Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

The overall goal of this methodology, is to develop a modular chemo-enzymatic approach to the synthesis of N-glycans by constructing a new glycan microarray on a aluminum oxide coated glass slide for detecting heteroglycan binding of HIV antibodies. This methods can help answer key questions in glycobiology for identification of cell virus specific glycans for much development. The major advantage of this method is to detect weak binding and identify a mixture of sugars that bind to a protein such as, antibody from HIV patient.

The implications of this technique extend to a diagnosis of influenza infections because sialic catabolist express on human airways epithelial cells and issues are common while the symptoms. Therefore, glycoarray will be an ideal tool to determine specificity of wild subtypes. Though this method can provide inside into the glycan specificities of HIV antibodies it can also be applied to other systems such as, diagnosis of various cancers based on the binding pattern of human serum antibodies toward human specific serum antigens printed on the surface.

To begin this procedure, add 2-5 micromoles of previously prepared glycan and a five milliliter round bottom flask. Add a magnetic stir bar to the flask and cap it with a rubber septum. Add 400 microliters of freshly dried DMF and 10-25 micromoles of the phosphonic acid linker to the flask.

Then, stir the reaction mixture at 800 rpm for five hours. Once the reaction is complete, evaporate the solvent using a rotary evaporator at five to 10 millibar vacuum pressure and 40 degrees Celsius. Following this, dissolve the dried reaction mixture and 0.5 milliliter of water and load on top of polyacrylamide gel bed.

Elute the products using water and collect one to two milliliter fractions. To check the collected fractions by TLC, spot each fraction on a TLC plate and dip the plate in 0.25 molars Cerium Ammonium Molybdate. Heat the stained plate with a hot plate to visualize the product.

After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Next, dissolve the dried product in two milliliters of water and add Palladium Hydroxide. Attach a hydrogen containing balloon to the flask and stir the reaction mixture at 800 rpm for 15 hours under hydrogen atmosphere.

Once the reaction is complete, filter the mixture through a Celite Bed and wash with two milliliters of methanol and two milliliters of distilled deionized water. Then, evaporate the solvent using a rotary evaporator at 300 millibar vacuum pressure and 40 degree Celsius. Following this, dissolve the crud mixture in approximately one milliliter of water and load it on top of a polyacrylamide gel bed.

Elute the product with water and collect one to two milliliter fractions. After combining the product containing fractions and freezing the solution biofolize to obtain the desired product as a white powder. Once the product is dry, characterize it by enimar and mass spectroscopy using Deuterium Oxide as the solvent.

For surface anodization, set a temperature controlled incubator to four degree Celsius. Then transfer a previously prepared 0.3 molar aqueous oxalic acid solution to a 500 milliliter beaker containing a magnetic stir bar. Place a 10 centimeter long platinum rod as a cathode into the solution.

Keep stirring the oxalic acid at 300 rpm throughout the anodization process. In the lab tracer software, click on the setup button and then click on function choice Sweep Voltage. Set the start and stop voltage to 25.8 volts.

The number of points to 100, the compliance to one, and the sweep delay to 1200 milliseconds. Then, click okay. Clamp an aluminum coated glass slide facing toward the cathode.

Click on the run test button and observe the current measurement. After surface anodization, wash the glass slide thoroughly with double distilled water and purge dry with nitrogen gas. Store the glass slide in a 30%relative humidity chamber until further use.

For fabrication, prepare 100 microliters solutions of all monovalent glycans in ethanol glycol at 10 millimolar concentration. Dilute the glycan solutions with printing buffer to make 100 micromolar solutions. For the Heteroligand Study, add five microliters of MAN-5 Glycan to five microliters of each glycan solution.

Transfer the prepared glycans into the 384 well microplate for microarray printing step. After inserting a microplate on the printer array tech, print microarrays by robotic pin by depositing 0.6 nanoliters of the glycan solutions onto each aluminum coated glass slide. Next prepare 70 microliters of primary antibody PG-9 and PTSB buffer containing 3%BSA.

Then prepare 120 microliters of secondary fluorescent tag antibody Donkey and Anti-human Immunoglobulin G and PBST buffer containing 3%BSA in the dark. Mix PG9 and the secondary fluorescent tag antibody in a 1:1 ratio. Then incubate the premixed antibodies for 30 minutes at four degrees Celsius.

Following incubation, load an aluminum coated glass slide into the slide incubation chamber, which is divided into 16 wells. Transfer 100 microliters of the premixed antibodies to the glycan array. After incubating at four degrees Celsius for 16 hours remove the premixed antibodies from the glycan array with a pipette and then remove the slide incubation chamber.

Wash the glass slide in PBST buffer and deionized water. Then spin dry the glass slide at 2000 times gravity. Next, open the microarray image analysis software.

Insert the glass dried slide into the array scanner with the arrayed features facing down. Click on the settings menu. Set the image resolution to five micrometers per pixel.

And set the wavelength at 635 nanometers with PMT 450 and power to 100%Click on the scan button to start imaging the slide. Finally click the file icon to save the scan image in TIF format for image analysis. In this study, a wide array of N-Glycans were synthesized using a modular chemo enzymatic strategy.

To demonstrate the effectiveness of the strategy a biantennary isometric structure was synthesized by chemoenzymatic methods. Enzymatic galactosylation for disaccharide 1 followed by fucosylation of trisaccharide 2 afforded the desired tetrasaccharide 3. Per satylation and reducing end modifications of building block two and three, afforded to desire molecule four and five.

Stereoselective three O-glycosalyation of module four of trisaccharide six provided hexasaccharide seven. De-protection and reaction before module five yielded dedecasaccharide nine. Global detection afforded glycan 10 which was characterized by enimar and mass spectroscopy.

Glycans containing a Pentylamine tail at the reducing end were modified with phosphonic acid linkers and attached to the aluminum glass coated surface through phospinide chemistry. HIV-1 broadly neutralizing antibody PG9 was screened for it's glycan specificity using homo and heteroglycan arrays to demonstarte for the first time it's ineteraction with adjacent heteroglycans in the V1/V2 loop of HIV1 PG120 surface. Once mastered, this technique can be done in 24 hours from glycan immobilization to beta analysis if it was performed properly.

While attempting this procedure, it is important to remember to use freshly anodized aluminum coated glass slide for Glycan 10. For laying this procedure are the med techs slides. Determination of the body constant can be performed in order to quantify the binding interactions between carbohydrate and protein of interest.

After the development of this technique, it paved the way for researchers to better understand the role of carbohydrate in infectious disease in Kenya and help develop new therapies against diseases. Don't forget that working with toxic agents can be extremely hazardess and precautions such as wearing of the clothes, glasses and masks should always be taken when preforming experiments.