Glycosides having chromophores as substrates for sensitive enzyme analysis. II. Synthesis of phenolindophenyl-.BETA.-D-glucopyranosides having an electron-withdrawing substituent as substrates for .BETA.-glucosidase.

Tokutake, Shoichi; Kasai, Kouichi; Tomikura, T.; Yamaji, Nobuyuki; Kato, Motohiko

doi:10.1248/cpb.38.3466

Cited by 18 publications

(8 citation statements)

References 1 publication

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The favorable spectroscopic properties of resorufin have been previously harnessed for the production of sensitive in vitro substrates for esterases and glycosidases (Tokutake et al, 1990;Beisson et al, 2000;Coleman et al, 2007), including single-molecule detection (English et al, 2006;Gorris et al, 2007). Indeed, earlier work has highlighted the utility of resorufin b-glycosides for imaging the activity of GHs, including exo-b-galactosidase in yeast (Saccharomyces cerevisiae; Wittrup and Bailey, 1988) and exo-b-glucosidase in animal cells (Hays et al, 1998).…”

Section: Discussionmentioning

confidence: 99%

“…Thus, a library of resorufinyl b-glycosides, each designed to detect a specific glycosidase in planta on the basis of saccharide specificity, can be produced using the method presented herein. Indeed, the simple monosaccharide congeners Glcp-b-Res, Galp-b-Res, and GlcAp-b-Res are known (Tokutake et al, 1990;English et al, 2006), while during the course of this study, the synthesis of resorufinyl b-cellobioside ( Fig. 1 [2]) was presented together with its use for cellulase detection in vitro (Coleman et al, 2007).…”

Section: Discussionmentioning

confidence: 99%

See 1 more Smart Citation

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Ibatullin

Banasiak²,

Baumann³

et al. 2009

Plant Physiol.

View full text Add to dashboard Cite

There currently exists a diverse array of molecular probes for the in situ localization of polysaccharides, nucleic acids, and proteins in plant cells, including reporter enzyme strategies (e.g. protein-glucuronidase fusions). In contrast, however, there is a paucity of methods for the direct analysis of endogenous glycoside hydrolases and transglycosidases responsible for cell wall remodeling. To exemplify the potential of fluorogenic resorufin glycosides to address this issue, a resorufin b-glycoside of a xylogluco-oligosaccharide (XXXG-b-Res) was synthesized as a specific substrate for in planta analysis of XEH activity. The resorufin aglycone is particularly distinguished for high sensitivity in muro assays due to a low pK a (5.8) and large extinction coefficient (« 62,000 M 21 cm 21 ), long-wavelength fluorescence (excitation 571 nm/emission 585 nm), and high quantum yield (0.74) of the corresponding anion. In vitro analyses demonstrated that XXXG-b-Res is hydrolyzed by the archetypal plant XEH, nasturtium (Tropaeolum majus) NXG1, with classical Michaelis-Menten substrate saturation kinetics and a linear dependence on both enzyme concentration and incubation time. Further, XEH activity could be visualized in real time by observing the localized increase in fluorescence in germinating nasturtium seeds and Arabidopsis (Arabidopsis thaliana) inflorescent stems by confocal microscopy. Importantly, this new in situ XEH assay provides an essential complement to the in situ xyloglucan endotransglycosylase assay, thus allowing delineation of the disparate activities encoded by xyloglucan endotransglycosylase/ hydrolase genes directly in plant tissues. The observation that XXXG-b-Res is also hydrolyzed by diverse microbial XEHs indicates that this substrate, and resorufin glycosides in general, may find broad applicability for the analysis of wall restructuring by polysaccharide hydrolases during morphogenesis and plant-microbe interactions.

show abstract

Section: Discussionmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

Ibatullin

Banasiak²,

Baumann³

et al. 2009

Plant Physiol.

View full text Add to dashboard Cite

show abstract

“…Catalysis of the dealkylation of resorufin ethers by haemoglobin has been investigated (10). A ͱ-D-glucopyranoside of resorufin has been developed as a substrate for ͱ-glucosidase (9). Resorufin acetate has been used as a substrate for hydrolytic enzymes, both as a means of assaying very low enzyme concentrations (6) and in order to probe the mechanism of action of such enzymes (1-3).…”

Section: Discussionmentioning

confidence: 99%

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Kitson

1998

Bioorganic Chemistry

View full text Add to dashboard Cite

“…Enzymatic consumption and release of 2 have been demonstrated to function as useful indicator reactions for spectrophotometric and fluorometric analyses, since the dye exhibits strong absorption (l max 571 nm, e 4-7ϫ10 4 ) and emission (excitation maximum at 563 nm and emission maximum at 587 nm at pH 7.4) at wavelengthsϾ550 nm, where potential interference in analysis of colored or turbid serum components can be avoided. [25][26][27][28][29][30][31][32][33][34][35] These observations imply that the transformation of 1 to 2 as an indicator reaction for glucose analysis using only GOD takes full advantage of 2 as an analytically useful dye.…”

Section: Resultsmentioning

confidence: 99%

“…As mentioned above, several analytical methods with spectrophotometry or fluorometry have been designed based on enzymatic release of 2 from its derivatives such as 1. [25][26][27][28][29][30][31][32][33][34][35] However, to our knowledge, generation of 2 from 1 has been used as an indicator reaction only for studies of esterase activity of cytosolic aldehyde dehydrogenase and chymotrypsin. [20][21][22][23] Thus, this is the first report to show that transformation of 1 to 2 is induced by H 2 O 2 .…”

Section: Resultsmentioning

confidence: 99%

Hydrogen Peroxide-Induced Deacetylation of Acetyl Resorufin as a Novel Indicator Reaction for Fluorometric Detection of Glucose Using Only Glucose Oxidase.

Maeda

Matsu-Ura

Nishida

et al. 2001

CHEMICAL & PHARMACEUTICAL BULLETIN

View full text Add to dashboard Cite

Spectrophotometric or fluorometric methods with glucose oxidase (GOD)-peroxidase (POD)-chromogen(s) systems are well known as useful tools for determination of glucose. [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15] Indicator reactions in these methods are based on oxidative formation of a colored or fluorescent substance from a chromogen(s) with H 2 O 2 , generated through GODcatalyzed oxidation of glucose, in the presence of POD. These color reactions are well-characterized chemical processes. However, the POD-dependent indicator reactions are known to be inevitably disturbed by electron donors present in biological samples such as ascorbic acid, uric acid, and bilirubin.3) Major processes of interference by these compounds are as follows: 1) reduction of oxidatively formed colored or fluorescent substance to its original chromogen(s); and 2) competition with a chromogen in reduction of H 2 O 2 . Several methodologies have been developed for elimination of such interference in glucose determination with GOD-POD-chromogen(s) systems.3) However, the most straightforward approach to glucose determination free from such interference is design of a novel indicator reaction without recourse to redox reactions coupled with H 2 O 2 and POD. To our knowledge, only a few methods with non-redox color reactions for glucose determination using only GOD have been reported. These methods utilize transformation of a vanadium(V), 16) titanium(IV) 17) or dinuclear iron(III) 18) complex to its adduct with H 2 O 2 accompanied by a bathochromic shift as a spectrophotometric indicator reaction. As expected, it was demonstrated that glucose determination with vanadium(V) and titanium(IV) complexes was not affected by various substances usually present in serum or added to test solution. 16,17) However, spectrophotometric or fluorometric determination of glucose with high accuracy should use formation of a colored or fluorescent substance from a chromogen rather than a color change of a dye as an indicator reaction.Recently, resorufin (2) was shown to be able to reoxidize a reduced form of GOD at 37°C, being transformed to a colorless dihydro derivative, although the reductive bleaching of the dye is of no use for GOD-based determination of glucose as an indicator reaction. 19) This finding prompted us to examine how resorufin derivatives such as acetyl resorufin (1) would behave in GOD-catalyzed oxidation of glucose, and it was found that 1 behaves in a manner different from 2 in the enzymatic reaction. Here, we report that deacetylation of non-fluorescent 1 to fluorescent 2 is induced by H 2 O 2 generated in GOD-catalyzed oxidation of glucose, and the transformation is promising as an indicator reaction for fluorometric determination of glucose without significant effects of ascorbic acid, uric acid, or bilirubin. ExperimentalMaterials GOD from Aspergillus niger (EC 1.1.3.4) and glucose were used as supplied from Wako Pure Chemical Industries, Ltd. Acetyl resorufin (1) [20][21][22][23] was prepared by reaction of resorufin sod...

show abstract

Glycosides having chromophores as substrates for sensitive enzyme analysis. II. Synthesis of phenolindophenyl-.BETA.-D-glucopyranosides having an electron-withdrawing substituent as substrates for .BETA.-glucosidase.

Cited by 18 publications

References 1 publication

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

A Real-Time Fluorogenic Assay for the Visualization of Glycoside Hydrolase Activity in Planta

The Oxidative Addition Reaction between Compounds of Resorufin (7-Hydroxy-3H-phenoxazin-3-one) and 2-Mercaptoethanol

Hydrogen Peroxide-Induced Deacetylation of Acetyl Resorufin as a Novel Indicator Reaction for Fluorometric Detection of Glucose Using Only Glucose Oxidase.

Contact Info

Product

Resources

About