A Reversible Multi‐Stimuli‐Responsive Fluorescence Probe and the Design for Combinational Logic Gate Operations

Here, a novel multi‐stimuli‐responsive fluorescence probe is developed by incorporating spiropyran group into the coumarin‐substituted polydiacetylene (PDA) vesicles. The fluorescence of PDA can be turned on upon heating, and can be quenched upon exposure to UV light irradiation or pH stimuli owing to the fluorescene resonance energy transfer (FRET) between the red‐phase PDA and the open merocyanine (MC) form of spiropyran. Moreover, we have designed and experimentally realized a set of logic gate operations for the first time based on the fluorescence modulation of the designed system upon thermal, photo, and pH stimuli. This novel type of resettable logic gates augur well for practical applications in information storage, optical recording, and sensing in complicated microenvironments.

     

Three Complementary One-Pot Four-Component Reaction Sequences for Rapid,General and Direct Spiropyran Synthesis

Three one-pot three-step four-component reaction sequences for spiropyran synthesis have been developed, based around three different methods for in situ generation of 1,2,3-trisubstituted indoles (indole N-alkylation, Fischer indolisation and indole C-alkylation). The reaction sequences give access to a broad swathe of spiropyran structures. Each sequence is operationally straightforward, rapid and high yielding. We have synthesized 58 structurally diverse spiropyrans bearing a wide range of useful functional groups, and their utility were demonstrated in the targeted synthesis of potential ratiometric fluorescence probes for metal ions and spiropyran-cholesterol conjugates. Finally, we showed that our one-pot N-alkylation-C-alkylation-condensation sequence can underpin combinatorial spiropyran synthesis through generation of a 16-member spiropyran library.     

A Novel Fluorescence Probe for the Reversible Detection of Bisulfite and Hydrogen Peroxide Pair in Vitro and in Vivo

The detection of changes in the reactive oxygen species (ROS)/reactive sulfur species (RSS) couple is important for studying the cellular redox state. Herein, we developed a 1,8-naphthalimide-based fluorescence probe ( NI ) for the reversible detection of bisulfite (HSO₃⁻) and hydrogen peroxide (H₂O₂) in vitro and in vivo. NI has been designed with a reactive ethylene unit which specifically reacts with HSO₃⁻ by a Michael addition reaction mechanism, resulting in the quenching of yellow fluorescence at 580 nm and the appearing of green fluorescence at 510 nm upon excitation at 500 nm and 430 nm, respectively. The addition product ( NI−HSO₃ ) could be specifically oxidized to form the original C=C bond of NI , recovering the fluorescence emission and color. The detection limits of NI for HSO₃⁻ and NI−HSO₃ for H₂O₂ were calculated to be 2.05 μM and 4.23 μM, respectively. The reversible fluorescence response of NI towards HSO₃⁻/H₂O₂ couple can be repeated for at least five times. NI is reliable at a broad pH range (pH 3.0–11.5) and features outstanding selectivity, which enabled its practical applications in biological and food samples. Monitoring the reversible and dynamic inter-conversion between HSO₃⁻ and H₂O₂ in vitro and in vivo has been verified by fluorescence imaging in live HeLa cells, adult zebrafish and nude mice. Moreover, NI has been successfully applied to detect of HSO₃⁻ levels in food samples.     

Probing Metal Ion Complexation of Ligands with Multiple Metal Binding Sites: The Case of Spiropyrans

Among known molecular switches, spiropyrans attract considerable interest because of their reversible responsiveness to external stimuli and the deep conformational and electronic changes that characterize the switching process between the two isomeric forms [spiropyran (SP) and merocyanine (MC)]. Metal coordination is one of the most interesting aspects of spiropyrans for its potential in sensing, catalysis, and medicinal chemistry, but little is known about the details surrounding spiropyran–metal ion binding. We investigated the interplay between an N‐modified 8‐methoxy‐6‐nitrospiropyran (SP‐E), designed to provide appropriate molecular flexibility and a range of competing/collaborative metal binding sites, with Mg²⁺, Cu²⁺ and Zn²⁺, which were chosen for their similar coordination geometry preferences while differing in their hard/soft character. The formed molecular complexes were studied by means of UV/Vis, fluorescence, and NMR spectroscopies and mass spectrometry, and the crystal structure of the SP‐E–Cu complex was also obtained. The results indicate that the Mg²⁺, Zn²⁺ and Cu²⁺ complexes have identical coordination stoichiometry. Furthermore, the Mg²⁺ and Zn²⁺ complexes display fluorescence properties in solution and visible‐light responsiveness. These results provide important spectroscopic and structural information that can serve as a foundation for rational design of spiropyran‐based smart materials for metal sensing and scavenging applications.     

Synthesis of high‐molecular‐weight linear methacrylate copolymers with spiropyran side groups: Conformational changes of single molecules in solution and on surfaces

P(BMA‐co‐HEMA‐spiropyran) was synthesized by reversible addition fragmentation chain transfer (RAFT) polymerization of butyl methacrylate (BMA) and 2‐(trimethylsilyloxy)‐ethyl methacrylate (HEMA‐TMS), removal of the TMS‐protective groups, and the polymer analogous esterification of the hydroxyethyl side chains with a spiropyran containing a carboxylic acid group. UV‐induced conformational changes of the synthesized macromolecules and low‐molecular‐weight spiropyran molecules were studied. Rate constants and half‐life times of the ring closure reaction from zwitterionic merocyanine to the spiropyran species were determined in the presence and absence of mica‐dispersed particles in toluene both with the free spiropyran and the polymer‐bound spiropyran. Scanning force microscopy was used to visualize the conformation of spiropyran‐decorated single macromolecular chains and agglomerated polymer‐bound merocyanine adsorbed on mica. © 2009 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 47: 1274–1283, 2009     

光谱法研究噻菁染料与血清蛋白的相互作用

本文通过吸收和荧光光谱法研究了一种噻菁染料与人血清蛋白及牛血清蛋白的相互作用。吸收光谱数据表明,与血清蛋白结合后,噻菁染料单体的吸收峰发生红移,同时强度也有很大变化;还通过吸收光谱计算确定了噻菁染料与血清蛋白的结合位点数（ n ）。与人血清蛋白或牛血清蛋白结合后,噻菁染料的荧光量子产率增加。分析噻菁染料的荧光强度随溶液中血清蛋白浓度的变化得到了二者反应的表观结合常数（ K _a）和自由能变化（ ΔG ）。根据表观结合常数（ K _a）可以判断,人血清蛋白比牛血清蛋白与噻菁染料的结合更强。     

Synthesis and spectral investigations of a new dyad with spiropyran and fluorescein units: toward information processing at the single molecular level

A new dyad 1 with two spiropyran units as the photochromic acceptors and one fluorescein unit as the fluorescent donor was synthesized and characterized. External inputs (ultraviolet light, visible light, and proton) induce the reversible changes of the structure and, concomitantly, the absorption spectrum of dyad 1 due to the presence of two spiropyran units. Only the absorption spectrum of the ME form of the spiropyran units in dyad 1 has large spectral overlap with the fluorescence spectrum of the fluorescein unit. Thus, the fluorescence intensity of dyad 1 is modulated by reversible conversion among the three states of the photochromic spiropyran units and the fluorescence resonance energy transfer (FRET) between the ME form and the fluorescein unit. Based on the fact that dyad 1 could "read out" three external input signals (ultraviolet light, visible ligh,t and proton) and "write" a compatible specific output signal (fluorescence intensity), dyad 1 described here can be considered to perform an integrated circuit function with one OR and one AND interconnected logic gates. The present results demonstrate an efficient strategy for elaborating and transmitting information at the single molecular level.     

Features of the Interaction of Alcian Blue with Gels Based on Copolymer of <Emphasis Type="Italic">N</Emphasis>-Vinylcaprolactam and Methacrylic Acid

The interaction of N-vinylcaprolactam–methacrylic acid copolymer hydrogel and N-vinylcaprolactam homopolymer hydrogel with alcian blue in aqueous solutions is studied. It is shown that copolymer gel are thermo- and pH-sensitive. Dye sorption by the copolymer gel and gel swelling degree depends on the pH of the solution. The features of the system behavior resulting from the pH sensitivity of both the gel and the dye are shown. The thermocollapse of gels incubated in aqueous solutions of alcian blue is studied. It is shown that the transition temperature of N-vinylcaprolactam–sodium methacrylate copolymer gel to the collapsed state decreases with the dye absorption. The temperature-induced collapse of homopolymer gels in dye solutions is reversible, while the copolymer gel mass restores incompletely during the return temperature decrease. The dye absorption by the copolymer gel leads to decreased hysteresis.     

Spiropyran Photoswitches in the Context of DNA: Synthesis and Photochromic Properties

A new design is presented for the incorporation of spiropyran photoswitches into nucleic acids by oligonucleotide solid phase synthesis. This design enables interaction of the 6‐nitrospirobenzopyran (NitroBIPS) photoswitch with the adjacent nucleobases and leaves the photochemical properties of NitroBIPS intact. UV/Vis spectroscopy and HPLC revealed that NitroBIPS incorporated into DNA consists of up to 40 % merocyanine in its thermal equilibrium and undergoes reversible switching between the photoisomeric spiropyran (SP) and merocyanin (MC) state by alternating excitation using visible light or heat for at least fifteen switching cycles. Exchanging the chromene part of NitroBIPS on the DNA level gives access to differently substituted spiropyran derivatives allowing the screening for spiropyrans with suitable properties in a straightforward manner. Thus, by incorporating the highly hydrolysis‐stable pyrido‐spiropyran derivative PyBIPS pure light‐triggered reversible switching of a spiropyran in DNA has been realized for the first time. Therefore, this design represents a new useful platform for investigating the photochromic behavior of different spiropyran photoswitches in a nucleic acid environment and for using spiropyrans to induce light‐ or heat‐triggered changes in conformations or in fluorescence quenching properties of oligonucleotides.     

Design and proof of reversible micelle‐to‐vesicle multistimuli‐responsive morphological regulations

Multistimuli‐responsive precise morphological control over self‐assembled polymers is of great importance for applications in nanoscience as drug delivery system. A novel pH, photoresponsive, and cyclodextrin‐responsive block copolymer were developed to investigate the reversible morphological transition from micelles to vesicles. The azobenzene‐containing block copolymer poly(ethylene oxide)‐b‐poly(2‐(diethylamino)ethyl methacrylate‐co‐6‐(4‐phenylazo phenoxy)hexyl methacrylate) [PEO‐b‐P(DEAEMA‐co‐PPHMA)] was synthesized by atom transfer radical polymerization. This system can self‐assemble into vesicles in aqueous solution at pH 8. On adjusting the solution pH to 3, there was a transition from vesicles to micelles. The same behavior, that is, transition from vesicles to micelles was also realizable on addition of β‐cyclodextrin (β‐CD) to the PEO‐b‐P(DEAEMA‐co‐PPHMA) solution at pH 8. Furthermore, after β‐CD was added, alternating irradiation of the solution with UV and visible light can also induce the reversible micelle‐to‐vesicle transition because of the photoinduced trans‐to‐cis isomerization of azobenzene units. The multistimuli‐responsive precise morphological changes were studied by laser light scattering, transmission electron microscopy, and UV–vis spectra. © 2011 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem, 2011     

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 λ_2PA=550–1000 nm, observing 2PA cross sections of σ_2PA=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.     

一种新型pH荧光探针：中位-吡啶取代的硼二吡咯亚甲基(BDP)染料的合成、结构与光谱性质研究

本文报道了一种新型中位-吡啶取代的硼-二吡咯亚甲基(BDP)染料的合成与晶体结构表征,发现中位-吡啶环与因达省平面几乎正交,其二面角为88.2°。同时对它的吸收和稳态荧光性质研究表明：当溶液的酸性增强时,化合物的荧光减弱,pK_a为2.24。该化合物在可见光激发下,可以作为比较灵敏的pH荧光探针。     

Reversible photoswitching aggregation and dissolution of spiropyran- functionalized copolymer and light-responsive FRET process

Well-defined, reversibly light-responsive amphiphilic diblock copolymer grafted with spiropyran, was prepared by reversible addition–fragmentation chain transfer(RAFT) polymerization. The copolymer self-assembles into polymeric micelles in water and exhibits reversible dissolution and re-aggregation characteristics upon ultraviolet(UV) and visible(Vis)-light irradiation. The fluorescence response of spiropyran immobilized onto the copolymer was light switchable. When nitrobenzoxadiazolyl derivative(NBD) dyes are encapsulated into the core of the micelles, a reversible, light-responsive, dual-color fluorescence resonance energy transfer(FRET) system is constructed and processed, which is well regulated by alternatively UV/vis irradiation. We anticipate these photoswitchable and FRET lighting up nanoparticles will be useful in drug delivery and cell imaging or tracking synchronously.     

pH和温度敏感型聚合物荧光探针的合成及应用

以萘酰亚胺(NI)、N-异丙基丙烯酰胺(NIPAM)和多面体齐聚倍半硅氧烷(POSS)为原料,通过可逆加成断裂自由基聚合(RAFT)方法,设计合成了具有p H响应和温度敏感的聚合物荧光探针Poly(POSS-NINIPAM)。通过核磁共振波谱仪(NMR)、荧光发射光谱仪和质谱仪(MS)等技术手段表征了Poly(POSS-NINIPAM)的p H和温度的响应性能。结果表明,在p H值5.8~8.0范围内,探针Poly(POSS-NI-NIPAM)的荧光强度变化明显,与p H值呈现良好的线性相关(λem=520 nm,p Ka=6.51),对p H和温度的响应均呈现出良好的循环检测性能,可用于检测活细胞He La的p H值变化。探针Poly(POSS-NI-NIPAM)在检测生物样本中p H/温度变化方面具有潜在的应用前景。     

Recognition-mediated contrasting fluorescence behaviour of 4′,6-diamidino-2-phenylindole (DAPI): probing the pKa of p-sulfonatocalix[4/6]arenes

Abstract

The interaction between a biologically important dye, 4′,6-diamidino-2-phenylindole (DAPI) and a macrocyclic host, p-sulfonatocalix[6]arene (SCX6) has been investigated using ground-state absorption, steady-state as well as time-resolved fluorescence measurements at two preset pH conditions: 2.5 and 6. Acknowledging the complexation with SCX6 at both the pHs with strong binding, DAPI shows spectral shifts in the absorption and emission spectra; however, strikingly, the fluorescence intensity behaviour is seen contrastingly opposite. At lower pH (~2.5), DAPI displays six-fold intensity, whereas drastic quenching in the fluorescence intensity is recorded at pH 6. Detailed spectroscopic analysis revealed that the SCX6 with phenolate ion stabilised at the lower rim, prevailing at pH 6, efficiently quench the excited state of DAPI, presumably through an electron transfer process, whereas at pH 2.5, the undissociated phenolic groups extend the dye a favourable encapsulation to prevent the excited-state protonation process resulting in remarkable enhancement in the fluorescence. This interesting host–guided fluorescence response with pH is worth as fluorescence on-off switch at lower pH conditions as well as the DAPI system offers a good optical probe for the easy determination of the pK_a of p-sulfonatocalixarenes by visible fluorescence measurements.     

INVESTIGATION OF LIGHT-INDUCED CONFORMATION CHANGES IN SPIROPYRAN-MODIFIED SUCCINYLATED POLY(l-LYSINE)

Abstract— To determine the maximum range of coupling between side-chain photochromism and polypeptide conformation change, we modified the carboxylate side chains of succinylated poly(l -lysine) with a spiropyran to form polypeptide I. The extent of modification was determined to be 35.5%. The spacer group length between the polypeptide a-carbon and the dye was 12 atoms, providing minimum polypeptide-dye interaction. Conformation changes were monitored by circular dichroism as a function of light adaptation and solvent composition (hexafluoroisopropanol [HFIP] vs trifluoroethanol [TFE]). Under all solvent compositions, the dark-adapted dye was in the merocyanine form. Light adaptation by visible light converted the dye to the spiropyran form. When dissolved in TFE, I adopted a helical conformation insensitive to light adaptation. With increasing percentage HFIP, a solvent-induced helix-to-coil transition was observed around 80% (vol/vol) HFIP. At 100% HFIP, both light- and dark-adapted forms of I were in the coil state. Near the midpoint of the solvent-induced helix-to-coil transition, light adaptation caused conformation changes. Applying helix-to-coil transition theory, we measured a statistically significant difference in coil segment-HFIP binding constant for light- v.v dark-adapted solutions (6.38 ± 0.03 M^-1vs 6.56 ± 0.03 M^-1), but not for the nucleation parameter σ (1.2 ± 0.4 10^-3 v.v 1.3 ± 0.3 × 10^-3). The small binding constant difference translated to a light-induced binding energy difference of 17 cal/mol/monomer. Near the midpoint of the helix-to-coil transition, collective interactions between monomer units made possible the translation of a small energy difference (less than RT) into large macromolecular conformation changes. This work parallels similar behavior observed in poly(isocyanate) (Green, M. M. et al. J. Am. Chem. Soc. 115 , 4941–4942, 1993). The subtle differences in dye-backbone interaction in I suggested a maximum coupling distance (12 atoms) beyond which polypeptide conformation and dye state are uncoupled.     

Thioflavin T hydroxylation at basic pH and its effect on amyloid fibril detection

The fluorescent dye thioflavin T (ThT) is commonly used for in situ amyloid fibril detection. In this work, we focused on the spectroscopic properties and chemical stability of ThT in aqueous solution as a function of pH, temperature, and dye concentration. A reversible hydroxylation process occurs in alkaline solutions, which was characterized using a combination of UV-vis absorption spectroscopy, proton NMR, and density functional theory (DFT). On the basis of these studies, we propose a chemical structure for the hydroxylated form. Finally, by means of fluorescence spectroscopy, ThT hydroxylation effects on in situ amyloid detection have been investigated, providing new insights on the efficiency of the ThT assay for quantitative fibril evaluation at basic pH.     

新型螺吡喃衍生物: 离子传感和分子水平的信息处理

为实现金属离子检测和分子水平的信息处理, 合成了一类新型的含有功能配位基团的螺吡喃衍生物(SP1-SP4). 研究发现: 在没有UV光照的条件下, 金属离子可以促进螺吡喃(SP2和SP4)开环并形成稳定可逆的络合物(MC-Mⁿ⁺). 紫外-可见吸收光谱研究表明, 在UV光照前加入不同的金属离子会引起SP2 和SP4 的光学性质的特征变化, 因此提供了一种简易的通过裸眼就能辨别金属离子的比色方法. 荧光光谱研究表明, 这类化合物能够高灵敏高选择性地检测锌离子. 此外, 基于吸收光谱和荧光光谱的变化, 这类螺吡喃衍生物可以用于构建组合的逻辑门, 执行分子水平的信息处理, 从而展现了其在化学或环境传感和未来的分子计算机领域的潜在应用前景.     

Dye/gelled colloidal photonic crystal composites and their reversible pH-responsive optical behaviors

We introduced dyes, the colors of which are variable by changing the pH of their medium solutions, into gelled colloidal photonic crystals (GCPCs) and investigated their reversible pH-responsive optical behaviors. When the extinction position of the dye overlaps with the Bragg peak of the GCPC, the Bragg-diffracted light can be trapped in the GCPC; when the extinction position of the dye is shifted out of the Bragg peak of the GCPC, the Bragg diffracted light is free and can escape out of the GCPC. The optical behaviors of dye/gelled colloidal photonic crystal composites are reversible to the pH changes of their surrounding medium.     

Photo-, thermally, and pH-responsive microgels

Microgels with photo-, thermally, and pH-responsive properties in aqueous suspension have been synthesized and characterized using dynamic light scattering and UV-visible spectroscopy. The new route involved first preparing poly(N-isopropylacrylamide) (PNIPAM)-allylamine copolymer microgels and a spiropyran photochrome (SP) bearing a carboxylic acid group. Then the functionalized spiropyran was coupled to the microgel via an amide bond. The dark-equilibrated gel particles feature spiropyran molecules in the polar, merocyanine form. After irradiation of visible light, the particle size becomes smaller because spiropyran changes to the relatively nonpolar, closed spiro form. The PNIPAM-SP microgels undergo a volume phase transition in water from a swollen state to a collapsed state with increasing temperature under all light conditions. However, the transition temperature range of the PNIPAM-SP is much broader than that for the PNIPAM without SP. The PNIPAM-SP microgels are monodisperse and self-assemble into a crystalline lattice while in suspension. The UV-visible spectra of an aqueous suspension of PNIPAM-SP microgel in the dark-adapted, merocyanine form showed both an absorption peak around 512 nm due to the merocyanine (giving a reddish color to the suspension) and two sharp peaks from Bragg diffraction of colloidal crystallites. Upon visible irradiation, the 512-nm band bleached significantly due to spiropyran photoisomerization. The spiropyran photoisomerization and accompanying color changes of the suspension were reversible upon alternating dark, UV, and visible light irradiation. Due to the residues of amine groups, the swelling capability of PNIPAM-SP microgels reduces as the pH value is changed from 7 to 10.