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We report self-assembled polymeric nanovesicles composed of 1,4-bis{4′-[N,N-bis(6′′-trimethylammoniumhexyl)amino]styryl}benzene tetrabromide (C1), poly[(ethylene oxide)-block-(sodium 2-acrylamido-2-methyl-1-propane sulfonate)] (Em-An), and hexadecyltrimethylammonium bromide (C16). Transmission electron microscopy (TEM) micrographs confirm the vesicular bilayer structures and atomic force microscopy (AFM) images show that the C1/Em-An/C16 complexes form spherical nanostructures with a particle size ranging from 40 to 80 nm. The encapsulation of C1 inside the nanovesicles enhances ∼2-fold the fluorescence quantum yield (η) and two-photon action cross-section (ηδ, where δ is the two-photon absorption cross-section), and allows internalization into the cells, as revealed by the bright two-photon microscopy (TPM) images of human cervical epithelioid carcinoma (HeLa) cells labeled with the nanovesicles. Moreover, nanovesicles containing a chemotherapeutic drug and a neutral molecule can also be prepared. Furthermore, the C1/vesicular complex is disassembled under acidic conditions, highlighting its potential as a pH-responsive smart nanocarrier for the intracellular drug delivery. These results suggest a new possibility of using nanovesicles as efficient two-photon probes for TPM imaging and possibly as nanocarriers for intracellular drug delivery.

Aims Establishing probe-based analysis is important for developing multi-photon microscopy (MPM) to make an early diagnosis of colon neoplasm and assess its antioxidant status. Cu(I) and Zn(II) ions are trace elements which roll as cofactors of antioxidant, superoxide dismutase. However, there have been no reports on the features of MPM image using probe of Cu(I) and Zn(II) ions. Our main objective in this study was application of newly developed multi-photon probe (MP) probe on Cu(I) and Zn(II) ions as a tool to assess antioxidative status of colon neoplasm. Methods This study was a pilot study. Colon cancer cell lines (ACT116 and HT-29), NIH3T3 cells and tissues of normal colon mucosa and colon neoplasm obtained during colonoscopic biopsy from 17 patients were stained with MP probes for Cu(I) and Zn(II) (ACu1 and AZn1). Cu(I)/Zn(II) levels in the cells and tissues were determined by detecting MP-excited fluorescence by MPM. Results MPM images of cells stained with MP probes revealed that Cu(I) was more abundant in ACT116 and HT-29 cells than in NIH3T3 cells, while Zn(II) was more abundant in NIH3T3 cells than in ACT116 and HT-29 cells. Normal tissues had a defined texture, whereas adenoma/adenocarcinoma tissues were amorphous. The level of Cu(I) increased and that of Zn(II) decreased with the transition from normal to adenoma to adenocarcinoma tissue. Conclusion MPM can be used to determine the relative Cu(I)/Zn(II) levels in cells and colon tissues by using ACu1 and AZn1 as MPM probes. These finding are new research tools for gastroenterologists to assess antioxidant status of colon neoplasm.

We developed two-photon (TP) probes for DNA (ABI-Nu), cytoplasm (Pyr-CT) and mitochondria (BF-MT). We found that ABI-Nu binds to AT in the minor groove, while ABI-Nu and BF-MT are effective for tracking in the cytoplasm and mitochondria, respectively. These probes showed very large effective two-photon action cross section values of 2230, 1555 and 790 Göppert-Mayer units (1 GM = 10−50 cm4 s photon−1molecule−1) at 740 nm with emission maxima at 473, 561 and 560 nm, respectively, in each organelle. Using these probes, we quantitatively estimated the mean nuclear area and the ratios of nuclei to cytoplasm and mitochondria to nuclei in human colon tissues by dual-colour two-photon microscopy imaging within 2 h after biopsy. The mean nuclear area and the nuclei to cytoplasm and mitochondria to cytoplasm ratios increased in the following order: normal colon mucosa

A naphthalimide-based water soluble Zn2+ sensor bearing polyamino carboxylate as a metal chelating moiety was synthesized and its application for the selective detection of Zn2+ ion in 100% aqueous solution is demonstrated. The newly developed fluorescent sensor exhibits selective binding affinity towards Zn2+ and pH insensitivity in biologically relevant range. Confocal fluorescent microscopy images indicate that this probe works effectively for intracellular Zn2+ imaging with high cell permeability.

We report two-photon Lysotrackers (CLT-blue and CLT-yellow) that can be excited by 750-840 nm femtosecond laser pulses and emit at 470 and 550 nm, respectively. They can be easily loaded into cells and tissue slices for visualization of lysosomes in live cells and tissues for a long period of time through two-photon microscopy. When combined with appropriate two-photon probes for other biological targets, these novel probes would greatly facilitate the two-photon microscopy colocalization experiments.

We report a ratiometric two-photon probe (SSH-Mito) for mitochondrial thiols. This probe shows a marked blue-to-yellow emission color change in response to RSH, a significant two-photon cross section, good mitochondrial thiol selectivity, low cytotoxicity, and insensitivity to pH over the biologically relevant pH range, allowing the direct visualization of RSH levels in live cells as well as in living tissues at 90-190 μm depth without interference from other biologically relevant species through the use of two-photon microscopy.

Nitrile-forming eliminations from (1) promoted by in 70 mol % MeCN(aq) have been studied kinetically. When X = , the reactions exhibited second-order kinetics as well as Brnsted = 0.63 and = 0.34-0.46, and an E2 mechanism is evident. As the leaving group was made poorer (X = H, Cl, and ), Brnsted value increased from 0.63 to 0.85-0.89 without much change in the value E2, indicating that structure of the transition state changed to an E1cb-like with extensive bond cleavage, significant negative charge development at the -carbon, and limited -OAr bond cleavage.

We report a two-photon (TP) probe (ACaLN) for near-membrane Ca(2+) that shows a 13-fold TP excited fluorescence (TPEF) enhancement in response to Ca(2+), dissociation constants (K(d)(TP)) of (1.9±0.2) μM, pH-insensitivity at the biologically relevant pH, and can detect near-membrane Ca(2+) in live cells for more than 1500 s and in living tissues at 120 μm depth without interference from other metal ions. Comparison with existing probes provides a useful strategy for the design of efficient TP probes for the near-membrane metal ions.

A series of Zn(2+)-selective two-photon fluorescent probes (AZnM1-AZnN) that had a wide range of dissociation constants (K(d) (TP) =8 nm-12 μM) were synthesized. These probes showed appreciable water solubility (>3 μM), cell permeability, high photostability, pH insensitivity at pH>7, significant two-photon action cross-sections (86-110 GM) upon complexation with Zn(2+), and can detect the Zn(2+) ions in HeLa cells and in living tissue slices of rat hippocampal at a depth of >80 μm without mistargeting and photobleaching problems. These probes can potentially find application in the detection of various amounts of Zn(2+) ions in live cells and intact tissues.

The lo domains, also called lipid rafts, are believed to serve as a platform to support various cellular processes such as signal transduction, membrane trafficking, pathogen invasion, cholesterol homeostasis, neurodegenerative diseases, and angiogenesis. The existence of lipid rafts has been demonstrated in mammalian cells and in model membranes by using various techniques such as atomic force microscopy, fluorescence microscopy, and coherent anti-Stokes Raman scattering microscopy. To understand their roles in cell biology, it is crucial to visualize such domains in the live cells and tissues. An ideal tool for this purpose is two-photon microscopy (TPM) that utilizes two near-infrared photons for excitation. The advantages of TPM include the capability of visualizing the real-time activities of the biological targets in live cells and intact tissues at >100 mm depth with minimum interference due to the tissue preparation artifacts that can extend ~70 mm from the tissue surface. For maximum utilization of TPM, many TP probes for specific applications are needed. However, TP probes for such applications are rare. Earlier, we reported a membrane TP polarity probe, CL, which showed several advantages over laurdan including greater sensitivity to the solvent polarity, brighter TPM image, and more precise reflection of the cell environment. We also developed a TP turn-on probe, CL2, which showed larger TP action cross-section and higher sensitivity to the polarity of the environment than CL. This allowed selective detection of the lipid rafts in live cells and tissues by TPM. Unfortunately, CL2 had one shortcoming; it internalized into the cytoplasm upon prolonged incubation with cells, causing a blurred TPM image. To overcome this problem, we have designed a new TP turn-on (off-on) probe (SL2) that has sodium sulfonate in the head group and exclusively stains the plasma membrane. Herein, we report that SL2 can detect the lipid rafts in the live cells and intact tissues without internalization problems by simply collecting the TP excited fluorescence (TPEF) by using TPM. The preparation of SL2 is given in the Supporting Information. The solubility of SL2 in water was 7 mm, which is sufficient to stain the cells (Figure S1 in the Supporting Information). The change of the head group from a carboxylic acid (CL2, 4 mm) to a sulfonate increased the water solubility by ~ twofold. The absorption and emission spectra of SL2 showed gradual bathochromic shifts with increasing solvent polarity (Figure 1A and Table S1) indicating the charge-transfer character of the emitting state. The shifts were larger for the emission than for the absorption spectra (21 vs. 126 nm). Moreover, the fluorescence quantum yield (F) increased by 22-fold as the solvent was changed from EtOH/H2O (F=0.04) to THF (F=0.88). CL2 showed similar behavior except that fluorescence enhancement factor (FEF) was lower [FEF(CL2)=16 versus FEF(SL2)= 22] (Table S1). Hence, the fluorescence of SL2 is more sensitive to the polarity of the environment than that of CL2. The fluorescence intensity of SL2 showed similar decrease with the hydrophilicity of the large unilamellar vesicles (LUVs) in the order, 1,2-dipalmitoyl-sn-glycero-3-phosphocholine/cholesterol (DPPC/CHL (40 mol%), lo)>DOPC/sphingomyelin/CHL (1:1:1, raft mixture)>1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC, ld ; Figure S2). The relative fluorescence intensities of SL2 in DPPC/CHL/raft mixture/DOPC in the 420–520 nm range are 7.0/4.8/1.0, respectively; SL2 emits sevenfold stronger fluorescence in the lo than in the ld domain. The slightly weaker fluorescence in the raft mixture than in DPPC/CHL can be explained if SL2 exists in 63/37 ratio in the lo/ld domains of the raft mixture with 7/1 intensity ratio (vide supra). A similar result was reported for CL2 (lo/ld=86:14). [12] This indicates high preference of these probes to reside in the lo domain, which can be attributed to the more favorable hydrophobic interactions with sphingomyelin when compared to DOPC. The sensitivity of the laurdan derivatives on the polarity of the environment originates from the intramolecular charge [a] C. S. Lim, J. H. Lee, Dr. Y. S. Tian, Prof. B. R. Cho Department of Chemistry, Korea University 1-Anamdong, Seoul, 136-701 (Korea) Fax: (+82)2-3290-3544 E-mail : chobr@korea.ac.kr [b] H. J. Kim, Prof. H. M. Kim Division of Energy Systems Research, Ajou University Suwon, 443-749 (Korea) E-mail : kimhm@ajou.ac.kr [c] Dr. C. H. Kim, Prof. T. Joo Department of Chemistry, Pohang University of Science and Technology Pohang, 790-784 (Korea) E-mail : thjoo@postech.ac.kr Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbic.201000609.

The first example of cyclometalated platinum(II)-containing rhodamine probe (1) with two-photon induced luminescent properties was synthesized and investigated for mercury detection. A highly selective color change of 1, from light yellow to pink, is observed only in the presence of Hg2+ due to the formation of 1,3,4-oxadiazole ring in 2. This selectivity of Hg2+ with color changes can be observed easily by the naked-eye. Meanwhile, a remarkable turn-on and selective 20-fold fluorescent enhancement of 1 upon binding with Hg2+ over the other tested metal ions was observed. The water-soluble probe 1 was successfully applied in the visualizing of the site of Hg2+ accumulation as well as estimating of trace amounts of mercury ions in live HeLa cells by two-photon microscopy.

Since detergent-resistant lipid rafts play important roles in the signal transduction for myogenesis, their comprehensive proteomic analysis could provide new insights to understand their function in myotubes. Here, the detergent-resistant lipid rafts were isolated from C2C12 myotubes and analyzed by capillary RPLC/MS/MS. Among the 327 proteins (or protein groups) identified, 28% were categorized to the plasma membrane or raft proteins, 29% to mitochondria, 20% to microsomal proteins, 10% to other proteins, and 13% to unknown proteins. The localization of oxidative phosphorylation (OXPHOS) complexes in the sarcolemma lipid rafts was further confirmed from C2C12 myotubes by cellular fractionation, surface-biotin labeling, immunofluorescence, and lipid raft fractionation. After adding exogenous cytochrome c, the sarcolemma isolated from myotubes had an ability to consume oxygen in the presence of NADH or succinate. The generation of NADH-dependent extracellular superoxide was increased by inhibiting or downregulating OXPHOS I, III, and IV in myotubes, indicating that OXPHOS proteins are major sources for extracellular ROS in skeletal muscle. With all these data, we can conclude that OXPHOS proteins are associated with the sarcolemma lipid rafts during C2C12 myogenesis to generate extracellular ROS.

A combination of a strong electron-withdrawing β-aryl group and anion-solvating protic solvent was required for the mecha-nistic change.We were interested in learning if a change to the E1cB mechanism could be realized in aprotic solvent by making the β-aryl group even more electron-withdrawing. To explore such possibility, we have now studied the reactions of (

dicating a higher sensitivity of ACaCL to the solvent polarity. This can be attributed to the polar amide group between the re-ceptor and fluorophore in ACaCL that can be better solvated by the polar solvent in the excited state, thereby enhancing the intramolecular charge transfer (ICT).When Ca

607 Background: In cancer cells, lysosomal pH decreases along with a concomitant increase in lysosomal volume and cathepsin expression levels. Lysosomes also play crucial roles in cancer progression following their release into the extracellular space. Since cancer cells invade a tissue by secreting degradative enzymes, the extracellular pH of tumor tissues becomes acidic. However, to date, there has been no report on the use of multi-photon microscopy (MPM) probes to image human colon cancer tissues. Methods: We have developed multi-photon (MP) pH-sensitive probes (BH-2 and BHEt-1) that exhibit absorption and emission maxima at 370 and 466 nm, and TP absorption cross-section values of 51 and 61 GM (1 GM = 10−50 cm4 s/photon), respectively, at 750 nm and pH 3.0 in a universal buffer (0.1 M citric acid, 0.1 M KH2PO4, 0.1 M Na2B4O7, 0.1 M Tris, 0.1 M KCl)/1,4-dioxane (7/ 3) solution. Results: The TPM images of CCD-18co (a normal colon cell line) and HCT116 cells (a colon cancer cell line) labeled with BH-2 were too dim to be distinguished. When the same cells were labeled with BHEt-1, however, the MPM image of the HCT116 cells was much brighter than that of CCD-18co cells, and the relative proportion of the acidic vesicles (Pacid) of the former was 5-fold larger than that of latter. BHEt-1 could also differentiate HepG2 cells (a human liver cancer cell line) from LX-2 cells (a human hepatic stellate cell line) with a 6-fold larger P acid value. Human colon cancer tissues labeled with BHEt-1 showed similar results, demonstrating much brighter MPM images and 6-fold larger Pacid values compared to normal tissue. Conclusions: These results suggest the potential utility of BHEt-1 for molecular image analysis of colon cancer tissues using MPM.

We briefly describe the advantages and limitations of label-free multiphoton microscopy and probe-labeled two-photon microscopy for the endomicroscopic diagnosis. The two methods are complementary and more information can be collected from tissues by combining the two methods. Therefore, parallel efforts should be directed to the development of both label-free MPM and probe-labeled TPM as the diagnostic tool.

Lipid rafts are plasma membrane platforms mediating signal transduction pathways for cellular proliferation, differentiation and apoptosis. Here, we show that membrane fluidity was increased in HeLa cells following treatment with ginsenoside Rh2 (Rh2), as determined by cell staining with carboxy-laurdan (C-laurdan), a two-photon dye designed for measuring membrane hydrophobicity. In the presence of Rh2, caveolin-1 appeared in non-raft fractions after sucrose gradient ultracentrifugation. In addition, caveolin-1 and GM1, lipid raft landmarkers, were internalized within cells after exposure to Rh2, indicating that Rh2 might disrupt lipid rafts. Since cholesterol overloading, which fortifies lipid rafts, prevented an increase in Rh2-induced membrane fluidity, caveolin-1 internalization and apoptosis, lipid rafts appear to be essential for Rh2-induced apoptosis. Moreover, Rh2-induced Fas oligomerization was abolished following cholesterol overloading, and Rh2-induced apoptosis was inhibited following treatment with siRNA for Fas. This result suggests that Rh2 is a novel lipid raft disruptor leading to Fas oligomerization and apoptosis.

The development of two-photon materials has been growing very fast during the last decade due to the potential applications in optical limiting, 3D microfabrication, and multiphoton microscopy. In this article, the structure-two-photon absorption property relationships of dipolar, quadrupolar, and octupolar molecules as well as the derivatives of triphenylamine, [2.2]paracyclophanes, multi-annulenes, and porphyrins are briefly reviewed to provide a guideline for the design of efficient two-photon materials.

The polymerization reaction of 4-[(n-butylsulfinyl)methyl]-4'-(chloromethyl)benzene (1a) with t-BuONa in CH 2 Cl 2 /monomethylformamide (MMF) (4/6) was studied kinetically. The reaction proceeds in two steps; the formation of a p-quinodimethane intermediate (M) followed by the polymerization of M. Results of kinetic studies and H-D exchange experiments reveal that the 1,6-elimination proceeds by the (Elcb)i rr mechanism. The rate of the disappearance of M was increased by the carbanion and S 2 O 8 2- , inhibited by TEMPO and unaltered by the addition of HCl. From these results, a free radical polymerization mechanism is proposed.

Two-photon fluorescent probes for the cellular membrane, derived from 6-acyl-2-aminonaphthalene as the fluorophore and hexanoyl (CH), lauryl (CL), and stearyl (CS) groups as the receptor, have been synthesized. Their photophysical properties and utility as membrane probes were also studied. Whereas CH cannot be used as a membrane probe due to its high water solubility, CL and CS are useful two-photon probes for membrane lateral heterogeneity, as they can easily stain cells, emit fluorescence with high sensitivity to the environment polarity, and are capable of imaging the membrane lateral heterogeneity in live cells. Moreover, CS is more likely to be located in the plasma membrane due to its negligible water solubility. Our results show that the liquid ordered-like domain covers 31-35% of the cellular surface.

To identify RNA motifs interacting with the stem-loop RNA which plays an important role in the synthesis of gag-pol fusion protein of HIV-1, a SELEX (Systematic Evolution of Ligands by Exponential Enrichment) was performed. RNA aptamers binding to the stem-loop sequence at the gag-pol junction of HIV-1 RNA were selected from a 30-mer random sequence library through 13 rounds of selection. Some of resulting RNA aptamers contained the consensus sequences AAGGGA and GUUGAU. The stem-loop RNA ligand interacted with synthetic RNA fragment containing two conserved sequence motifs AAGGGA and GUUGAU but did not bind to DNA oligonucleotides having the sequences identical to the RNA fragment. These results suggest that a specific tertiary interaction rather than Watson-Crick base-pairing is involved in the complex formation between RNA aptamer and the stem-loop RNA.

Considerable efforts are being made to develop octupolar nonlinear optical materials for possible application in optical and electro-optical devices. [1–3] An advantage of such molecules in comparison to the more conventional dipolar molecules is the existence of a broader range of tensorial coefficients, providing optimum nonlinear efficiency with a polarization-independent second-harmonic response with respect to the incident light. Great progress has been made by optimization at the molecular level, as exemplified by the established structure–property relationship of two-dimensional octupoles and the development of highly efficient molecules. [3–5] Recently, we reported that 1,3,5-tricyano-2,4,6tris(p-diethylaminostyryl)benzene (TTB) produces non-centrosymmetric crystals that show very large second-harmonic generation (SHG). [1] To integrate such materials in optical devices, it is essential to prepare thin films showing large SHG and high thermal stability. As electric poling is not possible because of the lack of a ground-state dipole moment, it is not readily obvious how to align octupolar molecules non-centrosymmetrically in a thin film. Although optical poling in polymers has been proposed, it requires photoisomerizable molecular functions and resonant excitation of the molecules by intense laser pulses, which may be a limitation for industrial application. An ideal solution to this problem would be to prepare self-oriented films without applying external fields. For this purpose, we have studied thin films of TTB in a polymethylmetacrylate (PMMA) matrix prepared by using free

Ketene-forming eliminations from C4H3(S)CH2C(0)0-C6H3-2-X-4-N02 (1) promoted by R 2 NH in MeCN have been studied kinetically. The reactions are second-order and exhibit Bronsted β=0.51-0.62 and lβlgl= 0.47-0.53. Hence, an E2 mechanism is evident. The Bronsted β increased from 0.33 to 0.53 and |βlg| remained nearly the same by the change of the base-solvent from Bz(i-Pr)NH/Bz(i-Pr)NH 2 + in 70 mol% MeCN(aq) to Bz(i-Pr)NH-MeCN, indicating a change to a more symmetrical transition state with similar extents of Cβ-H and C α -OAr bond cleavage. When the /?-aryl group was changed from thienyl to phenyl in MeCN, the /? value increased from 0.53 to 0.73 and lβ lg l decreased from 0.53 to 0.43. This indicates that the transition state became skewed toward more Cβ-H bond breaking with less C α -OAr bond cleavage. Noteworthy is the greater double bond stabilizing ability of the thienyl group in the ketene-forming transition state.

The lipid-rafts hypothesis proposes that naturally occurring lipid aggregates exist in the plane of membrane that are involved in signal transduction, protein sorting, and membrane transport. To understand their roles in cell biology, a direct visualization of such domains in living cells is essential. For this purpose, 6-dodecanoyl-2-(dimethylamino)naphthalene (laurdan), a membrane probe that is sensitive to the polarity of the membrane, has often been used. We have synthesized and characterized 6-dodecanoyl-2-[N-methyl-N-(carboxymethyl)amino]naphthalene (C-laurdan), which has the advantages of greater sensitivity to the membrane polarity, a brighter two-photon fluorescence image, and reflecting the cell environment more accurately than laurdan. Lipid rafts can be visualized by two-photon microscopy by using C-laurdan as a probe. Our results show that the lipid rafts cover 38 % of the cell surface.

The Kin.46 kinase ribozyme was previously selected from a random-sequence RNA library that was loosely based on the sequence of a known ATP aptamer, based on its ability to catalyze transfer of the thiophosphate from ATPgS to its own 5' hydroxyl end. The reaction requires an oligonucleotide effector that is complementary to the 3' end of the ribozyme and that served as reverse transcription primer during the amplification steps of the original selection for activity (Fig. 1). Omitting the oligonucleotide reduces the observed catalytic rate constant (kobs) by 10 3 to 10-fold, indicating that the oligo acts as an allosteric effector that is necessary for full catalytic activity. The activator helix is separated from the substrate-binding internal guide sequence by a 5nt “linker” that appears to form long-range baseparing interaction with nucleotides within the catalytic core (S. Rhee, unpublished results). Binding of small ligands, such as metal ions and ATP, can also potentially influence the folded structure of the RNA. Divalent metal ions are well known for their abilities to stabilize nucleic acid secondary and tertiary structures, and for their potential catalytic roles in ribozyme-catalyzed reactions. For example, we recently found that topologically rearranged versions of Kin.46 exhibit markedly altered Mg dependence. Similarly, nucleotide binding by aptamers nucleates formation of the folded structure and induces order in the NTP binding pocket (adaptive binding). ATP recognition by Kin.46 is clearly distinct from ATP recognition by the original aptamer; key nucleotides required for ATP recognition are mutated in Kin.46, and the values of Km ATPgS and Km ATP for Kin.46 (~3 mM) are >10 times higher than Kd ATP for the aptamer (~1 μM). In this work, fluorescence emission from cy5-labeled ribozymes was monitored in order to investigate conformational dynamics as a function of ATP concentration and Mg ion. Truncated versions of Kin.46 ribozyme were obtained by internal cleavage or deletions (Fig. 1). Ribozyme130 was derived from the Kin.46 by just cleaving the most right-side loop, ribozyme119 was obtained by deletion of the loop and

Ketene-forming eliminations from C4H3(S)CH2C(O)O-C6H3-2-X-4-NO2 (1) promoted by R2NH in MeCN have been studied kinetically. The reactions are second-order and exhibit Bronsted β = 0.51-0.62 and |βlg| = 0.47-0.53. Hence, an E2 mechanism is evident. The Bronsted β increased from 0.33 to 0.53 and |βlg| remained nearly the same by the change of the base-solvent from Bz(i-Pr)NH/Bz(i-Pr)NH2 + in 70 mol% MeCN(aq) to Bz(i-Pr)NH-MeCN, indicating a change to a more symmetrical transition state with similar extents of Cβ-H and Cα-OAr bond cleavage. When the β-aryl group was changed from thienyl to phenyl in MeCN, the β value increased from 0.53 to 0.73 and |βlg| decreased from 0.53 to 0.43. This indicates that the transition state became skewed toward more Cβ-H bond breaking with less Cα-OAr bond cleavage. Noteworthy is the greater double bond stabilizing ability of the thienyl group in the ketene-forming transition state.

Novel two-photon (TP) probes were developed for lysosomes (PLT-yellow) and mitochondria (BMT-blue and PMT-yellow). These probes emitted strong TP-excited fluorescence in cells at widely separated wavelength regions and displayed high organelle selectivity, good cell permeability, low cytotoxicity, and pH insensitivity. The BMT-blue and PLT-yellow probes could be utilized to detect lysosomes and mitochondria simultaneously in live tissues by using dual-color two-photon microscopy, with minimum interference from each other.

Two-photon absorption cross-section of a series of dyes (1a–3a) in polymer thin films was determined by measuring the femtosecond laser ablation threshold. The ablation threshold decreased gradually when the dopant increased. The two-photon absorption cross-section of the dye molecules dispersed in the polymer film was estimated by using the theoretical relationship between the ablation threshold of the blended polymeric thin films and the dye concentration. The relative values of two-photon absorption cross-section are in good agreement with those measured in solution. On the other hand, the absolute values are smaller than the latter.

A series of donor-acceptor substituted stilbene and diphenylacetylene derivatives and their octupolar analogues have been synthesized and the linear and nonlinear optical properties (beta) studied by both experiments and theoretical calculation. The lambda(max) of the dipoles increases with the conjugation length and is always larger when the C=C bond is used, instead of the C[triple bond]C bond, as the conjugation bridge. Although the lambda(max) values of the octupoles show no clear trend, they are much larger than those of the dipoles. The beta(0) values of the dipoles increase with conjugation length and as the conjugation bridge is changed from the C[triple bond]C to C=C bond. This increase is accompanied by an increase in either lambda(max) or the oscillator strength. Similarly, the beta(0) values of the octupoles increase with the conjugation length and with a change in the donor in the order: NEt2N(i-amyl)PhNPh2. Moreover, beta(yyy)/beta(zzz) ratios are in the range of 1.6-3.9 and decrease with the conjugation length. Beta values calculated by the finite-field and sum-over-states methods are in good agreement with the experimental data. Also, there is a parallel relationship between the calculated beta values and bond length alternation (BLA). From these results, the origin of the larger beta values for octupoles than for dipoles is assessed.

In vitro selection was used to isolate Mg 2 + -dependent self-cleaving ribozymes with cis-cleavage activity from a pre-tRNA library having 40-mer random sequences attached to 5'-end of E. coli tRNA P h c . After 8 rounds ofSELEX (Systematic Evolution of Ligands by Exponential Enrichment), RNA molecules which can self-cleave at the high concentration of Mg 2 + were isolated. The selected ribozymes can carry out the self-cleavage reaction in the presence of 100 mM Mg 2 + but not in 10 mM Mg 2 + . The cleavage sites of the ribozymes are located at +3 and +4 of tRNA P h e , compared with +1 position of 5'-end cleavage site of pre-tRNA by RNase P. New RNA constructs deprived of its D stem-loop, anticodon stem-loop, variable loop and T stem-loop, respectively showed the cleavage specificity identical to a ribozyme having the intact tRNA structure. Also, the new ribozyme fused with both a ribozyme and tRNA L e u showed the cleavage activities at the various sites within its sequences, different from two sites of position +3 and +4 observed in the ribozyme with tRNA P h c . Our results suggest that the selected ribozyme is not structural-specific for tRNA.

Elimination reactions of have been studied under various conditions. When X was moderately electron-withdrawing, Y = H, and base-solvent was NH-MeCN, the reaction proceeded by the E2 mechanism via an E1cb-like transition state. Concave downward curve was noted in the Hammett plots. When X = 4-, Y = Cl, , and the base-solvent was in 70 mol % MeCN(aq), the reaction proceeded by the E2 mechanism. The mechanism changed to a competing E2 and E1cb when X = 4- and Y = H, MeO, and to the E1cb when X = 2,4-(, and Y = . From these results, a plausible pathway of the change of the mechanism from E2 to the E1cb extreme is proposed.

Recently, the development and use of single precursors for metal organic chemical vapor deposition (MOCVD) has been extensively focused on application to electronic devices. However, systematic design and synthesis of organic ligands appropriate for preparation of metal complexes with relevant chemical and physical properties, such as stability, volatility, and resistance to air and moisture have not received much attention. Generalization to prepare organic ligands for MOCVD precursors is especially important because the large scale synthesis of the metal complexes exclusively depends on the potential usefulness of the ligands. It is desirable for metal complexes to be in monomeric forms because mononuclear complexes generally show high vapor pressures and low sublimation or boiling temperatures. To do so, the coordinated atom of ligands should be crowded in its own environment with some substituents by keeping the coordinated atoms from bonding to another metal through bridging configuration. Therefore, pendant groups should be introduced into organic ligands. Some research results have been reported on the synthesis and application of organic ligands containing pendant groups. Herrmann reported his pioneering works on the synthesis of donor-functionalized alcohols and their application as bidentate ligands for the preparation of metal complexes. A variety of donor-functionalized alcohols which contain two alkyls on the α–carbon of the hydroxyl group were prepared and used to give metal complexes such as M(mmp)4 (mmp = 1-methoxy-2-methyl-2-propanolate; M = Zr, Hf), M(mmp)3 (M = Cr, Bi), 9 M(dmamp)3 (dmamp = 1-dimethylamino-2-methyl-2-propanolate; M = Y, Lu), Cu(dmamp)2. 12 The ligands in the complexes have pendant groups capable of coordinating to metals as bidentate ligands. Some of trifunctional alcohols have been used for the preparation and homoleptic complexes of alkaline-earth elements as well as transition metals. For larger cations such as Ba and Sr, it is necessary to introduce a neutral ancillary ligand possessing a sufficient number of donor sites or to incorporate a donor-functionalized feature into the anionic ligand to satisfy the multiple coordination requirement. Chi reported the successful preparation of alkalineearth metal complexes using a multi-donor site monoalcohol. Herein we disclose the synthesis and characterization of novel alcohols, which have pendant groups to stabilize the metal compounds by coordinating to the metal center in the formation of metal complexes. New alkoxyalcohols were prepared by the nucleophilic addition ring opening reaction between alkoxide anions and a highly reactive alkene oxide (epoxide).

A series of dipolar (1), bis-dipolar (2), and octupolar molecules (3) containing 1, 2, and 6 dipolar units within a molecule has been synthesized and their hyperpolarizabilities were analyzed. The βHRS increases in the order, 1

First hyperpolarizabilities (β) of the 1,3,5-tricyano-2,4,6-tris(styryl)benzene derivatives were studied by an ab initio method (HF/6–31G). The β values of these molecules increase with increase in donor strength, reaching a maximum value of 262 × 10−30 esu with an oxyanion as the donor. Also, the electronic charge is more delocalized, and the HOMO–LUMO energy gap (ΔE) and the bond length alternation (BLA) decrease with the same variation of the donor. Noteworthy is the excellent linear relationship between β and 1/ΔE2 and σgas+. These results may serve as a useful guideline for the design of two-dimensional octupoles with large first hyperpolarizabilities. Copyright © 2004 John Wiley & Sons, Ltd.

One of thesesequences, an 18-mer containing four guanine-tracts, 5'-GGGAGGGGCGGGUCUGGG-3' is associated with the 5'-UTR of the oncogenic N-ras sequence. According to a cell-free translation system coupled to a reporter gene assay, theN-ras G-quadruplex can inhibit gene expression at thetranslational level.

Bongrae ChoDepartment of Applied Chemistry, Cheongju University, Cheongju 360-764, Korea. E-mail: brcho@cju.ac.krReceived March 14, 2013, Accepted March 27, 2013Key Words : Sequence motif, G-rich sequence RNA, 5'-UTR, RNA aptamer, SELEXGuanine-rich tracts are observed in terminal segments ofeukaryotic genomes.

Two-photon-absorption (TPA) properties of multipolar molecules were studied using femtosecond z -scan method. We measured the z -scan curves at 800 nm, 1 kHz using 150 fs-laser pulse. The z -scan curves were measured for two kinds of multipolar molecules: three different quadrupole molecules (Q1, Q2, Q3) and three different octupolar molecules (O1, O2, O3). Each multipolar molecule has same molecular skeleton but the lengths of the dipolar subunits or the donor strengths are different each other. Their TPA cross-sections were obtained from the magnitudes of the transmittance dip in the measured z -scan curves: 233 (Q1), 127 (Q2), 164 (Q3) and 381 (O1), 230 (O2), 85 (O3) cm 4 /(photon/s) for quadrupole and octupolar molecules, respectively. These results are ascribed to the fact that TPA cross-sections of the multipolar molecules are raised by increasing the number of the alternating double bonds in the dipolar subunits or by enlarging the electron-donating strength of the substituent.

A series of 9,10-bis(arylethynyl)anthracene derivatives (1−8) have been synthesized and their two-photon absorption (TPA) cross sections were measured by nanosecond fluorescence measurement and femtosecond Z-scan methods. The λmax values of these molecules are very similar and the values are near 800 nm. The /MW of the dipole increases with the conjugation length, whereas those for the quadrupoles decrease slightly with MW. Also, the /MW of the octupole is smaller than the dipole. In all cases, /MW of the quadrupoles are larger than the dipoles. Moreover, the results of the femtosecond Z-scan experiment indicate significant contribution by the excited-state absorption in . Noteworthy is the good agreement between the structure−TPA property relationships determined by both methods.

First hyperpolarizabilities (β) of triazine derivatives were studied by the ab initio method (HF/6–31G). The β values of these molecules increase with a stronger donor and as the conjugation length increases, probably because the electronic charge becomes more delocalized and the HOMO–LUMO energy gap (ΔE) and the bond length alternation (BLA) decrease with variation of the chromophore structure. Also, the susceptibility of β to the donor strength is found to be larger for a more elongated substrate. Noteworthy is the excellent linear relationship between β and BLA and also the gas-phase substituent constants (σ+gas). This result may serve as a useful guideline for the design of two-dimensional octupoles with large first hyperpolarizabilities. Copyright © 2004 John Wiley & Sons, Ltd.

A series of 1,3,5-tricyano-2,4,6-tris(styryl)benzene derivatives (1,2) and dendrimers (3) have been synthesized and their first hyperpolarizabilities (β) were determined. Whereas the λmax values of 1–3 are similar, the oscillator strength increases with the molecular size. For all compounds, the Stokes shifts are significantly larger when NAr2 is used as the donor. The β(0) values of 1–3 range from 252 to 507 × 10–30 esu and increases with the number of octupolar units in the molecule. When the octupolar molecules are linked to the 1,3,5-position of the benzene ring by flexible ether linkages, the β(0) value increases by 1.7 fold. On the other hand, octupolar monodendron based on the nitrogen atom core and 1,3,5-tricyano-2,4,6-tris(p-diarylaminostyryl)benzene moieties at the periphery exhibits β(0) = 507 × 10–30 esu, which is one of the largest β(0) values reported for the octupolar molecules.