Conjugation through Si-O-Si bonds, silsesquioxane (SQ) half cage copolymers, extended examples via SiO0.5/SiO1.5 units: multiple emissive states in violation of Kasha's rule.

We previously reported that phenyl- and vinyl-silsesquioxanes (SQs), [RSiO1.5]8,10,12 (R = Ph or vinyl) functionalized with three or more conjugated moieties show red-shifted absorption- and emission features suggesting 3-D conjugation via a cage centered LUMOs. Corner missing [PhSiO1.5]7(OSiMe3)3 and edge opened, end capped [PhSiO1.5]8(OSiMe2)2 (double decker, DD) analogs also offer red shifted spectra again indicating 3-D conjugation and a cage centered LUMO. Copolymerization of DD [PhSiO1.5]8(OSiMevinyl)2 with multiple R-Ar-Br gives copolymers with emission red-shifts that change with degree of polymerization (DP), exhibit charge transfer to F4TNCQ and terpolymer averaged red-shifts suggesting through chain conjugation even with two (O-Si-O) end caps possibly via a cage centered LUMO. Surprisingly, ladder (LL) SQ, (vinylMeSiO2)[PhSiO1.5]4(O2SiMevinyl) copolymers offer emission red-shifts even greater for analogous copolymers requiring a different explanation. Here we assess the photophysical behavior of copolymers of a more extreme SQ form: the half cage [PhSiO1.5]4(OSiMe2Vinyl)4, Vy4HC SQs. We again see small red-shifted absorptions coupled with significant red-shifted emissions, even with just a half cage, thus further supporting the existence of pπ-dπ and/or σ*-π* conjugation through Si-O-Si bonds and contrary to most traditional views of Si-O-Si linked polymers. These same copolymers donate an electron to F4TCNQ generating the radical anion, F4TCNQ-. as further proof of conjugation. Column chromatographic separation of short from longer chain oligomers reveals a direct correlation between DP and emission λmax red-shifts as another indication of conjugation. Further, one- and two-photon absorption and emission spectroscopy reveals multiple excited fluorescence-emitting states in a violation of Kasha's rule wherein emission occurs only from the lowest excited state. Traditional modeling studies again find HOMO LUMO energy levels residing only on the aromatic co-monomers rather than through Si-O-Si bonds as recently found in related polymers.

Phase-separated droplets swim to their dissolution.

Biological macromolecules can condense into liquid domains. In cells, these condensates form membraneless organelles that can organize chemical reactions. However, little is known about the physical consequences of chemical activity in and around condensates. Working with model bovine serum albumin (BSA) condensates, we show that droplets swim along chemical gradients. Active BSA droplets loaded with urease swim toward each other. Passive BSA droplets show diverse responses to externally applied gradients of the enzyme's substrate and products. In all these cases, droplets swim toward solvent conditions that favor their dissolution. We call this behavior "dialytaxis", and expect it to be generic, as conditions which favor dissolution typically reduce interfacial tension, whose gradients are well-known to drive droplet motion through the Marangoni effect. These results could potentially suggest alternative physical mechanisms for active transport in living cells, and may enable the design of fluid micro-robots.

Strongly Polarized π-Extended 1,4-Dihydropyrrolo[3,2-b]pyrroles Fused with Tetrazolo[1,5-a]quinolines.

A straightforward route to 1,4-dihydropyrrolo[3,2-b]pyrroles comprised of two electron-withdrawing quinoline or tetrazolo[1,5-a]quinoline scaffolds has been developed. The versatile multicomponent reaction affording 1,4-dihydropyrrolo[3,2-b]pyrroles combined with intramolecular direct arylation enables assembly of these products in just three steps from anilines with overall yields exceeding 30%. The planarized, ladder-type heteroacenes possess up to 14 conjugated rings. These nominally quadrupolar materials exhibit efficient fluorescence with wavelengths spanning most of the visible spectrum from green-yellow for the dyes possessing biaryl bridges and orange-red for the fully fused systems. In many cases, the fluorescence quantum yields are large, the solvatofluorochromic effects are strong, and the fluorescence is maintained even in crystalline state. Analysis of the electronic structure of these molecular architectures using quantum chemical methods suggests that the character and position of the flanking heterocycle determine the shape of HOMO and LUMO and their extension to N-aryl substituents, influencing the values of molar absorption coefficient. An experimental study of the two-photon absorption (2PA) properties has revealed that it occurs in the 700-800 nm range with apparent deviation from the Laporte parity selection rule, which may be attributed to Hertzberg-Teller contribution to vibronically allowed 2PA transition.

Surface Passivation Method for the Super-repellence of Aqueous Macromolecular Condensates.

Solutions of macromolecules can undergo liquid-liquid phase separation to form droplets with ultralow surface tension. Droplets with such low surface tension wet and spread over common surfaces such as test tubes and microscope slides, complicating in vitro experiments. The development of a universal super-repellent surface for macromolecular droplets has remained elusive because their ultralow surface tension requires low surface energies. Furthermore, the nonwetting of droplets containing proteins poses additional challenges because the surface must remain inert to a wide range of chemistries presented by the various amino acid side chains at the droplet surface. Here, we present a method to coat microscope slides with a thin transparent hydrogel that exhibits complete dewetting (contact angles θ ≈ 180°) and minimal pinning of phase-separated droplets in aqueous solution. The hydrogel is based on a swollen matrix of chemically cross-linked polyethylene glycol diacrylate of molecular weight 12 kDa (PEGDA), and can be prepared with basic chemistry laboratory equipment. The PEGDA hydrogel is a powerful tool for in vitro studies of weak interactions, dynamics, and the internal organization of phase-separated droplets in aqueous solutions.

Slow light and photon echoes in the 7F0→5D0 transition of Sm2+ in single-crystal BaFCl.

Slow light effects induced by transient spectral hole-burning in the 7F0→5D0 transition of Sm2+ in BaFCl at 688 nm are reported and a probe pulse delay of 1.25 µs was observed through a 5 mm thick crystal. This delay corresponds to a reduction of the group velocity vG of the transmitted light to ∼4000 m/s. An analysis of the dependence of the slow light effect on the probe pulse timing indicates some broadening of the spectral hole caused by relatively fast excitation energy transfer. We also demonstrate two-pulse (2PE) and (three-pulse) stimulated photon echoes (SPE) for the first time for Sm2+ in the solid state and a homogenous linewidth of 16 kHz (∼2.5·10-8 nm) was obtained at 1.8 K. The echoes in the optically dense medium were very efficient and revealed spectral diffusion on the 100-µs time scale possibly due to flipping of the fluorine and chlorine nuclear spins in the environment of the Sm2+ ions. Furthermore, the SPE also indicates relatively fast energy transfer, commensurate with the hole decay.

On-off-on Control of Molecular Inversion Symmetry via Multi-stage Protonation: Elucidating Vibronic Laporte Rule.

The Laporte rule dictates that one- and two-photon absorption spectra of inversion-symmetric molecules should display alternatively forbidden electronic transitions; however, for organic fluorophores, drawing clear distinction between the symmetric- and non-inversion symmetric two-photon spectra is often obscured due to prevalent vibronic interactions. We take advantage of consecutive single- and double-protonation to break and then reconstitute inversion symmetry in a nominally symmetric diketopyrrolopyrrole, causing large changes in two-photon absorption. By performing detailed one- and two-photon titration experiments, with supporting quantum-chemical model calculations, we explain how certain low-frequency vibrational modes may lead to apparent deviations from the strict Laporte rule. As a result, the system may be indeed considered as an on-off-on inversion symmetry switch, opening new avenues for two-photon sensing applications.

Photophysical and Electrochemical Properties of Push-Pull Oligo(ferrocenyl-phenyleneethynylene)s: Supramolecular Orders in Molecular Films.

We report on the optoelectronic properties of a series of unsymmetrical π-conjugated phenyleneethynylene macromolecules bearing ferrocene (Fc) as the electron-donor group (D), (benzyl) benzoate (Bz) or benzoic acid (Ac) as the electron attractor group (A) and connected through 2,5-di(alcoxy) phenyleneethynylene(s) (nPE) with n = 1, 2, 3 as π-conjugated bridges. In the series, by increasing the distance between the electron-attracting and electron-donor groups, the push-pull effect decreases. The intramolecular charge transfer (D → π → A) was evaluated by static and dynamic spectroscopy, electrochemistry, and density functional theory (DFT) theoretical calculations. The longest oligomer Fc3PEBz formed the best optical quality films. A study at the atomic level by scanning tunneling microscopy (STM) revealed that the molecules self-assemble on highly ordered pyrolytic graphite (HOPG) in domains with a short-range order. Films are mesoporous and the molecules arrange in a lamellar-like pattern, with an edge-on conformation with respect to HOPG, where the conjugated backbones lie parallel to the surface. Two different assemblies were identified in the monoatomic film, which depends on the ferrocene-ferrocene or benzyl-benzyl interactions.

Novel Lipophilic Fluorophores with Highly Acidity-Dependent Two-Photon Response.

Lipophilic fluorophores are widely implemented in nonlinear microscopy; however, few existing membrane-specific probes combine the high brightness of two-photon excited fluorescence (2PEF) with pH sensitivity. Herein we describe four novel two-photon excited fluorophores, based on a coumarin 151 core structure, where lipophilicity is induced by a covalently attached phosphazene moiety. Changing the environmental acidity using trifluoromethanesulfonic (triflic) acid leads to profound changes in the linear fluorescence and 2PEF characteristics, due to chromophores' switching between neutral- and protonated forms. We characterize this dependence by measuring the two-photon absorption (2PA) spectra over the region λ2PA =550-1000 nm, observing 2PA cross sections of σ2PA =10-20 GM, with an associated 2PEF brightness of 10-13 GM, in neutral solutions of both acetonitrile and n-octanol. Although quantum chemical modelling and NMR measurements show that, at high chromophore concentrations, protonation may be accompanied by a dimerization process, these dimers likely do not form at the lower concentrations used in optical spectroscopy.

Sustained enzymatic activity and flow in crowded protein droplets.

Living cells harvest energy from their environments to drive the chemical processes that enable life. We introduce a minimal system that operates at similar protein concentrations, metabolic densities, and length scales as living cells. This approach takes advantage of the tendency of phase-separated protein droplets to strongly partition enzymes, while presenting minimal barriers to transport of small molecules across their interface. By dispersing these microreactors in a reservoir of substrate-loaded buffer, we achieve steady states at metabolic densities that match those of the hungriest microorganisms. We further demonstrate the formation of steady pH gradients, capable of driving microscopic flows. Our approach enables the investigation of the function of diverse enzymes in environments that mimic cytoplasm, and provides a flexible platform for studying the collective behavior of matter driven far from equilibrium.

Going beyond the borders: pyrrolo[3,2-b]pyrroles with deep red emission.

A two-step route to strongly absorbing and efficiently orange to deep red fluorescent, doubly B/N-doped, ladder-type pyrrolo[3,2-b]pyrroles has been developed. We synthesize and study a series of derivatives of these four-coordinate boron-containing, nominally quadrupolar materials, which mostly exhibit one-photon absorption in the 500-600 nm range with the peak molar extinction coefficients reaching 150 000, and emission in the 520-670 nm range with the fluorescence quantum yields reaching 0.90. Within the family of these ultrastable dyes even small structural changes lead to significant variations of the photophysical properties, in some cases attributed to reversal of energy ordering of alternate-parity excited electronic states. Effective preservation of ground-state inversion symmetry was evidenced by very weak two-photon absorption (2PA) at excitation wavelengths corresponding to the lowest-energy, strongly one-photon allowed purely electronic transition. π-Expanded derivatives and those possessing electron-donating groups showed the most red-shifted absorption- and emission spectra, while displaying remarkably high peak 2PA cross-section (σ 2PA) values reaching ∼2400 GM at around 760 nm, corresponding to a two-photon allowed higher-energy excited state. At the same time, derivatives lacking π-expansion were found to have a relatively weak 2PA peak centered at ca. 800-900 nm with the maximum σ 2PA ∼50-250 GM. Our findings are augmented by theoretical calculations performed using TD-DFT method, which reproduce the main experimental trends, including the 2PA, in a nearly quantitative manner. Electrochemical studies revealed that the HOMO of the new dyes is located at ca. -5.35 eV making them relatively electron rich in spite of the presence of two B--N+ dative bonds. These dyes undergo a fully reversible first oxidation, located on the diphenylpyrrolo[3,2-b]pyrrole core, directly to the di(radical cation) stage.

The golgin family exhibits a propensity to form condensates in living cells.

The Golgi is surrounded by a ribosome-excluding matrix. Recently, we reported that the cis-Golgi-localized golgin GM130 can phase-separate to form dynamic, liquid-like condensates in vitro and in vivo. Here, we show that the overexpression of each of the remaining cis (golgin160, GMAP210)- and trans (golgin97, golgin245, GCC88, GCC185)-golgins results in novel protein condensates. Focused ion beam scanning electron microscopy (FIB-SEM) images of GM130 condensates reveal a complex internal organization with branching aqueous channels. Pairs of golgins overexpressed in the same cell form distinct juxtaposed condensates. These findings support the hypothesis that, in addition to their established roles as vesicle tethers, phase separation may be a common feature of the golgin family that contributes to Golgi organization.

Liquid-liquid phase separation of the Golgi matrix protein GM130.

Golgins are an abundant class of peripheral membrane proteins of the Golgi. These very long (50-400 nm) rod-like proteins initially capture cognate transport vesicles, thus enabling subsequent SNARE-mediated membrane fusion. Here, we explore the hypothesis that in addition to serving as vesicle tethers, Golgins may also possess the capacity to phase separate and, thereby, contribute to the internal organization of the Golgi. GM130 is the most abundant Golgin at the cis Golgi. Remarkably, overexpressed GM130 forms liquid droplets in cells analogous to those described for numerous intrinsically disordered proteins with low complexity sequences, even though GM130 is neither low in complexity nor intrinsically disordered. Virtually pure recombinant GM130 also phase-separates into dynamic, liquid-like droplets in close to physiological buffers and at concentrations similar to its estimated local concentration at the cis Golgi.

Stereochemical Effects on Platinum Acetylide Two-Photon Chromophores.

A series of cis-platinum(II) acetylide complexes containing two-photon-absorbing chromophores have been synthesized and characterized to explore the effects of stereochemistry on the nonlinear absorption properties. The molecules feature 4-(phenylethynyl)phenylethynylene (PE2), diphenylaminofluorene (DPAF), and benzothiazolylfluorene (BTF) ligands. The photophysical properties were investigated under one- and two-photon conditions and compared to the known trans analogues via UV-visible absorption, photoluminescence, femtosecond and nanosecond transient absorption (TA), nanosecond z-scan, and femtosecond two-photon absorption (2PA). The bent cis complexes exhibit blue shifts in the absorption, emission, femtosecond, and nanosecond TA spectra along with lower molar extinction coefficients and lower phosphorescence yields relative to the trans complexes suggesting less efficient Pt-induced spin-orbit coupling and intersystem crossing in the cis configuration. The cis chromophores are noncentrosymmetric and therefore show dipolar behavior with a pronounced 2PA in the 0-0 transition of the S0 → S1 band, while the trans complexes show quadrupolar behavior with a forbidden 0-0 transition. In the S0 → Sn region, both cis and trans complexes show intense two-photon-absorption bands (up to 3700 GM by the peak cross section for cis-BTF) which contain a significant contribution from the excited state absorption (S1 → Sn). All six complexes exhibit comparable nonlinear absorption response with a significant contribution from triplet-triplet absorption that slightly favors trans complexes but is more strongly dependent upon the structure of the π-conjugated chromophore.

S-Palmitoylation Sorts Membrane Cargo for Anterograde Transport in the Golgi.

While retrograde cargo selection in the Golgi is known to depend on specific signals, it is unknown whether anterograde cargo is sorted, and anterograde signals have not been identified. We suggest here that S-palmitoylation of anterograde cargo at the Golgi membrane interface is an anterograde signal and that it results in concentration in curved regions at the Golgi rims by simple physical chemistry. The rate of transport across the Golgi of two S-palmitoylated membrane proteins is controlled by S-palmitoylation. The bulk of S-palmitoylated proteins in the Golgi behave analogously, as revealed by click chemistry-based fluorescence and electron microscopy. These palmitoylated cargos concentrate in the most highly curved regions of the Golgi membranes, including the fenestrated perimeters of cisternae and associated vesicles. A palmitoylated transmembrane domain behaves similarly in model systems.

Two-photon absorption spectra of fluorescent isomorphic DNA base analogs.

Fluorescent DNA base analogs and intrinsic fluorophores are gaining importance for multiphoton microscopy and imaging, however, their quantitative nonlinear excitation properties have been poorly documented. Here we present the two-photon absorption (2PA) spectra of 2-aminopurine (2AP), 7-methyl guanosine (7MG), isoxanthopterin (IXP), 6-methyl isoxanthopterin (6MI), as well as L-tryptophan (L-trp) and 3-methylindole (3MI) in aqueous solution and some organic solvents measured in the wavelength range 550 - 810 nm using femtosecond two-photon excited fluorescence (2PEF) and nonlinear transmission (NLT) methods. The peak 2PA cross section values range from 0.1 GM (1 GM = 10-50 cm4 s photon-1) for 2AP to 2.0 GM for IXP and 7MG. Assuming typical excitation conditions for a scanning 2PEF microscope, we estimate a maximum image frame rate of ~175 frames per second (FPS).

Spontaneous Symmetry Breaking Facilitates Metal-to-Ligand Charge Transfer: A Quantitative Two-Photon Absorption Study of Ferrocene-phenyleneethynylene Oligomers.

Change of the permanent molecular electric dipole moment, Δµ, in a series of nominally centrosymmetric and noncentrosymmteric ferrocene-phenyleneethynylene oligomers was estimated by measuring the two-photon absorption cross-section spectra of the lower energy metal-to-ligand charge-transfer transitions using femtosecond nonlinear transmission method and was found to vary in the range up to 12 D, with the highest value corresponding to the most nonsymmetric system. Calculations of the Δµ performed by the TD-DFT method show quantitative agreement with the experimental values and reveal that facile rotation of the ferrocene moieties relative to the organic ligand breaks the ground-state inversion symmetry in the nominally symmetric structures.

Two Disease-Causing SNAP-25B Mutations Selectively Impair SNARE C-terminal Assembly.

Synaptic exocytosis relies on assembly of three soluble N-ethylmaleimide-sensitive factor attachment receptor (SNARE) proteins into a parallel four-helix bundle to drive membrane fusion. SNARE assembly occurs by stepwise zippering of the vesicle-associated SNARE (v-SNARE) onto a binary SNARE complex on the target plasma membrane (t-SNARE). Zippering begins with slow N-terminal association followed by rapid C-terminal zippering, which serves as a power stroke to drive membrane fusion. SNARE mutations have been associated with numerous diseases, especially neurological disorders. It remains unclear how these mutations affect SNARE zippering, partly due to difficulties to quantify the energetics and kinetics of SNARE assembly. Here, we used single-molecule optical tweezers to measure the assembly energy and kinetics of SNARE complexes containing single mutations I67T/N in neuronal SNARE synaptosomal-associated protein of 25kDa (SNAP-25B), which disrupt neurotransmitter release and have been implicated in neurological disorders. We found that both mutations significantly reduced the energy of C-terminal zippering by ~10 kBT, but did not affect N-terminal assembly. In addition, we observed that both mutations lead to unfolding of the C-terminal region in the t-SNARE complex. Our findings suggest that both SNAP-25B mutations impair synaptic exocytosis by destabilizing SNARE assembly, rather than stabilizing SNARE assembly as previously proposed. Therefore, our measurements provide insights into the molecular mechanism of the disease caused by SNARE mutations.

TD-DFT calculations of one- and two-photon absorption in Coumarin C153 and Prodan: attuning theory to experiment.

We use TD-DFT to calculate the one-photon absorption (1PA) and two-photon absorption (2PA) properties of C153 and Prodan in toluene and DMSO, and benchmark different methods relative to accurate experimental data available from the literature on these particular systems. As the first step, we modify the range-separated TD-DFT to provide the best prediction for the peak 1PA wavelength, and then apply the optimized functionals to achieve quantitative predictions of the corresponding two-photon absorption cross section, σ2PA, with an accuracy ∼10-20% in C153 and ∼20-30% in Prodan. To elucidate the origin of residual discrepancies between the theory and experimental observations, we invoked the two essential states model for σ2PA, which allows us to verify not only the transition wavelength and the σ2PA value, but also to quantitatively benchmark the calculation of key molecular parameters such as the transition dipole moment and the change of the permanent dipole moment. Such comprehensive cross-checking indicates that a larger discrepancy in Prodan is most likely caused by a noted failure of DFT to predict the relative intensity and relative ordering of closely lying excited states with different degrees of intramolecular charge transfer, which we further support by analyzing the predictions obtained by high-level coupled-cluster calculations in the gas phase. Our results highlight the utility of benchmarking the calculations not only relative to other theoretical methods, but also in comparison to the experimental measurements, wherever such data are available.

Two-Photon Spectroscopy of a Series of Platinum Acetylides: Conformation-Induced Ground-State Symmetry Breaking.

With the goal of elucidating electronic and conformational effects on structure-spectroscopic property relationships in platinum acetylides, we synthesized a series of nominally centrosymmetric chromophores trans-Pt(PBu3)2(C≡C-Phenyl-X)2, where X = diphenylamino (DPA), NH2, OCH3, t-Bu, CH3, H, F, benzothiazole (BTH), CF3, CN, and NO2. We collected one- and two-photon absorption spectra and also performed density functional theory (DFT) and time-dependent (TD) DFT calculations on the ground- and excited-state properties of these compounds. The DFT calculations revealed facile rotation between the two ligands, suggesting that the compounds exhibit nonplanar ground-state conformations in solution. TDDFT calculation of the S1 state energy and transition dipole moment for a nonplanar conformation gave good agreement with experiment. Two-photon absorption spectra obtained from these compounds allowed estimation of the change of permanent electric dipole moment upon vertical excitation from ground state to S1 state. The values are small Δµ < 1.0 D for neutral substituents such as CH3, H, and F but increase sharply to Δµ ≈ 11 D for electron-accepting NO2. When in a nonplanar conformation, the corresponding calculated Δµ values showed good agreement with the experimental data indicating that the two-photon spectra result from nonplanar ground-state conformations. Previously studied related chromophores having extended conjugation ( Rebane, A.; Drobizhev, M.; Makarov, N. S.; Wicks, G.; Wnuk, P.; Stepanenko, Y.; Haley, J. E.; Krein, D. M.; Fore, J. L.; Burke, A. R.; Slagle, J. E.; McLean, D. G.; Cooper, T. M. J. Phys. Chem. A 2014 , 118 , 3749 - 3759 ) show similar dependence of Δµ on the substituents, which allows us to conclude that the excited-state properties of these floppy chromophores are a function of the electronic properties of the substituents, ligand size, and nonplanar molecular conformation.

Ultra-slow light propagation by self-induced transparency in ruby in the superhyperfine limit.

Self-induced transparency is reported for circularly polarized light in the R1(-3/2) line of a 30 ppm ruby (α-Al2O3:Cr3+) at 1.7 K in a magnetic field of B‖c=4.5 T. In such a field and temperature, a 30 ppm ruby is in the so-called superhyperfine limit resulting in a long phase memory time, TM=50 µs, and a thousand-fold slower pulse propagation velocity of ∼300 m/s was observed, compared to the ∼300 km/s measured in the first observation of self-induced transparency (SIT) ∼50 years ago, that employed a ruby with a 500 ppm chromium concentration in zero field and at 4.2 K. To date, this is the slowest pulse propagation ever observed in a SIT experiment.