Novel sensitive high-throughput screening strategy for nitrilase-producing strains

Nitrilases have found wide use in the pharmaceutical industry for the production of fine chemicals, and it is important to have a method by which to screen libraries of isolated or engineered nitrilase variants (including bacteria and fungi). The conventional methods, such as high-performance liquid chromatography, liquid chromatography-mass spectrometry, capillary electrophoresis, or gas chromatography, are tedious and time-consuming. Therefore, a direct and sensitive readout of the nitrilase's activity has to be considered. In this paper, we report a novel time-resolved luminescent probe: o-hydroxybenzonitrile derivatives could be applied to detect the activity of the nitrilases. By the action of nitrilases, o-hydroxybenzonitrile derivatives can be transformed to the corresponding salicylic acid derivatives, which, upon binding Tb(3+), serve as a photon antenna and sensitize Tb(3+) luminescence. Because of the time-resolved property of the luminescence, the background from the other proteins (especially in the fermentation system) in the assay could be reduced and, therefore, the sensitivity was increased. Moreover, because the detection was performed on a 96- or 384-well plate, the activity of the nitrilases from microorganisms could be determined quickly. Based on this strategy, the best fermentation conditions for nitrilase-producing strains were obtained.     

Development of a dual functional luminescent sensor for zinc ion based on a peptidic architecture

A synthetic peptide bearing a lanthanide complex, TbOTZ exhibits a decrease of chromophore fluorescence and a concomitant luminescence enhancement due to sensitized Tb³⁺ upon Zn²⁺ binding. Thus, TbOTZ can be a valuable tool for ratiometric sensing of Zn²⁺ as well as for time-resolved fluorescence detection with a single molecule.     

High-performance liquid chromatographic determination of α-keto acids

A high-performance liquid chromatographic method has been developed for the determination of eight kinds of α-keto acids. These acids were derivatized with o-phenylenediamine into 2-quinoxalinol derivatives, which were extracted into chloroform. The quinoxalinol derivatives were separated by reversed-phase high-performance liquid chromatography using a 250 mm × 2.1 mm I.D. column packed with LiChrosorb RP-8 (5 μm). This method could be satisfactorily applied to urine samples without any prepurification.     

A method for hydrolyzing and determining the amino acid compositions of picomole quantities of proteins in less than 3 hours

A method is described for quantitatively hydrolyzing proteins in 45 min and for analyzing the hydrolysates by high-performance liquid chromatography in an additional 52 min. The α-amino acids were detected by the fluorescence of their o-phthaldialdehyde derivatives. Ten picomoles of each of the commonly occuring α-amino acids could be reliably determined. The method described yielded OPA-ethanethiolamino acid derivatives that were stable for $\text{1}\text{h}$ h and the HPLC method produced a better separation than previously published methods.     

A simple quantitative method for the determination of 3-fluorotyrosine substitution in proteins

A rapid quantitative method is described for determining 3-fluorotyrosine incorporation into proteins. Derivatives of tyrosine and 3-fluorotyrosine with o-phthalaldehyde are well separated from one another by a reverse-phase high-performance liquid chromatography system used for routine analyses of o-phthalaldehyde-amino acid derivatives. Since both amino acids are well resolved from all other derivatized amino acids, the method is useful for amino acid analyses of proteins. Determination of the fluorotyrosine content of proteins by this method involves a single separation step, is reproducible, and requires no corrections for stability or yield. Further, the o-phthalaldehyde derivatives of 5-fluorotryptophan, 2-fluorophenylalanine, 3-fluorophenylalanine, and 4-fluorophenylalanine can also be resolved. The method may be generally applicable to fluorinated aromatic amino acid-labeled proteins that are studied structurally and dynamically by nuclear magnetic resonance.     

Oxidation of N-β-alanyldopamine by insect cuticles and its role in cuticular sclerotization

The oxidation products formed when various types of insect cuticle were incubated with N-β-alanyldopamine (NBAD) have been studied by means of reversed phase high performance liquid chromatography, and compared to the corresponding products obtained when N-acetyldopamine (NADA) was incubated with the cuticles. The results indicate that NBAD is oxidized to o-quinone and quinone methide derivatives. In contrast, NADA can be oxidized by some cuticles not only to o-quinone and quinone methide derivatives, but it can also be desaturated to α,β-dehydro-N-acetyldopamine, a probable intermediate in β-sclerotization. Some implications for in vivo sclerotization are discussed.     

Tunable emission,energy transfer and charge compensation in SrMoO4:Sm3+,Tb3+,Na+ phosphor

A series of SrMoO₄:Sm³⁺,Tb³⁺,Na⁺ phosphors was synthesized using a high‐temperature solid‐state reaction method in air. On excitation at 290 nm, SrMoO₄:Sm³⁺,Tb³⁺ phosphor emitted light that varied systematically from green to reddish‐orange on changing the Sm³⁺ and Tb³⁺ ion concentrations. The emission intensities of SrMoO₄:Sm³⁺ and SrMoO₄:Sm³⁺,Tb³⁺ phosphors were increased two to four times due to charge compensation when Na⁺ was added as a charge compensator. The luminescence mechanism and energy transfer could be explained using energy‐level diagrams of the MoO₄^2– group, Sm³⁺ and Tb³⁺ ions. SrMoO₄:Sm³⁺,Tb³⁺,Na⁺ could be used as reddish‐orange phosphor in white light‐emitting diodes (LEDs) based on an ~ 405 nm near‐UV LED chip. This research is helpful in adjusting and improving the luminescence properties of other phosphors. Copyright © 2015 John Wiley & Sons, Ltd.     

The external K+ concentration and mutations in the outer pore mouth affect the inhibition of kv1.5 current by Ni2+

By examining the consequences both of changes of [K⁺]_o and of point mutations in the outer pore mouth, our goal was to determine if the mechanism of the block of Kv1.5 ionic currents by external Ni²⁺ is similar to that for proton block. Ni²⁺ block is inhibited by increasing [K⁺]_o, by mutating a histidine residue in the pore turret (H463Q) or by mutating a residue near the pore mouth (R487V) that is the homolog of Shaker T449. Aside from a slight rightward shift of the Q-V curve, Ni²⁺ had no effect on gating currents. We propose that, as with H_o⁺, Ni²⁺ binding to H463 facilitates an outer pore inactivation process that is antagonized by K_o⁺ and that requires R487. However, whereas H_o⁺ substantially accelerates inactivation of residual currents, Ni²⁺ is much less potent, indicating incomplete overlap of the profiles of these two metal ions. Analyses with Co²⁺ and Mn²⁺, together with previous results, indicate that for the first-row transition metals the rank order for the inhibition of Kv1.5 in 0 mM K_o⁺ is Zn²⁺ (K_D ~ 0.07 mM) ≥ Ni²⁺ (K_D ~ 0.15 mM) > Co²⁺ (K_D ~ 1.4 mM) > Mn²⁺ (K_D > 10 mM).     

Relationship between Acid Tolerance, Cytoplasmic pH, and ATP and H+-ATPase Levels in Chemostat Cultures of Lactococcus lactis

The acid tolerance response (ATR) of chemostat cultures of Lactococcus lactis subsp. cremoris NCDO 712 was dependent on the dilution rate and on the extracellular pH (pH_o). A decrease in either the dilution rate or the pH_o led to a decrease in the cytoplasmic pH (pH_i) of the cells, and similar levels of acid tolerance were observed at any specific pH_i irrespective of whether the pH_i resulted from manipulation of the growth rate, manipulation of the pH_o, or both. Acid tolerance was also induced by sudden additions of acid to chemostat cultures growing at a pH_o of 7.0, and this induction was completely inhibited by chloramphenicol. The end products of glucose fermentation depended on the growth rate and the environmental pH_o of the cultures, but neither the spectrum of end products nor the total rate of acid production correlated with a specific pH_i. The rate of ATP formation was not correlated with pH_i, but a good correlation between the cellular level of H⁺-ATPase and pH_i was observed. Moreover, an inverse correlation between the cytoplasmic levels of ATP and pH_i was established. Each pH_i below 6.6 was characterized by unique levels of ATR, H⁺-ATPase, and ATP. High levels of H⁺-ATPase also coincided with high levels of acid tolerance of cells in batch cultures induced with sublethal levels of acid. We concluded that H⁺-ATPase is one of the ATR proteins induced by acid pH_i through growth at an acid pH_o or a slow growth rate.     

Purification and Characterization of an Arginine Aminopeptidase from Lactobacillus sakei

An arginine aminopeptidase (EC 3.4.11.6) that exclusively hydrolyzes basic amino acids from the amino (N) termini of peptide substrates has been purified from Lactobacillus sakei. The purification procedure consisted of ammonium sulfate fractionation and three chromatographic steps, which included hydrophobic interaction, gel filtration, and anion-exchange chromatography. This procedure resulted in a recovery rate of 4.2% and a 500-fold increase in specific activity. The aminopeptidase appeared to be a trimeric enzyme with a molecular mass of 180 kDa. The activity was optimal at pH 5.0 and 37°C. The enzyme was inhibited by sulfhydryl group reagents and several divalent cations (Cu²⁺, Hg²⁺, and Zn²⁺) but was activated by reducing agents, metal-chelating agents, and sodium chloride. The enzyme showed a preference for arginine at the N termini of aminoacyl derivatives and peptides. The K_m values for Arg-7-amido-4-methylcoumarin (AMC) and Lys-AMC were 15.9 and 26.0 μM, respectively. The nature of the amino acid residue at the C terminus of dipeptides has an effect on hydrolysis rates. The activity was maximal toward dipeptides with Arg, Lys, or Ala as the C-terminal residue. The properties of the purified enzyme, its potential function in the release of arginine, and its further metabolism are discussed because, as a whole, it could constitute a survival mechanism for L. sakei in the meat environment.     

Activation of Calcineurin by the Trivalent Metal Terbium

As a possible probe for metal activation of calcineurin, Tb³⁺ was tested for effects on calcineurin activity. Calcineurin was activated by Tb³⁺ with the following kinetic parameters estimated: k _cat = 0.78 ± 0.02 sec^–1, K _m(pNPP) = 32.6 ± 1.8 mM, and K _act(Tb³⁺) = 0.08 ± 0.03 mM. Terbium luminescence was demonstrated in the presence of the heterodimer of calcineurin and exploited to localize the binding of exogenous metal to the enzyme active site. Exogenous Mn²⁺ reduced luminescence, although the affinity of calcineurin for Tb³⁺ seemed to be greater. Putative active-site ligands, such as para-nitrophenol and a synthetic peptide from the autoinhibitory region, reduced the luminescence of terbium. Collectively, these data suggested that Tb³⁺ was binding directly at the active site of calcineurin, with the corollary that exogenous activating metal (Mn²⁺) binds at the active site of the enzyme. These data support the hypothesis that activating, exogenous divalent metal participates directly in catalysis.     

Expression of the Escherichia coli pntA and pntB Genes, Encoding Nicotinamide Nucleotide Transhydrogenase, in Saccharomyces cerevisiae and Its Effect on Product Formation during Anaerobic Glucose Fermentation

We studied the physiological effect of the interconversion between the NAD(H) and NADP(H) coenzyme systems in recombinant Saccharomyces cerevisiae expressing the membrane-bound transhydrogenase from Escherichia coli. Our objective was to determine if the membrane-bound transhydrogenase could work in reoxidation of NADH to NAD⁺ in S. cerevisiae and thereby reduce glycerol formation during anaerobic fermentation. Membranes isolated from the recombinant strains exhibited reduction of 3-acetylpyridine-NAD⁺ by NADPH and by NADH in the presence of NADP⁺, which demonstrated that an active enzyme was present. Unlike the situation in E. coli, however, most of the transhydrogenase activity was not present in the yeast plasma membrane; rather, the enzyme appeared to remain localized in the membrane of the endoplasmic reticulum. During anaerobic glucose fermentation we observed an increase in the formation of 2-oxoglutarate, glycerol, and acetic acid in a strain expressing a high level of transhydrogenase, which indicated that increased NADPH consumption and NADH production occurred. The intracellular concentrations of NADH, NAD⁺, NADPH, and NADP⁺ were measured in cells expressing transhydrogenase. The reduction of the NADPH pool indicated that the transhydrogenase transferred reducing equivalents from NADPH to NAD⁺.     

Tubby-tagged balancers for the Drosophila X and second chromosomes

We generated FM7a and CyO balancer chromosomes bearing a Tubby¹ (Tb¹) dominant transgene. Flies heterozygous for these FM7a and CyO derivatives exhibit a phenotype undistinguishable from that elicited by the Tb¹ mutation associated with the TM6B balancer. We tested two of these Tb-bearing balancers (FM7-TbA and CyO-TbA) for more than 30 generations and found that the Tb¹ transgene they carry is stable. Thus, these new Tb-tagged balancers are particularly useful for balancing lethal mutations and distinguish homozygous mutant larvae from their heterozygous siblings.     

Bioenergetic Mechanism for Nisin Resistance, Induced by the Acid Tolerance Response of Listeria monocytogenes

This study examined the bioenergetics of Listeria monocytogenes, induced to an acid tolerance response (ATR). Changes in bioenergetic parameters were consistent with the increased resistance of ATR-induced (ATR⁺) cells to the antimicrobial peptide nisin. These changes may also explain the increased resistance of L. monocytogenes to other lethal factors. ATR⁺ cells had lower transmembrane pH (ΔpH) and electric potential (Δψ) than the control (ATR⁻) cells. The decreased proton motive force (PMF) of ATR⁺ cells increased their resistance to nisin, the action of which is enhanced by energized membranes. Paradoxically, the intracellular ATP levels of the PMF-depleted ATR⁺ cells were ~7-fold higher than those in ATR⁻ cells. This suggested a role for the F_oF₁ ATPase enzyme complex, which converts the energy of ATP hydrolysis to PMF. Inhibition of the F_oF₁ ATPase enzyme complex by N′-N′-1,3-dicyclohexylcarbodiimide increased ATP levels in ATR⁻ but not in ATR⁺ cells, where ATPase activity was already low. Spectrometric analyses (surface-enhanced laser desorption ionization-time of flight mass spectrometry) suggested that in ATR⁺ listeriae, the downregulation of the proton-translocating c subunit of the F_oF₁ ATPase was responsible for the decreased ATPase activity, thereby sparing vital ATP. These data suggest that regulation of F_oF₁ ATPase plays an important role in the acid tolerance response of L. monocytogenes and in its induced resistance to nisin.     

Fumarate-Mediated Inhibition of Erythrose Reductase, a Key Enzyme for Erythritol Production by Torula corallina

Torula corallina, a strain presently being used for the industrial production of erythritol, has the highest erythritol yield ever reported for an erythritol-producing microorganism. The increased production of erythritol by Torula corallina with trace elements such as Cu²⁺ has been thoroughly reported, but the mechanism by which Cu²⁺ increases the production of erythritol has not been studied. This study demonstrated that supplemental Cu²⁺ enhanced the production of erythritol, while it significantly decreased the production of a major by-product that accumulates during erythritol fermentation, which was identified as fumarate by instrumental analyses. Erythrose reductase, a key enzyme that converts erythrose to erythritol in T. corallina, was purified to homogeneity by chromatographic methods, including ion-exchange and affinity chromatography. In vitro, purified erythrose reductase was significantly inhibited noncompetitively by increasing the fumarate concentration. In contrast, the enzyme activity remained almost constant regardless of Cu²⁺ concentration. This suggests that supplemental Cu²⁺ reduced the production of fumarate, a strong inhibitor of erythrose reductase, which led to less inhibition of erythrose reductase and a high yield of erythritol. This is the first report that suggests catabolite repression by a tricarboxylic acid cycle intermediate in T. corallina.     

Potential of Candida utilis to ferment Ecklonia cava by-product for enhanced anti-methicillin-resistant Staphylococcus aureus (MRSA) activity

In order to utilize the by-product of Ecklonia cava (the remaining biomass of E. cava), microbial fermentation which may result in the production of bioactive compounds using the by-product was applied in this study. The fermentation broth of E. cava by-product fermented with Candida utilis showed enhanced antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA) and food-borne pathogenic bacteria compared to that of control. To perform a more detailed investigation on the antibacterial activity, the broth was extracted with methanol and further fractionated with organic solvents. After 1 day of fermentation, the ethyl acetate (EtOAc) fraction exhibited the highest anti-MRSA activity with minimum inhibitory concentration ranging from 64 to 256 μg mL^?1, suggesting that the fermentation of E. cava by-product with C. utilis could enhance antibacterial activity against MRSA. In addition, high-performance liquid chromatography analysis revealed that dieckol, eckol, eckstolonol, and triphlorethol-A contents in the EtOAc-soluble extract increased significantly. The anti-MRSA activity of E. cava by-product most probably originated from phlorotannins, and the fermentation of C. utilis may have stimulated the breakdown of phlorotannins or have increased the efficiency in extracting phlorotannins.     

NADP+ Reduction with Reduced Ferredoxin and NADP+ Reduction with NADH Are Coupled via an Electron-Bifurcating Enzyme Complex in Clostridium kluyveri

It was recently found that the cytoplasmic butyryl-coenzyme A (butyryl-CoA) dehydrogenase-EtfAB complex from Clostridium kluyveri couples the exergonic reduction of crotonyl-CoA to butyryl-CoA with NADH and the endergonic reduction of ferredoxin with NADH via flavin-based electron bifurcation. We report here on a second cytoplasmic enzyme complex in C. kluyveri capable of energetic coupling via this novel mechanism. It was found that the purified iron-sulfur flavoprotein complex NfnAB couples the exergonic reduction of NADP⁺ with reduced ferredoxin (Fd_red) and the endergonic reduction of NADP⁺ with NADH in a reversible reaction: Fd_red²⁻ + NADH + 2 NADP⁺ + H⁺ = Fd_ox + NAD⁺ + 2 NADPH. The role of this energy-converting enzyme complex in the ethanol-acetate fermentation of C. kluyveri is discussed.Clostridium kluyveri is unique in fermenting ethanol and acetate to butyrate, caproate, and H₂ (reaction 1) and in deriving a large (30%) portion of its cell carbon from CO₂. Both the energy metabolism and the pathways of biosynthesis have therefore been the subject of many investigations (for relevant literature, see references 12 and 27). (1)During growth of C. kluyveri on ethanol and acetate, approximately five ethanol and four acetate molecules are converted to three butyrate molecules and one caproate molecule (reaction 1a), and one ethanol molecule is oxidized to one acetate⁻, one H⁺, and two H₂ (reaction 1b) molecules (23, 31). How exergonic reaction 1a is coupled with endergonic reaction 1b and with ATP synthesis from ADP and P_i (ΔG^o′ = +32 kJ/mol) has remained unclear for many years. (1a) (1b)We recently showed (12) that, in Clostridium kluyveri, the exergonic reduction of crotonyl-coenzyme A (crotonyl-CoA) (E_o′ = −10 mV) with NADH (E_o′ = −320 mV) involved in reaction 1a is coupled with the endergonic reduction of ferredoxin (Fd_ox) (E_o′ = −420 mV) with NADH (E_o′ = −320 mV) involved in reaction 1b via the recently proposed mechanism of flavin-based electron bifurcation (7). The coupling reaction is catalyzed by the cytoplasmic butyryl-CoA dehydrogenase-EtfAB complex (reaction 2) (12): (2)The reduced ferredoxin (Fd_red²⁻) is assumed to be used for rereduction of NAD⁺ via a membrane-associated, proton-translocating ferredoxin:NAD oxidoreductase (RnfABCDEG) (reaction 3), and the proton motive force thus generated is assumed to drive the phosphorylation of ADP via a membrane-associated F₁F₀ ATP synthetase (reaction 4): (3) (4)The novel coupling mechanism represented by reactions 2 and 3 allowed for the first time the possibility of formulating a metabolic scheme for the ethanol-acetate fermentation that could account for the observed fermentation products and growth yields and thus for the observed ATP gains (27). One issue, however, remained open, namely, why the formation of butyrate from ethanol and acetate in the fermentation involves both an NADP⁺- and an NAD⁺-specific β-hydroxybutyryl-CoA dehydrogenase (16), considering that, in the oxidative part of the fermentation (ethanol oxidation to acetyl-CoA), only NADH is generated (8, 9, 13).The presence of a reduced ferredoxin:NADP⁺ oxidoreductase was proposed based on results of enzymatic studies performed 40 years ago. Cell extracts of Clostridium kluyveri were found to catalyze the formation of H₂ from NADPH in a ferredoxin- and NAD⁺-dependent reaction (34). The results were interpreted to indicate that C. kluyveri contains a ferredoxin-dependent hydrogenase and an NADPH:ferredoxin oxidoreductase with transhydrogenase activity. H₂ formation from NADPH was strictly dependent on the presence of NAD⁺ and was inhibited by NADH, inhibition being competitive with the presence of NAD⁺, indicating that ferredoxin reduction with NADPH is under the allosteric control of the NAD⁺/NADH couple. The cell extracts also catalyzed the NADH-dependent reduction of NADP⁺ with reduced ferredoxin (21, 34). Purification of the enzyme catalyzing these reactions was not achieved, and no function in the energy metabolism of C. kluyveri was assigned.In this communication, we report on the properties of the recombinant enzyme that catalyzes the NAD⁺-dependent reduction of ferredoxin with NADPH and the NADH-dependent reduction of NADP⁺ with reduced ferredoxin and show that the cytoplasmic heterodimeric enzyme couples the exergonic reduction of NADP⁺ with reduced ferredoxin with the endergonic reduction of NADP⁺ with NADH in a fully reversible reaction. The transhydrogenation reaction is endergonic, because in vivo the NADH/NAD⁺ ratio is generally near 0.3 and the NADPH/NADP⁺ ratio is generally above 1 (2, 30). (5)NADP⁺ reduction is most probably the physiological function of the enzyme, which is why we chose the abbreviation NfnAB (for NADH-dependent reduced ferredoxin:NADP⁺ oxidoreductase).     

Outersphere and innersphere coordinated metal ions in an aminoacyl-tRNA synthetase ribozyme

Metal ions are essential cofactors for various ribozymes. Here we dissect the roles of metal ions in an aminoacyl-tRNA synthetase-like ribozyme (ARS ribozyme), which was evolved in vitro. This ribozyme can charge phenylalanine on tRNA in cis, where it is covalently attached to the 5′-end of tRNA (i.e. a form of precursor tRNA), as well as in trans, where it can act as a catalyst. The presence of magnesium ion is essential for this ribozyme to exhibit full catalytic activity. Metal-dependent kinetics, as well as structural mappings using Tb³⁺ in competition with Mg²⁺ or Co(NH₃)₆³⁺, identified two potential metal-binding sites which are embedded near the tRNA-binding site. The high affinity metal-binding site can be filled with either Mg²⁺ or Co(NH₃)₆³⁺ and thus the activity relies on a metal ion that is fully coordinated with water or ammonium ions. This site also overlaps with the amino acid-binding site, suggesting that the metal ion plays a role in constituting the catalytic core. The weak metal-binding site is occupied only by a metal ion(s) that can form innersphere contacts with ligands in the ribozyme and, hence, Mg²⁺ can enhance ribozyme activity, but Co(NH₃)₆³⁺ cannot. The experiments described in this work establish the roles of metal ions that have distinct coordination properties in the ARS ribozyme.     

Responses of Listeria monocytogenes to Acid Stress and Glucose Availability Revealed by a Novel Combination of Fluorescence Microscopy and Microelectrode Ion-Selective Techniques

Fluorescence ratio imaging microscopy and microelectrode ion flux estimation techniques were combined to study mechanisms of pH homeostasis in Listeria monocytogenes subjected to acid stress at different levels of glucose availability. This novel combination provided a unique opportunity to measure changes in H⁺ at either side of the bacterial membrane in real time and therefore to evaluate the rate of H⁺ flux across the bacterial plasma membrane and its contribution to bacterial pH homeostasis. Responses were assessed at external pHs (pH_o) between 3.0 and 6.0 for three levels of glucose (0, 1, and 10 mM) in the medium. Both the intracellular pH (pH_i) and net H⁺ fluxes were affected by the glucose concentration in the medium, with the highest absolute values corresponding to the highest glucose concentration. In the presence of glucose, the pH_i remained above 7.0 within a pH_o range of 4 to 6 and decreased below pH_o 4. Above pH_o 4, H⁺ extrusion increased correspondingly, with the maximum value at pH_o 5.5, and below pH_o 4, a net H⁺ influx was observed. Without glucose in the medium, the pH_i decreased, and a net H⁺ influx was observed below pH_o 5.5. A high correlation (R = 0.75 to 0.92) between the pH_i and net H⁺ flux changes is reported, indicating that the two processes are complementary. The results obtained support other reports indicating that membrane transport processes are the main contributors to the process of pH_i homeostasis in L. monocytogenes subjected to acid stress.     

Synthesis and fluorescence properties of 1,2,4‐triazolo[3,4‐b]‐1,3,4‐thiadiazol derivatives and their terbium complexes

Eight novel 1,2,4‐triazolo[3,4‐b]‐1,3,4‐thiadiazol derivatives have been designed and synthesized, and their corresponding Tb³⁺ complexes were also prepared successfully. The fluorescence properties and fluorescence quantum yields of the target complexes were investigated, the results showed that the ligands were an efficient sensitizer for Tb³⁺ luminescence, and the target complexes exhibited characteristic fluorescence emissions of Tb³⁺ ion. The fluorescence intensity of the complex substituted by chlorine was stronger than that of other complexes. The substituents' nature has a great effect upon the electrochemical properties of the target complexes. The results showed that the introduction of the electron‐withdrawing groups tended to decrease the oxidation potential and highest occupied molecular orbital energy levels of the target Tb³⁺ complexes; however, introduction of the electron‐donating groups can increase the corresponding complexes' oxidation potential and highest occupied molecular orbital energy levels. Copyright © 2014 John Wiley & Sons, Ltd.