Multipolar symmetric squaraines with large two-photon absorption cross-sections in the NIR region

The two-photon absorption (TPA) properties of two extended symmetric squaraine dyes are thoroughly characterized from the experimental and quantum-chemical point of view. The two molecules are specially engineered to have a multipolar structure with increasing complexity, D-π-A-π-D and A'-π-D-π-A-π-D-π-A', respectively. The experimental TPA spectra, measured by means of the Z-scan technique in the femtoseconds regime, display considerably high values of TPA cross sections (σ(TPA)) for both molecules. In particular, the squaraine with the more extended structure shows the highest value of σ(TPA) ever reported for this class of molecules. CIS and TDDFT calculations of the one and two-photon absorption properties are carried out to clarify the origin of the observed TPA properties and fully characterize the electronic properties of these compounds. The calculations, in good agreement with the experimental data, suggest that the origin of this exceptionally high σ(TPA) can be ascribed to the presence of a peripheral A' group, that increases the density of excited states involved in the TPA process.     

Self-assembled nanochaperones enable the disaggregation of amyloid insulin fibrils

The deposition of highly ordered amyloid fibrils is recognized as a hallmark of amyloidosis diseases such as Alzheimer’s disease and Parkinson’s disease.Disaggregating the amyloid fibrils is considered as one of the effective strategies for the control and treatment of amyloidosis diseases.In this article,by simulating the function of natural molecular chaperones,co-assembled block copolymer micelles with coordination groups of nitrilotriacetic acid(NTA)and hydrophobic microdomains of poly(Nisopropylacrylamide)(PNIPAM)on the surface were used as nanochaperones(n Chaps)to disaggregate amyloid insulin fibrils.Zinc ions chelated by NTA can bind the histidine imidazole residues while the PNIPAM microdomains can interact with the exposed hydrophobic sites on the amyloid insulin fibrils,which synergistically perturb the stability of amyloid insulin fibrils,loosen their structure,and finally promote their disaggregation.A combination of characterizations with fluorescence spectroscopy,transmission electron microscopy(TEM),dynamic hight scattering(DLS),and quartz crystal microbalance(QCM)demonstrated that mature amyloid insulin fibrils were completely disaggregated after incubating with n Chaps for 90 h.This study may provide a promising strategy for the development of n Chaps for the treatment of amyloidosis diseases.     

Microfluidic self-assembly of insulin monomers into amyloid fibrils on a solid surface

We report the self-assembly of insulin monomers into amyloid fibrils within microchannels. To demonstrate the microfluidic amyloid formation and fibril growth on a solid surface, we seeded the internal surfaces of the microchannels with insulin monomers via N-hydroxysuccinimide ester activation and continuously flushed a fresh insulin solution through the microchannels. According to our analysis using optical and fluorescence microscopy, insulin amyloid preferentially formed in the center of the microchannels and, after reaching a certain density, spread to the side walls of the microchannels. By using ex situ atomic force microscopy, we observed the growth of amyloid fibrils inside the microchannels, which occurred at a much higher rate than that in bulk systems. After 12 h of incubation, insulin formed amyloid spherulites having "Maltese cross" extinction patterns within the microchannels according to the polarized microscopic analysis. Microfluidic amyloid formation enabled low consumption of reagents, reduction of incubation time, and simultaneous observation of amyloid formation under different conditions. This work will contribute to the rapid analysis of amyloid formation associated with many protein misfolding diseases.     

Extremely strong near-IR two-photon absorption in conjugated porphyrin dimers: quantitative description with three-essential-states model

Two-photon absorption spectra (2PA) of a series of conjugated dimers and the corresponding monomer were studied in the near-IR region. All of the dimers show very large peak cross section values, sigma(2) = (3-10) x 10(3) GM (1 GM = 1 x 10(-50) cm(4) s photon(-1)), which is several hundred times larger than that obtained for the corresponding monomer in the same region. We explain such dramatic cooperative enhancement by a combination of several factors, such as strong enhancement of the lowest one-photon Q-transition, better resonance conditions in the three-level system, dramatic enhancement of the excited-state singlet-singlet transition, and parallel arrangement of consecutive transitions in dimers, as compared to perpendicular arrangement in the monomer. We show that the absolute values of the 2PA cross section in these molecules are quantitatively described by a quantum-mechanical expression, derived for the three-level model. We also demonstrate the possibility of singlet oxygen generation upon one- and two-photon excitation of these dimers, which makes them particularly attractive for photodynamic therapy.     

Long-wavelength boradiazaindacene derivatives with two-photon absorption activity and strong emission: versatile candidates for biological imaging applications

Novel D-π-D-type boradiazaindacene dyes exhibit considerable two-photon absorption cross-section and strong red emission. Cell stained with these dyes show bright intracellular fluorescence. These properties qualify them as competitive candidates for fluorescent bioimaging applications     

Substrate binding and sequence preference of the proteasome revealed by active-site-directed affinity probes

Background: The proteasome is a multicatalytic protease complex responsible for most cytosolic protein breakdown. The complex has several distinct proteolytic activities that are defined by the preference of each for the carboxyterminal (P1) amino acid residue. Although mutational studies in yeast have begun to define substrate specificities of individual catalytically active β subunits, little is known about the principles that govern substrate hydrolysis by the proteasome.Results: A series of tripeptide and tetrapeptide vinyl sulfones were used to study substrate binding and specificity of the proteasome. Removal of the aromatic amino-terminal cap of the potent tripeptide vinyl sulfone proteasome inhibitor 4-hydroxy-3-iodo-2-nitrophenyl-leucinyl-leucinyl-leucine vinyl sulfone resulted in the complete loss of binding and inhibition. Addition of a fourth amino acid (P4) to the tri-leucine core sequence fully restored inhibitory potency. 1251-labeled peptide vinyl sulfones were also used to examine inhibitor binding and to determine the correlation of subunit modification with inhibition of peptidase activity. Changing the amino acid in the P4 position resulted in dramatically different profiles of β-subunit modification.Conclusions: The P4 position, distal to the site of hydrolysis, is important in defining substrate processing by the proteasome. We observed direct correlations between subunit modification and inhibition of distinct proteolytic activities, allowing the assignment of activities to individual β subunits. The ability of tetrapeptides, but not tripeptide vinyl sulfones, to act as substrates for the proteasome suggests there could be a minimal length requirement for hydrolysis by the proteasome. These studies indicate that it is possible to generate inhibitors that are largely specific for individual β subunits of the proteasome by modulation of the P4 and carboxy-terminal vinyl sulfone moieties.     

Acetylene bridged porphyrin-monophthalocyaninato ytterbium(III) hybrids with strong two-photon absorption and high singlet oxygen quantum yield

Several acetylene bridged porphyrin-monophthalocyaninato ytterbium(III) hybrids, PZn-PcYb, PH(2)-PcYb and PPd-PcYb, have been prepared and characterized by (1)H and (31)P NMR, mass spectrometry, and UV-vis spectroscopy. Their photophysical and photochemical properties, especially the relative singlet oxygen ((1)O(2)) quantum yields and the two-photon absorption cross-section (σ(2)), were investigated. These three newly synthesized compounds exhibited very large σ(2) values and substantial (1)O(2) quantum yields upon photo-excitation, making them potential candidates as one- and two-photon photodynamic therapeutic agents.     

Photochemical production of nitric oxide via two-photon excitation with NIR light

Herein we demonstrate the successful photochemical generation of nitric oxide via two-photon excitation (TPE) from the supramolecular complex PPIX-RSE ({mu-S,mu-S'-protoporphyrin-IX-bis(2-thioethyl)diester]tetranitrosyl-diiron). The TPE fluorescence spectra indicate efficient energy transfer from the PPIX antennae to the iron sulfur nitrosyl cluster. Further evidence of NO release is demonstrated using a nitric oxide specific electrode and ESI+ MS.     

Measurement of strong nonlinear absorption in stilbene-chloroform solutions,explained by the superposition of two-photon absorption and one-photon absorption from the excited state

A strong nonlinear absorption is measured when ruby laser light passes through a solution of stilbene in chloroform. The experimental results are interpreted by the use of the following model: two-photon absorption (TPA) is followed by one-photon absorption (OPA) by the excited molecules. The solution of the corresponding system of rate equations and fitting of parameters to the experimental curve give the TPA cross section σ⁽²⁾ = 8 × 10^?49 cm⁴ sec/photon and the product σ⁽¹⁾ τ₁₀ = 3 × 10^?26 cm² sec. Measurements at other frequencies are in agreement with this model.     

Functionalization of multiwalled carbon nanotubes and their pH-responsive hydrogels with amyloid fibrils

New biocompatible, pH-responsive, and fully fibrous hydrogels have been prepared based on amyloid fibrils hybridized and gelled by functionalized multiwalled carbon nanotubes (MWNTs) far below the gelling concentration of amyloid fibrils. Sulfonic functional groups were introduced on the surfaces of MWNTs either by a covalent diazonium reaction or by physical π-π interactions. The presence of the isoelectric point of amyloid fibrils allows a reversible gelling behavior through ionic interactions with functionalized MWNTs.     

Wolff Rearrangement of 2-diazo-1(2H)-naphthalenone induced by nonresonant two-photon absorption of NIR radiation

The irradiation of 2-diazo-1(2H)-naphthalenone (1), the common component of positive photoresists, with 800 nm pulses of ultrafast laser results in Wolff rearrangement via nonresonant two-photon absorbance of light. The 10% conversion of starting material resulting in the formation of methyl 1H-indene-3-carboxylate (2) was achieved after 11 min of irradiation of the methanol solution of 1 with an unfocused beam of a Ti:Sapphire laser operating at 1 kHz. The two-photon cross-section of the diazonaphthoquinone 1 at 800 nm was calculated to be sigma = 2.2 x 10-51 cm4 s photon-1 (0.2 GM).     

Organelle-specific detection of phosphatase activities with two-photon fluorogenic probes in cells and tissues

Two-photon fluorescence microscopy (TPFM) provides key advantages over conventional fluorescence imaging techniques, namely, increased penetration depth, lower tissue autofluorescence and self-absorption, and reduced photodamage and photobleaching and therefore is particularly useful for imaging deep tissues and animals. Enzyme-detecting, small molecule probes provide powerful alternatives over conventional fluorescent protein (FP)-based methods in bioimaging, primarily due to their favorable photophysical properties, cell permeability, and chemical tractability. In this article, we report the first fluorogenic, small molecule reporter system (Y2/Y1) capable of imaging endogenous phosphatase activities in both live mammalian cells and Drosophila brains. The one- and two-photon excited photophysical properties of the system were thoroughly investigated, thus confirming the system was indeed a suitable Turn-ON fluorescence pair for TPFM. To our knowledge, this is the first enzyme reporting two-photon fluorescence bioimaging system which was designed exclusively from a centrosymmetric dye possessing desirable two-photon properties. By conjugation of our reporter system to different cell-penetrating peptides (CPPs), we were able to achieve organelle- and tumor cell-specific imaging of phosphatase activities with good spatial and temporal resolution. The diffusion problem typically associated with most small molecule imaging probes was effectively abrogated. We further demonstrated this novel two-photon system could be used for imaging endogenous phosphatase activities in Drosophila brains with a detection depth of >100 μm.     

Intermolecular structure determination of amyloid fibrils with magic-angle spinning and dynamic nuclear polarization NMR

We describe magic-angle spinning NMR experiments designed to elucidate the interstrand architecture of amyloid fibrils. Three methods are introduced for this purpose, two being based on the analysis of long-range (13)C-(13)C correlation spectra and the third based on the identification of intermolecular interactions in (13)C-(15)N spectra. We show, in studies of fibrils formed by the 86-residue SH3 domain of PI3 kinase (PI3-SH3 or PI3K-SH3), that efficient (13)C-(13)C correlation spectra display a resonance degeneracy that establishes a parallel, in-register alignment of the proteins in the amyloid fibrils. In addition, this degeneracy can be circumvented to yield direct intermolecular constraints. The (13)C-(13)C experiments are corroborated by (15)N-(13)C correlation spectra obtained from a mixed [(15)N,(12)C]/[(14)N,(13)C] sample which directly quantify interstrand distances. Furthermore, when the spectra are recorded with signal enhancement provided by dynamic nuclear polarization (DNP) at 100 K, we demonstrate a dramatic increase (from 23 to 52) in the number of intermolecular (15)N-(13)C constraints detectable in the spectra. The increase in the information content is due to the enhanced signal intensities and to the fact that dynamic processes, leading to spectral intensity losses, are quenched at low temperatures. Thus, acquisition of low temperature spectra addresses a problem that is frequently encountered in MAS spectra of proteins. In total, the experiments provide 111 intermolecular (13)C-(13)C and (15)N-(13)C constraints that establish that the PI3-SH3 protein strands are aligned in a parallel, in-register arrangement within the amyloid fibril.     

Synthesis and two-photon absorption of triphenylbenzene-cored dendritic chromophores

The synthesis and the characterization of triphenylbenzene-cored dendrimers of first- and second-generation are described. Their absorption, photoluminescence as well as their two-photon absorption properties have been investigated and compared to a quadrupolar counterpart. These molecules combine wide transparency in the visible range and high two-photon absorption cross-sections that increase with the generation, making these dendrimers promising systems for optical power limiting applications.     

Synthesis and two-photon absorption property of phenylacetylene macrocycles

The first synthesis of phenylacetylene macrocycle with large two-photon absorption cross section is reported.     

Use of entrapment and high-performance affinity chromatography to compare the binding of drugs and site-specific probes with normal and glycated human serum albumin

Protein entrapment and high-performance affinity chromatography were used with zonal elution to examine the changes in binding that occurred for site-specific probes and various sulfonylurea drugs with normal and glycated forms of human serum albumin (HSA). Samples of this protein in a soluble form were physically entrapped within porous silica particles by using glycogen-capped hydrazide-activated silica; these supports were then placed into 1.0 cm × 2.1 mm inner diameter columns. Initial zonal elution studies were performed using (R)-warfarin and l-tryptophan as probes for Sudlow sites I and II (i.e., the major drug binding sites of HSA), giving quantitative measures of binding affinities in good agreement with literature values. It was also found for solutes with multisite binding to the same proteins, such as many sulfonylurea drugs, that this method could be used to estimate the global affinity of the solute for the entrapped protein. This entrapment and zonal approach provided retention information with precisions of ±0.1–3.3% (± one standard deviation) and elution within 0.50–3.00 min for solutes with binding affinities of 1?×?10⁴–3?×?10⁵ M^?1. Each entrapped-protein column was used for many binding studies, which decreased the cost and amount of protein needed per injection (e.g., the equivalent of only 125–145 pmol of immobilized HSA or glycated HSA per injection over 60 sample application cycles). This method can be adapted for use with other proteins and solutes and should be valuable in high-throughput screening or quantitative studies of drug–protein binding or related biointeractions.