Competitive inhibition of mushroom tyrosinase by 4-substituted benzaldehydes

A kinetic study of the inhibition of mushroom tyrosinase by 4-substituted benzaldehydes showed that these compounds behave as classical competitive inhibitors, inhibiting the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) by mushroom tyrosinase (o-diphenolase activity). The kinetic parameter (K(I)) characterizing this inhibition was evaluated for all of the seven compounds assayed. Cuminaldehyde showed the most potent inhibitory activity (K(I) = 9 microM). It also inhibited the oxidation of L-tyrosine by mushroom tyrosinase (o-monophenolase activity) in a competitive manner. The corresponding kinetic parameter for this inhibition was evaluated (K(I) = 0.12 mM).     

Kinetic study of the oxidation of gamma-L-glutaminyl-4-hydroxybenzene catalyzed by mushroom (Agaricus bisporus) tyrosinase.

Despite the importance of the substrate gamma-L-glutaminyl-4-hydroxybenzene (GHB) in the melanin biosynthesis pathway in mushrooms Agaricus bisporus, the kinetics of its oxidation catalyzed by tyrosinase has never been properly characterized. For this purpose GHB and its corresponding o-diphenol (GDHB) were isolated and purified from A. bisporus mushrooms. The kinetic constants that characterize the action of tyrosinase on GHB and GDHB are = 2.10 +/- 0.10 microM/min, = 0.30 +/- 0.03 mM, = 210.0 +/- 7.3 microM/min, and = 7.80 +/- 0.41 mM. The oxygen kinetic constants for tyrosinase in the presence of these compounds are = 3. 20 +/- 0.21 microM/min, = 1.50 +/- 0.12 microM, = 200.2 +/- 8.1 microM/min, and = 100.2 +/- 8.2 microM. These values were compared to those obtained for the pair L-tyrosine/L-DOPA. The kinetic and structural reaction mechanisms of tyrosinase were corroborated for these physiological phenolic compounds.     

Enantiomeric separation and toxicity of an organophosporus insecticide, pyraclofos

Despite the fact that the biological processes of chiral pesticides are enantioselective, knowledge of the toxicities of pyraclofos due to enantiospecificity is scarce. In this study, the optical isomers of pyraclofos were separated and their toxicities to butyrylcholinesterase (BChE) and Daphnia magna were assessed. Baseline resolution of the enantiomers was obtained on both Chiralcel OD and Chiralpak AD columns. The effect of the mobile phase composition and column temperature were then discussed. The resolved enantiomers were characterized by their optical rotation and circular dichroism signs. The anti-BChE tests demonstrated that (-)-pyraclofos was about 15 times more potent than its (+)-form. However, acute aquatic assays suggested that (+)-pyraclofos was about 6 times more toxic than its antipode. Moreover, the joint toxicity of pyraclofos enantiomers to D. magna was found to be an additive effect. These results demonstrated that the overall toxicity of pyraclofos should be assessed using the individual enantiomers.     

Single and joint acute toxicity of isocarbophos enantiomers to Daphnia magna

Although enantioselectivity in the toxicity of chiral pesticides has received considerable attention over recent years, how coexisting enantiomers interact with each other during their toxic action remains unknown. In this study, we attempted to resolve the enantiomers of a chiral organophosphate insecticide, isocarbophos, and investigated the acute toxicity of individual enantiomers and various enantiomer mixtures. Baseline enantiomeric separation of isocarbophos was achieved on a Chiralcel OD column with the mobile phase of n-hexane/isopropanol (90/10, v/v) at a flow rate of 0.8 mL/min. The resolved enantiomers were differentiated by their responses on a circular dichroism detector. The median lethal concentrations (LC 50) of racemate, (+)-enantiomer, and (-)-enantiomer of isocarbophos toward Daphnia magna were 13.9, 7.08, and 353 microg/L, respectively, after 48 h of static exposure, displaying a 50-fold difference between the enantiomers. Toxic unit (TU) analysis was employed to evaluate the joint toxicity of isocarbophos enantiomer mixtures. The calculated TU mix for the acute toxicity (48 h test) of various binary mixtures ranged from 0.83 to 1.04, suggesting a mode of additive effect. Further evaluation of available literature data for chiral organophosphorus insecticides showed that the joint toxicity of enantiomers may be additive, synergistic, and antagonistic. Therefore, when significant enantioselectivity exists for a chiral pesticide, it is important to also evaluate the interaction of enantiomers in the joint toxicity effect when enantiomers are present in a mixture.     

Characterization of the activity of tyrosinase on betaxanthins derived from (R)-amino acids

The activity of tyrosinase (EC 1.14.18.1) on selected (R)-betaxanthins is characterized in depth, demonstrating that the activity of the enzyme is not restricted to betaxanthins derived from (S)-amino acids. Conversion of (R)-tyrosine-betaxanthin [(R)-portulacaxanthin II] to the pigment (R)-dopaxanthin and its further oxidation to a series of products is described. Compound identity was studied by high performance liquid chromatography and electrospray ionization-mass spectrometry. The reaction rate on the (R)-isomer of dopaxanthin is 1.9-fold lower than that obtained for the (S)-isomer in previous studies. Tyrosinase showed stereospecificity in its affinity toward betaxanthins. The characterization of the activity of tyrosinase on (R)-betaxanthins reinforces the role of the enzyme in the biosynthetic scheme of betalains.     

Action of tyrosinase on ortho-substituted phenols: possible influence on browning and melanogenesis

The action of tyrosinase on ortho-substituted monophenols (thymol, carvacrol, guaiacol, butylated hydroxyanisole, eugenol, and isoeugenol) was studied. These monophenols inhibit melanogenesis because they act as alternative substrates to L-tyrosine and L-Dopa in the monophenolase and diphenolase activities, respectively, despite the steric hindrance on the part of the substituent in ortho position with respect to the hydroxyl group. We kinetically characterize the action of tyrosinase on these substrates and assess its possible effect on browning and melanognesis. In general, these compounds are poor substrates of the enzyme, with high Michaelis constant values, K(m), and low catalytic constant values, k(cat), so that the catalytic efficiency k(cat)/K(m) is low: thymol, 161 ± 4 M(-1) s(-1); carvacrol, 95 ± 7 M(-1) s(-1); guaiacol, 1160 ± 101 M(-1) s(-1).     

Gender-related differences in stereoselective degradation of flutriafol in rabbits

The stereoselective pharmacokinetics of flutriafol were investigated in male and female adult Japanese white rabbits. Following intravenous administration of rac-flutriafol to rabbits at 5 mg/kg (bd wt), the concentrations of the enantiomers in plasma were determined by a HLPC-UV method using a CDMPC-CSP chiral column. R-Flutriafol exhibited a shorter distribution half-life but a longer elimination half-life than the S-isomer. In female rabbits, the distribution half-lives of R- and S-flutriafol were found to be 0.09 and 0.18 h, respectively, significantly shorter than those in male rabbits, but the volume of distribution and elimination half-life for flutriafol enantiomers in both sexes of rabbit showed no significant differences. Female rabbits had a higher clearance for both flutriafol enantiomers. The protein binding value was high for both isomers, with enantioselectivity, but no gender difference. It was an important factor in modulating the disposition of flutriafol. Flutriafol concentrations in kidney, liver, fat, and lung were higher than in other tissues at 10 h after administration, and the concentrations of R-flutriafol were higher in all tissues than those of its antipode. However, gender difference in flutriafol residues in tissues was not observed. It is concluded that the stereoselectivity of flutriafol on distribution and elimination in rabbits mainly depends upon gender.     

手性异丙甲草胺在土壤中的选择性降解

Separation of chiral enantiomers and the dissipation of rac-metolachlor and S-metolachlor in soil were evaluated using achiral high-performance liquid chromatography （HPLC） and chiral gas chromatography （GC） methods. Under the experimental conditions the possible metabolite was considered to be N-（2-ethyl-6-methyl-phenyl）-2-hydroxy-acetamide. Because of the presence of two chiral elements （asymmetrically substituted carbon and chiral axis）, the baseline separation of metolachlor enantiomers was not achieved. S-metolachlor degraded faster in soil than rac-metolachlor. After a 42-day incubation, 73.4% of rac-metolachlor and 90.0% of S-metolachlor were degraded. However, due to the absence of biological processes the degradation process in sterilized soil showed no enantioselectivity. The results indicated that enantioselective degradations could greatly affect the environmental fate of metolachlor and should be considered when the environmental behavior of these compounds was assessed.     

手性除草剂2甲4氯丙酸对映体在拟南芥中吸收转运及亚细胞分布的差异研究

为探明不同手性构型的2甲4氯丙酸(mecoprop,MCPP)在双子叶植物中的吸收、代谢等差异,以手性¹⁴C-MCPP对映体为示踪剂,野生哥伦比亚型拟南芥为受试植物,研究实验室条件下不同手性构型的MCPP在拟南芥中的吸收转运与亚细胞分布特征。结果表明,拟南芥对¹⁴C-MCPP 2种对映体的吸收效率分别为90.45%(¹⁴C-R-MCPP)和85.75%(¹⁴C-S-MCPP),二者差异显著。施药后,2种构型的¹⁴C-MCPP均易向下运转,其中¹⁴C-R-MCPP在拟南芥植株中的转运过程更持久,转运到植株其他部位的比率相对更高。2种构型¹⁴C-MCPP在标记下位达到平衡时,¹⁴C-R-MCPP的分布比率为¹⁴C-S-MCPP的2.5倍。亚细胞分布方面,在拟南芥标记叶位细胞中各组分的分布比率均为胞浆>细胞壁>细胞器,在施药后144 h,¹⁴C-S-MCPP在细胞壁的分布比率较¹⁴C-R-MCPP高,分别为39.60%和28.74%,表明拟南芥叶组织细胞壁对¹⁴C-S-MCPP具有更强的固定能力;而在细胞器中,¹⁴C-R-MCPP的分布比率(10.55%)较¹⁴C-S-MCPP(4.11%)高,表明¹⁴C-R-MCPP相较于¹⁴C-S-MCPP更易富集于叶组织细胞的细胞器中,在同施药浓度下与靶标蛋白的接触频率更高。综上可知,手性激素型除草剂MCPP在双子叶植物模式植物拟南芥中的运转和分布存在显著的对映体选择性差异,映证了R-MCPP从吸收、转运、亚细胞分布的层面而言相较于其对映体S-MCPP具有更强的除草活性。     

Solid-phase synthesis of mimosine tetrapeptides and their inhibitory activities on neuraminidase and tyrosinase

Neuraminidase is a rational target for influenza inhibition, and the search for neuraminidase inhibitors has been intensified. Mimosine, a nonprotein amino acid, was for the first time identified as a neuraminidase inhibitor with an IC(50) of 9.8 ± 0.2 μM. It was found that mimosine had slow, time-dependent competitive inhibition against the neuraminidase. Furthermore, a small library of mimosine tetrapeptides (M-A(1)-A(2)-A(3)) was synthesized by solid-phase synthesis and was assayed to evaluate their neuraminidase and tyrosinase inhibitory properties. Most of the tetrapeptides showed better activities than mimosine. Mimosine-FFY was the best compound, and it exhibited 50% neuraminidase inhibition at a low micromolar range of 1.8 ± 0.2 μM, whereas for tyrosinase inhibition, it had an IC(50) of 18.3 ± 0.5 μM. The kinetic studies showed that all of the synthesized peptides inhibited neuraminidase noncompetitively with K(i) values ranging from 1.9 -to 7.2 μM. These results suggest that mimosine could be used as a source of bioactive compounds and may have possibilities in the design of drugs as neuraminidase and tyrosinase inhibitors.     

One- and two-dimensional direct chiral liquid chromatographic determination of mixtures of diclofop-Acid and diclofop-methyl herbicides

Simple one- and two-dimensional high-performance liquid chromatography (HPLC) methods for the simultaneous enantiomeric determination of alkyloxyphenoxypropionic acid herbicides is presented. Compounds studied were ( R, S)-2-[4-(2,4-dichlorophenoxy)phenoxy]propionic acid (diclofop-acid) and ( R, S)-2-[4-(2,4-dichlorophenoxy)]methyl propionate (diclofop-methyl). Mobile phases necessary to separate their enantiomers on an alpha1-acid glycoprotein chiral stationary phase are different; therefore, the simultaneous separation by an isocratic mode is not possible. The chiral separation method proposed involves a one-step gradient allowing for the simultaneous determination of both racemic enantiomers. Detection limits of the method were 0.03 mg/L for both diclofop-acid enantiomers and 0.14 and 0.15 mg/L for diclofop-methyl enantiomers, respectively. The two-dimensional method involves the use of two chromatographs in one achiral-chiral coupling. The LC-LC method is more suitable for complex samples because it involves an online cleanup effect. Detection limits were 1.25 and 1.87 mg/L for diclofop-acid and 2.70 and 3.02 mg/L for diclofop-methyl enantiomers, respectively. Accuracy, repeatability, and reproducibility have been studied in standard samples and a technical product.     

Enantiomeric resolution and growth-retardant activity in rice seedlings of uniconazole

The increasing application of chiral pesticides has enhanced interest in their enantioselectivity. However, little relevant information is currently available for enantioselective activity of chiral plant growth regulators. In an attempt to screen active enantiomers of uniconazole, this work investigated enantiomeric separation and the enantioselective effect of uniconazole on the growth of rice seedlings and cyanobacteria. Baseline resolution of uniconazole enantiomers was achieved on a Chiralpak AD column by chiral high-performance liquid chromatography (HPLC). The relationship among circular dichroism (CD), optical rotation (OR), and absolute configuration was successfully established by coupling of CD and OR detection. The t test at the 95% level of confidence indicated significant differences between the enantiomers in their retardant activity toward growth of rice seedlings and stimulation effect on growth of cyanobacteria, the natural biofertilizers in rice paddy fields. The S-(+)-enantiomer was more active than the R-(-)-enantiomer in retarding growth of rice seedlings and stimulating growth of Microcystis aeruginosa . This special enantiomeric selectivity was further elucidated by probing the binding mode of enantiomers to gibberellin (GA) 20-oxidase by molecular docking. The S-(+)-enantiomer was found to bind tightly with GA 20-oxidase. The results suggested that the S-(+)-enantiomer instead of a racemate of uniconazole should be used to improve rice seedling quality.     

Enantiomeric discrimination and quantification of the chiral organophosphorus pesticide fenamiphos in aqueous samples by a novel and selective ³¹P nuclear magnetic resonance spectroscopic method using cyclodextrins as chiral selector

A rapid, selective, and accurate quantitative 31P nuclear magnetic resonance (31P NMR) spectroscopy method was used for the chiral recognition of the racemic organophosphorus pesticide fenamiphos using chiral solvating agents (CSAs). Six neutral cyclodextrins (CDs) (α-CD, β-CD, methyl-β-CD, hydroxyethyl-β-CD, hydroxypropyl-β-CD, and hydroxypropyl-γ-CD) and two anionic CDs (carboxymethyl-β-CD and carboxyethyl-β-CD) were selected for these experiments. The shift displacement values (Δδ), after addition of each of the eight CDs in the highest possible molar ratio to a guest, were recorded. The results showed that β-CD and hydroxypropyl-β-CD were the best chiral solvating agents for the enantiomeric discrimination of fenamiphos. Two-dimension rotating frame nuclear Overhauser spectroscopy (ROESY) was used to investigate the structure of the β-CD-fenamiphos inclusion complex in aqueous solution. To determine the fenamiphos enantiomers, a calibration curve was drawn for two enantiomers over the range of 0.05-0.25 mg mL?1. The limits of detection (S/N = 3) were obtained as 0.0068 and 0.0060 mg mL?1 for fenamiphos enantiomers. The recovery studies were performed on aqueous real samples ranging from 94 to 107% with coefficients of variation of ≤ 9%.     

Enantioselective separation and phytotoxicity on rice seedlings of paclobutrazol

The environmental significance of enantioselectivity in chiral insecticides and herbicides has been widely studied. However, little information is currently available on the enantioselective behavior of chiral plant growth regulators. In this study, paclobutrazol enantiomers were resolved and prepared by chiral high-performance liquid chromatography with a Sino-chiral OJ column. The relationship among absolute configuration, optical activity and circular dichroism of paclobutrazol enantiomers was established. The enantioselective behavior of paclobutrazol, including enantioselective effect of paclobutrazol on the growth of rice seedlings and cyanobacteria and enantioselective loss of paclobutrazol in rice seedling growth media, in rice culture system was studied. The (2S,3S)-(-)-enantiomer was almost 3.1 times more active than the (2R,3R)-(+)-enantiomer toward shoot growth as measured by 7 day EC50 values. Enantioselectivity could not be determined with respect to root growth of rice seedlings because a typical dosage response was not observed in the range of the concentrations studied. The dissipation of paclobutrazol in rice growth medium is not enantioselective. Enantiomers and diastereoisomer of paclobutrazol all facilitated the growth of cyanobacteria, which increase the effectiveness of rice biofertilizers. The (2S,3S)-(-)-enantiomer showed stronger stimulatory activity on Microcystis aeruginosa cyanobacteria than the (2R,3R)-(+)-enantiomer, whereas the latter was a more potent stimulator of Anabaena sp. growth. These observations indicate that application of the (2S,3S)-(-)-enantiomer of paclobutrazol and Microcystis aeruginosa in rice cultivation is a good strategy for improving rice seedling performance.     

Flavonols from saffron flower: tyrosinase inhibitory activity and inhibition mechanism.

A common flavonol, kaempferol, isolated from the fresh flower petals of Crocus sativus L. (Iridaceae) was found to inhibit the oxidation of L-3,4-dihydroxyphenylalanine (L-DOPA) catalyzed by mushroom tyrosinase with an ID(50) of 67 microgram/mL (0.23 mM). Interestingly, its 3-O-glycoside derivatives did not inhibit this oxidation. The inhibition kinetics analyzed by a Lineweaver-Burk plot found kaempferol to be a competitive inhibitor, and this inhibitory activity presumably comes from its ability to chelate copper in the enzyme. This copper chelation mechanism can be applicable for all of the flavonols as long as their 3-hydroxyl group is free. However, quercetin, kaempferol, and galangin each affect the oxidation of L-tyrosine in somewhat different ways.     

Isolation and identification of the metolachlor stereoisomers using high-performance liquid chromatography, polarimetric measurements, and enantioselective gas chromatography

Because of the presence of two chiral elements (an asymmetrically substituted carbon and a chiral axis), the herbicide metolachlor consists of four stereoisomers stable at ambient temperature with aSS-, aRS-, aSR-, and aRR-configurations (aSS, the isomer with aS,1'S-configuration, etc.). Metolachlor, initially introduced into the market as the racemic product containing all four stereoisomers, is currently being replaced worldwide by S-metolachlor, the product enantiomerically enriched with the herbicidally active 1'S-isomers (aSS, aRS). The isomer-specific analysis of metolachlor requires not only enantioselective ("chiral") analytical techniques but also suitable reference compounds. In this study, two of the four metolachlor isomers were isolated from rac-metolachlor in enantio- (ee > 98%) and diastereomerically pure forms by a combination of achiral and chiral high-performance liquid chromatography (HPLC). The two isomers were identified as the aSS- and the aRR-isomers by polarimetric measurements, in reference to previous data. The two isomers were then thermally equilibrated to 1:1 mixtures of the aSS/aRS and aRR/aSR diastereomers, respectively, so that analytical data of all four metolachlor isomers became available; they were then used to identify these isomers in technical products by chiral high-resolution gas chromatography (HRGC). The kinetics of the thermally induced interconversion of the atropisomers was studied and the consequences, such as for GC analysis, are discussed. A comparison of on-column and split/splitless injection indicated that the latter technique results in significant isomerization prior to separation and, therefore, cannot be used for accurate isomer analysis.     

Enantioselective degradation and ecotoxicity of the chiral herbicide diclofop in three freshwater alga cultures

Aryloxyphenoxypropanoates are a class of chiral herbicides. They have a pair of enantiomers, only the R(+) form of which is herbicidally active. Diclofop, the model compound of these herbicides, is commercialized as the racemate of the ester form, diclofop-methyl, consisting of a 1:1 mixture of the enantiomers. This study evaluated the enantioselectivity in aquatic toxicity and biodegradation of diclofop and diclofop-methyl. The herbicidally inactive S(-) enantiomers of both diclofop-methyl and diclofop were similar to or higher than the corresponding R(+) forms in toxicity to algae, depending on specific species. Although no enantiomeric conversion occurred for diclofop-methyl and diclofop, the difference in the enantioselective degradation of these herbicides observed in algae cultures suggested that their application forms were an important factor determining their enantioselective environmental behavior. The cell permeability and heat treatment of algae revealed that the enantioselective degradation of diclofop in algae cultures was governed primarily by the facilitated uptake by algae, whereas the enantioselective toxicity was primarily governed by the passive uptake. These results suggested that the acute toxicity test such as the 96 h EC 50 was insufficient to assess the ecological risk of chiral pesticides because of the differential degradation as well as possibly differential action sites of enantiomers. From this study, it was concluded that the enantioselective degradation and toxicity of chiral herbicides may result in their ecotoxicological effects being difficult to predict and that specific attention should thus be paid to currently used racemic pesticides as less active or inactive enantiomers may pose higher ecological risks.     

Enantiomeric resolution of chiral pesticides by high-performance liquid chromatography

Successful enantiomeric separation of 10 chiral pesticides by high-performance liquid chromatography (HPLC) using cellulose-tris(3,5-dimethylphenylcarbamate) (CDMPC) chiral stationary phase (CSP) was performed. The mobile phase was n-hexane modified by ethanol, propanol, 2-propanol (IPA), butanol, or isobutanol. The effects of mobile phase composition and column temperature on the separation were investigated. Baseline separation was obtained with ethofumesate, fluroxypyr-meptyl, malathion, benalaxyl, diclofop-methyl, methamidophos, vinclozolin, and lactofen, whereas near baseline separation was obtained with profenofos and acetochlor. Butanol was the best modifier for benalaxyl; isobutanol was the best modifier for lactofen, malathion, diclofop-methyl, and ethofumesate; and IPA was the best modifier for the other five. Better separations were not always at low temperature. The elution orders of the eluting enantiomers were determined by a circular dichroism (CD) detector. The quantitative analysis methods for the enantiomers of ethofumesate, benalaxyl, and diclofop-methyl were established. Validation parameters include linearity, precision, and limit of detection (LOD). The enantiomeric residual analysis procedures in soil and water samples were also developed using acetone extraction and C(18) solid phase extraction. The methods were reliable for residual analysis of the enantiomers.