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Among near-infrared (NIR) dyes, squaraine derivatives are applied as efficient sensitizers in optoelectronic and biomedical devices due to their simple synthesis, intense absorption, and emission and exceptional photochemical stability. The fundamental understanding of the structure-property relationships of sensitizers provides the insight to increase the efficiency of such devices. Here, unsymmetrical squaraine derivatives ( ABSQs ) with donor-acceptor-donor (D-A-D') architectures having N , N -dimethyl amino anthracene and benzothiazole ( ABSQ-H ) halogenated with fluoride ( ABSQ-F ), chloride ( ABSQ-Cl ), and bromide ( ABSQ-Br ) were synthesized to understand the effect of halogen on the photophysical properties and intermolecular interaction dynamics with phenyl-C 61 -butyric acid methyl ester (PCBM), which is used widely as an electron acceptor in bulk heterojunction-based devices. Interestingly, ABSQ-H exhibited intense absorption (ε ∼ 6.72 × 10 4 M -1 cm -1 ) spectra centered at ∼660 nm. Upon halogen substitution, a bathochromic shift in the absorption spectra with an increase of molar absorptivity was observed (ε ∼ 8.59 × 10 4 M -1 cm -1 ), which is beneficial for NIR light harvesting. The femtosecond transient absorption spectra of ABSQs revealed that the polarity of the solvent controlled the excited-state relaxation dynamics. Upon addition of PCBM, the fluorescence intensity and dynamics of halogenated ABSQs were quenched, and the formation of a squaraine radical cation was observed, reflecting the occurrence of intermolecular charge-transfer dynamics between ABSQs and PCBM. Thus, the observation of a bathochromic shift with intense absorption and an efficient intermolecular interaction with PCBM upon halogenation of ABSQs provide a design strategy for the development of unsymmetrical squaraine derivatives for bulk heterojunction-based optoelectronic devices.

The role of excimer formation in inhibiting or enhancing the efficiency of the intramolecular singlet fission (iSF) process has been a subject of recent debate. Here, we investigated the effect of excimer formation on iSF dynamics by modifying its configuration by connecting pentacenes at various positions. Hence, pentacene dimers having slip-stacked ( 2,2' BP , J-type), oblique ( 2,6' BP ), and facial ( 6,6' BP , H-type) configurations were synthesized by covalently linking pentacenes at positions 2,2', 2,6', and 6,6', respectively, with an ethynyl bridge, and their ultrafast excited-state relaxation dynamics were characterized. Femtosecond time-resolved transient absorption spectra revealed that the efficiency of iSF dynamics decreased from slip-stacked (182%) to oblique configuration (97%),whereas in the 6,6' BP with facial configuration, strong electronic coupling led to the formation of excimers that decayed nonradiatively without formation of correlated triplet pairs. These studies reveal the formation of excimers by strong intrapentacene electronic coupling upon ultrafast excitation, preventing the efficient iSF process.

Recent advancements in a nanoarchitecture platform for safe and effective targeted phototherapy in a synergistic fashion is an absolute necessity in localized cancer therapy. Photothermal and photodynamic therapies (PTT and PDT) are considered as the most promising localized therapeutic intervention for cancer management as they have no long-term side effects and are minimally invasive and affordable. Herein, we have demonstrated a tailor-made nanotheranostic probe in which macrocyclic host cucurbituril [8] (CB[8]) is placed as a glue between two gold nanorods (GNRs) within ∼3 nm gaps in linear nanoassemblies with exquisitely sensitive plasmonics that exert combined phototherapy to investigate the therapeutic progression on human breast cancer cells. Photosensitizer methylene blue was positioned on CB[8] to impart the PDT effect, whereas GNR was responsible for PTT on a single laser trigger ensuring the synchronized phototherapy. Furthermore, the nanoconstruct was tagged with targeting anti-Her2 monoclonal antibody (MB-CB[8]@GNR-anti-Her2) for localized PTT and PDT on Her2 positive SKBR3 cells, subsequent cellular recognition by surface-enhanced Raman spectroscopy (SERS) platform, and further assessment of the combined intracellular phototherapy. Hence, the current strategy is definitely marked as a proof-of-concept straightforward approach that implies the perfect nature of the combined phototherapy to achieve an efficient cancer treatment.

Singlet fission (SF) improves the power conversion efficiency of optoelectronic devices by converting high-energy photons into two triplet excitons. SF dynamics and efficiency (Φ) are controlled by various factors. Here, the effect of planarity and length of the bridge in pentacene dimers on the intramolecular SF (iSF) process was investigated by synthesizing the dimers connected by bridges having fluorene ( FL - PD , planar), methyl-substituted biphenyl ( MBP - PD , twisted), and diphenyl acetylene ( DPA - PD , longer) groups and characterizing their excited-state relaxation dynamics using nanosecond and femtosecond pump-probe spectroscopy. Transient absorption studies reveal that iSF dynamics of FL - PD having a planar bridge are ∼787 times faster (187 ps) and exhibit higher Φ (198%) by feasible electronic coupling, compared to MBP - PD possessing a twisted bridge showing a low Φ of ∼16%. However compared to FL - PD , iSF dynamics of DPA - PD with an increase of bridge length are slower by an order (1.09 ns) and show comparable Φ of 185% through extended conjugation. Thus, the planarity and length of the bridge in pentacene dimers control the rate and efficiency of the iSF process.

The ubiquitous electron transfer heme protein, Cytochrome c (Cyt c) catalyzes the peroxidation of cardiolipin (CL) in the early stage of apoptosis, where Cyt c undergoes conformational changes leading to the partial unfolding of the protein. Here the interaction dynamics of Cyt c with liposomes having different charges [CL, - 2; POPG (2-Oleoyl-1-palmitoyl- sn -glycero-3-phospho-rac-(1-glycerol) sodium salt), -1; and POPC (2-Oleoyl-1-palmitoyl- sn -glycero-3-phosphocholine), 0] leading to various degrees of partial unfolding is investigated with steady state optical spectroscopy and femtosecond time-resolved pump-probe spectroscopy. The signature of the partial unfolding of the protein was observed in the absorption, fluorescence, and CD spectra of Cyt c-liposome complexes with an increase of lipid/protein (L/P) ratio, and the protein was refolded by the addition of 0.1 M of NaCl. The femtosecond transient absorption spectra of the complexes were measured by selectively exciting the heme and tryptophan (Trp) at 385 and 280 nm, respectively. Though significant changes were not observed in the excited state relaxation dynamics of the heme in liposomes by exciting at 385 nm, the 280 nm excitation exhibited a systematic increase of the excited state relaxation dynamics leading to the increase of lifetime of Trp and global conformational relaxation dynamics with the increase of anionic charge of the lipids. This reveals the decrease of efficiency of fluorescence resonance energy transfer from Trp to heme due to the increase of distance between them upon increase of partial unfolding of the proteins by liposomes. Such observation exhibits the Trp as a marker amino acid to reflect the dynamics of partial unfolding of the protein rising from the change in the tertiary structure and axial ligand interaction of the heme proteins in liposomes. The relaxation dynamics of the complexes in the presence of salt are similar to that of the protein alone, reflecting that the refolding of the protein and the interactions are dominated by electrostatic interaction rather than the hydrophobic interaction.

Red fluorescent proteins with a large Stokes shift offer a limited autofluorescence background and are used in deep tissue imaging. Here, by introducing the free amino group in Aequorea victoria, the electrostatic charges of the p-hydroxybenzylidene imidazolinone chromophore of green fluorescent protein (GFP) have been altered resulting in an unusual, 85 nm red-shifted fluorescence. The structural and biophysical analysis suggested that the red shift is due to positional shift occupancy of Glu222 and Arg96, resulting in extended conjugation and a relaxed chromophore. Femtosecond transient absorption spectra exhibited that the excited state relaxation dynamics of red-shifted GFP (rGFP) (τ 4 = 234 ps) are faster compared to the A. victoria green fluorescent protein (τ 4 = 3.0 ns). The nanosecond time-resolved emission spectra of rGFP reveal the continuous spectral shift during emission by a solvent reorientation in the chromophore. Finally, the molecular dynamics simulations revealed the rearrangement of the hydrogen bond interactions in the chromophore vicinity, reshaping the symmetric distribution of van der Waals space to fine tune the GFP structure resulting from highly red-shifted rGFP.

Hemin is a unique model compound of heme proteins carrying out variable biological functions. Here, the excited state relaxation dynamics of heme model compounds in the ferric form are systematically investigated by changing the axial ligand (Cl/Br), the peripheral substituent (vinyl/ethyl-meso), and the solvent (methanol/DMSO) using femtosecond pump-probe spectroscopy upon excitation at 380 nm. The relaxation time constants of these model compounds are obtained by global analysis. Excited state deactivation pathway of the model compounds comprising the decay of the porphyrin excited state (S*) to ligand to metal charge transfer state (LMCT, τ 1 ), back electron transfer from metal to ligand (MLCT, τ 2 ), and relaxation to the ground state through different electronic spin states of iron (τ 3 and τ 4 ) are proposed along with the vibrational cooling processes. This is based on the excited state absorption spectral evolution, similarities between the transient absorption spectra of the ferric form and steady state absorption spectra of the low-spin ferrous form, and the data analysis. The observation of an increase of all the relaxation time constants in DMSO compared to the methanol reflects the stabilization of intermediate states involved in the electronic relaxation. The transient absorption spectra of met-myoglobin are also measured for comparison. Thus, the transient absorption spectra of these model compounds reveal the involvement of multiple iron spin states in the electronic relaxation dynamics, which could be an alternative pathway to the ground state beside the vibrational cooling processes and associated with the inherent features of the heme b type.

Interaction of light with electron donor-acceptor π-conjugated systems leading to intramolecular charge transfer (ICT) plays an essential role in transformation of light energy. Here the cascade of photoinduced ICT processes is directly observed by investigating the excited state relaxation dynamics of cyano and mono/di methoxy substituted diphenyl acetylene derivatives using femtosecond pump-probe spectroscopy and nanosecond laser flash photolysis. The femtosecond transient absorption spectra of the chromophores upon ultrafast excitation reveal the dynamics of intermediates involved in transition from initially populated Frank-Condon state to local excited state (LE). It also provides the dynamic details of the transition from the LE to the charge transfer state yielding the formation of the radical ions. Finally, the charge transfer state decays to the triplet state by geminate charge recombination. The latter dynamics are observed in the nanosecond transient absorption spectra. It is found that excited state relaxation pathways are controlled by different stages of solvation and intramolecular relaxation depending on the solvent polarity. The twisted ICT state is more stabilized (978 ps) in acetonitrile than cyclohexane where major components of transient absorption originate from the S1 state.

Femtosecond vibrational coherence spectroscopy is used to investigate the low frequency vibrational dynamics of the electron transfer heme protein, cytochrome c (cyt c). The vibrational coherence spectra of ferric cyt c have been measured as a function of excitation wavelength within the Soret band. Vibrational coherence spectra obtained with excitation between 412 and 421 nm display a strong mode at ~44 cm(-1) that has been assigned to have a significant contribution from heme ruffling motion in the electronic ground state. This assignment is based partially on the presence of a large heme ruffling distortion in the normal coordinate structural decomposition (NSD) analysis of the X-ray crystal structures. When the excitation wavelength is moved into the ~421-435 nm region, the transient absorption increases along with the relative intensity of two modes near ~55 and 30 cm(-1). The intensity of the mode near 44 cm(-1) appears to minimize in this region and then recover (but with an opposite phase compared to the blue excitation) when the laser is tuned to 443 nm. These observations are consistent with the superposition of both ground and excited state coherence in the 421-435 nm region due to the excitation of a weak porphyrin-to-iron charge transfer (CT) state, which has a lifetime long enough to observe vibrational coherence. The mode near 55 cm(-1) is suggested to arise from ruffling in a transient CT state that has a less ruffled heme due to its iron d(6) configuration.

The equilibrium unfolding process of ferric horse heart cytochrome c (cyt c), induced by guanidinium hydrochloride (GdHCl), was studied using UV-vis absorption spectroscopy, resonance Raman spectroscopy, and vibrational coherence spectroscopy (VCS). The unfolding process was successfully fit using a three-state model which included the fully folded (N) and unfolded (U) states, along with an intermediate (I) assigned to a Lys bound heme. The VCS spectra revealed for the first time several low-frequency heme modes that are sensitive to cyt c unfolding: γ(a) (~50 cm(-1)), γ(b) (~80 cm(-1)), γ(c) (~100 cm(-1)), and ν(s)(His-Fe-His) at 205 cm(-1). These out-of-plane modes have potential functional relevance and are activated by protein-induced heme distortions. The free energies for the N-I and the I-U transitions at pH 7.0 and 20 °C were found to be 4.6 kcal/M and 11.6 kcal/M, respectively. Imidazole was also introduced to replace the methionine ligand so the unfolding can be modeled as a two-state system. The intensity of the mode γ(b)~80 cm(-1) remains nearly constant during the unfolding process, while the amplitudes of the other low frequency modes track with spectral changes observed at higher frequency. This confirms that the heme deformation changes are coupled to the protein tertiary structural changes that take place upon unfolding. These studies also reveal that damping of the coherent oscillations depends sensitively on the coupling between heme and the surrounding water solvent.

Vibrational coherence spectroscopy (VCS) is used to investigate the low-frequency dynamics of camphor-free and camphor-bound cytochrome P450(cam) (CYP 101) and its L358P mutant. The low-frequency heme vibrations are found to be perturbed upon binding to the electron transfer partner putidaredoxin (Pdx). A strong correlation between the "detuned" vibrational coherence spectrum, which monitors frequencies between 100 and 400 cm(-1), and the lower frequency part of the Raman spectrum is also demonstrated. The very low frequency region ≤200 cm(-1), uniquely accessed by open-band VCS measurements, reveals a mode near 103 cm(-1) in P450(cam) when camphor is not present in the distal pocket. This reflects the presence of a specific heme distortion, such as saddling or ruffling, in the substrate-free state where water is coordinated to the low-spin iron atom. Such distortions are likely to retard the rate of electron transfer to the substrate-free protein. The presence of strong mode near ∼33 cm(-1) in the camphor-bound form suggests a significant heme-doming distortion, which is supported by analysis using normal coordinate structural decomposition. Pdx also displays a strong coherent vibration near 30 cm(-1) that in principle could be involved in vibrational resonance with its electron transfer target. A splitting of the 33 cm(-1) feature and intensification of a mode near 78 cm(-1) appear when the P450(cam)/Pdx complex is formed. These observations are consistent with vibrational mixing and heme geometric distortions upon Pdx binding that are coincident with the increased thiolate electron donation to the heme. The appearance of a mode near 65 cm(-1) in the coherence spectra of the L358P mutant is comparable to the mode at 78 cm(-1) seen in the P450(cam)/Pdx complex and is consistent with the view that the heme and its environment in the L358P mutant are similar to the Pdx-bound native protein. Resonance Raman spectra are presented for both P450(cam) and the L358P mutant and the changes are correlated with an increased amount of thiolate electron donation to the heme in the mutant sample.

Vibrational coherence spectroscopy is used to study the low frequency dynamics of the truncated dimer of human cystathionine beta-synthase (CBS). CBS is a pyridoxal-5'-phosphate-dependent heme enzyme with cysteine and histidine axial ligands that catalyzes the condensation of serine and homocysteine to form cystathionine. A strong correlation between the "detuned" coherence spectrum (which probes higher frequencies) and the Raman spectrum is demonstrated, and a rich pattern of modes below 200 cm(-1) is revealed. Normal coordinate structural decomposition (NSD) of the ferric CBS crystal structure predicts the enhancement of normal modes with significant heme "doming", "ruffling", and "saddling" content, and they are observed in the coherence spectra near approximately 40, approximately 60, and approximately 90 cm(-1). When pH is varied, the relative intensities and frequencies of the low frequency heme modes indicate the presence of a unique protein-induced heme structural perturbation near pH 7 that differs from what is observed at higher or lower pH. For ferric CBS, we observe a new mode near approximately 25 cm(-1), possibly involving the response of the protein, which exhibits a phase jump of approximately pi for excitation on the blue and red side of the Soret band maximum. The low frequency vibrational coherence spectrum of ferrous CBS is also presented, along with our efforts to probe its NO-bound complex. The CO geminate rebinding kinetics of CBS are similar to the CO-bound form of the gene activator protein CooA, but with the appearance of a significant additional kinetic inhomogeneity. Analysis of this inhomogeneity suggests that it arises from the two subunits of CBS and leads to a factor of approximately 20 for the ratio of the average CO geminate rebinding rates of the two subunits.