Özet

Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Published: May 27, 2015
doi:

Özet

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.

Abstract

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 Zemplén.

Introduction

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.

Figure 1
Figure 1: Natural indigo precursors and formation of indigo dye by hydrolysis. Please click here to view a larger version of this figure.

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-β-ᴅ-glucopyranoside6 and 26% for (N-acetyl-5-bromo-4-chloro-indol-3-yl)-2,3,2',3',4'-penta-O-acetyl-β-ᴅ-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-β-ᴅ-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 β-galactosidase employing X-Gal.

Protocol

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 <50 ppm; Et2O <0.01%; CH2Cl2 <0.003%; THF <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…

Representative Results

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 °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…

Discussion

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 …

Açıklamalar

The authors have nothing to disclose.

Acknowledgements

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

Materials

Name of Material/ Equipment Company Catalog Number Comments/Description
Acetic anhydride Grüssing 10298 Corrosive, flammable
Acetonitrile Sigma-Aldrich 608-001-00-3 Harmful, flammable
Allyl alcohol Aldrich 453021 Harmful, dangerous for the environment
Amberlite IR-120 H+ Fluka 06428 Irritant
Bromoacetic acid Merck 802260 Corrosive, toxic, dangerous for the environment
4-Bromo-3-chloro-2-methylaniline ABCR AB 171687 Irritant
Dichloromethane ACROS 326850010 Harmful
Diethyl ether Grüssing 10274 Harmful, extremly flammable
Dimethylformamide ACROS 348430010 Harmful, flammable
Dimethylsulfoxide Sigma-Aldrich 41648
Ethyl acetate Sigma-Aldrich 607-022-00-5 Irritant, flammable
Ethylenediaminetetraacetic acid AppliChem A1103.0500 Irritant
ß1,3-Galactosidase, Recombinant, E. coli Calbiochem 345795
Hydrochloric acid VWR  20252.290 Corrosive
Magnesium sulfate hydrate Merck 105885
Methanol ACROS 326950010 Toxic, flammable
Morpholine Janssen Chimica 15.868.57 Corrosive, flammable
Peroleum ether Azelis 111053 Flammable, irritant, dangerous for the environment
Potassium carbonate Grüssing 12005 Corrosive
Potassium permanganate Grüssing 12056 Harmful, oxidising
Potassium tert-butoxide Merck 804918 Corrosive, flammable
Pyridine Sigma-Aldrich 613-002-00-7 Harmful, flammable
Silver acetate Fluka 85140 Irritant, dangerous for the environment
Sodium bicarbonate Grüssing 12144 Corrosive
Sodium hydride Merck 814552 Corrosive, flammable
Sodium hydroxide Riedel-de Häen S181200 Corrosive
Sodium methanolate Merck 806538 Corrosive, flammable
Sodium sulfate Grüssing 12175
Tetrabutylammonium hydrogensulfate Lancaster 5438 Harmful
Tetrahydrofurane Sigma-Aldrich 87371 Harmful, flammable
Tetrakis(triphenylphosphine)palladium(0) Sigma-Aldrich 216666
Triphosgene Fluka 15217 Toxic
Tris(hydroxymethyl)aminomethane hydrochloride  Sigma T-3253 Irritant

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Böttcher, S., Thiem, J. Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities. J. Vis. Exp. (99), e52442, doi:10.3791/52442 (2015).

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