Rapid One-step Enzymatic Synthesis and All-aqueous Purification of Trehalose Analogues

The overall goal of this method is to rapidly synthesize and purify trehalose analogues which have possible applications ranging from the detection of pathogenic bacteria to the preservation of bio materials. This is a fast and high-yielding method that provides access to analogues of the disaccharide trehalose. These compounds have various established and emerging applications in biomedical and biotechnological research.

This method consists of a one-step enzymatic synthesis and an all-aqueous purification. Together, these procedures help to address some of the drawbacks of traditional chemical syntheses of trehalose analogues which can be lengthy, low yielding and environmentally unfavorable. The implications of this method may extend toward the study and diagnosis of tuberculosis because modified trehalose analogues have been used for imaging microbacterium tuberculosis, the causative agent of tuberculosis.

This method also can help accelerate the synthesis and evaluation of trehalose analogues for other possible applications such as preservation of biological products and use as noncaloric sweeteners. Prior to beginning the procedure, prepare an overnight culture of TreT-expressing E.coli. To begin the TreT expression procedure, place 750 milliliters of TB growth medium in a 2, 800 milliliter Fernbach culture flask and autoclave it.

Add ampicillin to the broth to achieve a final concentration of 100 micrograms per milliliter. Obtain cuvettes for UV-vis spectroscopy and fill one cuvette with broth from the flask as a blank. Next, add three milliliters of the prepared TreT-expressing E.coli overnight culture to the flask.

Incubate the flask at 37 degrees Celsius while shaking at 200 rpm. Check the absorbance of the broth at 600 nanometers against the broth blank periodically. When the absorbance value at 600 nanometers is between 0.5 and 1.0, add 750 microliters of arabinose to the flask to induce TreT expression.

Incubate the culture at 37 degrees Celsius overnight while shaking at 200 rpm. Next, place the culture in a one liter polypropylene centrifuge bottle and centrifuge the culture at 4, 000 times G for 15 minutes at four degrees Celsius. Discard the supernatant and resuspend the pellet in 15 milliliters phosphate buffered saline.

Transfer the suspension to a conical centrifuge tube and centrifuge under the same conditions. Then discard the supernatant and if necessary, store the pellet at minus 80 degrees Celsius. Dissolve a protease inhibitor mini tablet in 20 milliliters of wash buffer.

Add the protease inhibitor solution to the pellet and vortex the mixture to resuspend the pellet. Transfer the suspension to a 100 milliliter beaker. Next, sonicate the suspension to lyse the cells positioning the probe about one centimeter from the bottom of the beaker.

Place the lysate in a metal conical tube and centrifuge the lysate at 15, 000 times G at four degrees Celsius for 60 minutes. Filter the lysate with a syringe filter. Isolate TreT by fast protein liquid chromatography and assess the purity with SDS page.

Then exchange TreT into Tris buffer by dialysis overnight at four degrees Celsius to form the TreT solution for trehalose analogue synthesis. Collect the blank from the dialysis washing. Determine the TreT concentration by UV-vis spectroscopy against the dialysis blank.

To begin synthesis of 6-Azido-trehalose using TreT on the 20 to 30 milligram scale, place 0.08 millimole 6-Azido-glucose, 0.16 millimole UDP-glucose and 0.08 millimole magnesium dichloride in a 15 millimeter conical tube. Add the TreT and Tris buffer. Dilute the mixture with Tris buffer as needed to achieve an enzyme concentration of 300 micrograms per milliliter in a final volume of four milliliters.

Cap and invert the tube to dissolve the solids. Incubate the reaction mixture for one hour at 70 degrees Celsius while shaking at 300 rpm. During the incubation, place three milliliters deionized water into a 10 kilodalton centrifugal filter unit and centrifuge at 3, 000 times G until all liquid is in the tube.

Repeat the wash twice more to ensure thorough rinsing of the filter unit. When the reaction is complete, cool the mixture to room temperature on ice. Then transfer the product mixture to the centrifugal filter unit.

Rinse the reaction tube with one milliliter deionized water three times and transfer each rinse to the centrifugal filter unit. Centrifuge the mixture at 3, 000 times G until all liquid has passed into the tube. Add three milliliters deionized water to the filter unit and centrifuge again under the same conditions.

Repeat the rinse as needed to maximize product recovery. Add three grams mixed bed ion exchange resin to the filtrate. Stir the mixture with a stir bar at room temperature for one hour.

After stirring, decant and filter the supernatant. Rinse the resin with five milliliters deionized water and filter the rinse. Repeat as needed to maximize product recovery and combine all filtrates.

Concentrate the combined filtrate. Then to check for unreacted 6-GlcAz precursor, prepare silica gel TLC plate with spots of 6-GlcAz, 6-TreAz, the concentrated product and a co-spot. Develop the plate with a mixture of n-butanol, ethanol and deionized water.

Once the plate is dry, visualize the spots by dipping the plate in 5%sulfuric acid and ethanol and heating the plate on a hot plate on high setting for about five minutes. If unreacted precursor is observed, purify the product mixture on a size exclusion column and repeat the TLC analysis. Once the pure trehalose analogue has been obtained, remove water to obtain the dried 6-TreAz product.

The TreT enzyme stereo selectively joins the glucose analogue and UDP-glucose to form the corresponding trehalose analogue. The reaction scope include stereo chemical deoxifluoro, thio and azido modifications. However, substituents at the four position on the ring are less well-tolerated.

Using this procedure, the glucose analogue 6-GlcAz was quantitatively converted to the trehalose analogue 6-TreAz in less than 60 minutes as confirmed by TLC. The pure product was isolated with the non-chromatographic spin dialysis ion exchange purification method and characterized by NMR in HPLC. The pure 6-TreAz solution was then used to perform azide labeling of the model bacterium M.smegmatis.

Subsequent fluorophore labeling by a click chemistry reaction with the cell surface azides showed that M.smegmatis with a functional trehalose transporter had been successfully labeled with azides whereas the strain without was not. Once mastered, the TreT catalyze reaction and subsequent purification can be performed in one to four hours depending on the number of rinse steps used to maximize production recovery. While attempting this procedure, it's important to remember that the reaction product should always be analyzed by NMR and TLC or HPLC to confirm its purity and structure.

Following this procedure, the synthesized trehalose analogue can be evaluated or employed as a bacterial imaging probe, metabolic tracer, enzyme inhibitor or biomolecule stabilizing agent. This method provides a way for researchers to prepare trehalose analogues with modifications such as stable isotope or radioisotope labels. Altogether, the compounds accessible through this method should be useful in tuberculosis research and other biomedical research areas.

After watching this video, you should have a good understanding of how to express and purify the TreT enzyme, use TreT to synthesize trehalose analogues in one step and purify the trehalose analogue products.