Support of this work by Glycom A/S, Copenhagen, Denmark, is gratefully acknowledged.

General remarks:
All reactions were carried out using a fume hood and appropriate personal protective equipment. All reagents and solvents (p.a.; water content for dry solvents: MeCN &#60;50 ppm; Et2O &#60;0.01%; CH2Cl2 &#60;0.003%; THF &#60;0.005%) were purchased from commercial sources and used as received. TLC was performed on Merck silica gel 60 F254 plates. Compounds were detected by UV and/or by treatment with EtOH/H2SO4 (9:1) and subsequent heating. Column chromatography was performed with Merck/Fluka silica gel 60 (230-400 mesh). Filtrations were performed using B&#252;chner funnel and aspirator pump. Solvents were removed employing a rotary evaporator (40 &#176;C, ~200 rmp). Products were dried in high vacuum (rotary vane pump). Reactions requiring dry solvents were carried out under argon atmosphere.
In the following the synthesis of X-Gal (16) employing 5-bromo-4-chloro-indoxylic acid allyl ester (12) is described. For further compounds with varied substitution patterns as well as methyl esters prepared according to this synthetic sequence see ref.17-19.
1. Acceptor: Synthesis of 5-Bromo-4-Chloro-Indoxylic Acid Allyl Ester18 (12)
<ol>
	<li>N-Acetylation: Synthesis of N-(4-bromo-3-chloro-2-methylphenyl)-acetamide (6)
	<ol>
		<li>Dissolve 10.0 g (45.7 mmol) of 4-bromo-3-chloro-2-methylaniline in 30 ml dichloromethane. 
		Caution! Dichloromethane is harmful and cool the solution in an ice-bath.</li>
		<li>Add 6.50 ml (68.7 mmol) acetic anhydride dropwise and allow the mixture to warm to room temperature. The mixture can be stirred for 8 hr or overnight. Caution! Acetic anhydride is corrosive and flammable.</li>
		<li>Remove the solvent and recrystallize from 9:1 ethyl acetate/methanol (yield: 97% (11.6 g, 44.2 mmol), colorless solid). 
		Caution! Ethyl acetate is irritant and flammable. Caution! Methanol is toxic and flammable.</li>
	</ol>
	</li>
	<li>Oxidation: Synthesis of N-acetyl-5-bromo-6-chloro-anthranilic acid (7)
	<ol>
		<li>Reflux a mixture of 2.30 g (8.76 mmol) of 6 (product obtained from step 1.1) and 2.10 g (8.52 mmol) MgSO4&#8226;7H2O in 40 ml water in a 3-neck round bottom flask equipped with cooler and dropping funnel.</li>
		<li>Add dropwise 50 ml saturated KMnO4 solution in water to the reaction mixture over a period of 3 hr. Then heat for 2 additional hr under reflux, ca. 135 &#176;C oil bath. Caution! Potassium permanganate is harmful and oxidising.</li>
		<li>Cool to room temperature and filter the pyrolusite (MnO2), which is formed during the reaction. 
		Caution! Pyrolusite is harmful.</li>
		<li>Add hydrochloric acid (37%) to the filtrate until pH 1, then filter the product and dry in vacuum, (yield: 70% (1.80 g, 6.15 mmol), colorless solid). 
		Caution! Hydrochloric acid is corrosive.</li>
	</ol>
	</li>
	<li>Deacetylation: Synthesis of 5-bromo-6-chloro-anthranilic acid (8)
	<ol>
		<li>Dissolve 3.90 g (13.3 mmol) of 7 (product obtained from step 1.2) in 70 ml of 1 N aqueous sodium hydroxide solution and heat at ca. 100 &#176;C (oil bath) for 5 hr. 
		Caution! Sodium hydroxide is corrosive.</li>
		<li>Cool to room temperature and add hydrochloric acid (37%) until pH 1, then filter the product with a B&#252;chner funnel and dry in vacuum, (yield: 88% (2.93 g, 11.7 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Anhydride formation: Synthesis of 5-bromo-6-chloro-isatoic anhydride (9)
	<ol>
		<li>Suspend 8.17 g (32.6 mmol) of 8 (product obtained from step 1.3) in 33 ml dry acetonitrile. 
		Caution! Acetonitrile is harmful and flammable.</li>
		<li>Stir at room temperature and add 5.3 ml and a solution of 3.22 g (10.8 mmol) triphosgene in 18.5 ml dry dichloromethane dropwise simultaneously over a period of 30 min. Then heated for 3 hr at 50 &#176;C (oil bath). Caution! Pyridine is harmful and flammable. 
		Caution! Triphosgene is toxic and can release phosgene.</li>
		<li>Remove about 75% of the solvent and quench the reaction by adding 100 ml distilled water and filter your product. Wash with a small amount of cold dichloromethane.</li>
		<li>Dry the product in vacuum, (yield: 88% (7.53 g, 27.2 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>N-Alkylation: Synthesis of 5-bromo-6-chloro-N-[(methoxycarbonyl)allyl]-isatoic anhydride (10)
	<ol>
		<li>Dissolve 25 g (90 mmol) of 9 (product obtained from step 1.4) in 250 ml anhydrous dimethylformamide. and cool with an ice-bath. 
		Caution! Dimethylformamide is harmful and flammable</li>
		<li>Add 1.15 equivalents of sodium hydride (60% in paraffin) portion wise. 
		Caution! Sodium hydride is corrosive and flammable; heavy reaction with water/ice.</li>
		<li>After 30 min of stirring at 0 &#176;C allow warming to room temperature and add 1.2 equivalents allyl bromoacetate dropwise.</li>
		<li>After stirring for 5 hr quench the reaction by adding 500 ml distilled water and filter your product.</li>
		<li>Wash with water (3 times, 100 ml) and dry the product in vacuum, (yield: 93% (31.5 g, 84.1 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Anhydride Opening: Synthesis of 5-bromo-6-chloro-N-(methoxycarbonylallyl)-anthranilic acid allyl ester (11)
	<ol>
		<li>Dissolve 10 g (27 mmol) of 10 (product obtained from step 1.5) in 100 ml allyl alcohol. 
		Caution! Allyl alcohol is harmful and dangerous for the environment.</li>
		<li>Add 350 mg (8.70 mmol) sodium hydride (60% in paraffin) portion wise.</li>
		<li>After stirring for 6.5 hr or overnight remove the solvent.</li>
		<li>Subject the crude product to column chromatography (column 30 x 6 cm; 240 g silica) petroleum ether/ethyl acetate 2:1; RF = 0.66), (yield: 80% (8.30 g, 21.3 mmol), yellow oil). 
		Caution! Petroleum ether is flammable, irritant and dangerous for the environment.</li>
	</ol>
	</li>
	<li>Dieckmann Condensation: Synthesis of 5-bromo-4-chloro-indoxylic acid allyl ester (12)
	<ol>
		<li>Mix 3.0 g (7.7 mmol) of 11(product obtained from step 1.6) and 1.75 g (15.6 mmol) of potassium tert-butoxide in 100 ml dry diethyl ether. 
		Caution! Potassium tert-butoxide is corrosive and flammable. 
		Caution! Diethyl ether is harmful and extremely flammable.</li>
		<li>Heat the mixture under reflux (cooler) for 2 hr at 40-45 &#176;C.</li>
		<li>Remove about 75% of the solvent, and add diluted hydrochloric acid (100 ml, 1 M). 
		Caution! Do not dry completely, decomposition can occur.</li>
		<li>Filter the product and dry in vacuum, (yield: 84% (5.14 g, 15.5 mmol), colorless to slightly green solid).</li>
	</ol>
	</li>
</ol>
2. Indoxyl-Glycoside: Synthesis of X-Gal17
<ol>
	<li>Phase Transfer Glycosylation: Synthesis of (5-bromo-4-chloro-indox-3-ylic acid allyl ester) 2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-galactopyranoside (14)
	<ol>
		<li>Mix 400 mg (1.21 mmol) of the indoxylic acid allylester 12 (product obtained from step 1.7), 410 mg (1.21 mmol) tetrabutylammonium hydrogensulfate and 500 mg (1.21 mmol) of the donor 2,3,4,6-tetra-O-acetyl-&#945;-&#7429;-galactopyranosyl bromide (&#945;-acetobromogalactose) in 10 ml dichloromethane. Caution! Tetrabutylammonium hydrogensulfate is harmful.</li>
		<li>Add 10 ml of an aqueous solution of potassium carbonate (1 M). 
		Caution! Potassium carbonate is corrosive.</li>
		<li>Stir at room temperature until complete consumption of the donor (TLC: petroleum ether/ethyl acetate).</li>
		<li>Separate the organic phase (separatory funnel), dry over Na2SO4 and remove the solvent.</li>
		<li>Subject the crude product to column chromatography (petroleum ether/ethyl acetate 1:1; RF = 0.33). (yield: 86% (690 mg, 1.04 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Ester Cleavage and Decarboxylation: Synthesis of (N-acetyl-5-bromo-4-chloro-indol-3-yl) 2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-galactopyranoside (15)
	<ol>
		<li>Dissolve 600 mg (0.908 mmol) of 14 (product obtained from step 2.1) in 15 ml dry tetrahydrofurane. 
		Caution! Tetrahydrofurane is harmful and flammable.</li>
		<li>Add 800 &#956;l morpholine and 105 mg (9.09 &#956;mol) tetrakis(triphenylphosphine)palladium(0) and stir overnight at room temperature. 
		Caution! Morpholine is flammable and corrosive.</li>
		<li>Remove the solvent.</li>
		<li>Add 400 mg (2.40 mmol) silver acetate, 800 mg (5.79 mmol) potassium carbonate and 10 ml acetic anhydride. 
		Caution! Silver acetate is irritant and dangerous for the environment.</li>
		<li>Heat at 90-100 &#176;C for 20 min.</li>
		<li>After cooling to room temperature dilute with dichloromethane.</li>
		<li>Wash the mixture twice with water and once with a diluted NaHCO3 solution (10%). 
		Caution! Sodium bicarbonate is corrosive.</li>
		<li>Dry the organic phase over Na2SO4 (filter) and remove the solvent.</li>
		<li>Subject the crude product to column chromatography (PE/EA 1:1; RF = 0.24), (yield: 88% (495 mg, 8.00 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Zempl&#233;n Deacetylation: Synthesis of (5-bromo-4-chloro-indol-3-yl)-&#946;-&#7429;-galactopyranoside (16)
	<ol>
		<li>Dissolve 396 mg (0.436 mmol) 15 (product obtained from step 2.2) in 10 ml methanol.</li>
		<li>Add a catalytic amount of sodium methanolate and stir overnight at room temperature. 
		Caution! Sodium methanolate is flammable and corrosive.</li>
		<li>Neutralize with Amberlite IR-120 H+ (filter) and concentrate. 
		Caution! Amberlite IR-120 H+ is irritant.</li>
		<li>Dry the product in vacuum, (yield: 87% (170 mg, 0.416 mmol), colorless solid).</li>
	</ol>
	</li>
</ol>
3. Illustrating Model Experiment: Activity proof of &#946;-galactosidase employing X-Gal
<ol>
	<li>Prepare a 0.5 mM solution of X-Gal in 500 &#956;l buffer (Tris-HCl 20 mM; pH 7.4; 50 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid; or any other suitable buffer for your enzyme) in an microcentrifuge tube (X-Gal can be dissolved in a few &#956;l of dimethylsulfoxide).</li>
	<li>Add 1 &#956;l &#946;,1-3-galactosidase (EC 3.2.1.23 10,000 U/ml; or any other suitable &#946;-galactosidase) and shake (600 rps) at 37 &#176;C.</li>
	<li>Follow the reaction. The indigo type dye is formed after a short period of time.</li>
</ol>

<ol>
	<li>Clark, R. J. H., Cooksey, C. J., Daniels, M. A. M., Withnall, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indigo,+Woad,+and+Tyrian+Purple:+Important+Vat+Dyes+from+Antique+to+the+Present.">Indigo, Woad, and Tyrian Purple: Important Vat Dyes from Antique to the Present.</a> Endeavour. 17, 191-199 (1993).</li><li>Hunger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Industrial Dyes: Chemistry, Properties, Applications. , (2003).</li><li>Kiernan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indigogenic+Substrates+for+Detection+and+Localization+of+Enzymes.">Indigogenic Substrates for Detection and Localization of Enzymes.</a> Biotechn. Histochem. 82, 73-103 (2007).</li><li>Barnett, R. J., Seligman, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+Demonstration+of+Esterases+by+Production+of+Indigo.">Histochemical Demonstration of Esterases by Production of Indigo.</a> Science. 114, 579-582 (1951).</li><li>Pearson, B., Andrews, M., Grose, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Histochemical+Demonstration+of+Mammalian+Glucosidase+by+Means+of+3-(5-Bromoindolyl)-&#946;-&#7429;-glucopyranoside.">Histochemical Demonstration of Mammalian Glucosidase by Means of 3-(5-Bromoindolyl)-&#946;-&#7429;-glucopyranoside.</a> Exp. Biol. Med. 108, 619-623 (1961).</li><li>Anderson, F. B., Leaback, D. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrates+for+the+Histochemical+Localization+of+some+Glycosidases.">Substrates for the Histochemical Localization of some Glycosidases.</a> Tetrahedron. 12, 236-239 (1961).</li><li>Van Dort, M. E., Lee, K. C., Hamilton, C. A., Rehemtulla, A., Ross, B. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radiosynthesis+and+Evaluation+of+5-[125I]Iodoindolyl-3-yl-&#946;-&#7429;-galactopyranoside+([125I]IBDG)+as+a+&#946;-Galactosidase+Imaging+Radioligand.">Radiosynthesis and Evaluation of 5-[125I]Iodoindolyl-3-yl-&#946;-&#7429;-galactopyranoside ([125I]IBDG) as a &#946;-Galactosidase Imaging Radioligand.</a> Mol. Imaging. 7, 187-197 (2008).</li><li>Yoshida, K., Iino, N., Koga, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheses+of+Halogen+Substituted+&#946;-&#7429;-Glucuronides+and+Their+Hydrolysis+by+Rabbit+Liver+&#946;-Glucoronidase.">Syntheses of Halogen Substituted &#946;-&#7429;-Glucuronides and Their Hydrolysis by Rabbit Liver &#946;-Glucoronidase.</a> Chem. Pharm. Bull. 32, 1759-1769 (1975).</li><li>Horwitz, J. P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Substrates+for+Cytochemical+Demonstartion+of+Enzyme+Activity+I.+Some+Substituted+3-Indolyl-&#946;-&#7429;-glycopyranosides.">Substrates for Cytochemical Demonstartion of Enzyme Activity I. Some Substituted 3-Indolyl-&#946;-&#7429;-glycopyranosides.</a> J. Med. Chem. 7, 574-575 (1964).</li><li>Eschenfelder, V., Brossmer, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=5-Bromo-indol-3-yl+5-Acetamido-3,5-dideoxy-&#945;-&#7429;-glycero-&#7429;-galactononulopyranosidic+Acid,+a+Novel+Chromogenic+Substrate+for+the+Staining+of+Sialidase+Activity.">5-Bromo-indol-3-yl 5-Acetamido-3,5-dideoxy-&#945;-&#7429;-glycero-&#7429;-galactononulopyranosidic Acid, a Novel Chromogenic Substrate for the Staining of Sialidase Activity.</a> Glycoconjugate J. 4, 171-178 (1987).</li><li>Fujii, I., Iwabuchi, Y., Teshima, T., Shiba, T., Kikuchi, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=X-Neu5Ac:+A+Novel+Substrate+for+Chromogenic+Assay+of+Neuraminidase+Activity+in+Bacterial+Expression+Systems.">X-Neu5Ac: A Novel Substrate for Chromogenic Assay of Neuraminidase Activity in Bacterial Expression Systems.</a> Bioorg. Med. Chem. 1, 147-149 (1993).</li><li>Berlin, W., Sauer, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+Color+Detection+of+&#945;-&#671;-Arabinofuranisodase,+a+&amp;#34;No-Background&amp;#34;+Reporter+Gene,+with+5-Bromo-3-indolyl-&#945;-&#671;-arabinofuranoside.">In situ Color Detection of &#945;-&#671;-Arabinofuranisodase, a &amp;#34;No-Background&amp;#34; Reporter Gene, with 5-Bromo-3-indolyl-&#945;-&#671;-arabinofuranoside.</a> Anal. Biochem. 243, 171-175 (1996).</li><li>Marmuse, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+Chromogenic+Substrates+for+Feruloyl+Esterases.">New Chromogenic Substrates for Feruloyl Esterases.</a> Org. Biomol. Chem. 6, 1208-1214 (2008).</li><li>Kaneko, S., Kiaoka, M., Kuno, A., Hayashi, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheses+of+4-Methylumbelliferyl-&#946;-&#7429;-Xylobioside+and+5-Bromo-3-Indolyl-&#946;-&#7429;-Xylobioside+for+Sensitive+Detection+of+Xylanase+Activity+on+Agar+Plates.">Syntheses of 4-Methylumbelliferyl-&#946;-&#7429;-Xylobioside and 5-Bromo-3-Indolyl-&#946;-&#7429;-Xylobioside for Sensitive Detection of Xylanase Activity on Agar Plates.</a> Biosci. Biotechnol. Biochem. 64, 741-745 (2000).</li><li>Robertson, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Syntheses+of+Glucosides.+Part+I.+The+Synthesis+of+Indican.">Syntheses of Glucosides. Part I. The Synthesis of Indican.</a> Chem. Soc. , 1937-1943 (1927).</li><li>Robertson, A., Waters, R. B. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthese+of+Glucosides.+Part+VII.+The+Synthesis+of+6-Bromoindican.">Synthese of Glucosides. Part VII. The Synthesis of 6-Bromoindican.</a> Chem. Soc. , 2239-2243 (1929).</li><li>B&#246;ttcher, S., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indoxylic+Acid+Esters+as+Convenient+Intermediates+Towards+Indoxyl+Glycosides.">Indoxylic Acid Esters as Convenient Intermediates Towards Indoxyl Glycosides.</a> Eur. J. Org. Chem. , 564-574 (2014).</li><li>B&#246;ttcher, S., Hederos, M., Champion, E., D&#233;k&#225;ny, G., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Efficient+Routes+to+Indoxyl+Glycosides+for+Monitoring+Glycosidase+Activities.">Novel Efficient Routes to Indoxyl Glycosides for Monitoring Glycosidase Activities.</a> Org. Lett. 15, 3766-3769 (2013).</li><li>B&#246;ttcher, S., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Preparation+of+Indoxyl-+and+Nitrophenyl+Glycosides+of+Lactosamine+and+Isolactosamine.">Facile Preparation of Indoxyl- and Nitrophenyl Glycosides of Lactosamine and Isolactosamine.</a> RSC Adv. 4, 10856-10861 (2014).</li></ol>

The authors declare that they have no competing financial interests.

Owing to poor yields and limitations, especially for glucose type structures and more complex saccharides, a novel synthetic approach towards indoxyl glycosides was developed. Indoxylic acid esters proved to be precious key intermediates and were obtained in a modular, scalable pathway. All steps are high yielding and due to cheap starting materials and easy workup multi-gram syntheses are possible. The advantage of the allyl ester approach is the blocking of the reactive 2-position. Thus yield decreasing side reactions ...

For a long time the production of indigo was an economically very important process. Before large scale chemical syntheses gave cheap access to indigo, precursors were obtained from natural sources since pre-Christian times. The cultivation of indigo providing plants (natural indigo) in Europe became unrewarding in the 17th century, as the amount of indigo precursors of the Indian indigo plant (0.2-0.8 %) is about 30 times higher. At the end of the 19th century chemical synthesis of indigo suppressed the conventional cultivation1,2.
Indigo precursors occurring naturally in plants include Indican (1), Insatan A (2) and Isatan B (3) (Figure 1). All of them consist of an indoxyl motive linked to a glycosyl residue. Cleavage of the glycosidic linkage, for example by enzymatic hydrolysis, leads to release of indoxyl (4). Indoxyl itself is almost colorless, but can be rapidly oxidized to form an indigo dye (5). This sensitive reaction has been adapted in biochemistry, histochemistry, bacteriology and molecular biology for monitoring enzyme activities. Activity screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) is possible. Depending of the residue (e.g., esters, glycosides, sulfates) linked to the indoxyl moiety, suitable substrates for different enzyme classes (e.g., esterases, glycosidases, sulfatases) have been developed3. In the following focus will be on formation and application of indoxyl glycosides.
<img alt="Figure 1" src="/files/ftp_upload/52442/52442fig1highres.jpg" width="500" /> 
Figure 1: Natural indigo precursors and formation of indigo dye by hydrolysis. <a href="https://www.jove.com/files/ftp_upload/52442/52442fig1highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The substitution pattern of the indoxyl moiety determines the color and physical properties of the resulting indigo dye. The most common substitution patterns are 5-bromo-4-chloro (abbreviated by X; greenish-blue), 5-bromo (blue) and 5-bromo-6-chloro (magenta), since these form the smallest dye particles, do not form granules and have the least diffusion from sites of hydrolysis. The last property is especially important for in vivo experiments3.
The first report of an indigogenic method for detection of esterase activity was published in 1951 by Barrnett and Seligman, who employed indoxyl acetate and butyrate4. About one decade later the indigogenic principle was adapted for localization of mammalian glucosidase5. Up to now several indoxyl glycosides have been developed even though their synthesis proved to be difficult. Most syntheses are based on employing an N-acetylated indoxyl as acceptor and the respective glycosyl halide donor6-14. Glycosylation is performed in acetone with sodium hydroxide. Under these conditions a number of side reactions occur, decreasing the yield significantly. Especially for glucose type structures very low glycosylation yields were reported (e.g., 15% for (N-acetyl-5-bromo-4-chloro-indol-3-yl)-2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-glucopyranoside6 and 26% for (N-acetyl-5-bromo-4-chloro-indol-3-yl)-2,3,2&#39;,3&#39;,4&#39;-penta-O-acetyl-&#946;-&#7429;-xylobioside14 in a more recent example). Through a novel approach, employing indoxylic acid esters, a considerable number of indoxyl glycosides were prepared in good yields (e.g., (N-acetyl-5-bromo-4-chloro-indol-3-yl)-2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-glucopyranoside 57% yield).
The following protocol describes the straightforward synthesis of indoxylic acid allyl ester (5-bromo-4-chloro) and based thereon the synthesis of an indoxyl glycoside (X-Gal). A simple model experiment shows the enzyme reactivity of &#946;-galactosidase employing X-Gal.

<table><tbody><tr><td>Acetic anhydride</td><td>Gr&amp;uuml;ssing</td><td>10298</td><td>Corrosive, flammable</td></tr><tr><td>Acetonitrile</td><td>Sigma-Aldrich</td><td>608-001-00-3</td><td>Harmful, flammable</td></tr><tr><td>Allyl alcohol</td><td>Aldrich</td><td>453021</td><td>Harmful, dangerous for the environment</td></tr><tr><td>Amberlite IR-120 H&lt;sup&gt;+&lt;/sup&gt;</td><td>Fluka</td><td>06428</td><td>Irritant</td></tr><tr><td>Bromoacetic acid</td><td>Merck</td><td>802260</td><td>Corrosive, toxic, dangerous for the environment</td></tr><tr><td>4-Bromo-3-chloro-2-methylaniline</td><td>ABCR</td><td>AB 171687</td><td>Irritant</td></tr><tr><td>Dichloromethane</td><td>ACROS</td><td>326850010</td><td>Harmful</td></tr><tr><td>Diethyl ether</td><td>Gr&amp;uuml;ssing</td><td>10274</td><td>Harmful, extremly flammable</td></tr><tr><td>Dimethylformamide</td><td>ACROS</td><td>348430010</td><td>Harmful, flammable</td></tr><tr><td>Dimethylsulfoxide</td><td>Sigma-Aldrich</td><td>41648</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma-Aldrich</td><td>607-022-00-5</td><td>Irritant, flammable</td></tr><tr><td>Ethylenediaminetetraacetic acid</td><td>AppliChem</td><td>A1103.0500</td><td>Irritant</td></tr><tr><td>&amp;beta;1,3-Galactosidase, recombinant, &lt;em&gt;E. coli&lt;/em&gt;</td><td>Calbiochem</td><td>345795</td><td></td></tr><tr><td>Hydrochloric acid</td><td>VWR</td><td>20252.290</td><td>Corrosive</td></tr><tr><td>Magnesium sulfate hydrate</td><td>Merck</td><td>105885</td><td></td></tr><tr><td>Methanol</td><td>ACROS</td><td>326950010</td><td>Toxic, flammable</td></tr><tr><td>Morpholine</td><td>Janssen Chimica</td><td>15.868.57</td><td>Corrosive, flammable</td></tr><tr><td>Peroleum ether</td><td>Azelis</td><td>111053</td><td>Flammable, irritant, dangerous for the environment</td></tr><tr><td>Potassium carbonate</td><td>Gr&amp;uuml;ssing</td><td>12005</td><td>Corrosive</td></tr><tr><td>Potassium permanganate</td><td>Gr&amp;uuml;ssing</td><td>12056</td><td>Harmful, oxidising</td></tr><tr><td>Potassium tert-butoxide</td><td>Merck</td><td>804918</td><td>Corrosive, flammable</td></tr><tr><td>Pyridine</td><td>Sigma-Aldrich</td><td>613-002-00-7</td><td>Harmful, flammable</td></tr><tr><td>Silver acetate</td><td>Fluka</td><td>85140</td><td>Irritant, dangerous for the environment</td></tr><tr><td>Sodium bicarbonate</td><td>Gr&amp;uuml;ssing</td><td>12144</td><td>Corrosive</td></tr><tr><td>Sodium hydride</td><td>Merck</td><td>814552</td><td>Corrosive, flammable</td></tr><tr><td>Sodium hydroxide</td><td>Riedel-de H&amp;auml;en</td><td>S181200</td><td>Corrosive</td></tr><tr><td>Sodium methanolate</td><td>Merck</td><td>806538</td><td>Corrosive, flammable</td></tr><tr><td>Sodium sulfate</td><td>Gr&amp;uuml;ssing</td><td>12175</td><td></td></tr><tr><td>Tetrabutylammonium hydrogensulfate</td><td>Lancaster</td><td>5438</td><td>Harmful</td></tr><tr><td>Tetrahydrofurane</td><td>Sigma-Aldrich</td><td>87371</td><td>Harmful, flammable</td></tr><tr><td>Tetrakis (triphenylphosphine)palladium(0)</td><td>Sigma-Aldrich</td><td>216666</td><td></td></tr><tr><td>Triphosgene</td><td>Fluka</td><td>15217</td><td>Toxic</td></tr><tr><td>Tris (hydroxymethyl)aminomethane hydrochloride</td><td>Sigma</td><td>T-3253</td><td>Irritant</td></tr></tbody></table>

synthesis of indoxyl-glycosides for detection of glycosidase activities

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are rapidly oxidized, for example by atmospheric oxygen, to indigo type dyes. This reaction enables fast and easy screening in vivo without isolation or purification of enzymes, as well as rapid tests on agar plates or in solution (e.g., blue-white screening, micro-wells) and is used in biochemistry, histochemistry, bacteriology and molecular biology. Unfortunately the synthesis of such substrates proved to be difficult, due to various side reactions and the low reactivity of the indoxyl hydroxyl function. Especially for glucose type structures low yields were observed. Our novel approach employs indoxylic acid ester as key intermediates. Indoxylic acid esters with varied substitution patterns were prepared on scalable pathways. Phase transfer glycosylations with those acceptors and peracetylated glycosyl halides can be performed under common conditions in high yields. Ester cleavage and subsequent mild silver mediated glycosylation yields the peracetylated indoxyl glycosides in high yields. Finally deprotection is performed according to Zempl&#233;n.

The very first syntheses of indicane and 5-bromo-indicane, were published by Robertson already in 1927 and 192915,16. By employing indoxylic acid methyl ester as acceptor, the reactive 2-position was blocked and thus side reactions were partially suppressed. Glycosylation in acetone/sodium hydroxide, following deprotection and decarboxylation (160 &#176;C, acetic anhydride, 1 hr) and finally deacetylation yielded Indicane and 5-bromo-indicane. Based on this concept we developed an improved synthesis of indoxyl...

Watch this Scientific Journal Video about Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities at JoVE.com

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

synthesis-indoxyl-glycosides-for-detection-glycosidase

Research

Education

JoVE Core

JoVE Journal

Organic Chemistry

Chemistry

Unit 1

&alpha;-Carbon Chemistry: Enols, Enolates, and Enamines

SCIENTISTS IN ACTION

6.6K Views.  University of Hamburg. Indoxyl glycosides are well-established and widely used tools for enzyme screening and enzyme activity monitoring. Especially for glucose type structures previous syntheses proved to be challenging and low yielding. Our novel approach employs indoxylic acid esters as precious intermediates to yield a considerable number of indoxyl glycosides in good yields.

Video: Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

High-throughput Screening of Carbohydrate-degrading Enzymes Using Novel Insoluble Chromogenic Substrate Assay Kits

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper and food industries. A deeper understanding of CAZymes is important from both fundamental biology and industrial standpoints. Vast numbers of CAZymes exist in nature (especially in microorganisms) and hundreds of thousands have been cataloged and described in the carbohydrate active enzyme database (CAZy). However, the rate of discovery of putative enzymes has outstripped our ability to biochemically characterize their activities. One reason for this is that advances in genome and transcriptome sequencing, together with associated bioinformatics tools allow for rapid identification of candidate CAZymes, but technology for determining an enzyme's biochemical characteristics has advanced more slowly. To address this technology gap, a novel high-throughput assay kit based on insoluble chromogenic substrates is described here. Two distinct substrate types were produced: Chromogenic Polymer Hydrogel (CPH) substrates (made from purified polysaccharides and proteins) and Insoluble Chromogenic Biomass (ICB) substrates (made from complex biomass materials). Both CPH and ICB substrates are provided in a 96-well high-throughput assay system. The CPH substrates can be made in four different colors, enabling them to be mixed together and thus increasing assay throughput. The protocol describes a 96-well plate assay and illustrates how this assay can be used for screening the activities of enzymes, enzyme cocktails, and broths.

A high-throughput assay for enzyme screening is described. This multiplexed ready-to-use assay kit comprises of pre-chosen Chromogenic Polymer Hydrogel (CPH) substrates and complex Insoluble Chromogenic Biomass (ICB) substrates. Target enzymes are polysaccharide degrading endo-enzymes and proteases.

فحص الإنتاجية العالية من الكربوهيدرات المهينة الانزيمات عن طريق رواية غير قابل للذوبان اللونية الركيزة الفحص أطقم

Carbohydrates active enzymes (CAZymes) have multiple roles in vivo and are widely used for industrial processing in the biofuel, textile, detergent, paper ...

Biochemistry

Rapid Colorimetric Assays to Qualitatively Distinguish RNA and DNA in Biomolecular Samples

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in potentially heterogeneous specimens. Nucleic acids can be detected via several established approaches, including analytical methods that are spectrophotometric (e.g., A260), fluorometric (e.g., binding of fluorescent dyes), or colorimetric (nucleoside-specific chromogenic chemical reactions).1 Though it cannot readily distinguish RNA from DNA, the A260/A280 ratio is commonly employed, as it offers a simple and rapid2 assessment of the relative content of nucleic acid, which absorbs predominantly near 260 nm and protein, which absorbs primarily near 280 nm. Ratios &lt; 0.8 are taken as indicative of 'pure' protein specimens, while pure nucleic acid (NA) is characterized by ratios &gt; 1.53.
However, there are scenarios in which the protein/NA content cannot be as clearly or reliably inferred from simple uv-vis spectrophotometric measurements. For instance, (i) samples may contain one or more proteins which are relatively devoid of the aromatic amino acids responsible for absorption at &asymp;280 nm (Trp, Tyr, Phe), as is the case with some small RNA-binding proteins, and (ii) samples can exhibit intermediate A260/A280 ratios (~0.8 &lt; ~1.5), where the protein/NA content is far less clear and may even reflect some high-affinity association between the protein and NA components. For such scenarios, we describe herein a suite of colorimetric assays to rapidly distinguish RNA, DNA, and reducing sugars in a potentially mixed sample of biomolecules. The methods rely on the differential sensitivity of pentoses and other carbohydrates to Benedict's, Bial's (orcinol), and Dische's (diphenylamine) reagents; the streamlined protocols can be completed in a matter of minutes, without any additional steps of having to isolate the components. The assays can be performed in parallel to differentiate between RNA and DNA, as well as indicate the presence of free reducing sugars such as glucose, fructose, and ribose (Figure 1).

A suite of colorimetric assays is described for rapidly distinguishing protein, RNA, DNA, and reducing sugars in potentially heterogeneous biomolecular samples.

فحوصات اللونية السريعة للتمييز نوعيا RNA والحمض النووي في العينات البيولوجية

Biochemical experimentation generally requires accurate knowledge, at an early stage, of the nucleic acid, protein, and other biomolecular components in ...

Spectrophotometric Methods for the Study of Eukaryotic Glycogen Metabolism

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains of &#945;1,4-linked glucose residues with branches introduced through the addition of &#945;1,6-linkages. Understanding how the synthesis and degradation of glycogen are regulated and how glycogen attains its characteristic branched structure requires the study of the enzymes of glycogen storage. However, the methods most commonly used to study these enzyme activities typically employ reagents or techniques that are not available to all investigators. Here, we discuss a battery of procedures that are technically simple, cost-effective, and yet still capable of providing valuable insight into the control of glycogen storage. The techniques require access to a spectrophotometer, operating in the range of 330 to 800 nm, and are described assuming that the users will employ disposable, plastic cuvettes. However, the procedures are readily scalable and can be modified for use in a microplate reader, allowing highly parallel analysis.

Techniques to measure the activity of key enzymes of glycogen metabolism are presented, using a simple spectrophotometer operating in the visible range.

الطرق الطيفية لدراسة استقلاب الجليكوجين حقيقي النواة

Glycogen is synthesized as a storage form of glucose by a wide array of organisms, ranging from bacteria to animals. The molecule comprises linear chains ...

<meta charset="utf8"></head><body>القياس الكمي للجلوكوز الأنزيمي Bergmeyer لتحليل الميكروبات والخميرة

Bergmeyer Glucose Quantification for Microbiological Samples

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, and, in some cases, how cells couple glucose metabolism to energetic metabolism. In addition, intracellular and extracellular glucose levels are indicative of the cellular metabolic status. Enzymatic techniques, such as Bergmeyer glucose quantification, are more accurate and sensitive than other techniques, such as dinitrosalicylic acid or fluorescence methods, which are usually utilized in microbiology. Although mainly used in the clinical area, Bergmeyer glucose quantification can also be applied to any cell but has not been reported in detail for bacteria, fungi, yeasts, or other microorganisms. 
Herein, we present a methodology to quantify glucose from bacteria and yeast samples using the enzymatic Bergmeyer glucose quantification method. The procedure involved the enzymes glucose oxidase, peroxidase, and o-dianisidine dihydrochloride incubated at 37 &#176;C for 20 min, followed by the addition of sulfuric acid. The absorbance is then measured at 545 nm. It is important to highlight that although this technique presents difficulties in measuring high concentrations of glucose (above 60 g/L), it is possible to measure concentrations below 50 g/L using dilution factors. This enzymatic approach is valuable for research and analysis in microbiology and other scientific areas. The precision and sensitivity of the method make it helpful for detecting even low concentrations of glucose in microbiological samples.

<meta charset="utf8"></head><body>تقدير الجلوكوز Bergmeyer للعينات الميكروبيولوجية

Bergmeyer glucose quantification is a spectrophotometric enzymatic technique mainly used for clinical tests that accurately and sensitively measure the amount of glucose. Herein, we present a protocol for using this method for microbiological samples.

تقدير الجلوكوز Bergmeyer للعينات الميكروبيولوجية

The glucose concentration is a key indicator of cellular metabolism and could indicate the total metabolism rate, an aberration in glucose metabolism, ...

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

تحديد وتوصيف ارتباط بالغليكوزيل باستخدام بروتين معين إندو وexoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

Biology

Biochemical Titration of Glycogen In vitro

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular metabolism. Many methods to detect glycogen already exist but only a few are quantitative. We describe here a method using the Abcam Glycogen assay kit, which is based on specific degradation of glycogen to glucose by glucoamylase. Glucose is then specifically oxidized to a product that reacts with the OxiRed probe to produce fluorescence. Titration is accurate, sensitive and can be achieved on cell extracts or tissue sections. However, in contrast to other techniques, it does not give information about the distribution of glycogen in the cell. As an example of this technique, we describe here the titration of glycogen in two cell lines, Chinese hamster lung fibroblast CCL39 and human colon carcinoma LS174, incubated in normoxia (21% O2) versus hypoxia (1% O2). We hypothesized that hypoxia is a signal that prepares cells to synthesize and store glycogen in order to survive1.

Biochemical Titration of Glycogen In vitro

We describe here an accurate, reproducible and convenient biochemical method for titration of glycogen in vitro. This technique uses the Abcam Glycogen assay kit and is based on successive hydrolysis of glycogen to glucose and glucose titration by fluorescence.

المعايرة الكيميائية الحيوية من الجليكوجين<em&gt; في المختبر</em

Glycogen is the main energetic polymer of glucose in vertebrate animals and plays a crucial role in whole body metabolism as well as in cellular ...

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate (CDAP)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be conveniently functionalized and linked to vaccine carrier proteins using 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), an easy-to-handle cyanylating reagent. CDAP activates polysaccharides by reacting with carbohydrate hydroxyl groups at pH 7-9. The stability and reactivity of CDAP are highly pH-dependent. The pH of the reaction also decreases during activation due to the hydrolysis of CDAP, which makes good pH control the key to reproducible activation. The original CDAP activation protocol was performed at room temperature in unbuffered pH 9 solutions.
Due to the rapid reaction under this condition (&#60;3 min) and the accompanying fast pH drop from the rapid CDAP hydrolysis, it was challenging to quickly adjust and maintain the target reaction pH in the short time frame. The improved protocol described here is performed at 0 &#176;C, which slows CDAP hydrolysis and extends the activation time from 3 min to ~15 min. Dimethylaminopyridine (DMAP) was also used as a buffer to pre-adjust the polysaccharide solution to the target activation pH before adding the CDAP reagent. The longer reaction time, coupled with the slower CDAP hydrolysis and the use of DMAP buffer, makes it easier to maintain the activation pH for the entire duration of the activation process. The improved protocol makes the activation process less frenetic, more reproducible, and more amenable to scaling up.

Activation and Conjugation of Soluble Polysaccharides using 1-Cyano-4-Dimethylaminopyridine Tetrafluoroborate CDAP

Proteins and amine-containing ligands can be covalently linked to polysaccharides activated by the cyanylation reagent, 1-cyano-4-dimethylaminopyridine tetrafluoroborate (CDAP), to form covalent protein (ligand)-polysaccharide conjugates. This article describes an improved protocol for carrying out controlled CDAP activation at 0 &#176;C and varying pH and performing subsequent conjugation of the activated polysaccharides.

تفعيل وارتقاط السكريات المتعددة القابلة للذوبان باستخدام 1-سيانو-4-ديميثيلامينوبيريدين تترافلوروبورات (CDAP)

Conjugate vaccines are remarkable advances in vaccinology. For the preparation of polysaccharide conjugate vaccines, the polysaccharides can be ...

Immunology and Infection

Automated Modular High Throughput Exopolysaccharide Screening Platform Coupled with Highly Sensitive Carbohydrate Fingerprint Analysis

Many microorganisms are capable of producing and secreting exopolysaccharides (EPS), which have important implications in medical fields, food applications or in the replacement of petro-based chemicals. We describe an analytical platform to be automated on a liquid handling system that allows the fast and reliable analysis of the type and the amount of EPS produced by microorganisms. It enables the user to identify novel natural microbial exopolysaccharide producers and to analyze the carbohydrate fingerprint of the corresponding polymers within one day in high-throughput (HT). Using this platform, strain collections as well as libraries of strain variants that might be obtained in engineering approaches can be screened. The platform has a modular setup, which allows a separation of the protocol into two major parts. First, there is an automated screening system, which combines different polysaccharide detection modules: a semi-quantitative analysis of viscosity formation via a centrifugation step, an analysis of polymer formation via alcohol precipitation and the determination of the total carbohydrate content via a phenol-sulfuric-acid transformation. Here, it is possible to screen up to 384 strains per run. The second part provides a detailed monosaccharide analysis for all the selected EPS producers identified in the first part by combining two essential modules: the analysis of the complete monomer composition via ultra-high performance liquid chromatography coupled with ultra violet and electrospray ionization ion trap detection (UHPLC-UV-ESI-MS) and the determination of pyruvate as a polymer substituent (presence of pyruvate ketal) via enzymatic oxidation that is coupled to a color formation. All the analytical modules of this screening platform can be combined in different ways and adjusted to individual requirements. Additionally, they can all be handled manually or performed with a liquid handling system. Thereby, the screening platform enables a huge flexibility in order to identify various EPS.

We present an automated modular high-throughput-method for the identification and characterization of microbial exopolysaccharides in small scale. This method combines a fast preselection to analyze the total amount of secreted polysaccharides with a detailed carbohydrate fingerprint to enable the fast screening of newly isolated bacterial strains or entire strain collections.

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Summary

Explore More Videos

Chapters in this video

فحص الإنتاجية العالية من الكربوهيدرات المهينة الانزيمات عن طريق رواية غير قابل للذوبان اللونية الركيزة الفحص أطقم

فحوصات اللونية السريعة للتمييز نوعيا RNA والحمض النووي في العينات البيولوجية

الطرق الطيفية لدراسة استقلاب الجليكوجين حقيقي النواة

تقدير الجلوكوز Bergmeyer للعينات الميكروبيولوجية

تقدير الجلوكوز Bergmeyer للعينات الميكروبيولوجية

تقدير الجلوكوز Bergmeyer للعينات الميكروبيولوجية

تحديد وتوصيف ارتباط بالغليكوزيل باستخدام بروتين معين إندو وexoglycosidases

المعايرة الكيميائية الحيوية من الجليكوجين

تفعيل وارتقاط السكريات المتعددة القابلة للذوبان باستخدام 1-سيانو-4-ديميثيلامينوبيريدين تترافلوروبورات (CDAP)

الآلي وحدات إنتاجية عالية Exopolysaccharide فحص منصة مقرونا حساسة للغاية تحليل الكربوهيدرات بصمات الأصابع