Синтез индоксил-гликозидов для обнаружения гликозидазой деятельности

Резюме

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.

Аннотация

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.

Введение

В течение долгого времени производство индиго был экономически очень важный процесс. Перед крупномасштабных химических синтезов дал дешевый доступ к индиго, предшественники были получены из природных источников с дохристианских времен. Выращивание индиго обеспечения растений (естественно индиго) в Европе стал неблагодарным в ^17-м веке, как количество индиго предшественники индийской индиго (0,2-0,8%) составляет около 30 раз выше. В конце 19-го века химического синтеза индиго подавил обычный выращивание ^1,2.

Indigo предшественники, происходящие в растениях включают индикана (1), Insatan A (2) и Isatan B (3) (рис 1). Все они состоят из индоксил мотива, связанного с гликозильного остатка. Расщепление гликозидной связи, например, с помощью ферментативного гидролиза, приводит к выделению индоксил (4). Сама индоксил практически бесцветны, но могут быть быстро окисляется с образованием индиго Краситель (5). Это чувствительная реакция была адаптирована в биохимии, бактериологии, гистохимии и молекулярной биологии для мониторинга активности ферментов. Скрининг активности в естественных условиях без выделения или очистки ферментов, а также экспресс-тесты на чашках с агаром или в растворе (например, бело-голубым скрининга, микро-скважин) возможно. В зависимости от остатка (например, сложные эфиры, гликозиды, сульфаты), связанного с индоксил фрагмента, пригодными субстратами в различных классов ферментов (например, эстераз, глюкозидазы, сульфатазы) были разработаны ^3. В следующем внимание будет уделено формированию и применению индоксил гликозидов.

Рисунок 1: Природные предшественники индиго и формирование красителя индиго путем гидролиза.цель = "_ пустое"> Пожалуйста, нажмите здесь, чтобы посмотреть большую версию этой фигуры.

Замещение картина индоксил фрагмента определяет цвет и физические свойства полученного красителя индиго. Наиболее распространенные шаблоны замещения 5-бром-4-хлор (сокращенно от X; зеленовато-голубой), 5-бром (синий) и 5-бром-6-хлор (пурпурный), так как они образуют мельчайшие частицы красителя, делают не образуют гранулы и имеют наименьший диффузию из участков гидролиза. Последнее свойство особенно важно для естественных условиях экспериментов в ^3.

Первый доклад о indigogenic метода для обнаружения эстеразы был опубликован в 1951 году и Barrnett Селигман, который занятого индоксил ацетат и бутират ^4. О десятилетие спустя indigogenic принцип был адаптирован для локализации глюкозидазы млекопитающих ^5. До сих пор несколько индоксил гликозидов были разработаны, хотя их синтез оказался трудно. Большинство синтезов на основе использования в -acetylated индоксил N в качестве акцептора и соответствующих гликозилгалогенидом донора ^6-14. Гликозилирование проводят в ацетоне с помощью гидроксида натрия. В этих условиях количество побочных реакций происходит, значительно уменьшая выход. Специально для глюкозы структур типа очень низкие выходы гликозилирования сообщалось (например, 15% (N-ацетил-5-бром-4-хлор-индол-3-ил) -2,3,4,6-тетра-О-ацетил -β-ᴅ-глюкопиранозид ⁶ и 26% (N-ацетил-5-бром-4-хлор-индол-3-ил) -2,3,2 ', 3', 4'-пента вывода -acetyl- β-ᴅ-xylobioside ¹⁴ в более недавний пример). Через новый подход, используя сложные эфиры indoxylic кислоты, значительное количество индоксил гликозидов были получены с хорошим выходом (например, (N-ацетил-5-бром-4-хлор-индол-3-ил) -2,3,4, 6-тетра-О-ацетил-β-ᴅ-глюкопиранозид выход 57%).

_content "> Следующий протокол описывает простой синтез indoxylic кислоты аллилового эфира (5-бром-4-хлор) и на их основе синтеза с индоксил гликозида (X-Gal). Простой модельный эксперимент показывает ферментную реактивность β- галактозидазы с использованием X-Gal.

протокол

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 Büchner funnel and aspirator pump. Solvents were removed employing a rotary evaporator (40 °C, ~200 rmp). Products were dried in high vacuum (rotary vane pump). Reactions requiring dry solvents were carried out under argon atmosphere.

In the following the synthesis of X-Gal (16) employing 5-bromo-4-chloro-indoxylic acid allyl ester (12) is described. For further compounds with varied substitution patterns as well as methyl esters prepared according to this synthetic sequence see ref.^17-19.

1. Acceptor: Synthesis of 5-Bromo-4-Chloro-Indoxylic Acid Allyl Ester¹⁸ (12)

1. N-Acetylation: Synthesis of N-(4-bromo-3-chloro-2-methylphenyl)-acetamide (6)
   1. Dissolve 10.0 g (45.7 mmol) of 4-bromo-3-chloro-2-methylaniline in 30 ml dichloromethane.
      Caution! Dichloromethane is harmful and cool the solution in an ice-bath.
   2. Add 6.50 ml (68.7 mmol) acetic anhydride dropwise and allow the mixture to warm to room temperature. The mixture can be stirred for 8 hr or overnight. Caution! Acetic anhydride is corrosive and flammable.
   3. Remove the solvent and recrystallize from 9:1 ethyl acetate/methanol (yield: 97% (11.6 g, 44.2 mmol), colorless solid).
      Caution! Ethyl acetate is irritant and flammable. Caution! Methanol is toxic and flammable.
2. Oxidation: Synthesis of N-acetyl-5-bromo-6-chloro-anthranilic acid (7)
   1. Reflux a mixture of 2.30 g (8.76 mmol) of 6 (product obtained from step 1.1) and 2.10 g (8.52 mmol) MgSO₄•7H₂O in 40 ml water in a 3-neck round bottom flask equipped with cooler and dropping funnel.
   2. Add dropwise 50 ml saturated KMnO₄ solution in water to the reaction mixture over a period of 3 hr. Then heat for 2 additional hr under reflux, ca. 135 °C oil bath. Caution! Potassium permanganate is harmful and oxidising.
   3. Cool to room temperature and filter the pyrolusite (MnO₂), which is formed during the reaction.
      Caution! Pyrolusite is harmful.
   4. Add hydrochloric acid (37%) to the filtrate until pH 1, then filter the product and dry in vacuum, (yield: 70% (1.80 g, 6.15 mmol), colorless solid).
      Caution! Hydrochloric acid is corrosive.
3. Deacetylation: Synthesis of 5-bromo-6-chloro-anthranilic acid (8)
   1. Dissolve 3.90 g (13.3 mmol) of 7 (product obtained from step 1.2) in 70 ml of 1 N aqueous sodium hydroxide solution and heat at ca. 100 °C (oil bath) for 5 hr.
      Caution! Sodium hydroxide is corrosive.
   2. Cool to room temperature and add hydrochloric acid (37%) until pH 1, then filter the product with a Büchner funnel and dry in vacuum, (yield: 88% (2.93 g, 11.7 mmol), colorless solid).
4. Anhydride formation: Synthesis of 5-bromo-6-chloro-isatoic anhydride (9)
   1. Suspend 8.17 g (32.6 mmol) of 8 (product obtained from step 1.3) in 33 ml dry acetonitrile.
      Caution! Acetonitrile is harmful and flammable.
   2. Stir at room temperature and add 5.3 ml and a solution of 3.22 g (10.8 mmol) triphosgene in 18.5 ml dry dichloromethane dropwise simultaneously over a period of 30 min. Then heated for 3 hr at 50 °C (oil bath). Caution! Pyridine is harmful and flammable.
      Caution! Triphosgene is toxic and can release phosgene.
   3. Remove about 75% of the solvent and quench the reaction by adding 100 ml distilled water and filter your product. Wash with a small amount of cold dichloromethane.
   4. Dry the product in vacuum, (yield: 88% (7.53 g, 27.2 mmol), colorless solid).
5. N-Alkylation: Synthesis of 5-bromo-6-chloro-N-[(methoxycarbonyl)allyl]-isatoic anhydride (10)
   1. Dissolve 25 g (90 mmol) of 9 (product obtained from step 1.4) in 250 ml anhydrous dimethylformamide. and cool with an ice-bath.
      Caution! Dimethylformamide is harmful and flammable
   2. Add 1.15 equivalents of sodium hydride (60% in paraffin) portion wise.
      Caution! Sodium hydride is corrosive and flammable; heavy reaction with water/ice.
   3. After 30 min of stirring at 0 °C allow warming to room temperature and add 1.2 equivalents allyl bromoacetate dropwise.
   4. After stirring for 5 hr quench the reaction by adding 500 ml distilled water and filter your product.
   5. Wash with water (3 times, 100 ml) and dry the product in vacuum, (yield: 93% (31.5 g, 84.1 mmol), colorless solid).
6. Anhydride Opening: Synthesis of 5-bromo-6-chloro-N-(methoxycarbonylallyl)-anthranilic acid allyl ester (11)
   1. Dissolve 10 g (27 mmol) of 10 (product obtained from step 1.5) in 100 ml allyl alcohol.
      Caution! Allyl alcohol is harmful and dangerous for the environment.
   2. Add 350 mg (8.70 mmol) sodium hydride (60% in paraffin) portion wise.
   3. After stirring for 6.5 hr or overnight remove the solvent.
   4. Subject the crude product to column chromatography (column 30 x 6 cm; 240 g silica) petroleum ether/ethyl acetate 2:1; R_F = 0.66), (yield: 80% (8.30 g, 21.3 mmol), yellow oil).
      Caution! Petroleum ether is flammable, irritant and dangerous for the environment.
7. Dieckmann Condensation: Synthesis of 5-bromo-4-chloro-indoxylic acid allyl ester (12)
   1. Mix 3.0 g (7.7 mmol) of 11(product obtained from step 1.6) and 1.75 g (15.6 mmol) of potassium tert-butoxide in 100 ml dry diethyl ether.
      Caution! Potassium tert-butoxide is corrosive and flammable.
      Caution! Diethyl ether is harmful and extremely flammable.
   2. Heat the mixture under reflux (cooler) for 2 hr at 40-45 °C.
   3. Remove about 75% of the solvent, and add diluted hydrochloric acid (100 ml, 1 M).
      Caution! Do not dry completely, decomposition can occur.
   4. Filter the product and dry in vacuum, (yield: 84% (5.14 g, 15.5 mmol), colorless to slightly green solid).

2. Indoxyl-Glycoside: Synthesis of X-Gal¹⁷

1. Phase Transfer Glycosylation: Synthesis of (5-bromo-4-chloro-indox-3-ylic acid allyl ester) 2,3,4,6-tetra-O-acetyl-β-ᴅ-galactopyranoside (14)
   1. Mix 400 mg (1.21 mmol) of the indoxylic acid allylester 12 (product obtained from step 1.7), 410 mg (1.21 mmol) tetrabutylammonium hydrogensulfate and 500 mg (1.21 mmol) of the donor 2,3,4,6-tetra-O-acetyl-α-ᴅ-galactopyranosyl bromide (α-acetobromogalactose) in 10 ml dichloromethane. Caution! Tetrabutylammonium hydrogensulfate is harmful.
   2. Add 10 ml of an aqueous solution of potassium carbonate (1 M).
      Caution! Potassium carbonate is corrosive.
   3. Stir at room temperature until complete consumption of the donor (TLC: petroleum ether/ethyl acetate).
   4. Separate the organic phase (separatory funnel), dry over Na₂SO₄ and remove the solvent.
   5. Subject the crude product to column chromatography (petroleum ether/ethyl acetate 1:1; R_F = 0.33). (yield: 86% (690 mg, 1.04 mmol), colorless solid).
2. Ester Cleavage and Decarboxylation: Synthesis of (N-acetyl-5-bromo-4-chloro-indol-3-yl) 2,3,4,6-tetra-O-acetyl-β-ᴅ-galactopyranoside (15)
   1. Dissolve 600 mg (0.908 mmol) of 14 (product obtained from step 2.1) in 15 ml dry tetrahydrofurane.
      Caution! Tetrahydrofurane is harmful and flammable.
   2. Add 800 μl morpholine and 105 mg (9.09 μmol) tetrakis(triphenylphosphine)palladium(0) and stir overnight at room temperature.
      Caution! Morpholine is flammable and corrosive.
   3. Remove the solvent.
   4. Add 400 mg (2.40 mmol) silver acetate, 800 mg (5.79 mmol) potassium carbonate and 10 ml acetic anhydride.
      Caution! Silver acetate is irritant and dangerous for the environment.
   5. Heat at 90-100 °C for 20 min.
   6. After cooling to room temperature dilute with dichloromethane.
   7. Wash the mixture twice with water and once with a diluted NaHCO₃ solution (10%).
      Caution! Sodium bicarbonate is corrosive.
   8. Dry the organic phase over Na₂SO₄ (filter) and remove the solvent.
   9. Subject the crude product to column chromatography (PE/EA 1:1; R_F = 0.24), (yield: 88% (495 mg, 8.00 mmol), colorless solid).
3. Zemplén Deacetylation: Synthesis of (5-bromo-4-chloro-indol-3-yl)-β-ᴅ-galactopyranoside (16)
   1. Dissolve 396 mg (0.436 mmol) 15 (product obtained from step 2.2) in 10 ml methanol.
   2. Add a catalytic amount of sodium methanolate and stir overnight at room temperature.
      Caution! Sodium methanolate is flammable and corrosive.
   3. Neutralize with Amberlite IR-120 H⁺ (filter) and concentrate.
      Caution! Amberlite IR-120 H⁺ is irritant.
   4. Dry the product in vacuum, (yield: 87% (170 mg, 0.416 mmol), colorless solid).

3. Illustrating Model Experiment: Activity proof of β-galactosidase employing X-Gal

1. Prepare a 0.5 mM solution of X-Gal in 500 μl buffer (Tris-HCl 20 mM; pH 7.4; 50 mM NaCl, 0.1 mM ethylenediaminetetraacetic acid; or any other suitable buffer for your enzyme) in an microcentrifuge tube (X-Gal can be dissolved in a few μl of dimethylsulfoxide).
2. Add 1 μl β,1-3-galactosidase (EC 3.2.1.23 10,000 U/ml; or any other suitable β-galactosidase) and shake (600 rps) at 37 °C.
3. Follow the reaction. The indigo type dye is formed after a short period of time.

Результаты

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...

Обсуждение

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 ...

Материалы

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