Research

Introduction

Research Question

How does attaching mPEG of varying lengths to VEMA polymers affect the properties of the resulting nanodiscs?

On the left there is a large polymer with a chain of methoxypolyethylene glycol attached to it (Structure described below). On the right of the arrow is a giant circle of a lipid bilayer with a few tubes wrapping around it. This is the nanodisc.

Figure 2. Ultimate experimental aims: (a) Synthesis of a mPEG-VEMA polymer and (b) Formation of nanodiscs

First, a loose protein not attached to anything is shown. Then, it shows a circular cluster of lipids with a red coil around it. Finally, it shows the first protein being inserted into this circle of lipids. This is showing how the nanodiscs form and ultimately look.

Figure 1. Nanodisc formation around a membrane protein(3).

Membrane Proteins and Nanodiscs

Due to their medicinal usefulness, we need to be able to study the structure and function of various membrane proteins.

Creating a native lipid environment has proven to be challenging(1).

One promising way of studying membrane proteins is through the use of nanodiscs, which can assemble around these proteins (Figure 1)(2).

methoxypolyethylene glycol. It has a hydroxy group on one end, followed by a repeating unit of two carbons and an oxygen, and capped off with a methyl (CH3) group.

Figure 3. The structure of methoxypolyethylene glycol

VEMA Polymers and Nanodisc Formation

The Konkolewicz group has previously synthesized polymers consisting of alternating units of Butyl Vinyl Ether, Maleic Anhydride, and Dodecyl Vinyl Ether(4)

These are VEMA polymers (Vinyl Ether Maleic Anhydride)

In the presence of lipids, these VEMA polymers created nanodiscs that are effectively forming a bilayer in which we can insert proteins of interest (4)

They allow for a much wider and more accurate array of characterization techniques compared to the use of detergents and styrene-maleic anhydride polymers(1,5,6)

A vinyl ether maleic anhydride polymer. It consists of a butyl vinyl ether component with a 4-carbon methoxy chain, followed by a maleic anhydride with a closed ring, finally followed by a dodecyl vinyl ether, which is a 10-carbon methoxy chain. Altogether this represents the repeating units in the VEMA polymer.

Figure 2. An illustration of a VEMA polymer(4)

Methoxypolyethylene Glycol (mPEG) Conjugation

The conjugation of strongly hydrophilic side chains to these VEMA backbones has not been studied in-depth.

mPEG (Figure 3) is a favorable candidate for conjugation due to its adjustable size, non-toxic nature, relative ease of use in synthetic reactions, and its singular hydroxyl group(7).

Methods

Step 1

RAFT Polymerization (to make VEMA polymer)

AIBN, PADTC, monomers of butyl vinyl ether, dodecyl vinyl ether, maleic anhydride, and 1,4-Dioxane were addend into a round bottom flask.

The flask was purged with N2 for 30 minutes.

It was then stirred overnight at 65oC.

Finally, it was precipitated over cold hexanes and dried overnight under vacuum.

Step 2

Hydrolysis

We added the step 1, VEMA polymer into a round bottom with water.

Then, we added excess NaOH (4M) dropwise into the mixture.

Next, we stirred it at 50oC for 24 hours

Finally, we precipitated out the desired product with HCl (1M) and vacuum filter.

Step 3

EDC Coupling

First, we added the hydrolyzed VEMA polymer (part 2 product), mPEG (with molecular weights 164.2, 350, and 550 g/mol), and DMAP into a roundbottom flask with chloroform.

Next, we stirred for 30 minutes at 0oC

Then, we added EDC into the reaction and stirred overnight starting at 0oC

Finally, I extracted with 2M HCl solution and dialyzed the product for 3 days.

A large beaker of water with a small bit of orangish-yellow sample hanging in a transparent membrane.

Figure 4. The Part 3 Set up of dialysis

Results

Synthesis of mPEG-VEMA Conjugates

I successfully synthesized the VEMA polymer (Figure 5, Figure 6), the hydrolyzed VEMA polymer, and the mPEG-VEMA polymer using mPEG of average molecular weights 164.2, 350, and 550 g/mol.

The step 3 EDC coupling was confirmed using NMR analysis, as shown under the section "NMR Analysis" below.

Interestingly, I saw a decrease in molecular weight as measured by GPC between the VEMA and hydrolyzed VEMA polymers (18000 g/mol -> 16000 g/mol)

VEMA polymer on far right. Arrow with H2O, NaOH, and 50 degrees Celsius. VEMA polymer with two carboxylic acids in place of the maleic anhydride. Arrow with DMAP, EDC, mPEG, and 0 degrees celsius. Final VEMA polymer with a mPEG molecule attached to one of the opened carboxylic acids from the second structure.

Figure 5. Synthetic scheme of mPEG-VEMA polymer (far right).

Figure 6. A precipitated VEMA Polymer

NMR Analysis

I used the in-common H1/H2 peak from original polymer as reference on the new polymer to quantify my reaction's progress.

Specifically, I took the integrals and looked at the ratios. Ideally, the ratio of H3:H1+H2 should be 2:3, but my use of excess mPEG drove this ratio up, since it has a tendency to double-conjugate.

I confirmed this ratio for the 165, 350, and 550 MW mPEGs.

An NMR signal. Around 0.9 parts per million (ppm), there is a peak that is only common to my original polymer. So, I can use that as a reference. At 4.15 ppm, there is a singlet peak which only can correspond to my product. The integral for the 0.9 ppm is 3.000 and for the 4.15 ppm is 2.682.

Figure 7. NMR Determination of mPEG-VEMA esterification. Ester at 4.15 ppm (s), Vinyl ether methyl peaks at 0.9 ppm (m).

What Does This Mean?

My target polymer was successfully created!

This is the final step before synthesizing nanodiscs, meaning that I will soon be collecting the data to answer my research question.

Additionally, we can now replicate the experiment on a larger scale to study the effects even further.

Conclusions / Next Steps

While I have not yet reached an answer to my above research question, I was at least able to synthesize the different polymers that will directly be used in the later formation of nanodiscs.

My next steps from here are to prepare the phospholipids and introduce them to my polymers, where I can analyze the size of the nanodiscs and take some pictures (TEM) of them.

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