Synthesis of molecularly imprinted dye‐silica nanocomposites with high selectivity and sensitivity: Fluorescent imprinted sensor for rapid and efficient detection of τ‐fluvalinate in vodka

An imprinted fluorescent sensor was fabricated based on SiO₂ nanoparticles encapsulated with a molecularly imprinted polymer containing allyl fluorescein. High fluorine cypermethirin as template molecules, methyl methacrylate as functional monomer, and allyl fluorescein as optical materials synthesized a core‐shell fluorescent molecular imprinted sensor, which showed a high and rapid sensitivity and selectivity for the detection of τ‐fluvalinate. The sensor presented appreciable sensitivity with a limit of 13.251 nM, rapid detection that reached to equilibrium within 3 min, great linear relationship in the relevant concentration range from 0 to 150 nM, and excellent selectivity over structural analogues. In addition, the fluorescent sensor demonstrated desirable regeneration ability (eight cycling operations). The molecularly imprinted polymers ensured specificity, while the fluorescent dyes provided the stabile sensitivity. Finally, an effective application of the sensor was implemented by the detection of τ‐fluvalinate in real samples from vodka. The molecularly imprinted fluorescent sensor showed a promising potential in environmental monitoring and food safety.     

Electronic Modifications of Fluorescent Cytidine Analogues Control Photophysics and Fluorescent Responses to Base Stacking and Pairing

The rational design of fluorescent nucleoside analogues is greatly hampered by the lack of a general method to predict their photophysics, a problem that is especially acute when base pairing and stacking change fluorescence. To better understand these effects, a series of tricyclic cytidine (tC and tC^O) analogues ranging from electron-rich to electron-deficient was designed and synthesized. They were then incorporated into oligonucleotides, and photophysical responses to base pairing and stacking were studied. When inserted into double-stranded DNA oligonucleotides, electron-rich analogues exhibit a fluorescence turn-on effect, in contrast with the electron-deficient compounds, which show diminished fluorescence. The magnitude of these fluorescence changes is correlated with the oxidation potential of nearest neighbor nucleobases. Moreover, matched base pairing enhances fluorescence turn-on for the electron-rich compounds, and it causes a fluorescence decrease for the electron-deficient compounds. For the tC^O compounds, the emergence of vibrational fine structure in the fluorescence spectra in response to base pairing and stacking was observed, offering a potential new tool for studying nucleic acid structure and dynamics. These results, supported by DFT calculations, help to rationalize fluorescence changes in the base stack and will be useful for selecting the best fluorescent nucleoside analogues for a desired application.     

Enzymatic synthesis of caged NADP cofactors: aqueous NADP photorelease and optical properties

The synthesis of caged NADP analogues 18, 19, and 20 has been accomplished by utilizing the transglycosidase activity of solubilized NAD glycohydrolase (porcine brain) to incorporate caged nicotinamides 2, 3, and 4 into NADP. The synthesis of several nicotinamides modified at the carboxamide with o-nitrobenzyl photolabile groups is demonstrated as well as their potential for enzymatic transglycosidation. These results further demonstrate the feasibility of direct enzymatic transglycosidation of sterically hindered substrates into NAD(P), although high nicotinamide analogue water solubility was found to be a necessary trait for yield enhancement with certain analogues. Caged analogues were surveyed under aqueous conditions for net NADP photorelease, while the UV and fluorescent properties of both analogues and their photobyproducts were assessed for compatibility with systems that rely on optical monitoring of enzyme activity. A highly water-soluble alpha-methyl-o-nitrobenzyl group 8 was developed for the synthesis of 20 in order to enhance net NADP photorelease. Compound 20 demonstrated a high 75% net NADP photoreleased without substantial UV optical blackening or fluorescent byproducts. Analogues 18 and 19 were shown by ESI/MALDI-MS to photogenerate primarily adducts of NADP with deleterious UV and fluorescent properties. Our work stresses the superior release properties conferred by alpha-methyl substitution on aqueous carboxamide photorelease from o-nitrobenzyl compounds.     

8-(p-CF3-cinnamyl)-modified purine nucleosides as promising fluorescent probes

Natural nucleotides are not useful as fluorescent probes because of their low quantum yields. Therefore, a common methodology for the detection of RNA and DNA is the application of extrinsic fluorescent dyes coupled to bases in oligonucleotides. To overcome the many limitations from which fluorescent nucleotide-dye conjugates suffer, we have developed novel purine nucleosides with intrinsic fluorescence to be incorporated into oligonucleotide probes. For this purpose we synthesized adenosine and guanosine fluorescent analogues 7-25, conjugated at the C8 position with aryl/heteroaryl moieties either directly, or via alkenyl/alkynyl linkers. Directly conjugated analogues 7-14, exhibited high quantum yields, φ >0.1, and short λ(em) (<385 nm). Alkynyl conjugated analogues 22-25, exhibited low quantum yields, φ <0.075, and λ(em)<385 nm. The alkenyl conjugated analogues 15-21, exhibited λ(em) 408-459 nm. While analogues 15,16, and 20 bearing an EDG on the aryl moiety, exhibited φ <0.02, analogues 17, and 21 with EWG on the aryl moiety, exhibited extremely high quantum yields, φ ≈ 0.8, suggesting better intramolecular charge transfer. We determined the conformation of selected adenosine analogues. Directly conjugated analogue 8 and alkynyl conjugated analogue 22, adapted the syn conformation, whereas alkenyl conjugated analogue 15 adapted the anti conformation. Based on the long emission wavelengths, high quantum yields, anti conformation and base-paring compatibility, we suggest analogues 17 and 21 for further development as fluorescent probes for the sensitive detection of genetic material.     

A microenvironment-sensitive fluorescent pyrimidine ribonucleoside analogue: synthesis, enzymatic incorporation, and fluorescence detection of a DNA abasic site

Base-modified fluorescent ribonucleoside-analogue probes are valuable tools in monitoring RNA structure and function because they closely resemble the structure of natural nucleobases. Especially, 2-aminopurine, a highly environment-sensitive adenosine analogue, is the most extensively utilized fluorescent nucleoside analogue. However, only a few isosteric pyrimidine ribonucleoside analogues that are suitable for probing the structure and recognition properties of RNA molecules are available. Herein, we describe the synthesis and photophysical characterization of a small series of base-modified pyrimidine ribonucleoside analogues derived from tagging indole, N-methylindole, and benzofuran onto the 5-position of uracil. One of the analogues, based on a 5-(benzofuran-2-yl)pyrimidine core, shows emission in the visible region with a reasonable quantum yield and, importantly, displays excellent solvatochromism. The corresponding triphosphate substrate is effectively incorporated into oligoribonucleotides by T7 RNA polymerase to produce fluorescent oligoribonucleotide constructs. Steady-state and time-resolved spectroscopic studies with fluorescent oligoribonucleotide constructs demonstrate that the fluorescent ribonucleoside photophysically responds to subtle changes in its environment brought about by the interaction of the chromophore with neighboring bases. In particular, the emissive ribonucleoside, if incorporated into an oligoribonucleotide, positively reports the presence of a DNA abasic site with an appreciable enhancement in fluorescence intensity. The straightforward synthesis, amicability to enzymatic incorporation, and sensitivity to changes in the microenvironment highlight the potential of the benzofuran-conjugated pyrimidine ribonucleoside as an efficient fluorescent probe to investigate nucleic acid structure, dynamics, and recognition events.     

Synthesis of fluorescently labeled UDP-GlcNAc analogues and their evaluation as chitin synthase substrates

[structure: see text] Chitin synthase (CS) polymerizes UDP-GlcNAc to form chitin (poly-beta(1,4)-GlcNAc), a key component of fungal cell wall biosynthesis. Little is known about the substrate specificity of chitin synthase or the scope of substrate modification the enzyme will tolerate. Following a previous report suggesting that 6-O-dansyl GlcNAc is biosynthetically incorporated into chitin, we became interested in developing an assay for CS activity based on incorporation of a fluorescent substrate. We describe the synthesis of two fluorescent UDP-GlcNAc analogues and their evaluation as chitin synthase substrates.     

Fluorometry of singlet oxygen generated via a photosensitized reaction using folic acid and methotrexate

A method for the fluorometry of singlet oxygen (¹O₂) using less fluorescent folic acid and its analogue, methotrexate (MTX), was examined. Folic acid and MTX were decomposed into a strongly fluorescent pteridine compound via a photosensitized reaction by ¹O₂-generating photosensitizers in a deuterium oxide solution. The fluorescence intensity increased in proportion to the irradiation time or the number of photons absorbed by the photosensitizer. This method using the fluorescence enhancement of these folic acid analogues can be applied to determine the quantum yield of ¹O₂ generated through a photosensitized reaction in deuterium oxide. The background fluorescence of MTX is quite smaller than that of folic acid, indicating that MTX can be used for the more sensitive detection of ¹O₂. Figure (DOC 37.0 KB)     

Probing the mechanism of transport and compartmentalisation of polyamines in mammalian cells.

BACKGROUND: Many mammalian cells possess an active polyamine uptake system but little is known about the molecular mechanism of this transporter. The fate of polyamines taken up from the medium and the relationship to polyamine homeostasis remains to be fully established. The aim of this study was to develop a range of modified polyamines, particularly ligands incorporating a fluorophore, to explore the structural tolerances of the polyamine transport system and to probe the intracellular location of polyamines acquired from the medium. RESULTS: We synthesised a wide range of polyamine analogues incorporating cytotoxic agents, fluorescent chromophores and bulky substituents. All of these analogues have been shown to be good competitive inhibitors of spermidine uptake in a range of mammalian cells. Direct evidence for uptake of the fluorescent polyamine analogues and their subcellular distribution was obtained from confocal laser scanning fluorescence microscopy, which showed that they accumulated in granular structures within the cytoplasm and not in the nucleus. We demonstrated that their uptake is through the polyamine transport system by showing that pretreatment with DFMO, a potent inhibitor of polyamine biosynthesis, led to enhanced uptake, and cells deficient in the polyamine transport system did not accumulate these polyamine analogues. CONCLUSIONS: The polyamine transport system has a surprisingly broad structural tolerance. Fluorophore-containing polyamine analogues derived from the extracellular pool are located in granular structures within the cytoplasm and not to any great extent in the nuclei of mammalian cells. These observations might be consistent with a mechanism involving receptor-mediated endocytosis, and the granular 'structures' seen might reflect polyamine compartmentalisation within vesicles.     

Sensitive assay for serum angiotensin-converting enzyme and separation of angiotensin analogues by high-performance liquid chromatography with fluorescence detection

A high-performance liquid chromatographic method is described for the assay of angiotensin-converting enzyme in human serum and for the separation of angiotensins and their analogues after pre-column fluorescence derivatization with benzoin. Angiotensin II, formed enzymatically from angiotensin I, is converted into a fluorescent derivative which is then separated isocratically from the substrate and biological substances in the enzyme reaction mixture on a reversed-phase column (TSK gel ODS-120T). The lower limit of detection for angiotensin II is 0.66 pmol per enzyme assay tube. The method is simple and sensitive, and requires as little as 5 microliter of human serum. Angiotensin analogues can also be separated and quantified by the chromatographic technique, and thus this method permits the use of the analogues of angiotensin I as substrates.     

Synthesis and activity of fluorescent isoprenoid pyrophosphate analogues

New fluorescent analogues of farnesol and geranylgeraniol have been prepared and then converted to the corresponding pyrophosphates. These analogues incorporate anthranylate or dansyl-like groups anchored to the terpenoid skeleton through amine bonds that would be expected to be relatively stable to metabolism. After addition of the alcohols or the pyrophosphates to the culture medium, their fluorescence is readily observed inside a human-derived leukemia cell line. Enzyme assays have revealed that the farnesyl pyrophosphate analogue is an inhibitor of FTase, while the corresponding alcohol is not. These results, together with Western blot analyses of cell lysates, indicate that the farnesyl pyrophosphate analogue penetrates the cells as an intact pyrophosphate and that it does so at a biologically relevant concentration.     

Membrane‐permeable Triphosphate Prodrugs of Nucleoside Analogues

The metabolic conversion of nucleoside analogues into their triphosphates often proceeds insufficiently. Rate‐limitations can be at the mono‐, but also at the di‐ and triphosphorylation steps. We developed a nucleoside triphosphate (NTP) delivery system (TriPPPro‐approach). In this approach, NTPs are masked by two bioreversible units at the γ‐phosphate. Using a procedure involving H‐phosphonate chemistry, a series of derivatives bearing approved, as well as potentially antivirally active, nucleoside analogues was synthesized. The enzyme‐triggered delivery of NTPs was demonstrated by pig liver esterase, in human T‐lymphocyte cell extracts and by a polymerase chain reaction using a prodrug of thymidine triphosphate. The TriPPPro‐compounds of some HIV‐inactive nucleoside analogues showed marked anti‐HIV activity. For cellular uptake studies, a fluorescent TriPPPro‐compound was prepared that delivered the triphosphorylated metabolite to intact CEM cells.     

Quadracyclic Adenine: A Non‐Perturbing Fluorescent Adenine Analogue

Fluorescent‐base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base‐pairing properties of a new environment‐sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid‐phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B‐form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2‐aminopurine, and the recently developed triazole adenine, qA shows highly specific base‐pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300 nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8 % with an emission maximum at 456 nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2‐aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid‐containing systems.     

Structure and fluorescence properties of indole cyanine and merocyanine dyes with partially locked polymethine chain

The fluorescent properties of a series of symmetric cationic cyanine dyes and merocyanines with trimethylene bridge connecting N-atom of the indole residue with α-position of the polymethine chain have been explored in detail. It is shown that the constraining group influences their fluorescent properties both by steric and electronic effects. It revealed an essential decrease of the fluorescence quantum yields of the constrained symmetric cyanine dyes in comparison with those of their analogues without bridge group in their chromophore. This effect diminishes essentially at polymethine chain lengthening. In the case of merocyanines the bridge group can cause a decrease as well a substantial increase of their fluorescence quantum yields depending on their type of solvatochromism and solvent polarity. The conclusions made on the basis of the experimental data are in good agreement with the results of semiempirical quantum-chemical calculations.     

Quadracyclic adenine: a non-perturbing fluorescent adenine analogue

Fluorescent-base analogues (FBAs) comprise a group of increasingly important molecules for the investigation of nucleic acid structure and dynamics as well as of interactions between nucleic acids and other molecules. Here, we report on the synthesis, detailed spectroscopic characterisation and base-pairing properties of a new environment-sensitive fluorescent adenine analogue, quadracyclic adenine (qA). After developing an efficient route of synthesis for the phosphoramidite of qA it was incorporated into DNA in high yield by using standard solid-phase synthesis procedures. In DNA qA serves as an adenine analogue that preserves the B-form and, in contrast to most currently available FBAs, maintains or even increases the stability of the duplex. We demonstrate that, unlike fluorescent adenine analogues, such as the most commonly used one, 2-aminopurine, and the recently developed triazole adenine, qA shows highly specific base-pairing with thymine. Moreover, qA has an absorption band outside the absorption of the natural nucleobases (>300?nm) and can thus be selectively excited. Upon excitation the qA monomer displays a fluorescence quantum yield of 6.8?% with an emission maximum at 456?nm. More importantly, upon incorporation into DNA the fluorescence of qA is significantly less quenched than most FBAs. This results in quantum yields that in some sequences reach values that are up to fourfold higher than maximum values reported for 2-aminopurine. To facilitate future utilisation of qA in biochemical and biophysical studies we investigated its fluorescence properties in greater detail and resolved its absorption band outside the DNA absorption region into distinct transition dipole moments. In conclusion, the unique combination of properties of qA make it a promising alternative to current fluorescent adenine analogues for future detailed studies of nucleic acid-containing systems.     

Naphthalenyl- and anthracenyl-ethynyl dT analogues as base discriminating fluorescent nucleosides and intramolecular energy transfer donors in oligonucleotide probes

Fluorescent thymidine analogues functionalised in the 5-position with the moieties naphthalenylethynyl (NeT), anthracenylethynyl (AeT) and anthracenylbuta-1,3-diynyl (AeeT) have been incorporated into oligonucleotides. The modified oligonucleotides undergo significant emission enhancement when hybridised to fully complementary strands and a decrease in fluorescence emission when the modified thymine is paired with guanine. Thus these analogues are potentially useful as base discriminating fluorescent nucleosides (BDFs). When a fluorescein dT monomer is incorporated into the same oligonucleotide strand as the modified base, energy transfer enhances the fluorescein emission, particularly upon duplex formation. These dual-labelled probes may be useful for genetic analysis to detect point mutations and SNPs and could provide multiplexing capability.     

Furan Decorated Nucleoside Analogues as Fluorescent Probes: synthesis, photophysical evaluation and site-specific incorporation

The synthesis and photophysical evaluation of modified nucleoside analogues in which a five-membered heterocycle (furan, thiophene, oxazole, and thiazole) is attached to the 5-position of 2′-deoxyuridine are reported. The furan-containing derivative is identified as the most promising responsive nucleoside of this family due to its emission quantum efficiency and degree of sensitivity to its microenvironment. The furan moiety was then attached to the 5-position of 2′-deoxycytidine as well as the 8-position of adenosine and guanosine. Photophysical evaluation of these four furan-containing nucleoside analogues reveals distinct differences in the absorption, emission, and quantum efficiency depending upon the class of nucleoside (pyrimidine or purine). Comparing the photophysical properties of all furan-containing nucleosides, identifies the furan thymidine analogue, 5-(fur-2-yl)-2′-deoxyuridine, as the best candidate for use as a responsive fluorescent probe in nucleic acids. 5-(Fur-2-yl)-2′-deoxyuridine was then converted to the corresponding phosphoramidite and site specifically incorporated into DNA oligonucleotides with greater than 88% coupling efficiency. Such furan-modified oligonucleotides form stable duplexes upon hybridization to their complementary DNA strands and display favorable fluorescent features.     

Sealed and temperature-controlled sample cell for inverted and confocal microscopes and fluorescence correlation spectroscopy

Precise temperature control of sample environments plays a key role while exploring biological systems or temperature-sensitive materials. We have developed a sample cell for inverted microscopes, which allows a temperature accuracy of ±0.05 K in a temperature range of 5 to 65 °C, with an absolute precession of ±0.1 K. Our sample cell is developed for requirements of single-molecule experiments, which comprises easy-to-clean and well-sealed devices to prevent solvent evaporation. The applied control algorithm permits a tunable independent setting of heat and cooling behavior and allows the application on microscopes without any objective heating. For measuring precise and absolute diffusion coefficients with two-focus fluorescence correlation spectroscopy, the exact control of the sample temperature is essential. We performed diffusion measurements of TetraSpeck 100-nm fluorescent latex particles and of temperature-sensitive microgels in aqueous solutions to demonstrate the excellent temperature stability and reproducibility of the device.     

Synthesis,modeling, and biological studies of new thiazole-pyrazole analogues as anticancer agents

A series of various substituted thiazole-pyrazole hybrids 5, 7, 8, and 9 were synthesized, and their chemical structures were confirmed by spectral data (infrared, ¹H & ¹³C NMR and Mass). The frontier molecular orbital structural and energetic properties of the targeting thiazole-pyrazole hybrids were explored using the DFT/B3LYP methodology. The data indicated that they had a low energy gap (ΔE_H-L), 1.51–2.42 eV, and may be sorted as 6 < 9 < 7 < 8 < 4 < 3 < 5. The synthesized thiazole-pyrazole hybrids were explored for their activities towards HepG2, MCF-7, and HCT-116 in contrast to doxorubicin. The newly synthesized thiazole-pyrazole analogues demonstrated an acceptable efficiency towards the HepG2 cancer cell line in accordance with this order: 8 > 9 > 7 > 6. Meanwhile, most of the synthesized analogues displayed a significant reduction for the activity of the CAIX inhibitor, with IC₅₀ = 0.071 ± 0.015 to 0.902 ± 0.043 µM. Likewise, they revealed an IC₅₀ = 0.119 ± 0.043 to 0.906 ± 0.04 µM for CAXII inhibitor. Moreover, the newly synthesized thiazole-pyrazole analogues were exposed to the theoretical molecular docking study with PDB:1RHJ as the crystal structure of caspase-3 to examine their antiapoptotic effect as well as their certain properties on the caspase-3 enzyme.     

Photophysical characters of rationally designed hetero-ring-expanded guanine analogues and effect of cytosine pairing

We present the results of the CIS and TDB3LYP calculations of the optical absorption and emission spectra of some newly designed guanine (G) analogues and their Watson-Crick base pairs. Compared with natural G, the onset absorption peaks of these newly designed analogues are red-shifted, while the fluorescence peaks are blue-shifted. In general, the first excited singlet states (pipi*) of these analogues are nonplanar for all bases considered here. But, the Stokes shifts for the designed G-analogues are much smaller than that of natural G, suggesting that they have stronger molecular rigidity and higher fluorescence quantum yields than those of natural G. The first excited states of these Watson-Crick base pairs essentially originate from those of their isolated purine moieties, as demonstrated from the S1 geometries of their Watson-Crick base pairs. For G and its analogues, A1 and A2 (they are ring-expanded with one-bond intercalation at the C5 site), the pairing with cytosine reduces the oscillator strengths of both the first absorption peak (by 27%-60%) and the fluorescent emission (by 19%-23%), while for the analogues A3, A4, and xG in which G is ring-expanded with a two-bond intercalation at the C5 site, the pairing, in contrast, increases the oscillator strengths of both the first absorption peak (by 11%-15%) and the fluorescent emission (by 3%-20%). These observations indicate that the pairing with cytosine can quench the fluorescence for G, A1, and A2 but enhance the fluorescence quantum yields for A3, A4, and xG. The significant shifts induced by ring-expansion in the ring-expanded G with a two-bond intercalation at the C5 site reveal a possibility for their fluorescent detections.