GFP), has revolutionised the field of bioimaging. The ongoing development of fluorescent proteins require a detailed understanding of the photophysics of the biochromophore, and how chemical derivatisation influences the excited state dynamics. Here, we investigate the photophysical properties associated with the S1 state of three alkylated derivatives of the chromophore in GFP, in the gas phase using time-resolved photoelectron imaging, and in water using femtosecond fluorescence upconversion. The gas-phase lifetimes (1.6-10 ps), which are associated with the intrinsic (environment independent) dynamics, are substantially longer than the lifetimes in water (0.06-3 ps), attributed to stabilisation of both twisted intermediate structures and conical intersection seams in the condensed phase. In the gas phase, alkylation on the 3 and 5 positions of the phenyl ring slows the dynamics due to inertial effects, while a 'pre-twist' of the methine bridge through alkylation on the 2 and 6 positions significantly shortens the excited state lifetimes. Formation of a minor, long-lived (≫ 40 ps) excited state population in the gas phase is attributed to intersystem crossing to a triplet state, accessed because of a T1/S1 degeneracy in the so-called P-trap potential energy minimum associated with torsion of the single-bond in the bridging unit connecting to the phenoxide ring. A small amount of intersystem crossing is supported through TD-DFT molecular dynamics trajectories and MS-CASPT2 calculations. No such intersystem crossing occurs in water at T = 300 K or in ethanol at T ≈ 77 K, due to a significantly altered potential energy surface and P-trap geometry.

Charge-Separated Reactive Intermediates from the UV Photodissociation of Chlorobenzene in Solution.

Although ultraviolet (UV)-induced photochemical cleavage of carbon-halogen bonds in gaseous halocarbons is mostly homolytic, the photolysis of chlorobenzene in solution has been proposed to produce a phenyl cation, c-C6H5+, which is a highly reactive intermediate of potential use in chemical synthesis and N2 activation. Any evidence for such a route to phenyl cations is indirect, with uncertainty remaining about the possible mechanism. Here, ultrafast transient absorption spectroscopy of UV-excited (λ = 240 and 270 nm) chlorobenzene solutions in fluorinated (perfluorohexane) and protic (ethanol and 2,2,2-trifluoroethanol) solvents reveals a broad electronic absorption band centered at 540 nm that is assigned to an isomer of chlorobenzene with both charge-separated and triplet-spin carbene character. This spectroscopic feature is weaker, or absent, when experiments are conducted in cyclohexane. The intermediate isomer of chlorobenzene has a solvent-dependent lifetime of 30-110 ps, determined by reaction with the solvent or quenching to a lower-lying singlet state. Evidence is presented for dissociation to ortho-benzyne, but the intermediate could also be a precursor to phenyl cation formation.

Influence of the Solvent Environment on the Ultrafast Relaxation Pathways of a Sunscreen Molecule Diethylamino Hydroxybenzoyl Hexyl Benzoate.

The excited-state dynamics of photoexcited diethylamino hydroxybenzoyl hexyl benzoate (DHHB), a UVA absorber widely used in sunscreen formulations, are studied with transient electronic and vibrational absorption spectroscopy methods in four different solvents. In the polar solvents methanol, dimethyl sulfoxide (DMSO), and acetonitrile, strong stimulated emission (SE) is observed at early time delays after photoexcitation at a near-UV wavelength of λex = 360 nm, and decays with time constants of 420 fs in methanol and 770 fs in DMSO. The majority (∼95%) of photoexcited DHHB returns to the ground state with time constants of 15 ps in methanol and 25 ps in DMSO. In the nonpolar solvent cyclohexane, ∼ 98% of DHHB photoexcited at λex = 345 nm relaxes to the ground state with a ∼ 10 ps time constant, and the SE is weak. DHHB preferentially adopts an enol form in its ground S0 state, but excited state absorption (ESA) bands seen in TEAS are assigned to both the S1-keto and S1-enol forms, indicating a role for ultrafast intramolecular excited state hydrogen transfer (ESHT). This ESHT is inhibited by polar solvents. The two S1 tautomers decay with similar time scales to the observed recovery of ground state population. For molecules that avoid ESHT, torsion around a central C-C bond minimizes the S1-enol energy, quenches the SE, and is proposed to lead to a conical intersection with the S0 state that mediates the ground state recovery. A competing trans-enol isomeric photoproduct is observed as a minor competitor to parent recovery in polar solvents. Evidence is presented for triplet (T1) enol production in polar solvents, and for T1 quenching by octocrylene, a common UVB absorber sunscreen additive. The T1 keto form is observed in cyclohexane solution.

Effects of ring-strain on the ultrafast photochemistry of cyclic ketones.

Ring-strain in cyclic organic molecules is well-known to influence their chemical reactivity. Here, we examine the consequence of ring-strain for competing photochemical pathways that occur on picosecond timescales. The significance of Norrish Type-I photochemistry is explored for three cyclic ketones in cyclohexane solutions at ultraviolet (UV) excitation wavelengths from 255-312 nm, corresponding to an π* ← n excitation to the lowest excited singlet state (S1). Ultrafast transient absorption spectroscopy with broadband UV/visible probe laser pulses reveals processes common to cyclobutanone, cyclopentanone and cyclohexanone, occurring on timescales of ≤1 ps, 7-9 ps and >500 ps. These kinetic components are respectively assigned to prompt cleavage of an α C-C bond in the internally excited S1-state molecules prepared by UV absorption, vibrational cooling of these hot-S1 molecules to energies below the barrier to C-C bond cleavage on the S1 state potential energy surface (with commensurate reductions in the energy-dependent α-cleavage rate), and slower loss of thermalized S1-state population. The thermalized S1-state molecules may competitively decay by activated reaction over the barrier to α C-C bond fission on the S1-state potential energy surface, internal conversion to the ground (S0) electronic state, or intersystem crossing to the lowest lying triplet state (T1) and subsequent C-C bond breaking. The α C-C bond fission barrier height in the S1 state is significantly reduced by the ring-strain in cyclobutanone, affecting the relative contributions of the three decay time components which depend systematically on the excitation energy above the S1-state energy barrier. Transient infra-red absorption spectra obtained after UV excitation identify ring-opened ketene photoproducts of cyclobutanone and their timescales for formation.