Metal‐Free Facile Synthesis of Multisubstituted 1‐Aminoisoquinoline Derivatives with Dual‐State Emissions

Although isoquinoline is a good traditional fluorescent structural unit, most of its derivatives emit fluorescence in solution and a few of them can emit solid‐state fluorescence as well. Herein, a series of multisubstituted 1‐aminoisoquinoline derivatives were synthesized by a simple reaction of a readily available 4H‐pyran derivative and secondary amines. The reaction had advantages of metal‐free, mild conditions, simple operation, and good yields, which was realized by a ring‐opening and sequential ring‐closing mechanism. These 1‐aminoisoquinoline derivatives were found to exhibit interesting dual‐state emissions. In the solution, they emitted strong blue fluorescence at about 458 nm. In the solid state, they emitted solid‐state blue fluorescence at 444–468 nm with high fluorescence quantum yields of 40.3–98.1%. Crystal structural analyses indicated that solid‐state emissions of these compounds originated from twisted molecular conformations and the resultant loose stacking arrangements. Furthermore, their solid‐state fluorescence wavelengths were demonstrated to depend on molecular conformations rather than stacking arrangements. The discovery of these 1‐aminoisoquinolines with multiple reaction sites provides new possibilities for the development of solid‐state fluorescent materials based on the traditional isoquinoline skeleton.     

8-Vinylguanine nucleo amino acid: a fluorescent PNA building block

Attachment of a vinyl group at guanine position 8 provides fluorescent properties of the nucleobase. Therefore, 8-vinylguanine was introduced as a 2-aminoethylglycine peptide nucleic acid (PNA) building block. Incorporation of the guanine analog in short PNA sequences by Fmoc solid phase peptide synthesis allowed the differentiation between hybridization states of specific double strands with DNA, RNA, and PNA as well as quadruplex forming RNA/PNA oligomers based on fluorescence intensity.     

Modulation of pyrene fluorescence in DNA probes depends upon the nature of the conformationally restricted nucleotide

The DNA probes (ODNs) containing a 2'-N-(pyren-1-yl)-group on the conformationally locked nucleosides [2'-N-(pyren-1-yl)carbonyl-azetidine thymidine, Aze-pyr (X), and 2'-N-(pyren-1-yl)carbonyl-aza-ENA thymidine, Aza-ENA-pyr (Y)], show that they can bind to complementary RNA more strongly than to the DNA. The Aze-pyr (X) containing ODNs with the complementary DNA and RNA duplexes showed an increase in the fluorescence intensity (measured at lambda em approximately 376 nm) depending upon the nearest neighbor at the 3'-end to X [dA ( approximately 12-20-fold) > dG ( approximately 9-20-fold) > dT ( approximately 2.5-20-fold) > dC ( approximately 6-13-fold)]. They give high fluorescence quantum yields (Phi F = 0.13-0.89) as compared to those of the single-stranded ODNs. The Aza-ENA-pyr (Y)-modified ODNs, on the other hand, showed an enhancement of the fluorescence intensity only with the complementary DNA (1.4-3.9-fold, Phi F = 0.16-0.47); a very small increase in fluorescence is also observed with the complementary RNA (1.1-1.7-fold, Phi F = 0.17-0.22), depending both upon the site of the Y modification introduced as well as on the chemical nature of the nucleobase adjacent to the modification site into the ODN. The fluorescence properties, thermal denaturation experiments, absorption, and circular dichroism (CD) studies with the X- and Y-modified ODNs in the form of matched homo- and heteroduplexes consistently suggested (i) that the orientation of the pyrene moiety is outside the helix of the nucleic acid duplexes containing a dT-d/rA base pair at the 3'-end of the modification site for both X and Y types of modifications, and (ii) that the microenvironment around the pyrene moiety in the ODN/DNA and ODN/RNA duplexes is dictated by the chemical nature of the conformational constraint in the sugar moiety, as well as by the nature of neighboring nucleobases. The pyrene fluorescence emission in both X and Y types of the conformationally restricted nucleotides is found to be sensitive to a mismatched base present in the target RNA: (i) The X-modified ODN showed a decrease ( approximately 37-fold) in the fluorescence intensity (measured at lambda em approximately 376 nm) upon duplex formation with RNA containing a G nucleobase mismatch (dT-rG pair instead of dT-rA) opposite to the modification site. (ii) In contrast, the Y-modified ODN in the heteroduplex resulted in a approximately 3-fold increase in the fluorescence intensity upon dT-rG mismatch, instead of matched dT-rA pair, in the RNA strand. Our data corroborate that the pyrene moiety is intercalated in the X-modified mismatched ODN/RNA (G mismatch) heteroduplex as compared to that of the Y-modified ODN/RNA (G mismatch) heteroduplex, in which it is located outside the helix.     

A Multicolor Large Stokes Shift Fluorogen-Activating RNA Aptamer with Cationic Chromophores

Large Stokes shift (LSS) fluorescent proteins (FPs) exploit excited state proton transfer pathways to enable fluorescence emission from the phenolate intermediate of their internal 4-hydroxybenzylidene imidazolone (HBI) chromophore. An RNA aptamer named Chili mimics LSS FPs by inducing highly Stokes-shifted emission from several new green and red HBI analogues that are non-fluorescent when free in solution. The ligands are bound by the RNA in their protonated phenol form and feature a cationic aromatic side chain for increased RNA affinity and reduced magnesium dependence. In combination with oxidative functionalization at the C2 position of the imidazolone, this strategy yielded DMHBO⁺, which binds to the Chili aptamer with a low-nanomolar K_D. Because of its highly red-shifted fluorescence emission at 592 nm, the Chili–DMHBO⁺ complex is an ideal fluorescence donor for Förster resonance energy transfer (FRET) to the rhodamine dye Atto 590 and will therefore find applications in FRET-based analytical RNA systems.     

Multilabeled pyrene-functionalized 2'-amino-LNA probes for nucleic acid detection in homogeneous fluorescence assays

Homogeneous fluorescence assays for detection of nucleic acids are widely used in biological sciences. Typically, probes such as molecular beacons that rely on distance-dependent fluorescence quenching are used for such assays. Less attention has been devoted to tethering a single kind of fluorophores to oligonucleotides and exploiting hybridization-induced modulation of fluorescence intensity for nucleic acid detection. Herein, thermal denaturation experiments and fluorescence properties of oligodeoxyribonucleotides containing one or more 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA monomer(s) X are described. These pyrene-functionalized 2'-amino-LNAs display large increases in thermal stability against DNA/RNA complements with excellent Watson-Crick mismatch discrimination. Upon duplex formation of appropriately designed 2'-N-(pyren-1-yl)carbonyl-2'-amino-LNA probes and complementary DNA/RNA, intensive fluorescence emission with quantum yields between 0.28 and 0.99 are observed. Quantum yields of such magnitudes are unprecedented among pyrene-labeled oligonucleotides. Molecular modeling studies suggest that the dioxabicyclo[2.2.1]heptane skeleton and amide linkage of monomer X fix the orientation of the pyrene moiety in the minor groove of a nucleic acid duplex. Interactions between pyrene and nucleobases, which typically lead to quenching of fluorescence, are thereby reduced. Duplexes between multiple modified probes and DNA/RNA complements exhibit additive increases in fluorescence intensity, while the fluorescence of single stranded probes becomes increasingly quenched. Up to 69-fold increase in fluorescence intensity (measured at lambda(em) = 383 nm) is observed upon hybridization to DNA/RNA. The emission from duplexes of multiple modified probes and DNA/RNA at concentrations down to less than 500 nM can easily be seen by the naked eye using standard illumination intensities.     

A switch-on fluorescence assay for bacterial β-lactamases with amyloid fibrils as fluorescence enhancer and visual tool

Herein is described the development of a novel switch-on fluorescence assay for detecting β-lactamases. The fluorescence assay comprises two components: solid beads coated with a β-lactam antibiotic, which is linked to an environment-sensitive fluorophore (dansylaminothiophenol, DTA), and amyloid fibrils of hen lysozyme (acting as fluorescence enhancer and visual tool). In the presence of the clinically significant TEM-1 β-lactamase, the DTA-antibiotic complex on the solid beads is hydrolyzed, thus releasing the DTA dye into solution. The DTA dye is only weakly fluorescent in solution but gives strong green fluorescence upon binding to lysozyme fibrils. These strongly fluorescent DTA-bound fibrils can be easily visualized by the naked eye upon illumination of the sample with a simple UV lamp. The fluorescence assay can detect TEM-1 at low concentration (0.01 nM). In contrast, no observable fluorescence appears when the fluorescence assay is performed on samples without the TEM-1 β-lactamase.     

In Situ Spatial Complementation of Aptamer‐Mediated Recognition Enables Live‐Cell Imaging of Native RNA Transcripts in Real Time

Direct cellular imaging of the localization and dynamics of biomolecules helps to understand their function and reveals novel mechanisms at the single‐cell resolution. In contrast to routine fluorescent‐protein‐based protein imaging, technology for RNA imaging remains less well explored because of the lack of enabling technology. Herein, we report the development of an aptamer‐initiated fluorescence complementation (AiFC) method for RNA imaging by engineering a green fluorescence protein (GFP)‐mimicking turn‐on RNA aptamer, Broccoli, into two split fragments that could tandemly bind to target mRNA. When genetically encoded in cells, endogenous mRNA molecules recruited Split‐Broccoli and brought the two fragments into spatial proximity, which formed a fluorophore‐binding site in situ and turned on fluorescence. Significantly, we demonstrated the use of AiFC for high‐contrast and real‐time imaging of endogenous RNA molecules in living mammalian cells. We envision wide application and practical utility of this enabling technology to in vivo single‐cell visualization and mechanistic analysis of macromolecular interactions.     

Two color RNA intercalating probe for cell imaging applications

Here we report on a phenanthridine derivative which has a covalently linked fluorescein molecule in order to increase the light absorption and hence fluorescence signal intensity when bound to duplex RNA. Steady-state fluorescence shows that the energy transfer efficiency from the fluorescein to the phenanthridine fluorophore is approximately 77%, which results in the probe being over 5x brighter than other phenanthridine derivatives when bound to RNA. Due to the relatively long lifetime (approximately 20 ns) of the probe, time-resolved fluorescence is used to increase the signal to background ratio in cell growth medium from 7 (steady-state value) to over 40. Moreover, fluorescence images of cells containing the probe show that the fluorescein signal is readily apparent along with that of the intercalated fluorophore, allowing this probe to be used as a dual color probe which simultaneously reports the probes' location and that of RNA.     

Aggregation-Induced Emission with Large Redshift in 2,7-Diphenyl-fluorenone:Reality or Artifact?

The luminescence property of 2, 7-diphenyl-fluorenone (DPFO) was previously reported to be very unusual with a large aggregation-induced effect associated with a fluorescence redshift of 150 nm. The phenomenon is reexamined in this work. It is found that the abnormal observations are caused by the presence of a trace amount of impurity 2, 7-diphenyl-fluorene (DPF) in the as-synthesized DPFO. The pure DPFO molecule does have an intense fluorescence (FL) in solid (528 nm), about 4-5 times larger than in its dilute dichloromethane solutions (542 nm), but with a blueshifted rather than redshifted FL wavelength in solid. The enormous FL enhancement and redshifted FL wavelength of the as-synthesized DPFO solid are due to the presence of impurity DPF. The FL of DPF is much stronger than that of DPFO in dilute solutions and it also has shorter FL wavelengths. In a dilute solution of DPFO with a trace amount of DPF (~1%), the dominant FL peaks are from DPF. Because the electronic absorption peaks of DPF overlaps with DPFO, the electronic energy of DPF can transfer to DPFO. The energy transfer is faster with the increase of concentration because DPF and surrounding DPFO molecules become closer, which quenches the FL of DPF (356 and 372 nm) and enhances the FL of DPFO (542 nm in solution and 528 nm in solid). Therefore, at high concentrations or in solids, only peak at about 542 or 528 nm shows up, and peaks at 356 and 372 nm disappear.     

A Quadruplex‐Based,Label‐Free,and Real‐Time Fluorescence Assay for RNase H Activity and Inhibition

We demonstrate a unique quadruplex‐based fluorescence assay for sensitive, facile, real‐time, and label‐free detection of RNase H activity and inhibition by using a G‐quadruplex formation strategy. In our approach, a RNA–DNA substrate was prepared, with the DNA strand designed as a quadruplex‐forming oligomer. Upon cleavage of the RNA strand by RNase H, the released G‐rich DNA strand folds into a quadruplex in the presence of monovalent ions and interacts with a specific G‐quadruplex binder, N‐methyl mesoporphyrin IX (NMM); this gives a dramatic increase in fluorescence and serves as a reporter of the reaction. This novel assay is simple in design, fast in operation, and is more convenient and promising than other methods. It takes less than 30 min to finish and the detection limit is much better or at least comparable to previous reports. No sophisticated experimental techniques or chemical modification for either RNA or DNA are required. The assay can be accomplished by using a common spectrophotometer and obviates possible interference with the kinetic behavior of the catalysts. Our approach offers an ideal system for high‐throughput screening of enzyme inhibitors and demonstrates that the structure of the G‐quadruplex can be used as a functional tool in specific fields in the future.     

THE SOLID STATE LUMINESCENCE OF ESTROGENS

Abstract. The luminescent behavior of certain estrogens in the solid state is discussed. Spectra of solid films of estradiol and estriol are red shifted compared to that in liquid solutions with estriol shifted to a lesser extent. The red shifts are attributed to intermolecular hydrogen bonding, this conclusion being based on IR spectra and X-ray data. The calculated distance over which hydrogen bonding takes place is 2.755 Å in estradiol and 2.638 Å in estriol. The increased shift in the spectrum of estradiol is believed to be due to an additional hydrogen bond with a water molecule present in the solid film (O—O distance 2.793 Å). In estrone the weak fluorescence present in liquid solution is absent in solid film, in contrast to 3-desoxyestrone where fluorescence is preserved. Out of three possible forms of crystalline estrone only two can form hydrogen bonds, based on O-O distance calculations. In equilenin, which is highly fluorescent in liquid solution, no fluorescence is detected in solid film. This again is attributed to hydrogen bonding between the hydroxyl group of the β-naphtholic chromophore and the carbonyl group in the C(17) position. Dihydroequilenin which lacks the carbonyl group and equilenin dissolved in a host crystal retain their fluorescence.     

A theoretical approach for designing fluorescent reagentless biosensors: The optical model

In this paper a mathematical model describing the analytical signal obtained in fluorescence sensors is presented and compared with other commonly used models. The model starts from the Kubelka-Munk theory for solid surfaces but incorporates new theoretical improvements, being principally: (a) the increase in the effective optical pathlength due to the Scattering Induced Path Variation (SIPV), the incorporation of this parameter allows us to deduce that the fluorescence intensity from solid surfaces does not linearly change with the fluorophore concentrations; (b) the influence of the inner filter effect and how the error can be rectified and (c) the calculation of the scattering coefficients in sensor films for this kind of sensor. From this model it is possible to predict the effect of the fluorophore concentration, the sensor film scattering coefficient and the sample inner filter effect on the fluorescence signal. The conclusions obtained can be extended to other types of fluorescence measurements from solid surfaces.     

双态发光三苯胺基有机硼配合物的设计、合成与应用

以三(4-溴苯)胺、4-氨基苯硼酸频哪醇酯、4-二乙氨基水杨醛和三氟化硼乙醚溶液为原料,经过Suzuki偶联反应、缩合反应和配位反应,设计、合成了一种新型三枝结构的三苯胺有机硼配合物(TPAB),使用 ¹H和 ¹³C NMR对 TPAB的结构进行了表征,通过紫外可见吸收光谱和荧光发射光谱详细研究了TPAB溶液和固体态的光物理性能以及不同的外部条件对其发光性能的影响。发现 TPAB溶液和固体态都具有较强的荧光发射,在四氢呋喃溶液中的吸收峰位于 417 nm,发射峰位于 548 nm,荧光量子产率为 40.49%,荧光寿命为 1.72 ns;TPAB 固体的荧光发射峰位于 582 nm,荧光量子产率为 11.43%,荧光寿命为 0.72ns,表明TPAB具有优良的双光发光性能。此外,TPAB具有良好的发光稳定性,不受pH、金属离子、氨基酸和压力的影响。基于化合物优异的发光性能,将其应用于荧光细胞成像,在肝癌细胞(HepG2)中表现出良好的单光子和双光子荧光成像效果。     

Fluorescence Emission of Self-assembling Amyloid-like Peptides: Solution versus Solid State

Analysis of the intrinsic UV-visible fluorescence exhibited by self-assembling amyloid-like peptides in solution and in solid the state highlights that their physical state has a profound impact on the optical properties. In the solid state, a linear dependence of the fluorescence emission peaks as a function of excitation wavelength is detected. On the contrary, an excitation-independent emission is observed in solution. The present findings constitute a valuable benchmark for current and future explanations of the fluorescence emission by amyloids.     

双态发光三苯胺基有机硼配合物的设计、合成与应用

以三(4-溴苯)胺、4-氨基苯硼酸频哪醇酯、4-二乙氨基水杨醛和三氟化硼乙醚溶液为原料,经过Suzuki偶联反应、缩合反应和配位反应,设计、合成了一种新型三枝结构的三苯胺有机硼配合物(TPAB),使用¹H和¹³C NMR对TPAB的结构进行了表征,通过紫外可见吸收光谱和荧光发射光谱详细研究了TPAB溶液和固体态的光物理性能以及不同的外部条件对其发光性能的影响。发现TPAB溶液和固体态都具有较强的荧光发射,在四氢呋喃溶液中的吸收峰位于417 nm,发射峰位于548 nm,荧光量子产率为40.49%,荧光寿命为1.72 ns; TPAB固体的荧光发射峰位于582 nm,荧光量子产率为11.43%,荧光寿命为0.72ns,表明TPAB具有优良的双态发光性能。此外,TPAB具有良好的发光稳定性,不受pH、金属离子、氨基酸和压力的影响。基于化合物优异的发光性能,将其应用于荧光细胞成像,在肝癌细胞(HepG2)中表现出良好的单光子和双光子荧光成像效果。     

Fluorodynamers Displaying Tunable Fluorescence on Constitutional Exchanges in Solution and at Solid Film–Solution Interface

Dynamic covalent polymers—dynamers—are adaptive materials that offer timely variant adaptive macroscopic organization across extended scales. In the current study, imine exchange reactions and fluorescence transfer can occur at the interfaces between various solutions and solid state dynameric films. The fluorescence quenching upon imine formations for designed fluorogen was successfully demonstrated, and this tunable fluorescence was further used to study the re-composition of a solid film. Moreover, the dynamic covalent films also exhibited responsiveness to competing amines and acid/base conditions, both in solutions and solid film–solution interface. This work can provide more insights into interface dynamic chemistry and holds great potential for further applications in optical and biomedical materials.     

COMPARISON OF THE SOLID-MATRIX LUMINESCENCE PROPERTIES and PHOTOPHYSICAL PARAMETERS OF TWO PRODUCTS FROM BENZO(a)PYRENE-DNA ADDUCTS

Abstract The solid-matrix luminescence properties and several calculated photophysical parameters of two important products from the benzo(a)pyrene-DNA adducts were compared. The products were benzo(a)pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (I-1) and 7R,8S,9S-trihydroxy-10R-(A^p-deoxyguanosyl-3'-phosphate)-7,8,9,1O-tetrahydroben-zo(a)pyrene (BPDE-dG). The solid-matrix luminescence data were obtained for 1–1and BPDE-dG adsorbed on two different solid matrices, namely, 1% a-cyclodextrin (CD)/NaCl and 25% trehalose/NaCl and at two different temperatures (93 K and 296 K). The 25% trehalose/NaCl gave higher fluorescence and phosphorescence quantum yields from both 1–1and BPDE-dG in contrast to the 1%α-CD/NaCl matrix. The BPDE-dG showed lower fluorescence quantum yields on the solid matrices compared to 1–1. The lower fluorescence quantum yields for BPDE-dG were attributed to a photoinduced electron transfer mechanism. In contrast to the room-temperature solution fluorescence of BPDE-dG, BPDE-dG gave rather high fluorescence quantum yields at room temperature when adsorbed on the two solid matrices. From solid-matrix luminescence quantum yields and solid-matrix luminescence lifetimes, many photophysical parameters were calculated and compared. Several differences among the rate constants were noted with the two solid matrices for BPDE-dG and 1–1. For example, BPDE-dG showed internal conversion at 296 K with 25% trehalose/NaCl, but no internal conversion was observed at 93 K with this solid matrix. Also, for BPDE-dG the phosphorescence rate constants at 296 K and 93 K. were very small with 25% trehalose/NaCl, but the intersystem crossing rate constants from the triplet state to the ground state were very large. The results from this work show how solid-matrix luminescence can be employed to obtain unique luminescence information from the 1–1and BPDE-dG systems.     

COMPARISON OF THE SOLID-MATRIX LUMINESCENCE PROPERTIES AND PHOTOPHYSICAL PARAMETERS OF TWO PRODUCTS FROM BENZO(a)PYRENE-DNA ADDUCTS

Abstract: The solid-matrix luminescence properties and several calculated photophysical parameters of two important products from the benzo(a)pyrene-DNA adducts were compared. The products were benzo(a)pyrene-r-7,t-8,9,c-10-tetrahydrotetrol (I-1) and 7R,8S,9S-trihydroxy-10-( N ²-deoxyguanosyl-3'-phosphate)-7,8,9,10-tetrahydroben-zo(a)pyrene (BPDE-dG). The solid-matrix luminescence data were obtained for I-1 and BPDE-dG adsorbed on two different solid matrices, namely, 1%α-cyclodextrin (CD)/NaCl and 25% trehalose/NaCl and at two different temperatures (93 K and 296 K). The 25% trehalose/NaCl gave higher fluorescence and phosphorescence quantum yields from both I-1 and BPDE-dG in contrast to the 1%α-CD/NaCl matrix. The BPDE-dG showed lower fluorescence quantum yields on the solid matrices compared to I-1. The lower fluorescence quantum yields for BPDE-dG were attributed to a photoinduced electron transfer mechanism. In contrast to the room-temperature solution fluorescence of BPDE-dG, BPDE-dG gave rather high fluorescence quantum yields at room temperature when adsorbed on the two solid matrices. From solid-matrix luminescence quantum yields and solid-matrix luminescence lifetimes, many photophysical parameters were calculated and compared. Several differences among the rate constants were noted with the two solid matrices for BPDE-dG and I-1. For example, BPDE-dG showed internal conversion at 296 K with 25% trehalose/NaCl, but no internal conversion was observed at 93 K with this solid matrix. Also, for BPDE-dG the phosphorescence rate constants at 296 K and 93 K were very small with 25% trehalose/NaCl, but the intersystem crossing rate constants from the triplet state to the ground state were very large. The results from this work show how solid-matrix luminescence can be employed to obtain unique luminescence information from the I-1 and BPDE-dG systems.