Facile and Efficient Preparation of Tri-component Fluorescent Glycopolymers via RAFT-controlled Polymerization

The overall goal of this procedure is the well-controlled synthesis of fluorescent statistical linear glyco polymers using reversible addition fragmentation, chain transfer or raft polymerization. This is accomplished by first synthesizing the glyco monomer two lacto bio ethyl meth acrylamide, or L-A-E-M-A. The second step is to use raft polymerization of L-A-E-M-A-N two amino ethyl meth acrylamide, or A EMA and N two hydroxyethyl acrylamide or a GAA to synthesize the corresponding low dispersity glyco polymer.

Next, the glyco polymer is modified on one of its primary amines by reaction with carboxy fluorescein xito middle ester to produce a fluorescein ated glyco polymer. The final step is to bind the fluorescein native glyco polymer to lectin coated arose beads with known carbohydrate binding specificity. Ultimately, fluorescent microscopy is used to analyze the binding of the glyco polymers to the arose beads, confirming that the glyco polymer possesses the carbohydrate of interest and is labeled with a fluorescent tag.

The advantage of RAF polymerization over existing methods that employ conventional free radical polymerizations is that the wrath process is a control polymerization technique that minimizes the number of initiator derived chains. This results in polymers of predetermined molecular weights and very narrow poly dispersity rack. Polymerizations can be applied to a defined mixture of monomers for the preparation of specific fluorescent glyco polymers.

These glyco polymers have defined binding abilities that can be verified by the use of lectin coated beads. The efficient preparation of fluorescent glyco polymers should prove to be of great value to glyco biology research in the future. To begin dissolve two grams of lacto bionic acid in three milliliters of anhydrous methanol.

Next, slowly add absolute ethanol dropwise to the solution until it just turns cloudy. Use a rotary evaporator to remove all the solvents. Repeat this process three times to produce 1.94 grams of lacto bioo one five lactone in 98%yield.

Add a solution of one gram of lacto bio lactone in three milliliters of methanol to a solution of A EMA and MEHQ in two milliliters of methanol. Next, add one milliliter of triethylamine and stir the mixture at room temperature. After 48 hours, add 20 milliliters of deionized water to the mixture and transfer it to a rotary evaporator.

Evaporate until completely dry. Dissolve the residue in 20 milliliters of deionized water and apply the solution to the top of a 10 by 20 millimeter anion exchange column. Place a beaker containing one milligram of MEHQ underneath the outlet and elute the column into the beaker.

After drawing the solution in a rotary evaporator, dissolve the residue in 20 milliliters of deionized water and add one milligram aliquots of ion exchange resin. Until an an hydrant test is negative, performed in hydrant tests by removing one microliter of the solution and applying it to a thin layer chromatography plate. Spray the plate with a 2%anhy in ethanol solution and heat at 90 degrees Celsius for one minute on a hot plate.

The endpoint has been reached when no blue color forms. Next, filter the solution through a fritted glass funnel into a test tube. Freeze the solution at negative 80 degrees Celsius and then transfer it to a lyophilizer to be freeze dried.

Dissolve the residue in a minimum amount of methanol and precipitate the product by adding minus 20 degrees Celsius and anhydrous acetone. Collect the precipitated L-A-E-M-A by filtration through a fritted glass funnel. Transfer the funnel and precipitate to a vacuum desiccate for drying first at 0.5 grams of approximately 150 mesh aluminum oxide nanoparticles to one milliliter of commercial grade HEAA in a two milliliter test tube.

Centrifuge the tube at 300 times G for 30 seconds and use the top layer as inhibitor free HEAA carefully dissolve 32.8 milligrams of L-A-E-M-A, 1.7 milligrams of a EMA and 27.5 microliters of inhibitor free HEAA and 0.4 milliliters of deionized water and transfer the solution to a well cleaned one milliliter flank tube. Next, add 50 microliters of raft reagent and 50 microliters of initiator reagent to the tube and mix by gently tapping the side of the tube with the valve closed. Connect the tube to a vacuum line and freeze the contents using a dry ice ethanol bath.

Then reduce the tube pressure to tend to 50 millitorr with a pump and close the valve. Remove the dry ice bath and allow the contents to thaw. Repeat the freeze, vacuum and thaw cycle twice more carefully.

Inspect the tube to ensure the contents are fully dissolved and gently vortex if needed. Before seating, remove the closed tube from the vacuum line and place it into a transparent plastic vacuum bag. Evacuate the air from the bag with a plastic hand pump.

Then place the bag into a covered water bath at 70 degrees Celsius for 24 hours. Next, remove the tube from the bag and transfer its contents to a dialysis bag with a molecular weight cutoff of 3, 500 Daltons. Place the dialysis bag into a beaker and dialyze against two liters of deionized water for 24 hours.

Transfer the dialysis bag contents to a test tube and place it in a freezer at negative 80 degrees Celsius. Once frozen, transfer to a lyophilizer and freeze dry. To obtain the fluffy white glyco polymer PMA LA EMA first, prepare a solution of five milligrams of PMA LA EMA in 0.9 milliliters of PBS.

Next, slowly add 0.6 milligrams of carboxy fluorescein cin, middle eter in 100, microliters of dimethylformamide to the rapidly stirred solution, the micro tube and foil, and stir slowly for 16 hours at room temperature, transfer the solution into a dialysis bag with a molecular weight cutoff of 3, 500 Daltons. Place the bag into a beaker and dialyze against two liters of deionized water for 16 hours while keeping it protected from the light. Transfer the dialysis bag contents to a test tube.

After freezing the solution at negative 80 degrees Celsius, freeze dry. To obtain the fluffy yellow fluoresce labeled P-M-A-L-A-E-M-A, glyco polymer, determine the number average molecular weight, the weight, average molecular weight, and the dispersity of the glyco polymers with an HPLC system. First at 1.5 milliliters of PBS to a 50 microliter suspension of ery Krista galley lectin coated aro beads in a centrifuge tube.

Then centrifuge the tube at 300 times G for one minute. Next, discard the supernatant and repeat the wash twice more before finally adding 0.5 milliliters of PBS. Add a solution of three micrograms of the fluorescein labeled PMA LA EMA in six microliters of PBS to the tube containing the lectin coated beads.

Use fluorescein labeled P-M-A-G-A-E-M-A as a negative control. Next, wrap the tubes in foil and incubate them at room temperature for one hour. Centrifuge the tube for one minute and discard the supernatant.

Then reus, suspend the beads in 1.5 milliliters of fresh PBS and repeat the wash three times. Finally, reus suspend the beads in 0.2 milliliters of PBS Transfer four microliters of the suspended beads into a well of an immunofluorescence microscope. Slide and cover with a glass cover slip record.

Images of the slide using a fluorescent microscope fitted with a 10 x objective FITC filter and camera. The glyco polymers were analyzed by gel permeation chromatography. Polymers synthesized without using raft have a much higher dispersity than those made using raft.

When labeled with fluorescein, the physical appearance of the glyco polymer changes from white to a strong yellow color shown under UV light. A solution of unlabeled polymer remains dark while the fluorescein labeled polymer glows Bright green confirming the presence of the label specific binding to lectin coated beads only occurs with polymers carrying pendant sugar residues beads glow green when bound, while they remain dark when treated with polymers lacking pendant sugar residues. This raft based polymerization technique permits great flexibility in the type of glyco polymers that can be synthesized.

Subsequent labeling of the glyco polymers can also be achieved with a large variety of fluorescent reagents, capable of reacting with primary means in an aqueous environment. Researchers in the field of glyco biology can use this technique to readily obtain highly versatile and defined glyco polymers. These glyco polymers can be used as tools to explore the carbohydrate binding specificities, such as those observed in the respiratory track of patients with cystic fibrosis or those involved in many types of human cancers.