Simultaneous High-Throughput Monitoring of Urethane Reactions Using Near-Infrared Hyperspectral Imaging.

High-throughput (HTP) research is becoming more widely utilized due to its advantages in rapid screening of large parameter space. When HTP is used for reaction screening, often only the end products are analyzed by off-line techniques, leaving behind valuable process information. Information-rich spectroscopy tools have remained under-utilized in HTP workflows. In this study, near-infrared (NIR) hyperspectral imaging (HSI) is demonstrated to be a versatile and accurate tool that can simultaneously monitor multiple reactions, opening up future opportunities to maximize information extraction from such HTP reaction screening experiments. Model urethane reactions are used here to demonstrate the concept, and the general approach can be widely applied to any reactions involving NIR-active functional groups. The fast speed and accurate chemical information made possible by NIR HSI are expected be another important addition to the toolkit of HTP research.

Structural Tuning of Quantum Decoherence and Coherent Energy Transfer in Photosynthetic Light Harvesting.

Photosynthetic organisms capture energy from solar photons by constructing light-harvesting proteins containing arrays of electronic chromophores. Collective excitations (excitons) arise when energy transfer between chromophores is coherent, or wavelike, in character. Here we demonstrate experimentally that coherent energy transfer to the lowest-energy excitons is principally controlled in a light-harvesting protein by the temporal persistence of quantum coherence rather than by the strength of vibronic coupling. In the peridinin-chlorophyll protein from marine dinoflagellates, broad-band two-dimensional electronic spectroscopy reveals that replacing the native chlorophyll a acceptor chromophores with chlorophyll b slows energy transfer from the carotenoid peridinin to chlorophyll despite narrowing the donor-acceptor energy gap. The formyl substituent on the chlorophyll b macrocycle hastens decoherence by sensing the surrounding electrostatic noise. These findings demonstrate how quantum coherence enhances the efficiency of energy transfer despite being very short lived in light-harvesting proteins at physiological temperatures.

Fluorescence and Excited-State Conformational Dynamics of the Orange Carotenoid Protein.

The orange carotenoid protein (OCP) mediates nonphotochemical quenching (NPQ) mechanisms in cyanobacteria. A bound ketocarotenoid serves as a sensor of midvisible light intensity and as a quencher of phycocyanobilin excitons in the phycobilisome. The photochemical mechanism that triggers conversion of the protein from a resting, orange state (OCPO) to an active, red state (OCPR) after optical preparation of the S2 state of the carotenoid remains an open question. We report here that the fluorescence spectrum and quantum yield of the bound carotenoids in OCPO report important details of the motions that follow optical preparation of the S2 state. The fluorescence spectra from OCPO preparations containing 3'-hydroxyechinenone (3hECN) or canthaxanthin (CAN) are markedly mirror asymmetric with respect to the absorption line shape and more than an order of magnitude more intense than for carotenoids in solution. Further, 3hECN exhibits a narrower fluorescence line shape and a larger quantum yield than CAN because its excited-state motions are hindered by a hydrogen bonding interaction between the 3'-hydroxyl group on its ß2 ring and Leu37 in the N-terminal domain. These results show that large-amplitude motions of the carotenoid's ß2-cyclohexene ring and of the conjugated polyene backbone initiate photochemistry in OCPO.

Quantum Coherent Excitation Energy Transfer by Carotenoids in Photosynthetic Light Harvesting.

It remains an open question whether quantum coherence and molecular excitons created by delocalization of electronic excited states are essential features of the mechanisms that enable efficient light capture and excitation energy transfer to reaction centers in photosynthetic organisms. The peridinin-chlorophyll a protein from marine dinoflagellates is an example of a light-harvesting system with tightly clustered antenna chromophores in which quantum coherence has long been suspected, but unusually it features the carotenoid peridinin as the principal light absorber for mid-visible photons. We report that broad-band two-dimensional electronic spectroscopy indeed reveals the initial presence of exciton relaxation pathways that enable transfer of excitation from peridinin to chlorophyll a in <20 fs, but the quantum coherence that permits this is very short-lived. Strongly coupled excited-state vibrational distortions of the peridinins trigger a dynamic transition of the electronic structure of the system and a rapid conversion to incoherent energy transfer mechanisms.

Excitation Energy Transfer by Coherent and Incoherent Mechanisms in the Peridinin-Chlorophyll a Protein.

Excitation energy transfer from peridinin to chlorophyll (Chl) a is unusually efficient in the peridinin-chlorophyll a protein (PCP) from dinoflagellates. This enhanced performance is derived from the long intrinsic lifetime of 4.4 ps for the S2 (11Bu+) state of peridinin in PCP, which arises from the electron-withdrawing properties of its carbonyl substituent. Results from heterodyne transient grating spectroscopy indicate that S2 serves as the donor for two channels of energy transfer: a 30 fs process involving quantum coherence and delocalized peridinin-Chl states and an incoherent, 2.5 ps process initiated by dynamic exciton localization, which accompanies the formation of a conformationally distorted intermediate in 45 fs. The lifetime of the S2 state is lengthened in PCP by its intramolecular charge-transfer character, which increases the system-bath coupling and slows the torsional motions that promote nonradiative decay to the S1 (21Ag-) state.

Torsional Dynamics and Intramolecular Charge Transfer in the S2 (1(1)Bu(+)) Excited State of Peridinin: A Mechanism for Enhanced Mid-Visible Light Harvesting.

Of the carotenoids known in photosynthetic organisms, peridinin exhibits one of the highest quantum efficiencies for excitation energy transfer to chlorophyll (Chl) a acceptors. The mechanism for this enhanced performance involves an order-of-magnitude slowing of the S2 (1(1)Bu(+)) → S1 (2(1)Ag(-)) nonradiative decay pathway compared to carotenoids lacking carbonyl substitution. Using femtosecond transient grating spectroscopy with optical heterodyne detection, we have obtained the first evidence that the nonradiative decay of the S2 state of peridinin is promoted by large-amplitude torsional motions. The decay of an intermediate state termed Sx, which we assign to a twisted form of the S2 state, is substantially slowed by solvent friction in peridinin due to its intramolecular charge transfer (ICT) character.

Femtosecond Heterodyne Transient Grating Studies of Nonradiative Deactivation of the S2 (1(1)Bu(+)) State of Peridinin: Detection and Spectroscopic Assignment of an Intermediate in the Decay Pathway.

Femtosecond heterodyne transient grating spectroscopy was employed to investigate the nonradiative decay pathway from the S2 (1(1)Bu(+)) state to the S1 (2(1)Ag(-)) state of peridinin in methanol solution. Just as previously observed by this laboratory for ß-carotene in benzonitrile, the real (absorption) and imaginary (dispersion) components of the transient grating signal obtained with Fourier transform spectral interferometry from peridinin exhibit ultrafast responses indicating that S2 state decays in 12 fs to produce an intermediate state, Sx. The excited state absorption spectrum from the Sx state of peridinin, however, is found to be markedly blue-shifted from that of ß-carotene because it makes a substantial contribution to the signal observed with 40 fs, 520 nm pulses. The results of a global target analysis and numerical simulations using nonlinear response functions and the multimode Brownian oscillator model support the assignment of Sx to a displaced conformation of the S2 state rather than to a vibrationally excited (or hot) S1 state. The Sx state in peridinin is assigned to a structure with a distorted conjugated polyene backbone moving past an activation-energy barrier between planar and twisted structures on the S2 potential surface. The lengthened lifetime of the Sx state of peridinin in methanol, 900 ± 100 fs, much longer than that typically observed for carotenoids lacking carbonyl substituents, ∼150 fs, can be attributed to the slowing of torsional motions by solvent friction. In peridinin, the system-bath coupling is significantly enhanced over that in ß-carotene solution most likely due to the intrinsic intramolecular charge transfer character it derives from the electron withdrawing nature of the carbonyl substituent. An important additional implication is that the Sx state, and the distorted structures reached subsequently along the torsional gradient on the S2 potential surface, may serve as the principal excitation energy transfer donors to chlorophyll a in the peridinin-chlorophyll a protein from dinoflagellates.

Femtosecond Heterodyne Transient-Grating Studies of Nonradiative Decay of the S2 (1(1)Bu(+)) State of ß-Carotene: Contributions from Dark Intermediates and Double-Quantum Coherences.

Femtosecond transient-grating spectroscopy with heterodyne detection was employed to characterize the nonradiative decay pathway in ß-carotene from the S2 (1(1)Bu(+)) state to the S1 (2(1)Ag(-)) state in benzonitrile solution. The results indicate definitively that the S2 state populates an intermediate state, Sx, on an ultrafast time scale prior to nonradiative decay to the S1 state. Numerical simulations using the response function formalism and the multimode Brownian oscillator model were used to fit the absorption and dispersion components of the transient-grating signal with a common set of parameters for all of the relevant Feynman pathways, including double-quantum coherences. The requirement for inclusion of the Sx state in the nonradiative decay pathway is the observed fast rise time of the dispersion component, which is predominantly controlled by the decay of the stimulated emission signal from the optically prepared S2 state. The finding that the excited-state absorption spectrum from the Sx state is significantly red-shifted from that of S2 and S1 leads to a new assignment for the spectroscopic origin of the Sx state. Rather than assigning Sx to a discrete electronic state, such as the (1)Bu(-) state suggested in previous work, it is proposed that the Sx state corresponds to a transition-state-like structure on the S2 potential surface. In this hypothesis, the 12 fs time constant for the decay of the S2 state corresponds to a vibrational displacement of the C-C and C═C bond-length alternation coordinates of the conjugated polyene backbone from the optically prepared Franck-Condon structure to a potential energy barrier on the S2 surface that divides planar and torsionally displaced structures. The lifetime of the Sx state would be associated with a subsequent relaxation along torsional coordinates over a steep potential energy gradient toward a conical intersection with the S1 state. This hypothesis leads to the idea that twisted structures with intramolecular charge-transfer character along the S2 torsional gradient are active in excitation energy-transfer mechanisms to (bacterio)chlorophyll acceptors.

Vibrationally Coherent Preparation of the Transition State for Photoisomerization of the Cyanine Dye Cy5 in Water.

Femtosecond pump-continuum probe spectroscopy with impulsive excitation was employed to observe coherent wavepacket motions of the cyanine dye Cy5 in water that promote photoisomerization after optical preparation of the first excited singlet state, S1. The chief component in the excited-state vibrational coherence is a resonance Raman-inactive, 273 cm(-1) mode of mixed carbon-carbon bond length alternation and out-of-plane or twisting character. The ultrafast (30 fs) damping of these motions arises from trajectories that irreversibly cross the transition state barrier; after several recurrences to the transition state region, vibrational cooling traps a significant fraction of the excited-state molecules in the planar, Franck-Condon region of the potential energy surface. Motion in the 273 cm(-1) promoting mode is apparently launched by a change in conformation of the conjugated polyene backbone during the first few vibrations of the high-frequency C-C and C═C bond length alternation coordinates that principally contribute to the initial displacement from the Franck-Condon structure. To our knowledge, this work provides the first direct observations of the intramolecular redistribution of excited-state potential energy into reactive motions that are rapidly damped by transition state barrier-crossing events leading to photoisomerization in a conjugated polyene. These results provide insight into the vibrational dynamics that contribute to the photoisomerization of retinal protonated Schiff bases in the rhodopsins and to the formation of intramolecular charge transfer character in carotenoids in photosynthetic light-harvesting proteins.

Excited state conformational dynamics in carotenoids: dark intermediates and excitation energy transfer.

A consideration of the excited state potential energy surfaces of carotenoids develops a new hypothesis for the nature of the conformational motions that follow optical preparation of the S2 (1(1)Bu(+)) state. After an initial displacement from the Franck-Condon geometry along bond length alternation coordinates, it is suggested that carotenoids pass over a transition-state barrier leading to twisted conformations. This hypothesis leads to assignments for several dark intermediate states encountered in femtosecond spectroscopic studies. The Sx state is assigned to the structure reached upon the onset of torsional motions near the transition state barrier that divides planar and twisted structures on the S2 state potential energy surface. The X state, detected recently in two-dimensional electronic spectra, corresponds to a twisted structure well past the barrier and approaching the S2 state torsional minimum. Lastly, the S(∗) state is assigned to a low lying S1 state structure with intramolecular charge transfer character (ICT) and a pyramidal conformation. It follows that the bent and twisted structures of carotenoids that are found in photosynthetic light-harvesting proteins yield excited-state structures that favor the development of an ICT character and optimized energy transfer yields to (bacterio)chlorophyll acceptors.