Watching Excited State Dynamics with Optical and X-ray Probes: The Excited State Dynamics of Aquocobalamin and Hydroxocobalamin.

Femtosecond time-resolved X-ray absorption (XANES) at the Co K-edge, X-ray emission (XES) in the Co Kß and valence-to-core regions, and broadband UV-vis transient absorption are combined to probe the femtosecond to picosecond sequential atomic and electronic dynamics following photoexcitation of two vitamin B12 compounds, hydroxocobalamin and aquocobalamin. Polarized XANES difference spectra allow identification of sequential structural evolution involving first the equatorial and then the axial ligands, with the latter showing rapid coherent bond elongation to the outer turning point of the excited state potential followed by recoil to a relaxed excited state structure. Time-resolved XES, especially in the valence-to-core region, along with polarized optical transient absorption suggests that the recoil results in the formation of a metal-centered excited state with a lifetime of 2-5 ps. This combination of methods provides a uniquely powerful tool to probe the electronic and structural dynamics of photoactive transition-metal complexes and will be applicable to a wide variety of systems.

Transient vibration and product formation of photoexcited CS2 measured by time-resolved x-ray scattering.

We have observed details of the internal motion and dissociation channels in photoexcited carbon disulfide (CS2) using time-resolved x-ray scattering (TRXS). Photoexcitation of gas-phase CS2 with a 200 nm laser pulse launches oscillatory bending and stretching motion, leading to dissociation of atomic sulfur in under a picosecond. During the first 300 fs following excitation, we observe significant changes in the vibrational frequency as well as some dissociation of the C-S bond, leading to atomic sulfur in the both 1D and 3P states. Beyond 1400 fs, the dissociation is consistent with primarily 3P atomic sulfur dissociation. This channel-resolved measurement of the dissociation time is based on our analysis of the time-windowed dissociation radial velocity distribution, which is measured using the temporal Fourier transform of the TRXS data aided by a Hough transform that extracts the slopes of linear features in an image. The relative strength of the two dissociation channels reflects both their branching ratio and differences in the spread of their dissociation times. Measuring the time-resolved dissociation radial velocity distribution aids the resolution of discrepancies between models for dissociation proposed by prior photoelectron spectroscopy work.

Ultrafast excited state dynamics of provitamin D3 and analogs in solution and in lipid bilayers.

The photochemical ring-opening reaction of 7-dehydrocholesterol (DHC, provitamin D3) is responsible for the light-initiated formation of vitamin D3 in mammalian skin membranes. Visible transient absorption spectroscopy was used to explore the excited state dynamics of DHC and two analogs: ergosterol (provitamin D2) and DHC acetate free in solution and confined to lipid bilayers chosen to model the biological cell membrane. In solution, the excited state dynamics of the three compounds are nearly identical. However, when confined to lipid bilayers, the heterogeneity of the lipid membrane and packing forces imposed on the molecule by the lipid alter the excited state dynamics of these compounds. When confined to lipid bilayers in liposomes formed using DPPC, two solvation environments are identified. The excited state dynamics for DHC and analogs in fluid-like regions of the liposome membrane undergo internal conversion and ring-opening on 1 ps-2 ps time scales, similar to those observed in isotropic solution. In contrast, the excited state lifetime of a subpopulation in regions of lower fluidity is 7 ps-12 ps. The long decay component is unique to these liposomes and results from the structural properties of the lipid bilayer. Additional measurements in liposomes prepared with lipids having slightly longer or shorter alkane tails support this conclusion. In the lipid environments studied, the longest lifetimes are observed for DHC. The unsaturated sterol tail of ergosterol and the acetate group of DHC acetate disrupt the packing around the molecule and permit faster internal conversion and relaxation back to the ground state.

Exceptional Photochemical Stability of the Co-C Bond of Alkynyl Cobalamins, Potential Antivitamins B12 and Core Elements of B12-Based Biological Vectors.

Alkynylcorrinoids are a class of organometallic B12 derivatives, recently rediscovered for use as antivitamins B12 and as core components of B12-based biological vectors. They feature exceptional photochemical and thermal stability of their characteristic extra-short Co-C bond. We describe here the synthesis and structure of 3-hydroxypropynylcobalamin (HOPryCbl) and photochemical experiments with HOPryCbl, as well as of the related alkynylcobalamins: phenylethynylcobalamin and difluoro-phenylethynylcobalamin. Ultrafast spectroscopic studies of the excited state dynamics and mechanism for ground state recovery demonstrate that the Co-C bond of alkynylcobalamins is stable, with the Co-N bond and ring deformations mediating internal conversion and ground state recovery within 100 ps. These studies provide insights required for the rational design of photostable or photolabile B12-based cellular vectors.

Primed for Efficient Motion: Ultrafast Excited State Dynamics and Optical Manipulation of a Four Stage Rotary Molecular Motor.

All isomers of a four stage rotary molecular motor, dimethyl-tetrahydro-bi(cyclopenta[α]napthal-enylidene), are studied with ultrafast transient absorption spectroscopy. Single and two pulse excitations (pump and delayed repump with a different wavelength) are used to optically probe the excited state dynamics. These measurements demonstrate that this motor is not only designed for unidirectional isomerization, but is also "primed" for efficient rotary motion. The yield for photoisomerization from the stable P-cis isomer to the metastable M-trans isomer is 85% ± 10%, while the yield for the undesired back reaction is ca. 0.08 (+0.02, -0.05). The yield for photoisomerization from stable P-trans to the metastable M-cis isomer is ca. 85% ± 3% and the yield for the back reaction is 15% ± 3%. Excitation of P-trans in the lowest singlet state results in formation of a dark state on a 3.6 ps time scale and formation of the M-cis isomer on a ca. 12 ps time scale. Excitation of P-cis in the lowest singlet state results in formation of a dark state on ca. 13 ps time scale and formation of the M-trans isomer on a 71 ps time scale. Excitation of either isomer at 269 nm, higher in the excited state manifold, accesses additional excited state pathways, but does not change the ultimate product formation. This result suggests that pulse sequences accessing higher excited states may provide a tool to manipulate the molecular motor. Pulse sequences using a 269 nm pump pulse and a 404 nm repump pulse are able to increase the yield of the P-cis to M-trans reaction but only decrease the yield of the P-trans to M-cis reaction. These pulse sequences are unable to access reaction pathways that bypass the helix inversion step, although other wavelengths and time delays might yet provide optical control of the entire reaction cycle. We propose intermediates and candidate conical intersections between all four isomers.

Off to the Races: Comparison of Excited State Dynamics in Vitamin B12 Derivatives Hydroxocobalamin and Aquocobalamin.

Ultrafast time-resolved spectroscopy was used to study the photochemistry of hydroxocobalamin (HOCbl) and aquocobalamin (H2OCbl+) in solution. Spectroscopic measurements and TD-DFT simulations provide a consistent picture of the spectroscopy and photochemistry. Excitation of H2OCbl+ results in formation of an excited state followed by rapid internal conversion to the ground state (0.35 ± 0.15 ps) through an S1/S0 seam at a slightly elongated Co-O bond length and a significantly elongated Co-NIm bond length. In contrast, the initial elongation of the axial bonds in HOCbl is followed by contraction to an excited state minimum with bonds slightly shorter than those in the ground state. Internal conversion to the ground state follows on a picosecond time scale (5.3 ± 0.4 ps). For both compounds, photodissociation forming cob(II)alamin and hydroxyl radicals (∼1.5% yield) requires excitation to highly excited states. Dissociation is mediated by competition between internal conversion to the S1 surface and prompt bond cleavage.

Ultrafast X-ray Absorption Near Edge Structure Reveals Ballistic Excited State Structural Dynamics.

Polarized ultrafast time-resolved X-ray absorption near edge structure (XANES) allows characterization of excited state dynamics following excitation. Excitation of vitamin B12, cyanocobalamin (CNCbl), in the αß-band at 550 nm and the γ-band at 365 nm was used to uniquely resolve axial and equatorial contributions to the excited state dynamics. The structural evolution of the excited molecule is best described by a coherent ballistic trajectory on the excited state potential energy surface. Prompt expansion of the Co cavity by ca. 0.03 Å is followed by significant elongation of the axial bonds (>0.25 Å) over the first 190 fs. Subsequent contraction of the Co cavity in both axial and equatorial directions results in the relaxed S1 excited state structure within 500 fs of excitation.

Polarized XANES Monitors Femtosecond Structural Evolution of Photoexcited Vitamin B12.

Ultrafast, polarization-selective time-resolved X-ray absorption near-edge structure (XANES) was used to characterize the photochemistry of vitamin B12, cyanocobalamin (CNCbl), in solution. Cobalamins are important biological cofactors involved in methyl transfer, radical rearrangement, and light-activated gene regulation, while also holding promise as light-activated agents for spatiotemporal controlled delivery of therapeutics. We introduce polarized femtosecond XANES, combined with UV-visible spectroscopy, to reveal sequential structural evolution of CNCbl in the excited electronic state. Femtosecond polarized XANES provides the crucial structural dynamics link between computed potential energy surfaces and optical transient absorption spectroscopy. Polarization selectivity can be used to uniquely identify electronic contributions and structural changes, even in isotropic samples when well-defined electronic transitions are excited. Our XANES measurements reveal that the structural changes upon photoexcitation occur mainly in the axial direction, where elongation of the axial Co-CN bond and Co-NIm bond on a 110 fs time scale is followed by corrin ring relaxation on a 260 fs time scale. These observations expose features of the potential energy surfaces controlling cobalamin reactivity and deactivation.

Toward the Design of Photoresponsive Conditional Antivitamins B12: A Transient Absorption Study of an Arylcobalamin and an Alkynylcobalamin.

Cobalamins are of widespread importance in biology. Both of the cofactors essential for human metabolism, the organocobalamins coenzyme B12 and methylcobalamin, are highly photolabile, as are other alkylcobalamins. The alkynylcobalamin phenylethynylcobalamin (PhEtyCbl) and the arylcobalamin 4-ethylphenylcobalamin (EtPhCbl) with "atypical" Co-C-bonds to unsaturated carbons, were recently designed as metabolically inert cobalamins, classified as "antivitamins B12". The further development of an ideal light-activated or "conditional" antivitamin B12 would require it to be readily converted by light into an active B12 vitamin form. Very photolabile "antivitamins B12" would also represent particularly useful scaffolds for therapeutic light-activated reagents. Here, the photoactive arylcobalamin EtPhCbl and the remarkably photostable alkynylcobalamin PhEtyCbl are examined using femtosecond to picosecond UV-visible transient absorption spectroscopy. PhEtyCbl undergoes internal conversion to the ground state with near unit quantum yield on a time scale < 100 ps and an activation energy of 12.6 ± 1.4 kJ/mol. The arylcobalamin EtPhCbl forms an excited state with a ca. 247 ps lifetime. This excited state branches between internal conversion to the ground state and formation of a long-lived base-off species with a quantum yield of ∼9%. Anaerobic steady state photolysis of "light-sensitive" EtPhCbl results in the formation of cob(II)alamin, but only with quantum yield <1%. Hence, our studies suggest that suitably modified arylcobalamins may be a rational basis for the design of photoresponsive "antivitamins B12".

Energy cascades, excited state dynamics, and photochemistry in cob(III)alamins and ferric porphyrins.

Porphyrins and the related chlorins and corrins contain a cyclic tetrapyrrole with the ability to coordinate an active metal center and to perform a variety of functions exploiting the oxidation state, reactivity, and axial ligation of the metal center. These compounds are used in optically activated applications ranging from light harvesting and energy conversion to medical therapeutics and photodynamic therapy to molecular electronics, spintronics, optoelectronic thin films, and optomagnetics. Cobalt containing corrin rings extend the range of applications through photolytic cleavage of a unique axial carbon-cobalt bond, permitting spatiotemporal control of drug delivery. The photochemistry and photophysics of cyclic tetrapyrroles are controlled by electronic relaxation dynamics including internal conversion and intersystem crossing. Typically the electronic excitation cascades through ring centered ππ* states, ligand to metal charge transfer (LMCT) states, metal to ligand charge transfer (MLCT) states, and metal centered states. Ultrafast transient absorption spectroscopy provides a powerful tool for the investigation of the electronic state dynamics in metal containing tetrapyrroles. The UV-visible spectrum is sensitive to the oxidation state, electronic configuration, spin state, and axial ligation of the central metal atom. Ultrashort broadband white light probes spanning the range from 270 to 800 nm, combined with tunable excitation pulses, permit the detailed unravelling of the time scales involved in the electronic energy cascade. State-of-the-art theoretical calculations provide additional insight required for precise assignment of the states. In this Account, we focus on recent ultrafast transient absorption studies of ferric porphyrins and corrin containing cob(III)alamins elucidating the electronic states responsible for ultrafast energy cascades, excited state dynamics, and the resulting photoreactivity or photostability of these compounds. Iron tetraphenyl porphyrin chloride (Fe((III))TPPCl) exhibits picosecond decay to a metal centered d → d* (4)T state. This state decays on a ca. 16 ps time scale in room temperature solution but persists for much longer in a cryogenic glass. The photoreactivity of the (4)T state may lead to novel future applications for these compounds. In contrast, the nonplanar cob(III)alamins contain two axial ligands to the central cobalt atom. The upper axial ligand can be an alkyl group as in the two biologically active coenzymes or a nonalkyl ligand such as -CN in cyanocobalamin (vitamin B12) or -OH in hydroxocobalamin. The electronic structure, energy cascade, and bond cleavage of these compounds is sensitive to the details of the axial ligand. Nonalkylcobalamins exhibit ultrafast internal conversion to a low-lying state of metal to ligand or ligand to metal charge transfer character. The compounds are generally photostable with ground state recovery complete on a time scale of 2-7 ps in room temperature aqueous solution. Alkylcobalamins exhibit ultrafast internal conversion to an S1 state of d/π → π* character. Most compounds undergo bond cleavage from this state with near unit quantum yield within ∼100 ps. Recent theoretical calculations provide a potential energy surface accounting for these observations. Conformation dependent mixing of the corrin π and cobalt d orbitals plays a significant role in the observed photochemistry and photophysics.

Probing the Biexponential Dynamics of Ring-Opening in 7-Dehydrocholesterol.

Our prior discovery of a novel biexponential photochemical ring-opening in 7-dehydrocholesterol (DHC) to previtamin D3 [ Tang J. Chem. Phys. 2011 , 134 , 104503 ] is further explored with ultrafast transient absorption spectroscopy, and the results are compared with recently reported high-level theoretical calculations. Three types of experiments are reported. First, variation of the excitation wavelength from 297 to 266 nm leaves the excited state dynamics unaffected. The biexponential decay of the excited state absorption is independent of excitation wavelength with time constants of 0.57 ± 0.06 and 1.88 ± 0.09 ps, in excellent agreement with the results reported earlier (0.56 ± 0.06 and 1.81 ± 0.15 ps) following excitation at 266 nm. Second, variation of the chirp of the excitation pulse influences the relative amplitude of the fast and slow decay components but has no influence on the photoproduct yield. Third, a 545 nm pulse delayed by 0.64 ps with respect to the initial 266 nm pulse was used to perturb the "slow" population and probe the influence of additional electronic or vibrational energy on the reaction process. The results show ultrafast internal conversion Sn → S1 on a ca. 150 fs time scale but no subsequent effect on the reaction dynamics. The experiments reported here are consistent with the recent state averaged complete active space self-consistent field ab initio multiple spawning (SA-CASSCF-AIMS) calculations of Snyder et al. [ J. Phys. Chem. Lett. 2016 , 7 , 2444 ] that assign the biexponential decay to nonequilibrium dynamics related to the opening and closing motion of the cyclohexadiene ring moiety on the excited state surface. These new experiments support the model prediction that the biexponential dynamics does not involve multiple minima and demonstrate the direction for new experimental designs to manipulate the product yields and pathways.

Ultrafast polyene dynamics: the ring opening of 1,3-cyclohexadiene derivatives.

The light activated ring-opening reaction of the 1,3-cyclohexadiene chromophore finds application in optical control, optical switching, optical memory, light activated molecular machines, photobiology, photochromic materials, and conformation-specific photocatalysts. The development of ultrafast spectroscopic methods and powerful computational methods have accelerated the understanding and facilitated the application of this important chromophore in a wide range of systems. Here we look at the current state of theoretical and experimental understanding for the ring-opening reaction of the isolated cyclohexadiene molecule and the ring-opening reactions of substituted cyclohexadienes, including fulgides, diarylethenes, and provitamin D.

Ultrafast X-ray Absorption Spectroscopy Reveals Excited-State Dynamics of B12 Coenzymes Controlled by the Axial Base.

Polarized time-resolved X-ray absorption spectroscopy at the Co K-edge is used to probe the excited-state dynamics and photolysis of base-off methylcobalamin and the excited-state structure of base-off adenosylcobalamin. For both molecules, the final excited-state minimum shows evidence for an expansion of the cavity around the Co ion by ca. 0.04 to 0.05 Å. The 5-coordinate base-off cob(II)alamin that is formed following photodissociation has a structure similar to that of the 5-coordinate base-on cob(II)alamin, with a ring expansion of 0.03 to 0.04 Å and a contraction of the lower axial bond length relative to that in the 6-coordinate ground state. These data provide insights into the role of the lower axial ligand in modulating the reactivity of B12 coenzymes.

Structural Evolution of Photoexcited Methylcobalamin toward a CarH-like Metastable State: Evidence from Time-Resolved X-ray Absorption and X-ray Emission.

CarH is a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light via formation of a metastable excited state. Aside from AdoCbl bound to CarH, methylcobalamin (MeCbl) is the only other example─to date─of photoexcited cobalamins forming metastable excited states with lifetimes of nanoseconds or longer. The UV-visible spectra of the excited states of MeCbl and AdoCbl bound to CarH are similar. We have used transient Co K-edge X-ray absorption and X-ray emission spectroscopies in conjunction with transient absorption spectroscopy in the UV-visible region to characterize the excited states of MeCbl. These data show that the metastable excited state of MeCbl has a slightly expanded corrin ring and increased electron density on the cobalt, but only small changes in the axial bond lengths.

Solvent dependent branching between C-I and C-Br bond cleavage following 266 nm excitation of CH2BrI.

It is well known that ultraviolet photoexcitation of halomethanes results in halogen-carbon bond cleavage. Each halogen-carbon bond has a dominant ultraviolet (UV) absorption that promotes an electron from a nonbonding halogen orbital (nX) to a carbon-halogen antibonding orbital (σ*C-X). UV absorption into specific transitions in the gas phase results primarily in selective cleavage of the corresponding carbon-halogen bond. In the present work, broadband ultrafast UV-visible transient absorption studies of CH2BrI reveal a more complex photochemistry in solution. Transient absorption spectra are reported spanning the range from 275 nm to 750 nm and 300 fs to 3 ns following excitation of CH2BrI at 266 nm in acetonitrile, 2-butanol, and cyclohexane. Channels involving formation of CH2Br + I radical pairs, iso-CH2Br-I, and iso-CH2I-Br are identified. The solvent environment has a significant influence on the branching ratios, and on the formation and stability of iso-CH2Br-I. Both iso-CH2Br-I and iso-CH2I-Br are observed in cyclohexane with a ratio of ~2.8:1. In acetonitrile this ratio is 7:1 or larger. The observation of formation of iso-CH2I-Br photoproduct as well as iso-CH2Br-I following 266 nm excitation is a novel result that suggests complexity in the dissociation mechanism. We also report a solvent and concentration dependent lifetime of iso-CH2Br-I. At low concentrations the lifetime is >4 ns in acetonitrile, 1.9 ns in 2-butanol and ~1.4 ns in cyclohexane. These lifetimes decrease with higher initial concentrations of CH2BrI. The concentration dependence highlights the role that intermolecular interactions can play in the quenching of unstable isomers of dihalomethanes.

Transient X-ray fragmentation: probing a prototypical photoinduced ring opening.

We report the first study of UV-induced photoisomerization probed via core ionization by an x-ray laser. We investigated x-ray ionization and fragmentation of the cyclohexadiene-hexatriene system at 850 eV during the ring opening. We find that the ion-fragmentation patterns evolve over a picosecond, reflecting a change in the state of excitation and the molecular geometry: the average kinetic energy per ion fragment and H(+)-ion count increase as the ring opens and the molecule elongates. We discuss new opportunities for molecular photophysics created by optical pump x-ray probe experiments.

Ligand recruitment and spin transitions in the solid-state photochemistry of Fe(III)TPPCl.

We report evidence for the formation of long-lived photoproducts following excitation of iron(III) tetraphenylporphyrin chloride (Fe(III)TPPCl) in a 1:1 glass of toluene and CH(2)Cl(2) at 77 K. The formation of these photoproducts is dependent on solvent environment and temperature, appearing only in the presence of toluene. No long-lived product is observed in neat CH(2)Cl(2) solvent. A 2-photon absorption model is proposed to account for the power-dependent photoproduct populations. The products are formed in a mixture of spin states of the central iron(III) metal atom. Metastable six-coordinate high-spin and low-spin complexes and a five-coordinate high-spin complex of iron(III) tetraphenylporphyrin are assigned using structure-sensitive vibrations in the resonance Raman spectrum. These species appear in conjunction with resonantly enhanced toluene solvent vibrations, indicating that the Fe(III) compound formed following photoexcitation recruits a toluene ligand from the surrounding environment. Low-temperature transient absorption (TA) measurements are used to explain the dependence of product formation on excitation frequency in this photochemical model. The six-coordinate photoproduct is initially formed in the high-spin Fe(III) state, but population relaxes into both high-spin and low-spin state at 77 K. This is the first demonstration of coupling between the optical and magnetic properties of an iron-centered porphyrin molecule.

Control of retinal isomerization in bacteriorhodopsin in the high-intensity regime.

A learning algorithm was used to manipulate optical pulse shapes and optimize retinal isomerization in bacteriorhodopsin, for excitation levels up to 1.8 x 10(16) photons per square centimeter. Below 1/3 the maximum excitation level, the yield was not sensitive to pulse shape. Above this level the learning algorithm found that a Fourier-transform-limited (TL) pulse maximized the 13-cis population. For this optimal pulse the yield increases linearly with intensity well beyond the saturation of the first excited state. To understand these results we performed systematic searches varying the chirp and energy of the pump pulses while monitoring the isomerization yield. The results are interpreted including the influence of 1-photon and multiphoton transitions. The population dynamics in each intermediate conformation and the final branching ratio between the all-trans and 13-cis isomers are modified by changes in the pulse energy and duration.

Time-resolved spectroscopy: Advances in understanding the electronic structure and dynamics of cobalamins.

Time resolved spectroscopy provides unique insight into the structure and function of cobalamins. In these experiments, the cobalamin is initially excited by a short "pump" pulse in the UV-visible region and then characterized at some later time using a short "probe" pulse. The emphasis in this chapter is on both UV-visible and X-ray probe pulses, with a particular focus on the unique information provided by the latter. The principles of time-resolved spectroscopy are reviewed, with an emphasis on ultrafast measurements (time scales less than ~10ps) to characterize short-lived cobalamin excited states. Several practical considerations are discussed, with a focus on the technical details that are necessary to obtain high quality, interpretable data. These include sample delivery, polarization, and excitation power. Some of the theoretical approaches to interpreting data are discussed.