Probing the Formation and Conformational Relaxation of Previtamin D3 and Analogues in Solution and in Lipid Bilayers.

The photosynthesis of vitamin D3 in mammalian skin results from UV-B irradiation of provitamin D3 (7-dehydrocholesterol, DHC) at ca. 290 nm. Upon return to the ground state, the hexatriene product, previtamin D3, undergoes a conformational equilibration between helical gZg and more planar tZg and tZt forms. The helical gZg forms provide a pathway for the formation of vitamin D3 via a [1,7]-sigmatropic hydrogen shift. Steady state photolysis and UV transient absorption spectroscopy are combined to explore the conformational relaxation of previtamin D3 formed from DHC in isotropic solution and confined to lipid bilayers chosen to model the biological cell membrane. The results are compared with measurements for two analogues: previtamin D2 formed from ergosterol (provitamin D2) and previtamin D3 acetate formed from DHC acetate. The resulting spectral dynamics are interpreted in the context of simulations of optical excitation energy and oscillator strength as a function of conformation. In solution, the relaxation dynamics and steady state product distributions of the three compounds are nearly identical, favoring tZg forms. When confined to lipid bilayers, the heterogeneity and packing forces alter the conformational distributions and enhance the population of a gZg conformer capable of vitamin D formation.

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.

The Photoactive Excited State of the B12-Based Photoreceptor CarH.

We have used transient absorption spectroscopy in the UV-visible and X-ray regions to characterize the excited state of CarH, a protein photoreceptor that uses a form of B12, adenosylcobalamin (AdoCbl), to sense light. With visible excitation, a nanosecond-lifetime photoactive excited state is formed with unit quantum yield. The time-resolved X-ray absorption near edge structure difference spectrum of this state demonstrates that the excited state of AdoCbl in CarH undergoes only modest structural expansion around the central cobalt, a behavior similar to that observed for methylcobalamin rather than for AdoCbl free in solution. We propose a new mechanism for CarH photoreactivity involving formation of a triplet excited state. This allows the sensor to operate with high quantum efficiency and without formation of potentially dangerous side products. By stabilizing the excited electronic state, CarH controls reactivity of AdoCbl and enables slow reactions that yield nonreactive products and bypass bond homolysis and reactive radical species formation.

Ultrafast Excited State Dynamics and Fluorescence from Vitamin B12 and Organometallic [Co]-C≡C-R Cobalamins.

Cobalamins are cobalt-centered cyclic tetrapyrrole ring-based molecules that provide cofactors for exceptional biological processes and possess unique and synthetically tunable photochemistry. Typical cobalamins are characterized by a visible absorption spectrum consisting of peaks labeled α, ß, and sh. The physical basis of these peaks as having electronic origin or as a vibronic progression is ambiguous despite much investigation. Here, for the first time, cobalamin fluorescence is identified in several derivatives. The fluorescence lifetime is ca. 100-200 fs with quantum yields on the order of 10-6-10-5 because of rapid population of "dark" excited states. The results are compared with the fluorescent analogue with zinc replacing the cobalt in the corrin ring. Analysis of the breadth of the emission spectrum provides evidence that a vibrational progression in a single excited electronic state makes the dominant contribution to the visible absorption band.

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.

Ultrafast XANES Monitors Femtosecond Sequential Structural Evolution in Photoexcited Coenzyme B12.

Polarized X-ray absorption near-edge structure (XANES) at the Co K-edge and broadband UV-vis transient absorption are used to monitor the sequential evolution of the excited-state structure of coenzyme B12 (adenosylcobalamin) over the first picosecond following excitation. The initial state is characterized by sub-100 fs sequential changes around the central cobalt. These are polarized first in the y-direction orthogonal to the transition dipole and 50 fs later in the x-direction along the transition dipole. Expansion of the axial bonds follows on a ca. 200 fs time scale as the molecule moves out of the Franck-Condon active region of the potential energy surface. On the same 200 fs time scale there are electronic changes that result in the loss of stimulated emission and the appearance of a strong absorption at 340 nm. These measurements provide a cobalt-centered movie of the excited molecule as it evolves to the local excited-state minimum.

Probing the Excited State of Methylcobalamin Using Polarized Time-Resolved X-ray Absorption Spectroscopy.

We use picosecond time-resolved polarized X-ray absorption near-edge structure (XANES) measurements to probe the structure of the long-lived photoexcited state of methylcobalamin (MeCbl) and the cob(II)alamin photoproduct formed following photoexcitation of adenosylcobalamin (AdoCbl, coenzyme B12). For MeCbl, we used 520 nm excitation and a time delay of 100 ps to avoid the formation of cob(II)alamin. We find only small spectral changes in the equatorial and axial directions, which we interpret as arising from small (<∼0.05 Å) changes in both the equatorial and axial distances. This confirms expectations based on prior UV-visible transient absorption measurements and theoretical simulations. We do not find evidence for the significant elongation of the Co-C bond reported by Subramanian [ J. Phys. Chem. Lett. 2018 , 9 , 1542 - 1546 ] following 400 nm excitation. For AdoCbl, we resolve the difference XANES contributions along three unique molecular axes by exciting with both 540 and 365 nm light, demonstrating that the spectral changes are predominantly polarized along the axial direction, consistent with the loss of axial ligation. These data suggest that the microsecond "recombination product" identified by Subramanian et al. is actually the cob(II)alamin photoproduct that is produced following bond homolysis of MeCbl with 400 nm excitation. Our results highlight the pronounced advantage of using polarization-selective transient X-ray absorption for isolating structural dynamics in systems undergoing atomic displacements that are strongly correlated to the exciting optical polarization.

Multispectral multidimensional spectrometer spanning the ultraviolet to the mid-infrared.

Multidimensional spectroscopy is the optical analog to nuclear magnetic resonance, probing dynamical processes with ultrafast time resolution. At optical frequencies, the technical challenges of multidimensional spectroscopy have hindered its progress until recently, where advances in laser sources and pulse-shaping have removed many obstacles to its implementation. Multidimensional spectroscopy in the visible and infrared (IR) regimes has already enabled respective advances in our understanding of photosynthesis and the structural rearrangements of liquid water. A frontier of ultrafast spectroscopy is to extend and combine multidimensional techniques and frequency ranges, which have been largely restricted to operating in the distinct visible or IR regimes. By employing two independent amplifiers seeded by a single oscillator, it is straightforward to span a wide range of time scales (femtoseconds to seconds), all of which are often relevant to the most important energy conversion and catalysis problems in chemistry, physics, and materials science. Complex condensed phase systems have optical transitions spanning the ultraviolet (UV) to the IR and exhibit dynamics relevant to function on time scales of femtoseconds to seconds and beyond. We describe the development of the Multispectral Multidimensional Nonlinear Spectrometer (MMDS) to enable studies of dynamical processes in atomic, molecular, and material systems spanning femtoseconds to seconds, from the UV to the IR regimes. The MMDS employs pulse-shaping methods to provide an easy-to-use instrument with an unprecedented spectral range that enables unique combination spectroscopies. We demonstrate the multispectral capabilities of the MMDS on several model systems.

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.

Electronic Interactions in the Bacterial Reaction Center Revealed by Two-Color 2D Electronic Spectroscopy.

The bacterial reaction center (BRC) serves as an important model system for understanding the charge separation processes in photosynthesis. Knowledge of the electronic structure of the BRC is critical for understanding its charge separation mechanism. While it is well-accepted that the "special pair" pigments are strongly coupled, the degree of coupling among other BRC pigments has been thought to be relatively weak. Here we study the W(M250)V mutant BRC by two-color two-dimensional electronic spectroscopy to correlate changes in the Q x region with excitation of the Q y transitions. The resulting Q y-Q x cross-peaks provide a sensitive measure of the electronic interactions throughout the BRC pigment network and complement one-color 2D studies in which such interactions are often obscured by energy transfer and excited-state absorption signals. Our observations should motivate the refinement of electronic structure models of the BRC to facilitate improved understanding of the charge separation mechanism.

Strongly coupled bacteriochlorin dyad studied using phase-modulated fluorescence-detected two-dimensional electronic spectroscopy.

Fluorescence-detected two-dimensional electronic spectroscopy (F-2DES) projects the third-order non-linear polarization in a system as an excited electronic state population which is incoherently detected as fluorescence. Multiple variants of F-2DES have been developed. Here, we report phase-modulated F-2DES measurements on a strongly coupled symmetric bacteriochlorin dyad, a relevant 'toy' model for photosynthetic energy and charge transfer. Coherence map analysis shows that the strongest frequency observed in the dyad is well-separated from the excited state electronic energy gap, and is consistent with a vibrational frequency readily observed in bacteriochlorin monomers. Kinetic rate maps show a picosecond relaxation timescale between the excited states of the dyad. To our knowledge this is the first demonstration of coherence and kinetic analysis using the phase-modulation approach to F-2DES.

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.

Primary processes in the bacterial reaction center probed by two-dimensional electronic spectroscopy.

In the initial steps of photosynthesis, reaction centers convert solar energy to stable charge-separated states with near-unity quantum efficiency. The reaction center from purple bacteria remains an important model system for probing the structure-function relationship and understanding mechanisms of photosynthetic charge separation. Here we perform 2D electronic spectroscopy (2DES) on bacterial reaction centers (BRCs) from two mutants of the purple bacterium Rhodobacter capsulatus, spanning the Q y absorption bands of the BRC. We analyze the 2DES data using a multiexcitation global-fitting approach that employs a common set of basis spectra for all excitation frequencies, incorporating inputs from the linear absorption spectrum and the BRC structure. We extract the exciton energies, resolving the previously hidden upper exciton state of the special pair. We show that the time-dependent 2DES data are well-represented by a two-step sequential reaction scheme in which charge separation proceeds from the excited state of the special pair (P*) to P+HA- via the intermediate P+BA- When inhomogeneous broadening and Stark shifts of the B* band are taken into account we can adequately describe the 2DES data without the need to introduce a second charge-separation pathway originating from the excited state of the monomeric bacteriochlorophyll BA*.

Stimulated Emission Enhancement Using Shaped Pulses.

Controlling stimulated emission is of importance because it competes with absorption and fluorescence under intense laser excitation. We performed resonant nonlinear optical spectroscopy measurements using femtosecond pulses shaped by π- or π/2-step phase functions and carried out calculations based on density matrix representation to elucidate the experimental results. In addition, we compared enhancements obtained when using other pulse shaping functions (chirp, third-order dispersion, and a time-delayed probe). The light transmitted through the high optical density solution was dominated by an intense stimulated emission feature that was 14 times greater for shaped pulses than for transform limited pulses. Coherent enhancement depending on the frequency, temporal, and phase characteristics of the shaped pulse is responsible for the experimental observations.

Controlling S2 Population in Cyanine Dyes Using Shaped Femtosecond Pulses.

Fast population transfer from higher to lower excited states occurs via internal conversion (IC) and is the basis of Kasha's rule, which states that spontaneous emission takes place from the lowest excited state of the same multiplicity. Photonic control over IC is of interest because it would allow direct influence over intramolecular nonradiative decay processes occurring in condensed phase. Here we tracked the S2 and S1 fluorescence yield for different cyanine dyes in solution as a function of linear chirp. For the cyanine dyes with polar solvation response IR144 and meso-piperidine substituted IR806, increased S2 emission was observed when using transform limited pulses, whereas chirped pulses led to increased S1 emission. The nonpolar solvated cyanine IR806, on the other hand, did not show S2 emission. A theoretical model, based on a nonperturbative solution of the equation of motion for the density matrix, is offered to explain and simulate the anomalous chirp dependence. Our findings, which depend on pulse properties beyond peak intensity, offer a photonic method to control S2 population thereby opening the door for the exploration of photochemical processes initiated from higher excited states.

Investigating the role of human serum albumin protein pocket on the excited state dynamics of indocyanine green using shaped femtosecond laser pulses.

Differences in the excited state dynamics of molecules and photo-activated drugs either in solution or confined inside protein pockets or large biological macromolecules occur within the first few hundred femtoseconds. Shaped femtosecond laser pulses are used to probe the behavior of indocyanine green (ICG), the only Food and Drug Administration (FDA) approved near-infrared dye and photodynamic therapy agent, while free in solution and while confined inside the pocket of the human serum albumin (HSA) protein. Experimental findings indicate that the HSA pocket hinders torsional motion and thus mitigates the triplet state formation in ICG. Low frequency vibrational motion of ICG is observed more clearly when it is bound to the HSA protein.

Polyatomic molecules under intense femtosecond laser irradiation.

Interaction of intense laser pulses with atoms and molecules is at the forefront of atomic, molecular, and optical physics. It is the gateway to powerful new tools that include above threshold ionization, high harmonic generation, electron diffraction, molecular tomography, and attosecond pulse generation. Intense laser pulses are ideal for probing and manipulating chemical bonding. Though the behavior of atoms in strong fields has been well studied, molecules under intense fields are not as well understood and current models have failed in certain important aspects. Molecules, as opposed to atoms, present confounding possibilities of nuclear and electronic motion upon excitation. The dynamics and fragmentation patterns in response to the laser field are structure sensitive; therefore, a molecule cannot simply be treated as a "bag of atoms" during field induced ionization. In this article we present a set of experiments and theoretical calculations exploring the behavior of a large collection of aryl alkyl ketones when irradiated with intense femtosecond pulses. Specifically, we consider to what extent molecules retain their molecular identity and properties under strong laser fields. Using time-of-flight mass spectrometry in conjunction with pump-probe techniques we study the dynamical behavior of these molecules, monitoring ion yield modulation caused by intramolecular motions post ionization. The set of molecules studied is further divided into smaller sets, sorted by type and position of functional groups. The pump-probe time-delay scans show that among positional isomers the variations in relative energies, which amount to only a few hundred millielectronvolts, influence the dynamical behavior of the molecules despite their having experienced such high fields (V/Å). High level ab initio quantum chemical calculations were performed to predict molecular dynamics along with single and multiphoton resonances in the neutral and ionic states. We propose the following model of strong-field ionization and subsequent fragmentation for polyatomic molecules: Single electron ionization occurs on a suboptical cycle time scale, and the electron carries away essentially all of the energy, leaving behind little internal energy in the cation. Subsequent fragmentation of the cation takes place as a result of further photon absorption modulated by one- and two-photon resonances, which provide sufficient energy to overcome the dissociation energy. The proposed hypothesis implies the loss of a photoelectron at a rate that is faster than intramolecular vibrational relaxation and is consistent with the observation of nonergodic photofragmentation of polyatomic molecules as well as experimental results from many other research groups on different molecules and with different pulse durations and wavelengths.

Solvent Environment Revealed by Positively Chirped Pulses.

The spectroscopy of large organic molecules and biomolecules in solution has been investigated using various time-resolved and frequency-resolved techniques. Of particular interest is the early response of the molecule and the solvent, which is difficult to study due to the ambiguity in assigning and differentiating inter- and intramolecular contributions to the electronic and vibrational populations and coherence. Our measurements compare the yield of fluorescence and stimulated emission for two laser dyes IR144 and IR125 as a function of chirp. While negatively chirped pulses are insensitive to solvent viscosity, positively chirped pulses are found to be uniquely sensitive probes of solvent viscosity. The fluorescence maximum for IR125 is observed near transform-limited pulses; however, for IR144, it is observed for positively chirped pulses once the pulses have been stretched to hundreds of femtoseconds. We conclude that chirped pulse spectroscopy is a simple one-beam method that is sensitive to early solvation dynamics.

Optical Response of Fluorescent Molecules Studied by Synthetic Femtosecond Laser Pulses.

The optical response of the fluorescent molecule IR144 in solution is probed by pairs of collinear pulses with intensity just above the linear dependence using two different pulse shaping methods. The first approach mimics a Michelson interferometer, while the second approach, known as multiple independent comb shaping (MICS), eliminates spectral interference. The comparison of interfering and non-interfering pulses reveals that linear interference between the pulses leads to the loss of experimental information at early delay times. In both cases, the delay between the pulses is controlled with attosecond resolution and the sample fluorescence and stimulated emission are monitored simultaneously. An out-of-phase behavior is observed for fluorescence and stimulated emission, with the fluorescence signal having a minimum at zero time delay. Experimental findings are modeled using a two-level system with relaxation that closely matches the phase difference between fluorescence and stimulated emission and the relative intensities of the measured effects.

Solvation Stokes-Shift Dynamics Studied by Chirped Femtosecond Laser Pulses.

The early optical dynamic response, resulting population, and electronic coherence are investigated experimentally and modeled theoretically for IR144 in solution. The fluorescence and stimulated emission response are studied systematically as a function of chirp. The magnitude of the chirp effect on fluorescence and stimulated emission is found to depend quadratically on pulse energy, even where excitation probabilities range from 0.02 to 5%, in the so-called "linear excitation regime". Interestingly, the shape of the chirp dependence on fluorescence and stimulated emission is found to be independent of pulse energy. The chirp dependence reveals dynamics related to solvent rearrangement following excitation and also depends on electronic relaxation of the chromophore. The experimental results are successfully simulated using a four-level model in the presence of inhomogeneous broadening of the electronic transitions.