Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Summary

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 17^th 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 cultivation^1,2.

Indigo precursors occurring naturally in plants include Indican (1), Insatan A (2) and ....

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) and subsequent heating. Column chromatography was performed with Merck/Fluka silica gel 60 (230-400 mesh). Filtrations were performed using Büc....

Results

The very first syntheses of indicane and 5-bromo-indicane, were published by Robertson already in 1927 and 1929^15,16. 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 glyc.......

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 .......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
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
β1,3-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