Name: synthesis of indoxyl-glycosides for detection of glycosidase activities
Uploaded: 2015-05-27T13:00:00
Description: Watch this Scientific Journal Video about Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities at JoVE.com

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...

<ol>
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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. 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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 border="0" cellpadding="0" cellspacing="0" width="983">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Name of Material/ Equipment</td>
			<td style="width:160px;">Company</td>
			<td style="width:119px;">Catalog Number</td>
			<td style="width:347px;">Comments/Description</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetic anhydride</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">10298</td>
			<td>Corrosive, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Acetonitrile</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">608-001-00-3</td>
			<td>Harmful, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Allyl alcohol</td>
			<td style="width:160px;">Aldrich</td>
			<td style="width:119px;">453021</td>
			<td>Harmful, dangerous for the environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Amberlite IR-120 H+</td>
			<td style="width:160px;">Fluka</td>
			<td style="width:119px;">06428</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Bromoacetic acid</td>
			<td style="width:160px;">Merck</td>
			<td style="width:119px;">802260</td>
			<td>Corrosive, toxic, dangerous for the environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-Bromo-3-chloro-2-methylaniline</td>
			<td style="width:160px;">ABCR</td>
			<td style="width:119px;">AB 171687</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dichloromethane</td>
			<td style="width:160px;">ACROS</td>
			<td style="width:119px;">326850010</td>
			<td>Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Diethyl ether</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">10274</td>
			<td>Harmful, extremly flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Dimethylformamide</td>
			<td style="width:160px;">ACROS</td>
			<td style="width:119px;">348430010</td>
			<td>Harmful, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Dimethylsulfoxide</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">41648</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Ethyl acetate</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">607-022-00-5</td>
			<td>Irritant, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ethylenediaminetetraacetic acid</td>
			<td style="width:160px;">AppliChem</td>
			<td style="width:119px;">A1103.0500</td>
			<td>Irritant</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#223;1,3-Galactosidase, Recombinant, E. coli</td>
			<td style="width:160px;">Calbiochem</td>
			<td style="width:119px;">345795</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Hydrochloric acid</td>
			<td style="width:160px;">VWR&#160;</td>
			<td style="width:119px;">20252.290</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Magnesium sulfate hydrate</td>
			<td style="width:160px;">Merck</td>
			<td style="width:119px;">105885</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Methanol</td>
			<td style="width:160px;">ACROS</td>
			<td style="width:119px;">326950010</td>
			<td>Toxic, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Morpholine</td>
			<td style="width:160px;">Janssen Chimica</td>
			<td style="width:119px;">15.868.57</td>
			<td>Corrosive, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Peroleum ether</td>
			<td style="width:160px;">Azelis</td>
			<td style="width:119px;">111053</td>
			<td>Flammable, irritant, dangerous for the environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium carbonate</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">12005</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Potassium permanganate</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">12056</td>
			<td>Harmful, oxidising</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium tert-butoxide</td>
			<td style="width:160px;">Merck</td>
			<td style="width:119px;">804918</td>
			<td>Corrosive, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Pyridine</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">613-002-00-7</td>
			<td>Harmful, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Silver acetate</td>
			<td style="width:160px;">Fluka</td>
			<td style="width:119px;">85140</td>
			<td>Irritant, dangerous for the environment</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium bicarbonate</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">12144</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium hydride</td>
			<td style="width:160px;">Merck</td>
			<td style="width:119px;">814552</td>
			<td>Corrosive, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium hydroxide</td>
			<td style="width:160px;">Riedel-de H&#228;en</td>
			<td style="width:119px;">S181200</td>
			<td>Corrosive</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium methanolate</td>
			<td style="width:160px;">Merck</td>
			<td style="width:119px;">806538</td>
			<td>Corrosive, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Sodium sulfate</td>
			<td style="width:160px;">Gr&#252;ssing</td>
			<td style="width:119px;">12175</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrabutylammonium hydrogensulfate</td>
			<td style="width:160px;">Lancaster</td>
			<td style="width:119px;">5438</td>
			<td>Harmful</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrahydrofurane</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">87371</td>
			<td>Harmful, flammable</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tetrakis(triphenylphosphine)palladium(0)</td>
			<td style="width:160px;">Sigma-Aldrich</td>
			<td style="width:119px;">216666</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:359px;">Triphosgene</td>
			<td style="width:160px;">Fluka</td>
			<td style="width:119px;">15217</td>
			<td>Toxic</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tris(hydroxymethyl)aminomethane hydrochloride&#160;</td>
			<td style="width:160px;">Sigma</td>
			<td style="width:119px;">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...

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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 ...

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

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