Graeme A. King

Exploring nuclear motion through conical intersections in the UV photodissociation of phenols and thiophenol.

High-resolution time-of-flight measurements of H atom products from photolysis of phenol, 4-methylphenol, 4-fluorophenol, and thiophenol, at many UV wavelengths (λphot), have allowed systematic study of the influence of ring substituents and the heteroatom on the fragmentation dynamics. All dissociate by XH (X = O, S) bond fission after excitation at their respective S1(1ππ*)–S0 origins and at all shorter wavelengths. The achieved kinetic energy resolution reveals population of selected vibrational levels of the various phenoxyl and thiophenoxyl coproducts, providing uniquely detailed insights into the fragmentation dynamics. Dissociation in all cases is deduced to involve nuclear motion on the 1πσ* potential energy surface (PES). The route to accessing this PES, and the subsequent dynamics, is seen to be very sensitive to λphot and substitution of the heteroatom. In the case of the phenols, dissociation after excitation at long λphot is rationalized in terms of radiationless transfer from S1 to S0 levels carrying sufficient OH stretch vibrational energy to allow coupling via the conical intersection between the S0 and 1πσ* PESs at longer OH bond lengths. In contrast, H + C6H5O(X2B1) products formed after excitation at short λphot exhibit anisotropic recoil-velocity distributions, consistent with prompt dissociation induced by coupling between the photoprepared 1ππ* excited state and the 1πσ* PES. The fragmentation dynamics of thiophenol at all λphot matches the latter behavior more closely, reflecting the different relative dispositions of the 1ππ* and 1πσ* PESs. Additional insights are provided by the observed branching into the ground (X2B1) and first excited (2B2) states of the resulting C6H5S radicals.

Revealing the competition between peeled ssDNA, melting bubbles, and S-DNA during DNA overstretching using fluorescence microscopy

Mechanical stress plays a key role in many genomic processes, such as DNA replication and transcription. The ability to predict the response of double-stranded (ds) DNA to tension is a cornerstone of understanding DNA mechanics. It is widely appreciated that torsionally relaxed dsDNA exhibits a structural transition at forces of ∼65 pN, known as overstretching, whereby the contour length of the molecule increases by ∼70%. Despite extensive investigation, the structural changes occurring in DNA during overstretching are still generating considerable debate. Three mechanisms have been proposed to account for the increase in DNA contour length during overstretching: strand unpeeling, localized base-pair breaking (yielding melting bubbles), and formation of S-DNA (strand unwinding, while base pairing is maintained). Here we show, using a combination of fluorescence microscopy and optical tweezers, that all three structures can exist, uniting the often contradictory dogmas of DNA overstretching. We visualize and distinguish strand unpeeling and melting-bubble formation using an appropriate combination of fluorescently labeled proteins, whereas remaining B-form DNA is accounted for by using specific fluorescent molecular markers. Regions of S-DNA are associated with domains where fluorescent probes do not bind. We demonstrate that the balance between the three structures of overstretched DNA is governed by both DNA topology and local DNA stability. These findings enhance our knowledge of DNA mechanics and stability, which are of fundamental importance to understanding how proteins modify the physical state of DNA.

Optical tweezers analysis of DNA-protein complexes.

Optical tweezers have grown to be one of the most powerful and versatile single-molecule methods for analysis of DNA-protein complexes. The power of optical tweezers lies primarily in its extremely high sensitivity and bandwidth in combination with a wide and biologically relevant force range. To accurately apply and measure a force in an optical tweezers assay, the biological system, a DNA molecule, is tethered on two opposite ends. Most commonly used are the single-beam and dual-beam force measuring optical tweezers. Extreme refinement of optical tweezers sensitivity has led to unprecedented mechanistic insight into the single-base-pair stepping of RNA polymerases during DNA transcription. Experiments on DNA repair, which involves the orchestrated action of many proteins, each with different physical characteristics, have showcased the versatility of optical tweezers assays and also illustrated the large advantages of concurrent visualization by fluorescence microscopy.

Dynamical insights into π1σ∗ state mediated photodissociation of aniline

This article reports a comprehensive study of the mechanisms of H atom loss in aniline (C(6)H(5)NH(2)) following ultraviolet excitation, using H (Rydberg) atom photofragment translational spectroscopy. N-H bond fission via the low lying (1)pi sigma(*) electronic state of aniline is experimentally demonstrated. The (1)pi sigma(*) potential energy surface (PES) of this prototypical aromatic amine is essentially repulsive along the N-H stretch coordinate, but possesses a shallow potential well in the vertical Franck-Condon region, supporting quasibound vibrational levels. Photoexcitation at wavelengths (lambda(phot)) in the range 293.859 nm > or = lambda(phot) > or = 193.3 nm yields H atom loss via a range of mechanisms. With lambda(phot) resonant with the 1(1)pi pi(*) <-- S(0) origin (293.859 nm), H atom loss proceeds via, predominantly, multiphoton excitation processes, resonantly enhanced at the one photon energy by the first (1)pi pi(*) excited state (the 1(1)pi pi(*) state). Direct excitation to the first few quasibound vibrational levels of the (1)pi sigma(*) state (at wavelengths in the range 269.513 nm > or = lambda(phot) > or = 260 nm) induces N-H bond fission via H atom tunneling through an exit barrier into the repulsive region of the (1)pi sigma(*) PES, forming anilino (C(6)H(5)NH) radical products in their ground electronic state, and with very limited vibrational excitation; the photo-prepared vibrational mode in the (1)pi sigma(*) state generally evolves adiabatically into the corresponding mode of the anilino radical upon dissociation. However, as the excitation wavelength is reduced (lambda(phot) < 260 nm), N-H bond fission yields fragments with substantially greater vibrational excitation, rationalized in terms of direct excitation to 1(1)pi pi(*) levels, followed by coupling to the (1)pi sigma(*) PES via a 1(1)pi pi(*)/(1)pi sigma(*) conical intersection. Changes in product kinetic energy disposal once lambda(phot) approaches approximately 230 nm likely indicate that the photodissociation pathways of aniline proceed via direct excitation to the (higher) 2(1)pi pi(*) state. Analysis of the anilino fragment vibrational energy disposal-and thus the concomitant dynamics of (1)pi sigma(*) state mediated photodissociation-provides a particularly interesting study of competing sigma(*) <-- pi and pi(*) <-- pi absorption processes and develops our appreciation of the photochemistry of aromatic amines. It also allows revealing comparisons with simple amines (such as ammonia and methylamine) as well as the isoelectronic species, phenol. This study yields a value for the N-H bond strength in aniline, D(0)(H-anilino) = 31630+/-40 cm(-1).

High resolution photofragment translational spectroscopy studies of the ultraviolet photolysis of phenol-d(5).

The dissociation dynamics of gas phase phenol-d(5) molecules (C(6)D(5)OH) following excitation at numerous wavelengths in the range 275 > or = lambda(phot) > or = 193.3 nm have been investigated using the techniques of H (Rydberg) atom photofragment translational spectroscopy and resonance enhanced multiphoton ionization spectroscopy. The results are compared with those from recent studies of the fully hydrogenated and fully deuterated isotopologues (C(6)H(5)OH and C(6)D(5)OD), and various halo- and methyl-substituted phenols. Analysis of the vibrational energy disposal within the phenoxyl-d(5) dissociation products identifies three distinct O-H bond fission pathways, involving nonadiabatic coupling to dissociative states of (1)pisigma* character, following initial pi* <-- pi excitation. Dissociation at lambda(phot) > 248 nm involves internal conversion (IC) to high vibrational levels of the electronic ground ((1)pipi) state and subsequent coupling to the lowest (1)pisigma* potential energy surface (PES) via a conical intersection (CI) between the (1)pipi/(1)pisigma* PESs at extended O-H bond lengths (R(O-H)). Once lambda(phot) < or = 248 nm, dissociation proceeds directly, via a (1)pipi*/(1)pisigma* CI. Both pathways yield phenoxyl-d(5) products in selected vibrational levels of the ground (X(2)B(1)) electronic state. The detailed energy disposal within the phenoxyl-d(5)(X) products shows many parallels with that deduced from companion studies of other phenol isotopologues and various substituted phenols, but a notable isotope effect is identified, thus providing yet greater insights into the factors controlling the vibrational energy disposal in the phenoxyl products. A hitherto unobserved O-H bond fission channel yielding phenoxyl-d(5) fragments in the electronically excited B(2)A(2) state is identified at the shortest excitation wavelength (lambda(phot) = 193.3 nm) and rationalized in terms of nonadiabatic coupling to, and subsequent dissociation on, the second excited (1)pisigma* PES. Selective deuteration as in phenol-d(5) causes little reduction in the intensity of the slower H atom products that are observed from all phenol systems, suggesting that C-H/D bond fission makes at most a minor contribution to this feature.

Near-UV photolysis of substituted phenols. Part II. 4-, 3- and 2-methylphenol.

The photodissociation of jet-cooled 4-, 3- and 2-methylphenol molecules has been investigated using the experimental techniques of resonance enhanced multiphoton ionisation and H (Rydberg) atom photofragment translational spectroscopy. O-H bond fission is found to occur, via a repulsive (1)pisigma state, in a manner analogous to that occurring in phenol and 4-fluorophenol. Excitation to the (1)pipi manifold results in H-atom loss either directly (via a (1)pipi/(1)pisigma conical intersection) or indirectly, following internal conversion to the ground state and subsequent coupling to the (1)pisigma state via a second conical intersection at extended O-H bond lengths. The resulting methylphenoxyl radicals are created with specific vibrational excitation, reflecting the nuclear distortions required to access the (1)pisigma potential energy surface and the geometry changes induced by subsequent H atom loss. The position of the methyl group on the benzene ring is observed to influence the product vibrational energy disposal-not least through its influence on the mode(s) that are activated as a result of coupling to the repulsive (1)pisigma state. O-H bond strengths are reported for 4-, 3- and 2-methylphenol. These are in good agreement with values derived from recent combustion calorimetry studies and serve to highlight the relative destabilisation of the radical caused by methyl substitution at the 3-position.

Controlling Electronic Product Branching at Conical Intersections in the UV Photolysis of para-Substituted Thiophenols

H (Rydberg) atom photofragment translation spectroscopy and high-level ab initio electronic structure calculations are used to explore the photodissociation dynamics of three para-substituted thiophenols (p-YPhSH; Y = CH(3), F, and MeO). UV excitation in the wavelength range 305 > λ(phot) > 240 nm results in S-H bond fission and formation of p-YPhS radicals in their ground (X̃(2)B(1)) and first excited (Ã(2)B(2)) electronic states; the X̃/Ã state product branching ratio, Γ, varies with para-Y substituent and excitation wavelength. Excitation at λ(phot) < 265 nm results in direct population of the dissociative 1(1)πσ* potential energy surface (PES). Γ falls across the series p-CH(3)PhSH > p-FPhSH > p-MeOPhSH. Branching is ultimately determined at the conical intersection (CI) formed by the 1(1)πσ* and ground (S(0)) PESs at extended R(S-H) bond length but is sensitively dependent on the orientation of the S-H bond (relative to the ring plane) in the S(0) molecules prior to photoexcitation. Excitation at λ(phot) > 265 nm populates quasi-bound levels of the respective 1(1)ππ* states, which predissociate rapidly by tunneling under the lower diabats of the 1(1)ππ*/1(1)πσ* CI at short R(S-H). Less extreme X̃/Ã product branching ratios are measured, implicating intramolecular vibrational redistribution within the photoexcited 1(1)ππ* molecules prior to their sampling the region of the 1(1)πσ*/S(0) CI.

Exploring the mechanisms of H atom loss in simple azoles: Ultraviolet photolysis of pyrazole and triazole

The photophysics of gas phase pyrazole (C(3)N(2)H(4)) and 2H-1,2,3-triazole (C(2)N(3)H(3)) molecules following excitation at wavelengths in the range 230 nm>or=lambda(phot)>or=193.3 nm has been investigated using the experimental technique of H (Rydberg) atom photofragment translational spectroscopy. The findings are compared with previous studies of pyrrole (C(4)N(1)H(5)) and imidazole (C(3)N(2)H(4)), providing a guide to H atom loss dynamics in simple N-containing heterocycles. CASPT2 theoretical methods have been employed to validate these findings. Photoexcitation of pyrazole at the longest wavelengths studied is deduced to involve pi( *)<--pi excitation, but photolysis at lambda(phot)</=214 nm is characterized by rapid N-H bond fission on a (1)pisigma( *) potential energy surface. The eventual pyrazolyl radical products are formed in a range of vibrational levels associated with both the ground ((2)A(2)) and first excited ((2)B(1)) electronic states as a result of nonadiabatic coupling at large N-H bond lengths. The excitation energy of the lowest (1)pisigma( *) state of pyrazole is found to be significantly higher in energy than that of pyrrole and imidazole. Similar studies of 2H-1,2,3-triazole reveal that the lowest (1)pisigma( *) state is yet higher in energy and not accessible following excitation at lambda(phot)>or=193.3 nm. The N-H bond strength of pyrazole is determined as 37 680+/-40 cm(-1), significantly greater than that of the N-H bonds in pyrrole and imidazole. The correlation between the photochemistry of azoles and the number and position of nitrogen atoms within the ring framework is discussed in terms of molecular symmetry and orbital electron density. A photodissociation channel yielding H atoms with low kinetic energies is also clearly evident in both pyrazole and 2H-1,2,3-triazole. Companion studies of pyrazole-d(1) suggest that these slow H atoms arise primarily from the N-H site, following pi( *)<--pi excitation, and subsequent internal conversion and/or unintended multiphoton absorption processes.

DNA Twist Stability Changes with Magnesium(2+) Concentration

To understand DNA elasticity at high forces (F>30u2009u2009pN), its helical nature must be taken into account, as a coupling between twist and stretch. The prevailing model, the wormlike chain, was previously extended to include this twist-stretch coupling. Motivated by DNAs charged nature, and the known effects of ionic charges on its elasticity, we set out to systematically measure the impact of buffer ionic conditions on twist-stretch coupling. After developing a robust fitting approach, we show, using our new data set, that DNAs helical twist is stabilized at high concentrations of the magnesium divalent cation. DNAs persistence length and stretch modulus are, on the other hand, relatively insensitive to the applied range of ionic strengths.

UV photolysis of 4-iodo-, 4-bromo-, and 4-chlorophenol: Competition between C-Y (Y = halogen) and O-H bond fission

The wavelength dependences of C-Y and O-H bond fission following ultraviolet photoexcitation of 4-halophenols (4-YPhOH) have been investigated using a combination of velocity map imaging, H Rydberg atom photofragment translational spectroscopy, and high level spin-orbit resolved electronic structure calculations, revealing a systematic evolution in fragmentation behaviour across the series Y = I, Br, Cl (and F). All undergo O-H bond fission following excitation at wavelengths λ ≲ 240 nm, on repulsive ((n∕π)σ∗) potential energy surfaces (PESs), yielding fast H atoms with mean kinetic energies ∼11,000 cm(-1). For Y = I and Br, this process occurs in competition with prompt C-I and C-Br bond cleavage on another (n∕π)σ∗ PES, but no Cl∕Cl∗ products unambiguously attributable to one photon induced C-Cl bond fission are observed from 4-ClPhOH. Differences in fragmentation behaviour at longer excitation wavelengths are more marked. Prompt C-I bond fission is observed following excitation of 4-IPhOH at all λ ≤ 330 nm; the wavelength dependent trends in I∕I∗ product branching ratio, kinetic energy release, and recoil anisotropy suggest that (with regard to C-I bond fission) 4-IPhOH behaves like a mildly perturbed iodobenzene. Br atoms are observed when exciting 4-BrPhOH at long wavelengths also, but their velocity distributions suggest that dissociation occurs after internal conversion to the ground state. O-H bond fission, by tunnelling (as in phenol), is observed only in the cases of 4-FPhOH and, more weakly, 4-ClPhOH. These observed differences in behaviour can be understood given due recognition of (i) the differences in the vertical excitation energies of the C-Y centred (n∕π)σ∗ potentials across the series Y = I < Br < Cl and the concomitant reduction in C-Y bond strength, cf. that of the rival O-H bond, and (ii) the much increased spin-orbit coupling in, particularly, 4-IPhOH. The present results provide (another) reminder of the risks inherent in extrapolating photochemical behaviour measured for one molecule at one wavelength to other (related) molecules and to other excitation energies.