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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Organic Chemistry

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Unit 1

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

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Synthesis of Hypervalent Iodonium Alkynyl Triflates for the Application of Generating Cyanocarbenes

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent reactions with azides to form cyanocarbene intermediates. The synthesis of hypervalent iodonium alkynyl triflates can be facile, but difficulties stem from their isolation and reactivity. In particular, the necessity to use filtration under inert atmosphere at -45 &deg;C for some HIATs requires special care and equipment. Once isolated, the compounds can be stored and used in reactions with azides to form cyanocarbene intermediates. 
The evidence for cyanocarbene generation is shown by visible extrusion of dinitrogen as well as the characterization of products that occur from O-H insertion, sulfoxide complexation, and cyclopropanation. A side reaction of the cyanocarbene formation is the generation of a vinylidene-carbene and the conditions to control this process are discussed. There is also potential to form a hypervalent iodonium alkenyl triflate and the means of isolation and control of its generation are provided. The O-H insertion reaction involves using a HIAT, sodium azide or tetrabutylammonium azide, and methanol as solvent/substrate. The sulfoxide complexation reaction uses a HIAT, sodium azide or tetrabutylammonium azide, and dimethyl sulfoxide as solvent. The cyclopropanations can be performed with or without the use of solvent. The azide source must be tetrabutylammonium azide and the substrate shown is styrene.

Herein is described the synthesis, isolation, and reactions of hypervalent iodonium alkynyl triflates (HIATs) with azides to generate cyanocarbenes. The procedures will involve air-sensitive and cryogen techniques, including cold filtration under an inert atmosphere. Handling and safety of the synthesized compounds will also be discussed.

The procedures described in this article involve the synthesis and isolation of hypervalent iodonium alkynyl triflates (HIATs) and their subsequent ...

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of trifluoromethoxylated aromatic compounds remains a formidable challenge in organic synthesis. Conventional approaches often suffer from poor substrate scope, or require use of highly toxic, difficult-to-handle, and/or thermally labile reagents. Herein, we report a user-friendly protocol for the synthesis of methyl 4-acetamido-3-(trifluoromethoxy)benzoate using 1-trifluoromethyl-1,2-benziodoxol-3(1H)-one (Togni reagent II). Treating methyl 4-(N-hydroxyacetamido)benzoate (1a) with Togni reagent II in the presence of a catalytic amount of cesium carbonate (Cs2CO3) in chloroform at RT afforded methyl 4-(N-(trifluoromethoxy)acetamido)benzoate (2a). This intermediate was then converted to the final product methyl 4-acetamido-3-(trifluoromethoxy)benzoate (3a) in nitromethane at 120 &#176;C. This procedure is general and can be applied to the synthesis of a broad spectrum of ortho-trifluoromethoxylated aniline derivatives, which could serve as useful synthetic building blocks for the discovery and development of new pharmaceuticals, agrochemicals, and functional materials.

Protocol for the Synthesis of Ortho-trifluoromethoxylated Aniline Derivatives

An operationally simple procedure for the synthesis of ortho-trifluoromethoxylated aniline derivatives via a two-step sequence of O-trifluoromethylation of N-aryl-N-hydroxyacetamide followed by thermally induced intramolecular OCF3-migration is reported.

Molecules bearing trifluoromethoxy (OCF3) group often show desired pharmacological and biological properties. However, facile synthesis of ...

Synthesis and Characterization of Supramolecular Colloids

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical properties for applications in photonics, drug delivery and coating technology. Here we present a new family of colloidal building blocks, coined supramolecular colloids, whose self-assembly is controlled through surface-functionalization with a benzene-1,3,5-tricarboxamide (BTA) derived supramolecular moiety. Such BTAs interact via directional, strong, yet reversible hydrogen-bonds with other identical BTAs. Herein, a protocol is presented that describes how to couple these BTAs to colloids and how to quantify the number of coupling sites, which determines the multivalency of the supramolecular colloids. Light scattering measurements show that the refractive index of the colloids is almost matched with that of the solvent, which strongly reduces the van der Waals forces between the colloids. Before photo-activation, the colloids remain well dispersed, as the BTAs are equipped with a photo-labile group that blocks the formation of hydrogen-bonds. Controlled deprotection with UV-light activates the short-range hydrogen-bonds between the BTAs, which triggers the colloidal self-assembly. The evolution from the dispersed state to the clustered state is monitored by confocal microscopy. These results are further quantified by image analysis with simple routines using ImageJ and Matlab. This merger of supramolecular chemistry and colloidal science offers a direct route towards light- and thermo-responsive colloidal assembly encoded in the surface-grafted monolayer.

A protocol for the synthesis and characterization of colloids coated with supramolecular moieties is described. These supramolecular colloids undergo self-assembly upon the activation of the hydrogen-bonds between the surface-anchored molecules by UV-light.

Control over colloidal assembly is of utmost importance for the development of functional colloidal materials with tailored structural and mechanical ...

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the development of antibody drug conjugates (ADCs) and nanomedicine, fluorescent microscopy and systems chemistry. For many of these applications, specificity of the bioconjugation method used is of prime concern. The Michael addition of maleimides with cysteine(s) on the target proteins is highly selective and proceeds rapidly under mild conditions, making it one of the most popular methods for protein bioconjugation.
We demonstrate here the modification of the only surface-accessible cysteine residue on yeast cytochrome c with a ruthenium(II) bisterpyridine maleimide. The protein bioconjugation is verified by gel electrophoresis and purified by aqueous-based fast protein liquid chromatography in 27% yield of isolated protein material. Structural characterization with MALDI-TOF MS and UV-Vis is then used to verify that the bioconjugation is successful. The protocol shown here is easily applicable to other cysteine - maleimide coupling of proteins to other proteins, dyes, drugs or polymers.

Synthesis of Protein Bioconjugates via Cysteine-maleimide Chemistry

This protocol details the important steps required for the bioconjugation of a cysteine containing protein to a maleimide, including reagent purification, reaction conditions, bioconjugate purification and bioconjugate characterization.

The chemical linking or bioconjugation of proteins to fluorescent dyes, drugs, polymers and other proteins has a broad range of applications, such as the ...

Hydroquinone Based Synthesis of Gold Nanorods

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the synthetic methods for the preparation of gold nanorods are still not fully optimized. In this paper we describe a new, highly efficient, two-step protocol based on the use of hydroquinone as a mild reducing agent. Our approach allows the preparation of nanorods with a good control of size and aspect ratio (AR) simply by varying the amount of hexadecyl trimethylammonium bromide (CTAB) and silver ions (Ag+) present in the "growth solution". By using this method, it is possible to markedly reduce the amount of CTAB, an expensive and cytotoxic reagent, necessary to obtain the elongated shape. Gold nanorods with an aspect ratio of about 3 can be obtained in the presence of just 50 mM of CTAB (versus 100 mM used in the standard protocol based on the use of ascorbic acid), while shorter gold nanorods are obtained using a concentration as low as 10 mM.

This paper describes a protocol for the synthesis of gold nanorods, based on the use of hydroquinone as reducing agent, plus the different mechanisms for controlling their size and aspect ratio.

Gold nanorods are an important kind of nanoparticles characterized by peculiar plasmonic properties. Despite their widespread use in nanotechnology, the ...

Synthesis of a Water-soluble Metal&#8211;Organic Complex Array

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as Ru(II), Pt(II), and Rh(III). The compound, named as a water-soluble metal-organic complex array (WSMOCA), is obtained through 1) the conventional solution-chemistry-based preparation of the corresponding metal complex monomers having a 9-fluorenylmethyloxycarbonyl (Fmoc)-protected amino acid moiety and 2) their sequential coupling together with other water-soluble organic building units on the surface-functionalized polymeric resin by following the procedures originally developed for the solid-phase synthesis of polypeptides, with proper modifications. Traces of reactions determined by mass spectrometric analysis at the representative coupling steps in stage 2 confirm the selective construction of a predetermined sequence of metal centers along with the peptide backbone. The WSMOCA cleaved from the resin at the end of stage 2 has a certain level of solubility in aqueous media dependent on the pH value and/or salt content, which is useful for the purification of the compound.

Synthesis of a Water-soluble Metal&amp;#8211;Organic Complex Array

A potential general method for the synthesis of water-soluble multimetallic peptidic arrays containing a predetermined sequence of metal centers is presented.

We demonstrate a method for the synthesis of a water-soluble multimetallic peptidic array containing a predetermined sequence of metal centers such as ...

Synthesis of Substrate-Bound Au Nanowires Via an Active Surface Growth Mechanism

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a challenge, as it requires asymmetric growth of symmetric crystals. Here, we report a distinctive synthesis of substrate-bound Au nanowires. This template-free synthesis employs thiolated ligands and substrate adsorption to achieve the continuous asymmetric deposition of Au in solution at ambient conditions. The thiolated ligand prevented the Au deposition on the exposed surface of the seeds, so the Au deposition only occurs at the interface between the Au seeds and the substrate. The side of the newly deposited Au nanowires is immediately covered with the thiolated ligand, while the bottom facing the substrate remains ligand-free and active for the next round of Au deposition. We further demonstrate that this Au nanowire growth can be induced on various substrates, and different thiolated ligands can be used to regulate the surface chemistry of the nanowires. The diameter of the nanowires can also be controlled with mixed ligands, in which another &#34;bad&#34; ligand could turn on the lateral growth. With the understanding of the mechanism, Au nanowire-based nanostructures can be designed and synthesized.

We report a solution-based method to synthesize substrate-bound Au nanowires. By tuning the molecular ligands used during the synthesis, the Au nanowires can be grown from various substrates with different surface properties. Au nanowire-based nanostructures can also be synthesized by adjusting the reaction parameters.

Advancing synthetic capabilities is important for the development of nanoscience and nanotechnology. The synthesis of nanowires has always been a ...

Synthesis of Functionalized Magnetic Nanoparticles, Their Conjugation with the Siderophore Feroxamine and its Evaluation for Bacteria Detection

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine, a siderophore recognized by Yersinia enterocolitica, using a succinyl moiety as a linker are described.
Magnetic nanoparticles (MNP) of magnetite (Fe3O4) were prepared by solvothermal method and coated with SiO2 (MNP@SiO2) using the St&#246;ber process followed by functionalization with APTES (MNP@SiO2@NH2). Then, feroxamine was conjugated with the MNP@SiO2@NH2 by carbodiimide coupling to give MNP@SiO2@NH2@Fa. The morphology and properties of the conjugate and intermediates were examined by eight different methods including powder X-Ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), Raman spectroscopy, X-ray photoelectron spectroscopy (XPS), transmission electron microscopy (TEM) and energy dispersive X-Ray (EDX) mapping. This exhaustive characterization confirmed the formation of the conjugate. Finally, in order to evaluate the capacity and specificity of the nanoparticles, they were tested in a capture bacteria assay using Yersinia enterocolitica.

This work describes protocols for the preparation of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with (3-aminopropyl)triethoxysilane (APTES) and its conjugation with deferoxamine using a succinyl moiety as a linker. A deep structural characterization description and a capture bacteria assay using Y. enterocolitica for all the intermediate nanoparticles and the final conjugate are also described in detail.

In the present work, the synthesis of magnetic nanoparticles, its coating with SiO2, followed by its amine functionalization with ...

Construction of Cyclic Cell-Penetrating Peptides for Enhanced Penetration of Biological Barriers

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper tumor cells, leading to tumor recurrence. To conquer the limited penetration of biological barriers, cell-penetrating peptides (CPPs) have been discovered with excellent membrane translocation ability and have emerged as useful molecular transporters for delivering various cargoes into cells. However, conventional linear CPPs generally show compromised proteolytic stability, which limits their permeability across biological barriers. Thus, the development of novel molecular transporters that can penetrate biological barriers and exhibit enhanced proteolytic stability is highly desired to promote drug delivery efficiency in biomedical applications. We have previously synthesized a panel of short cyclic CPPs with aromatic crosslinks, which exhibited superior permeability in cancer cells and tissues compared to their linear counterparts. Here, a concise protocol is described for the synthesis of the fluorescently labeled cyclic polyarginine R8 peptide and its linear counterpart, as well as key steps for investigating their cell permeability.

This protocol describes the synthesis of cyclic cell-penetrating peptides with aromatic cross-links and the evaluation of their permeability across biological barriers.

Cancer has been a grand challenge in global health. However, the complex tumor microenvironment generally limits the access of therapeutics to deeper ...

Aqueous Synthesis of Plasmonic Gold-Tin Alloy Nanoparticles

This protocol describes the synthesis of Au nanoparticle seeds and the subsequent formation of Au-Sn bimetallic nanoparticles. These nanoparticles have potential applications in catalysis, optoelectronics, imaging, and drug delivery. Previously, methods for producing alloy nanoparticles have been time-consuming, require complex reaction conditions, and can have inconsistent results. The outlined protocol first describes the synthesis of approximately 13 nm Au nanoparticle seeds using the Turkevich method. The protocol next describes the reduction of Sn and its incorporation into the Au seeds to generate Au-Sn alloy nanoparticles. The optical and structural characterization of these nanoparticles is described. Optically, prominent localized surface plasmon resonances (LSPRs) are apparent using UV-visible spectroscopy. Structurally, powder X-ray diffraction (XRD) reflects all particles to be less than 20 nm and shows patterns for Au, Sn, and multiple Au-Sn intermetallic phases. Spherical morphology and size distribution are obtained from transmission electron microscopy (TEM) imaging. TEM reveals that after Sn incorporation, the nanoparticles grow to approximately 15 nm in diameter.

Author Spotlight: Designing Sustainable Nanomaterials for Advancing Synthesis and Element Mixing

Here, the synthesis of gold (Au) seeds is described using the Turkevich method. These seeds are then used to synthesize gold-tin alloy (Au-Sn) nanoparticles with tunable plasmonic properties.

This protocol describes the synthesis of Au nanoparticle seeds and the subsequent formation of Au-Sn bimetallic nanoparticles. These nanoparticles have ...