Synthesis of Indoxyl-glycosides for Detection of Glycosidase Activities

The overall goal of this procedure is to present a novel synthetic approach for Endo Glycosides, including those with complex carbohydrate structures in good yields. This is accomplished by first synthesizing an inic acid allo Ester accepter, starting from methyl lene. The second step involves glycosyl, this accepter with an acetylated glycosyl hali donor.

Next, the allele ester's cleaved by a palladium catalyst and subsequent silver mediated decarboxylation is applied to create the acetylated inox glycoside. The final step is zeplan deacetylation to yield the unprotected inox glycoside. Ultimately, the prepared chromogenic substrate is used for a qualitative enzyme activity test.

The main advantage of this technique over existing methods is that in looksy glycosides of complex and labile carbohydrate structures can be produced easily With, we first had the idea for this method when we read a publication by Alexander Robertson from 1927, who developed the first synthesis of Inca employ inic acid methyl eta. Later methods used occi inox cell acceptors due to the better accessibility, but suffered of low yield and purification issues. To begin synthesis, a satellite, a halogen substituted lene as described in the text protocol.

Then in a three neck round bottom, flask reflux, 2.3 grams of the resulting halogenated, methyl ol acetamide in 40 milliliters of water with 2.1 grams of magnesium sulfate hep dehydrate over three hours. Dropwise add 50 milliliters of a saturated potassium permanganate solution in water, and then reflux the mixture for two additional hours at approximately 135 degrees Celsius and an oil bath. Cool the mixture to room temperature and then filter it to remove the pyro cyte byproduct, adjust a solution pH to one with 37%hydrochloric acid.

Next, filter the product from the solvent and dry it under a vacuum. After drying deci, isolate the product as described in the text protocol and then suspend eight point 17 grams of the resulting deci product in 33 milliliters of dry acetonide trial over 30 minutes simultaneously add Dropwise 5.3 milliliters of pyridine and a solution of three point 22 grams of tristine in 18.5 milliliters of dry di chloro methane to the solution. Then heat the reaction to 50 degrees Celsius in an oil bath.

After three hours, remove about 75%of the solvent and add 100 milliliters of distilled water to quench the reaction. Filter the product and then wash the filter with five to 10 milliliters of cold chloro methane. After washing vacuum dry the product alkylate this intermediate and then open the resulting anhydride as described in the text protocol.

Isolate the resulting product by column chromatography. Begin dickman condensation by mixing three grams of the product with 1.75 grams of potassium oxide in 100 milliliters of dry dathyl ether reflux mixture for two hours at 40 to 45 degrees Celsius. Next, remove approximately 75%of the solvent in vacuum and precipitate the product with 100 milliliters of one molar hydrochloric acid.

Filter the product and then dry it under vacuum. To begin synthesis of XG.Mix 400 milligrams of the indoc clic acid allyl ester with 410 milligrams of TBAs and 500 milligrams of alpha xto bromo galactose in 10 milliliters of chloro methane. Then add 10 milliliters of one molar aqueous potassium carbonate to the reaction.

Stir the reaction at room temperature for about three hours and monitor consumption of alpha aceto bromo actose by thin layer chromatography in a one-to-one solution of petroleum ether and ethyl acetate. After the alpha aceto bromo galactose is completely consumed, isolate the organic phase of the solution with a separatory funnel. Next, dry the organic phase over sodium sulfate to remove remaining water and then use column chromatography to purify the product as described in the text protocol to cleave off the allo Ester dissolve 600 milligrams of the galacto puric side in 15 milliliters of dry tetra hydro furin.

Remove the solvent by vacuum at 400 milligrams of silver acetate, 800 milligrams of potassium carbonate, and 10 milliliters of acetate anhydride to the product, and then heat the reaction to about 95 degrees Celsius for 20 minutes. Allow the reaction to cool to room temperature and then dilute the reaction with 50 milliliters of di chloro methane. To work up this reaction, wash the mixture twice with water and once with 10%sodium bicarbonate solution.

Dry the organic phase over sodium sulfate and then concentrate the solution. Use column chromatography to purify the resulting acetylated galacto puric side. Next, dissolve 396 milligrams of the purified material in 10 milliliters of methanol.

Add a spatulas tip's worth of sodium methylate, and then stir the reaction overnight. Use a cation exchange resin to neutralize the reaction and then concentrate the solution in vacuum. Finally, dry the product under vacuum to yield xal to test the validity of the synthetic route.

First, dissolve the xal in a few microliters of dimethyl sulf oxide. Then make a 0.5 millimolar stock solution of xal in a suitable buffer. Next, add one microliter of beta galacto and shake the mixture at 600 revolutions per second.

At 37 degrees Celsius, visually monitor the reaction for formation of a vibrant indigo dye. The formation of blue dye in solution clearly indicates enzyme activity in doxyl. Glycosides are important for monitoring glycosate enzyme activity, yet they are difficult to synthesize.

The synthetic approach presented here gives the final endo galacto puric side in a 66%overall yield. A significant improvement over previous methodology resulting in only 28%yield. The resulting in doxyl glycosides can be used to test the activity of pure or impure enzyme solutions.

In this example, the indigo color was observed only in wells containing beta galacto and was produced with as little as 0.1 millimolar of XG.Once the indoc cell acceptor is composed, this synthetic route to yield indoc cell glycosides can be done within two days if performed properly, After watching this video, you should have a good understanding of synthesis and utilization of inial glycosides. This synthetic root produces these compounds in good yield despite the complex structures.