סינתזה של Indoxyl-גליקוזידים לאיתור של פעילויות glycosidase

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

גליקוזידים Indoxyl הוכיחו להיות כלי רב ערך ורב-תכליתי לפעילות glycosidase הניטור. Indoxyls פורסם על ידי הידרוליזה אנזימטית ומהירות חמצון, למשל על ידי חמצן אטמוספרי, לאינדיגו צבעי סוג. תגובה זו מאפשרת הקרנה מהירה וקלה in vivo בלי בידוד או טיהור של אנזימים, כמו גם בדיקות מהירות על צלחות אגר או בפתרון (לדוגמא, כחולה-לבנה הקרנה, מיקרו-בארות) ומשמש בביוכימיה, היסטוכימיה, בקטריולוגיה ומולקולרית ביולוגיה. למרבה הצער הסינתזה של מצעים כגון הוכיחה להיות קשה, עקב בצד תגובות שונות ותגובתיות הנמוכה של פונקצית הידרוקסיל indoxyl. במיוחד למבנים מסוג גלוקוז תשואות נמוכות נצפו. הגישה החדשנית שלנו מעסיקה אסתר חומצת indoxylic כביניים מפתח. אסטרים חומצת Indoxylic עם דפוסי החלפה מגוונים הוכנו על מסלולי מדרגי. glycosylations העברת שלב עם אלה acceptors וperacetylatedניתן לבצע הלידים glycosyl בתנאים מקובלים בתשואות גבוהות. מחשוף אסתר וglycosylation כסף בתיווך קל לאחר מכן מניב גליקוזידים indoxyl peracetylated בתשואות גבוהות. לבסוף deprotection מתבצע על פי 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 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: 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 donor^6-14. 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 …

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