Fluorogenic stereochemical probes for transaldolases

Transaldolase catalyzes the transfer of dihydroxyacetone from, for example, fructose 6-phosphate to erythrose 4-phosphate. As a potential probe for assaying fluorescent transaldolase, 6-O-coumarinyl-fructose (1) was prepared in six steps from D-fructose. The corresponding 6-O-coumarinyl-5-deoxy derivative 2 was prepared stereoselectively from acrolein and tert-butyl acetate by a chemoenzymatic route involving Amano PS lipase for the kinetic resolution of tert-butyl 3-hydroxypent-4-enoate (7) and E. coli transketolase for assembly of the final product. The corresponding stereoisomer related to D-tagatose was obtained by a chemical synthesis starting from D-ribose. Indeed, transaldolases catalyze the retro-aldolization of substrate 1 to give dihydroxyacetone and 3-O-coumarinyl-glyceraldehyde. The latter primary product undergoes a beta-elimination in the presence of bovine serum albumin (BSA) to give the strongly fluorescent product umbelliferone. A similar reaction is obtained with the 5-deoxy analogue 2, but there is almost no reaction with its stereoisomer 3. The stereoselectivity of transaldolases can be readily measured by the relative rates of fluorescence development in the presence of the latter pair of diastereomeric substrates.     

Fluorogenic polypropionate fragments for detecting stereoselective aldolases

A series of fluorogenic polypropionate fragments has been prepared. These undergo retroaldolization to an intermediate aldehyde that liberates the fluorescent product umbelliferone by a secondary beta-elimination reaction. leading to a >20-fold increase in fluorescence (lambda(em) = 460 +/- 20 nm, lambdaex = 360 +/- 20 nm). By applying the principle of microscopic reversibility to the reversible aldol reaction, we can use these substrates to detect stereoselective aldolases. Test substrates are available to probe the classical cases of syn- and anti-selective aldolization (11a-d), Cram/ anti-Cram-selective aldolization (10a-d), and double stereoselective aldolization (3a-h). The selectivity of aldolase antibody 38C2 for these substrates is demonstrated as an example. The assay is suitable for high-throughput screening for catalysis in microtiter plates, and therefore provides a convenient tool for the isolation of new stereoselective aldolases from catalyst libraries.     

Azalysine analogues as probes for protein lysine deacetylation and demethylation

Reversible lysine acetylation and methylation regulate the function of a wide variety of proteins, including histones. Here, we have synthesized azalysine-containing peptides in acetylated and unacetylated forms as chemical probes of the histone deacetylases (HDAC8, Sir2Tm, and SIRT1) and the histone demethylase, LSD1. We have shown that the acetyl-azalysine modification is a fairly efficient substrate for the sirtuins, but a weaker substrate for HDAC8, a classical HDAC. In addition to deacetylation by sirtuins, the acetyl-azalysine analogue generates a novel ADP-ribose adduct that was characterized by mass spectrometry, Western blot analysis, and nuclear magnetic resonance spectroscopy. This peptide-ADP-ribose adduct is proposed to correspond to a derailed reaction intermediate, providing unique evidence for the direct 2'-hydroxyl attack on the O-alkylimidate intermediate that is formed in the course of sirtuin catalyzed deacetylation. An unacetylated azalysine-containing H3 peptide proved to be a potent inhibitor of the LSD1 demethylase, forming an FAD adduct characteristic of previously reported related structures, providing a new chemical probe for mechanistic analysis.     

Strategy for selective chemical cross-linking of tyrosine and lysine residues

Chemical cross-linking of proteins combined with mass spectral analysis is a powerful technique that can be utilized to yield protein structural information, such as the spatial arrangement of multi-protein complexes or the folding of monomeric proteins. The succinimidyl ester cross-linking reagents are commonly used to cross-link primary amine-containing amino acids (N-terminus and lysine). However, in this study they were used to react with tyrosines as well, which allowed for the formation of cross-links between two primary amines, one primary amine and one tyrosine, or two tyrosines. This result is extremely important to the chemical cross-linking community for two reasons: (1) all possible cross-linked residues must be considered when analyzing data from these experiments to generate correct distance constraints and structural information, and (2) utilizing the versatility of these cross-linking reagents allows more information content to be generated from a single cross-linking reagent, which may increase the number of cross-links obtained in the experiment. Herein, we study the reactivity of the succinimidyl ester labeling and cross-linking reagents with angiotensin I and oxidized insulin beta-chain. Using the succinimidyl acetate labeling reagent, the reactivity of the N-terminus was found to be greater than either lysine or tyrosine. However, a selectivity of the cross-linking reagent was observed for either tyrosine or lysine depending on the pH of the reaction solution. In acidic pH, it was observed that tyrosine was more reactive, while in alkaline pH lysine was more reactive. Exploiting this selectivity predominantly N-terminus-tyrosine or tyrosine-tyrosine cross-links were favored at acidic pH, while N-terminus-tyrosine or tyrosine-lysine cross-links were favored at alkaline pH.     

An Expeditious Approach towards the Synthesis and Application of Water-Soluble and Photostable Fluorogenic Chromones for DNA Detection

The intensive research for hybridization probes based on organic molecules with fluorogenic properties is currently attracting particular attention due to their potential to efficiently recognize different DNA conformations and the local environment. However, most established organic chromophores do not meet the requirements of this task, as they do not exhibit good brightness in aqueous buffer media, develop aggregation and/or are not easily conjugated to oligodeoxynucleotides (ODNs) while keeping their photophysics intact. Herein, an important modification strategy was employed for a well-known fluorophore, 2-(4-(diethylamino)phenyl)-3-hydroxychromone (dEAF). Although this push–pull dye absorbs intensively in the visible range and shows emission with large Stokes shifts in all organic solvents, it is strongly quenched in water. This Achilles’ heel prompted us to implement a new strategy to obtain a series of dyes that retain all the photophysical features of dEAF in water, conjugate readily with oligonucleotides, and furthermore demonstrate sensitivity to hydration, thus paving the way for a high-performance fluorogenic DNA hybridization probe.     

Design and synthesis of activity probes for glycosidases

[structure: see text] A new synthetic route was developed for the preparation of activity probe 1 for beta-glucosidase in this study. The key glycosidation step begins with benzyl p-hydroxyphenylacetate. Benzylic functionalization for the construction of the trapping device was achieved at later stages. Probe 1 was shown to be able to label the target enzyme. This cassette-like design offers great flexibility for future alterations. It would allow the synthetic scheme to expand to other glycosidase probes with different linker/reporter combinations.     

Phenothiazine-based oligomers as novel fluorescence probes for detecting vapor-phase nitro compounds

To meet the need for rapid and low-cost chemical sensing of explosive, new fluorescence chemosensors based on oligophenothiazines for probing vapor-phase nitro compounds have been developed. The phenothiazine-based trimer P3 and pentamer P5 have been synthesized via Heck and Wittig reactions by convergent approach. It was found that they can detect the vapors of nitro compounds, including p-nitrotoluene (p-NT), 2,4-dinitrotoluene (DNT), 2,4,6-trinitrotoluene (TNT) with good sensitivity and reversibility. And the sensor of P3 film gave a linear fluorescence quenching response to 7-800 ppb TNT with the detection limit of 4 ppb. For DNT vapor, a linear working range of the sensor was 2-24 ppm with the detection limit of 40 ppb. Meanwhile, the interferents, including common organic solvents, p-nitrophenol and 2,4-dinitrophenylhydrazine cannot lead to obvious fluorescence quenching, meaning that the film based on oligophenothiazines exhibited good specificity of fluorescence response to explosive. Based on the fluorescence lifetime and UV-vis absorption measurements, we suggested that the fluorescence quenching of oligophenothiazine-based films exposed to the vapors of nitro compounds was due to the formation of non-fluorescent charge-transfer complex between oligophenothiazine and nitro compounds.     

Theoretical study of lanthanide-based in vivo luminescent probes for detecting hydrogen peroxide

The 4f-4f emissions from lanthanide trication (Ln³⁺) complexes are widely used in bioimaging probes. The emission intensity from Ln³⁺ depends on the surroundings, and thus, the design of appropriate photo-antenna ligands is indispensable. In this study, we focus on two probes for detecting hydrogen peroxide, for which emission intensities from Tb³⁺ are enhanced chemo-selectively by the H₂O₂-mediated oxidation of ligands. To understand the mechanism, the Gibbs free energy profiles of the ground and excited states related to emission and quenching are computed by combining our approximation—called the energy shift method—and density functional theory. The different emission intensities are mainly attributed to different activation barriers for excitation energy transfer from the ligand-centered triplet (T1) to the Tb³⁺-centered excited state. Additionally, quenching from T1 to the ground state via intersystem crossing was inhibited by intramolecular hydrogen bonds only in the highly emissive Tb³⁺ complexes. © 2018 Wiley Periodicals, Inc.     

Screening method for detecting cross-contamination residues of tiamulin in swine feeds

A method was developed for the determination of tiamulin (TML), 14-deoxy-14-[(2-diethylaminoethyl)mercaptoacetoxy]mutilin hydrogen fumarate, a semisynthetic derivative of the naturally occurring antibiotic pleuromutilin produced by the fungus Pleurotus mutilis. This drug, with high activity against Gram-positive bacteria, some Gram-negative bacteria, and several strains of mycoplasms is administered to animals in food, drinking water, or by injection; however, its chemical structure causes problems in analysis of feeds. Although the molecule is charged below pH 8, attempts to analyze TML-containing extracts on ion-exchange columns or other polar stationary phases have failed. Additionally, TML shows no fluorescence activity and only poor UV activity. The present method consists of organic solvent extraction followed by liquid chromatography with UV detection. A low wavelength (208 nm) was used for detection. Limits of detection and quantitation, as well as data for recovery and repeatability obtained during characterization of the method, are described. The applicability of the optimized method was tested by analyzing commercial blank feeds processed after TML-medicated feeds.     

Synthesis of regular polypeptides including lysine,proline, and alanine residues

Summary From the epigeal part ofThermopsis alterniflora, in addition to the alkaloids found previously, argentine, argentamine, and a new alkaloid, dimethamine, which is a representative of the bimolecular series, have been isolated. The structure of 3-(3,4-dihydro-N-methylcytisin-5-yl)-N-methylcytisine has been proposed for dimethamine on the basis of IR, UV, mass, and NMR spectra and also from its conversion products.Institute of the Chemistry of Plant Substances, Academy of Sciences of the Uzbek SSR. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 218–222, March–April, 1972.     

Synthesis of regular polypeptides including lysine, proline, and alanine residues

Conclusions Polypeptides of the following sequences have been obtained: -Lys-Pro-,-Lys₂-Pro-, -Lys₃-Pro-,-Lys-Pro₂-, and -Lys-Ala-, with molecular weights of from 1000 to 14,000. It has been established that the use of pentachlorophenyl esters for polymerization in place of 2,4,5-trichlorophenyl esters leads to a considerable increase in the molecular weights of the polypeptides.Institute of Chemistry, Academy of Sciences of Tadzhik SSR. Institute of Molecular Biology, Academy of Sciences of the USSR. Translated from Khimiya Prirodnykh Soedinenii, No. 2, pp. 210–213, March–April, 1974.     

Some bactericidal properties of oligopeptides and regular polypeptides including lysine and ornithine residues

Summary Oligopeptides and polypeptides of the sequences Lys₆-, Ala-Lys₄-Ala, Lys₈-, Orn₂-Ala₂-, Lys₃-Pro-, Lys₃-Gly-, Orn₃-Gly-, Lys-Pro-, Lys-Pro₂-, Lys₂-Pro-, Lys₃-Ala- possess bactericidal properties in relation to a strain of the bacteriumStaphylococcus aureus.It has been shown that with an increase in the amount of lysine and ornithine residues in the polypeptide chain the bactericidal properties increase; proline residues play a fundamental part in the manifestation of these properties.Institute of Molecular Biology, Academy of Sciences of the USSR, Moscow. Institute of Chemistry, Academy of Sciences of Tadzhik SSR, Dushanbe. Translated from Khimiya Prirodnykh Soedinenii, No. 1, pp. 130–133, January–February, 1978.     

Dual-functional probes towards in vivo studies of brain connectivity and plasticity

A Gd(3+) based paramagnetic dextran conjugate has been developed, which enables the tracking of neuroanatomical connectivity in the brain by both MR and optical imaging. Cell studies and subsequent in vivo experiments in rodents demonstrate efficient internalisation and transport properties of the new tracer molecule.     

Enzyme-based electrochemical biosensor for sensitive detection of DNA demethylation and the activity of DNA demethylase

A novel electrochemical method is developed for detection of DNA demethylation and assay of DNA demethylase activity. This method is constructed by hybridizing the probe with biotin tagged hemi-methylated complementary DNA and further capturing streptavidin tagged alkaline phosphatase (SA-ALP) to catalyze the hydrolysis reaction of p-nitrophenyl phosphate. The hydrolysate of p-nitrophenol (PNP) is then used as electrochemical probe for detecting DNA demethylation and assaying the activity of DNA demethylase. Demethylation of target DNA initiates a degradation reaction of the double-stranded DNA (dsDNA) by restriction endonuclease of BstUI. It makes the failed immobilization of ALP, resulting in a decreased electrochemical oxidation signal of PNP. Through the change of this electrochemical signal, the DNA demethylation is identified and the activity of DNA demethylase is analyzed with low detection limit of 1.3 ng mL⁻¹. This method shows the advantages of simple operation, cheap and miniaturized instrument, high selectivity. Thus, it provides a useful platform for detecting DNA demethylation, analyzing demethylase activity and screening inhibited drug.     

Chiral interaction of sequential lysine polypeptides with methyl orange. Effects of distance between lysine residues and hydrophobic side chains

Two series of sequential poly(Lys-X) (X: Gly, Ala, and Ahx in series A; Gly, Ala, Leu and Phe in series B) have been synthesized. On the chiral interaction between cationic polypeptides and methyl orange (MO), the effects of the distance (series A) and of the hydrophobic side chains (series B) were examined by means of the absorption and induced circular dichroism (CD) spectroscopies. Dichroic bands associated with the blue shifted absorption peaks or shoulders of MO in the visible wavelength region were observed by complex formation between the polypeptides and the dye. The intensity of the induced CD was affected by the concentration of the complexes and time after preparing the complex solutions, suggesting the formation of the intermolecular aggregation in some instances. When MO molecules bound to lysine residues are apart from each other, the aggregation of the complexes is not marked. Roughly, the intensity of the induced CD decreases with increasing distance between the intramolecular lysine residues in series A polypeptides. When the hydrophobicity of the side chains is increased, the induced CD spectra of the series B polypeptide-MO complexes exhibits the inversion of the sign of the induced CD extrema.     

SERRS-based enzymatic probes for the detection of protease activity

Measurement of protease activity, for the first time using SERRS as a detection method, is reported herein. Synthetic introduction of phenylalanine to a benzotriazole azo dye allows the SERRS response to be "switched off" and subsequent exposure to protease restores the SERRS response. The substrates exhibit varying reactivity for a range of proteases and allow for in situ, real-time analysis of protease reactivity. A limit of detection for one protease, Subtilisin carlsberg, was investigated and was established to be 50 ng ml-1.     

Versatile synthesis of probes for high-throughput enzyme activity screening

Mass spectrometry based technologies are promising as generalizable high-throughput assays for enzymatic activity. In one such technology, a specialized enzyme substrate probe is presented to a biological mixture potentially exhibiting enzymatic activity, followed by an in situ enrichment step using fluorous interactions and nanostructure-initiator mass spectrometry. This technology, known as Nimzyme, shows great potential but is limited by the need to synthesize custom substrate analogs. We describe a synthetic route that simplifies the production of these probes by fashioning their perfluorinated invariant portion as an alkylating agent. This way, a wide variety of compounds can be effectively transformed into enzyme activity probes. As a proof of principle, a chloramphenicol analog synthesized according to this methodology was used to detect chloramphenicol acetyltransferase activity in cell lysate. This verifies the validity of the synthetic strategy employed and constitutes the first reported application of Nimzyme to a non-carbohydrate-active enzyme. The simplified synthetic approach presented here may help advance the application of mass spectrometry to high-throughput enzyme activity determination.

Use of peptide nucleic acid probes for detecting DNA single-base mutations by capillary electrophoresis

Peptide nucleic acid (PNA) oligomers can be used as probes in pre-gel hybridization experiments, as an alternative to Southern hybridization. In this technique, the PNA probe is hybridized to a cyanine-5 labeled DNA sample denatured at low ionic strength, and the mixture is directly injected for size separation into a capillary electrophoresis (CE) system equipped with laser-induced fluorescence (LIF) detector. The neutral backbone of PNA allows hybridization to occur at low ionic strength and assures an efficient CE separation of the PNA/DNA hybrids from both double-stranded and single-stranded DNA. We have used as a model system the cystic fibrosis R553X and R1162X single-base mutations and we have assessed the influence of various factors, such as temperature and denaturants concentration on DNA/PNA hybrid stability in order to achieve the high specificity required for a single base pair discrimination.