Spectrofluorometric determination of common epoxides with sodium sulfide and o-phthalaldehyde and taurine reagents

Sano, Akira; Takitani, Shoji

doi:10.1021/ac00285a039

Cited by 9 publications

(3 citation statements)

References 30 publications

(16 reference statements)

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“…For the analysis of whole body insect extracts the protocol described by Bergot et al [14] was 20-fold more efficient than methanol/hexane (data not shown). Quantification by derivatizing epoxide groups with fluorescent tags was previously reported by Sano and Takitani [45] ; their method was based on opening the epoxide ring with hydrogen sulfide in the presence of sodium [46] and converting the epoxide into a fluorescent isoindole adduct by treating the derivative with o -phthalaldehyde (OPA) and taurine [45] . Duchateau et al [47] reported a HPLC method with a detection limit of 2 pmol based in the same principle.…”

Section: Discussionmentioning

confidence: 99%

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags

2012

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BackgroundThe juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. The lipophilic nature of JHs and their precursors, in conjunction with their low concentration in tissues and susceptibility to degradation had made their quantification difficult. A variety of methods exist for JH quantification but few can quantify on the femtomole range. Currently applied methods are expensive and time consuming. In the present study we sought to develop a novel method for accurate detection and quantification of JHs and their precursors.MethodsA sensitive and robust method was developed to quantify the precursor, farnesoic acid (FA) and juvenile hormone III (JH III) in biological samples. The assay is based on the derivatization of analytes with fluorescent tags, with subsequent analysis by reverse phase high performance liquid chromatography coupled to a fluorescent detector (HPLC-FD). The carboxyl group of FA was derivatized with 4-Acetamido-7-mercapto-2,1,3-benzoxadiazole (AABD-SH). Tagging the epoxide group of JH III required a two-step reaction: the opening of the epoxide ring with sodium sulfide and derivatization with the fluorescent tag 4-(N,N-Dimethylaminosulfonyl)-7-(N-chloroformylmethyl-N-methylamino)-2,1,3-benzoxadiazole (DBD-COCl).ConclusionsThe method developed in the present study showed high sensitivity, accuracy and reproducibility. Linear responses were obtained over the range of 10–20 to 1000 fmols. Recovery efficiencies were over 90% for JH III and 98% for FA with excellent reproducibility.SignificanceThe proposed method is applicable when sensitive detection and accurate quantification of limited amount of sample is needed. Examples include corpora allata, hemolymph and whole body of female adult Aedes aegypti and whole body Drosophila melanogaster. A variety of additional functional groups can be targeted to add fluorescent tags to the remaining JH III precursors.

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Section: Discussionmentioning

confidence: 99%

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags

2012

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“…By use of a spectrofluorometric procedure, oxiranes have been determined at the ca. 0.1 mmol/100 juL level (99).…”

Section: Oxiranesmentioning

confidence: 92%

Functional group analysis

Smith¹,

Patterson²

1986

Anal. Chem.

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“…section 6.3). 477 Another approach for the indirect detection of epoxides is based on their conversion to diols and the subsequent application of diolsensitive assays. Doderer et al described a colorimetric HTA that could be applied for all epoxides with at least one hydrogen substituent.…”

Section: Indirect Detectionmentioning

confidence: 99%

Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity

et al. 2023

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Many successful stories in enzyme engineering are based on the creation of randomized diversity in large mutant libraries, containing millions to billions of enzyme variants. Methods that enabled their evaluation with high throughput are dominated by spectroscopic techniques due to their high speed and sensitivity. A large proportion of studies relies on fluorogenic substrates that mimic the chemical properties of the target or coupled enzymatic assays with an optical read-out that assesses the desired catalytic efficiency indirectly. The most reliable hits, however, are achieved by screening for conversions of the starting material to the desired product. For this purpose, functional group assays offer a general approach to achieve a fast, optical read-out. They use the chemoselectivity, differences in electronic and steric properties of various functional groups, to reduce the number of false-positive results and the analytical noise stemming from enzymatic background activities. This review summarizes the developments and use of functional group probes for chemoselective derivatizations, with a clear focus on screening for enzymatic activity in protein engineering.

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Spectrofluorometric determination of common epoxides with sodium sulfide and o-phthalaldehyde and taurine reagents

Cited by 9 publications

References 30 publications

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags

A Quantitative Assay for the Juvenile Hormones and Their Precursors Using Fluorescent Tags

Functional group analysis

Enlightening the Path to Protein Engineering: Chemoselective Turn-On Probes for High-Throughput Screening of Enzymatic Activity

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