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) and subsequent heating. Column chromatography was performed with Merck/Fluka silica gel 60 (230-400 mesh). Filtrations were performed using B&#252;chner funnel and aspirator pump. Solvents were removed employing a rotary evaporator (40 &#176;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)
<ol>
	<li>N-Acetylation: Synthesis of N-(4-bromo-3-chloro-2-methylphenyl)-acetamide (6)
	<ol>
		<li>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.</li>
		<li>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.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Oxidation: Synthesis of N-acetyl-5-bromo-6-chloro-anthranilic acid (7)
	<ol>
		<li>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&#8226;7H2O in 40 ml water in a 3-neck round bottom flask equipped with cooler and dropping funnel.</li>
		<li>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 &#176;C oil bath. Caution! Potassium permanganate is harmful and oxidising.</li>
		<li>Cool to room temperature and filter the pyrolusite (MnO2), which is formed during the reaction. 
		Caution! Pyrolusite is harmful.</li>
		<li>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.</li>
	</ol>
	</li>
	<li>Deacetylation: Synthesis of 5-bromo-6-chloro-anthranilic acid (8)
	<ol>
		<li>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 &#176;C (oil bath) for 5 hr. 
		Caution! Sodium hydroxide is corrosive.</li>
		<li>Cool to room temperature and add hydrochloric acid (37%) until pH 1, then filter the product with a B&#252;chner funnel and dry in vacuum, (yield: 88% (2.93 g, 11.7 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Anhydride formation: Synthesis of 5-bromo-6-chloro-isatoic anhydride (9)
	<ol>
		<li>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.</li>
		<li>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 &#176;C (oil bath). Caution! Pyridine is harmful and flammable. 
		Caution! Triphosgene is toxic and can release phosgene.</li>
		<li>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.</li>
		<li>Dry the product in vacuum, (yield: 88% (7.53 g, 27.2 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>N-Alkylation: Synthesis of 5-bromo-6-chloro-N-[(methoxycarbonyl)allyl]-isatoic anhydride (10)
	<ol>
		<li>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</li>
		<li>Add 1.15 equivalents of sodium hydride (60% in paraffin) portion wise. 
		Caution! Sodium hydride is corrosive and flammable; heavy reaction with water/ice.</li>
		<li>After 30 min of stirring at 0 &#176;C allow warming to room temperature and add 1.2 equivalents allyl bromoacetate dropwise.</li>
		<li>After stirring for 5 hr quench the reaction by adding 500 ml distilled water and filter your product.</li>
		<li>Wash with water (3 times, 100 ml) and dry the product in vacuum, (yield: 93% (31.5 g, 84.1 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Anhydride Opening: Synthesis of 5-bromo-6-chloro-N-(methoxycarbonylallyl)-anthranilic acid allyl ester (11)
	<ol>
		<li>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.</li>
		<li>Add 350 mg (8.70 mmol) sodium hydride (60% in paraffin) portion wise.</li>
		<li>After stirring for 6.5 hr or overnight remove the solvent.</li>
		<li>Subject the crude product to column chromatography (column 30 x 6 cm; 240 g silica) petroleum ether/ethyl acetate 2:1; RF = 0.66), (yield: 80% (8.30 g, 21.3 mmol), yellow oil). 
		Caution! Petroleum ether is flammable, irritant and dangerous for the environment.</li>
	</ol>
	</li>
	<li>Dieckmann Condensation: Synthesis of 5-bromo-4-chloro-indoxylic acid allyl ester (12)
	<ol>
		<li>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.</li>
		<li>Heat the mixture under reflux (cooler) for 2 hr at 40-45 &#176;C.</li>
		<li>Remove about 75% of the solvent, and add diluted hydrochloric acid (100 ml, 1 M). 
		Caution! Do not dry completely, decomposition can occur.</li>
		<li>Filter the product and dry in vacuum, (yield: 84% (5.14 g, 15.5 mmol), colorless to slightly green solid).</li>
	</ol>
	</li>
</ol>
2. Indoxyl-Glycoside: Synthesis of X-Gal17
<ol>
	<li>Phase Transfer Glycosylation: Synthesis of (5-bromo-4-chloro-indox-3-ylic acid allyl ester) 2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-galactopyranoside (14)
	<ol>
		<li>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-&#945;-&#7429;-galactopyranosyl bromide (&#945;-acetobromogalactose) in 10 ml dichloromethane. Caution! Tetrabutylammonium hydrogensulfate is harmful.</li>
		<li>Add 10 ml of an aqueous solution of potassium carbonate (1 M). 
		Caution! Potassium carbonate is corrosive.</li>
		<li>Stir at room temperature until complete consumption of the donor (TLC: petroleum ether/ethyl acetate).</li>
		<li>Separate the organic phase (separatory funnel), dry over Na2SO4 and remove the solvent.</li>
		<li>Subject the crude product to column chromatography (petroleum ether/ethyl acetate 1:1; RF = 0.33). (yield: 86% (690 mg, 1.04 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Ester Cleavage and Decarboxylation: Synthesis of (N-acetyl-5-bromo-4-chloro-indol-3-yl) 2,3,4,6-tetra-O-acetyl-&#946;-&#7429;-galactopyranoside (15)
	<ol>
		<li>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.</li>
		<li>Add 800 &#956;l morpholine and 105 mg (9.09 &#956;mol) tetrakis(triphenylphosphine)palladium(0) and stir overnight at room temperature. 
		Caution! Morpholine is flammable and corrosive.</li>
		<li>Remove the solvent.</li>
		<li>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.</li>
		<li>Heat at 90-100 &#176;C for 20 min.</li>
		<li>After cooling to room temperature dilute with dichloromethane.</li>
		<li>Wash the mixture twice with water and once with a diluted NaHCO3 solution (10%). 
		Caution! Sodium bicarbonate is corrosive.</li>
		<li>Dry the organic phase over Na2SO4 (filter) and remove the solvent.</li>
		<li>Subject the crude product to column chromatography (PE/EA 1:1; RF = 0.24), (yield: 88% (495 mg, 8.00 mmol), colorless solid).</li>
	</ol>
	</li>
	<li>Zempl&#233;n Deacetylation: Synthesis of (5-bromo-4-chloro-indol-3-yl)-&#946;-&#7429;-galactopyranoside (16)
	<ol>
		<li>Dissolve 396 mg (0.436 mmol) 15 (product obtained from step 2.2) in 10 ml methanol.</li>
		<li>Add a catalytic amount of sodium methanolate and stir overnight at room temperature. 
		Caution! Sodium methanolate is flammable and corrosive.</li>
		<li>Neutralize with Amberlite IR-120 H+ (filter) and concentrate. 
		Caution! Amberlite IR-120 H+ is irritant.</li>
		<li>Dry the product in vacuum, (yield: 87% (170 mg, 0.416 mmol), colorless solid).</li>
	</ol>
	</li>
</ol>
3. Illustrating Model Experiment: Activity proof of &#946;-galactosidase employing X-Gal
<ol>
	<li>Prepare a 0.5 mM solution of X-Gal in 500 &#956;l 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 &#956;l of dimethylsulfoxide).</li>
	<li>Add 1 &#956;l &#946;,1-3-galactosidase (EC 3.2.1.23 10,000 U/ml; or any other suitable &#946;-galactosidase) and shake (600 rps) at 37 &#176;C.</li>
	<li>Follow the reaction. The indigo type dye is formed after a short period of time.</li>
</ol>

<ol>
	<li>Clark, R. J. H., Cooksey, C. J., Daniels, M. A. M., Withnall, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indigo,+Woad,+and+Tyrian+Purple:+Important+Vat+Dyes+from+Antique+to+the+Present.">Indigo, Woad, and Tyrian Purple: Important Vat Dyes from Antique to the Present.</a> Endeavour. 17, 191-199 (1993).</li><li>Hunger, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Industrial Dyes: Chemistry, Properties, Applications. , (2003).</li><li>Kiernan, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indigogenic+Substrates+for+Detection+and+Localization+of+Enzymes.">Indigogenic Substrates for Detection and Localization of Enzymes.</a> Biotechn. Histochem. 82, 73-103 (2007).</li><li>Barnett, R. 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. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthese+of+Glucosides.+Part+VII.+The+Synthesis+of+6-Bromoindican.">Synthese of Glucosides. Part VII. The Synthesis of 6-Bromoindican.</a> Chem. Soc. , 2239-2243 (1929).</li><li>B&#246;ttcher, S., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Indoxylic+Acid+Esters+as+Convenient+Intermediates+Towards+Indoxyl+Glycosides.">Indoxylic Acid Esters as Convenient Intermediates Towards Indoxyl Glycosides.</a> Eur. J. Org. Chem. , 564-574 (2014).</li><li>B&#246;ttcher, S., Hederos, M., Champion, E., D&#233;k&#225;ny, G., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+Efficient+Routes+to+Indoxyl+Glycosides+for+Monitoring+Glycosidase+Activities.">Novel Efficient Routes to Indoxyl Glycosides for Monitoring Glycosidase Activities.</a> Org. Lett. 15, 3766-3769 (2013).</li><li>B&#246;ttcher, S., Thiem, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Facile+Preparation+of+Indoxyl-+and+Nitrophenyl+Glycosides+of+Lactosamine+and+Isolactosamine.">Facile Preparation of Indoxyl- and Nitrophenyl Glycosides of Lactosamine and Isolactosamine.</a> RSC Adv. 4, 10856-10861 (2014).</li></ol>

The authors declare that they have no competing financial interests.

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 are suppressed. Employing allyl esters allows selective cleavage, which is especially important on substrates containing a carboxyl function.
Phase transfer glycosylation of these acceptors was performed with the respective peracetylated glycosyl halide and succeeded in high yields under common conditions. Afterwards the allyl ester was cleaved selectively with a palladium catalyst, and the crude product was subjected to mild silver mediated decarboxylation. This is the key step of the synthesis. It was especially important to keep the reaction time short and the temperature as low as possible. The best conditions were found employing silver acetate. Finally the acetyl protecting groups were removed as usual according to Zempl&#233;n conditions. This way, we could obtain a number of indoxyl glycosides in the best yields to date.
A model experiment showed the enzymatic hydrolysis of the synthesized X-Gal employing a &#946;-galactosidase. The formation of the blue dye in solution clearly indicates enzyme activity. Further elaborations of this concept could deal with &#945;-glycosylations of the indoxylic acid allylesters.

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.remotexs.ntu.edu.sg/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 glycosides, employing indoxylic acid allyl esters as acceptors17-19. The acceptor synthesis starts with the respective substituted aniline derivative (5) (Figure 2). After acetylation of the amino function (6), oxidation with potassium permanganate yielded the N-acetylated anthranilic acid derivatives (7). Then the acetyl protecting group was removed (8) and by reaction with triphosgene the respective isatoic anhydride obtained (9). N-Alkylation could be easily performed by deprotonation with sodium hydride and subsequent treatment with allyl bromoacetate (10). The anhydride was then opened with allyl alcohol (11), and finally ring closure was performed in a Dieckmann condensation (12). With one exception (11), all compounds were purified by recrystallization, precipitation or washing. The indoxylic acid allyl ester was obtained in 30% overall yield (seven steps).
<img alt="Figure 2" src="/files/ftp_upload/52442/52442fig2highres.jpg" width="700" /> 
Figure 2: Synthetic sequence towards 5-bromo-4-chloro-indoxylic acid allyl ester. Reagents and conditions: (i) dichloromethane, acetic anhydride, 0 &#176;C - rt, 5 hr; (ii) H2O, MgSO4, KMnO4, reflux, 6 hr; (iii) sodium hydroxide (1 N), reflux, 5 hr; (iv) acetonitrile, dichloromethane, pyridine, triphosgene, 50 &#176;C, 4 hr; (v) dimethylformamide, sodium hydride (60% paraffin), allyl bromoacetate, 0 &#176;C - rt, 5 hr; (vi) allyl alcohol, sodium hydride, rt, overnight; (vii) diethyl ether, potassium tert-butoxide, reflux, 2 hr. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52442/52442fig2highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
After high yielding phase transfer glycosylation (14) of the indoxylic acid allyl ester (12) under common conditions with a peracetylated glycosyl halide (13), the allyl ester was cleaved selectively with a palladium catalyst and morpholine. The subsequent decarboxylation was performed under significant milder conditions (90-110 &#176;C, 20-30 min) without purification of the staring material (15). Finally the acetyl protecting groups were removed (16) according to Zempl&#233;n employing sodium methanolate in anhydrous methanol. This way a three step glycosylation procedure gave the final indoxyl galactopyranoside component (16) in 66% overall yield. In a former attempt the synthesis of a corresponding indoxyl galactopyranoside resulted in 28% yield6.
<img alt="Figure 3" src="/files/ftp_upload/52442/52442fig3highres.jpg" width="700" /> 
Figure 3: Synthetic sequence towards X-Gal. Reagents and conditions: (i) dichloromethane, potassium carbonate (1 M), tetrabutylammonium hydrogensulfate, rt, 3-5 hr (TLC); (ii) (a) tetrahydrofurane, morpholine, tetrakis(triphenylphosphine)palladium(0), rt, overnight; (b) acetic anhydride, silver acetate, potassium carbonate, 90-110 &#176;C, 20 min. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52442/52442fig3highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/52442/52442fig4highres.jpg" width="450" /> 
Figure 4: Blue-white screening: An agar plate containing the respective indoxyl glycoside is treated with bacteria colonies expressing the corresponding matching enzyme. The released indoxyl is oxidized to give the indigo dye which occurs as blue spots. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52442/52442fig4highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/52442/52442fig5highres.jpg" width="650" /> 
Figure 5: Enzyme activity monitoring in micro wells. The upper row contains 0.1 mM of X-glycoside, the lower row has a concentration of 1 mM of X-glycoside. The blue color occurs only in the wells containing the enzymes to cleave the glycosidic linkage of the X-glycoside. A concentration of 0.1 mM is already sufficient. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52442/52442fig5highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/52442/52442fig6highres.jpg" width="500" /> 
Figure 6: Enzyme activity monitoring in Eppendorf tubes. After a short period of time the blue color evolves and is getting more intense. In the end the indigo dye precipitates. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/52442/52442fig6highres.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities at JoVE.com

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.

グリコシダーゼ活性の検出のためのインドキシル配糖体の合成

Indoxyl glycosides proved to be valuable and versatile tools for monitoring glycosidase activities. Indoxyls are released by enzymatic hydrolysis and are ...

synthesis-indoxyl-glycosides-for-detection-glycosidase

Nanyang Technological University

Research

Education

JoVE Core

JoVE Journal

Organic Chemistry

Chemistry

Unit 1

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

SCIENTISTS IN ACTION

6.6K ビュー.  University of Hamburg. 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.

ビデオ: Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

Seeded Synthesis of CdSe/CdS Rod and Tetrapod Nanocrystals

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded synthesis strategy is used in which spherical seeds of CdSe are prepared first using a hot-injection technique. By controlling the crystal structure of the seed to be either wurtzite or zinc-blende, the subsequent hot-injection growth of CdS off of the seed results in either a rod-shaped or tetrapod-shaped nanocrystal, respectively. The phase and morphology of the synthesized nanocrystals are confirmed using X-ray diffraction and transmission electron microscopy, demonstrating that the nanocrystals are phase-pure and have a consistent morphology. The extinction coefficient and quantum yield of the synthesized nanocrystals are calculated using UV-Vis absorption spectroscopy and photoluminescence spectroscopy. The rods and tetrapods exhibit extinction coefficients and quantum yields that are higher than that of the bare seeds. This synthesis demonstrates the precise arrangement of materials that can be achieved at the nanoscale by using a seeded synthetic approach.

A protocol for the seeded synthesis of rod-shaped and tetrapod-shaped multicomponent nanostructures consisting of CdS and CdSe is presented.

のCdSe / CdSのロッドやテトラポッドナノ結晶のシードの合成

We demonstrate a method for the synthesis of multicomponent nanostructures consisting of CdS and CdSe with rod and tetrapod morphologies. A seeded ...

化学

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.

生成Cyanocarbenesの適用のための超原子価ヨードニウムアルキニルトリフラートの合成

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

Synthesis of Cd-free InP/ZnS Quantum Dots Suitable for Biomedical Applications

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, quantum dots should be highly fluorescent and small/monodisperse in size. While commonly used cadmium-based quantum dots possess these qualities, they are potentially toxic due to the possible release of Cd2+ ions through nanoparticle degradation. Indium-based quantum dots, specifically InP/ZnS, have recently been explored as a viable alternative to cadmium-based quantum dots due to their relatively similar fluorescence characteristics and size. The synthesis presented here uses standard hot-injection techniques for effective nanoparticle growth; however, nanoparticle properties such as size, emission wavelength, and emission intensity can drastically change due to small changes in the reaction conditions. Therefore, reaction conditions such temperature, reaction duration, and precursor concentration should be maintained precisely to yield reproducible products. Because quantum dots are not inherently soluble in aqueous solutions, they must also undergo surface modification to impart solubility in water. In this protocol, an amphiphilic polymer is used to interact with both hydrophobic ligands on the quantum dot surface and bulk solvent water molecules. Here, a detailed protocol is provided for the synthesis of highly fluorescent InP/ZnS quantum dots that are suitable for use in biomedical applications.

In this protocol, the synthesis of Cd-free InP/ZnS quantum dots (QDs) is detailed. InP-based QDs are gaining popularity due to the toxicity of Cd2+ ions that may be released through nanoparticle degradation. After synthesis, QDs are solubilized in water using an amphiphilic polymer for use in biomedical applications.

バイオメディカル応用のための適切なカドミウムフリーのInP / ZnSの量子ドットの合成

Fluorescent nanocrystals, specifically quantum dots, have been a useful tool for many biomedical applications. For successful use in biological systems, ...

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.

の合成のためのプロトコル<em&gt;オルソ</em&gt; -trifluoromethoxylatedアニリン誘導体

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.

超分子コロイドの合成とキャラクタリゼーション

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

HKUST-1 as a Heterogeneous Catalyst for the Synthesis of Vanillin

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately 200 different odorant compounds in addition to vanillin. The natural extraction of vanillin (from the orchid Vanilla planifolia, Vanilla tahitiensis and Vanilla pompon) represents only 1% of the worldwide production and since this process is expensive and very long, the rest of the production of vanillin is synthesized. Many biotechnological approaches can be used for the synthesis of vanillin from lignin, phenolic stilbenes, isoeugenol, eugenol, guaicol, etc., with the disadvantage of harming the environment since these processes use strong oxidizing agents and toxic solvents. Thus, eco-friendly alternatives on the production of vanillin are very desirable and thus, under current investigation. Porous coordination polymers (PCPs) are a new class of highly crystalline materials that recently have been used for catalysis. HKUST-1 (Cu3(BTC)2(H2O)3, BTC = 1,3,5-benzene-tricarboxylate) is a very well known PCP which has been extensively studied as a heterogeneous catalyst. Here, we report a synthetic strategy for the production of vanillin by the oxidation of trans-ferulic acid using HKUST-1 as a catalyst.

The conversion of trans-ferulic acid to vanillin was achieved by heterogeneous catalysis. HKUST-1 was employed in this synthesis and the essential step in the catalytic process was the generation of unsaturated metal sites. Thus, when the catalyst was activated under vacuum, full vanillin conversion (yield of 95%) was obtained.

HKUST-1バニリンの合成のための不均一触媒として、

Vanillin (4-hydoxy-3-methoxybenzaldehyde) is the main component of the extract of vanilla bean. The natural vanilla scent is a mixture of approximately ...

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.

タンパク質のバイオコンジュゲートの合成<em&gt;経由で</em&gt;システイン - マレイミドケミストリー

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.

金ナノロッドのヒドロキノンベースの合成

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.

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

水溶性有機金属錯体アレイの合成

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

Chemical Synthesis of Porous Barium Titanate Thin Film and Thermal Stabilization of Ferroelectric Phase by Porosity-Induced Strain

Barium titanate (BaTiO3, hereafter BT) is an established ferroelectric material first discovered in the 1940s and still widely used because of its well-balanced ferroelectricity, piezoelectricity, and dielectric constant. In addition, BT does not contain any toxic elements. Therefore, it is considered to be an eco-friendly material, which has attracted considerable interest as a replacement for lead zirconate titanate (PZT). However, bulk BT loses its ferroelectricity at approximately 130 &#176;C, thus, it cannot be used at high temperatures. Because of the growing demand for high-temperature ferroelectric materials, it is important to enhance the thermal stability of ferroelectricity in BT. In previous studies, strain originating from the lattice mismatch at hetero-interfaces has been used. However, the sample preparation in this approach requires complicated and expensive physical processes, which are undesirable for practical applications.
In this study, we propose a chemical synthesis of a porous material as an alternative means of introducing strain. We synthesized a porous BT thin film using a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles were used as an organic template. Through a series of studies, we clarified that the introduction of pores had a similar effect on distorting the BT crystal lattice, to that of a hetero-interface, leading to the enhancement and stabilization of ferroelectricity. Owing to its simplicity and cost effectiveness, this fabrication process has considerable advantages over conventional methods.

Here, we present a protocol for the synthesis of porous barium titanate (BaTiO3) thin film by a surfactant-assisted sol-gel method, in which self-assembled amphipathic surfactant micelles are used as an organic template.