Name: microwave-assisted functionalization of poly(ethylene glycol) and on-resin peptides for use in chain polymerizations and hydrogel formation
Uploaded: 2013-10-29T18:30:00
Duration: 15 min 33 s
Description: Watch this Scientific Journal Video about Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation at JoVE.com

This work was funded in part by a Howard Hughes Med-into-Grad fellowship (AVH), by start-up funds provided to Dr. Danielle Benoit from the University of Rochester&#160;and the Orthopaedic Research and Education Foundation/Musculoskeletal Transplant Foundation (OREF/MTF). The authors would like to thank Dr. James L. McGrath for the use of his equipment.

1. Microwave-assisted Synthesis of PEGDM
<ol>
	<li>To prevent contamination with water, pre-dry all glassware being used in an oven (&#62;60 &#186;C) for 1 hour. 
	Note: Required glassware includes: two 100-ml&#160;beakers, a 250-ml&#160;beaker, 3 spatulas, a 250-ml&#160;B&#252;chner flask, a 7-cm B&#252;chner funnel, a 10-cm watch glass.</li>
	<li>Pre-chill 100-150 ml anhydrous diethyl ether (74.12 g/mol) for precipitation subsequently performed at step 1.6 by pouring it into a beaker, covering the beaker with a watch glass, and placing the beaker into a recrystallization dish filled with ice. Move microwave and vortexer into a chemical fume hood. 
	Note: Diethyl ether may also be prechilled by placing the beaker in a chemical freezer. The lower diethyl ether temperature achieved by chilling in a freezer will increase the rate and efficiency of precipitation.</li>
	<li>In a small weigh boat, weigh out 5 g of poly(ethylene glycol) (PEG) of the molecular weight of your choice (1,000-100,000 Da).
	<ol>
		<li>If present, remove the plastic piece from the lid of the scintillation vial. Tare the vial, and dispense 10 molar excess of methacrylic anhydride into the vial (MA, 154.16 g/mol) per equation 1 in the hood. Add PEG to the scintillation vial. 
		<img alt="Equation 1" fo:content-width="200px" src="/files/ftp_upload/50890/50890eq1.jpg" width="250px" /> (1) 
		where <img alt="Equation 1.1" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq1.1.jpg" width="30px" /> is the mass of PEG in g, <img alt="Equation 1.2" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq1.2.jpg" width="40px" /> is the molecular weight of PEG in g/mol, <img alt="Equation 1.3" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq1.3.jpg" width="40px" /> is the number of terminal OH groups on the PEG, and <img alt="Equation 1.4" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq1.4.jpg" width="40px" /> is the molecular weight of MA in g/mol.</li>
	</ol>
	</li>
	<li>Loosely twist the cap onto the scintillation vial. Set the microwave to 5 min&#160;on maximum power. Wearing heat-resistant gloves, remove the vial from the microwave every 30 sec.
	<ol>
		<li>Fully tighten the cap and vortex for 30 sec. Repeat until the solution has been microwaved for the full 5 min. The cap may need to be replaced during the procedure due to cracking.</li>
	</ol>
	</li>
	<li>With the cap loosened, let the PEGDM cool to room temperature. Dissolve the PEGDM in a small amount (10-15 ml) of dichloromethane (DCM, 84.93 g/mol). 
	Note: It is recommended that the PEGDM be allowed to cool significantly (~5 min) prior to DCM addition, to prevent boiling of the DCM (a suspected carcinogen) due to residual heat. The PEGDM can be broken into small chunks using a spatula and vortexed to aid in dissolution.</li>
	<li>Precipitate the PEGDM in 10x excess ice-cold diethyl ether for 20 min. 
	Note: It may be necessary to scratch the beaker side with a spatula to initiate crystal formation to precipitate lower molecular weight PEGs (2,500 Da); however, PEG with a molecular weight below 1,000 Da will not precipitate despite scratching.</li>
	<li>Using a B&#252;chner funnel and flask, collect the PEGDM by vacuum filtration. Do not filter to complete dryness, as this will promote water adsorption to the PEGDM. 
	Note: If necessary for the specific vacuum system in use, a vacuum trap can be placed between the filtration setup and the vacuum source to protect the vacuum pump from damage by solvent vapors.</li>
	<li>Transfer the filtered PEGDM to a 50 ml&#160;conical tube with a large gauge needle pierced through the cap for ventilation. Store overnight in a vacuum chamber to dry.</li>
	<li>Redissolve PEGDM in DCM and reprecipitate (as in steps 1.5-1.7) as a final step to remove unreacted MA. Dry again as in step 1.8.</li>
</ol>
2. Characterization of PEGDM Functionalization
<ol>
	<li>Using deuterated chloroform (120.38 g/mol) as the solvent, prepare samples for 1H-NMR. Place a small sample of the PEGDM (&#8776;10 mg) in a scintillation vial with a small amount of the solvent (&#8776;1.0 ml). The target concentration is 10 mg/ml.
	<ol>
		<li>Once the sample is dissolved, transfer it to a clean NMR tube. The sample should fill the bottom 4-5 cm of the NMR tube.</li>
	</ol>
	</li>
	<li>Collect the proton NMR spectra. Our data is collected using a 400 MHz spectrometer. Run samples at room temperature for at least 64 scans to obtain sufficient data resolution.</li>
	<li>If NMR analysis (Figure 4) indicates PEGDM functionalization is less than 90%, the methacrylation procedure should be repeated. Adjust the mass of MA used to account for the reduced amount of unfunctionalized PEG.</li>
</ol>
Note: The percent of PEG functionalized into PEGDM can be calculated from the observed: theoretical ratio of terminal methacrylate protons (a, b and c) to central PEG protons (d) (Figure 4). For linear PEG, the theoretical number of central PEG protons is calculated by equation 2:
<img alt="Equation 2" fo:content-width="150px" src="/files/ftp_upload/50890/50890eq2.jpg" width="200px" /> (2) 
where <img alt="Equation 2.1" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq2.1.jpg" width="40px" /> is the molecular weight of the PEG in g/mol and <img alt="Equation 2.2" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq2.2.jpg" width="70px" /> is the molecular weight of a single PEG repeat (44 g/mol). For nonlinear PEG, this equation must be modified to reflect the specific branching structure (Figure 1). The percent functionalization can then be calculated using equation 3: 
<img alt="Equation 3" fo:content-width="150px" src="/files/ftp_upload/50890/50890eq3.jpg" width="300px" />(3) 
where <img alt="Equation 3.1" fo:content-width="30px" src="/files/ftp_upload/50890/50890eq3.1.jpg" width="40px" /> is the observed area under peak d (&#948;=6.63 ppm), <img alt="Equation 3.2" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq3.2.jpg" width="40px" /> is the observed area under the methacrylate proton peaks (a, &#948;=1.94 ppm; b and c, &#948;=5.57 and 6.12 ppm), and <img alt="Equation 3.3" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq3.3.jpg" width="40px" /> is the theoretical number of methacrylate protons (a=3*<img alt="Equation 1.3" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq1.3.jpg" width="60px" /> , b=1*<img alt="Equation 1.3" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq1.3.jpg" width="60px" /> and c=1*<img alt="Equation 1.3" fo:content-width="40px" src="/files/ftp_upload/50890/50890eq1.3.jpg" width="60px" /> - 6, 2 and 2 respectively for linear PEG). The percent functionalization calculation should be performed using peaks a, b and c separately, and then averaged to obtain an overall percent functionalization.
Note: Sufficiently functionalized PEGDM can then be dialyzed (in water, against water) and collected by lyophilization to remove residual methacrylic anhydride and methacrylic acid. The final product should be mixed with a small amount (0.01 wt%) of inhibitor such as citric acid or vitamin C and stored with desiccant at -20 &#176;C until use. The final PEGDM product can be used to produce hydrogels, as detailed in the JoVE article by Khetan and Burdick22.
3. Microwave-assisted Functionalization of On-resin Peptides
Note: Our peptides are synthesized using Fmoc-Gly-Wang resin, using an automated peptide synthesizer with UV monitoring, and 0.2 M amino acid solutions in N-methylpyrrolidone (NMP, 99.1 g/mol). 5% piperazine (86.1 g/mol) in dimethylformamide (DMF, 73.1 g/mol) is used for deprotection, 0.5 M O-Benzotriazole-N,N,N&#8217;,N&#8217;-tetramethyl-uronium-hexafluoro-phosphate (HBTU, 379.3 g/mol) in DMF is used as the activator, and 2 M diisopropylethylamine (DIEA, 129.3 g/mol) in NMP is used as the activator base. Peptides can also be obtained from a commercial peptide supplier. When using commercial sources it is critical that peptides are procured on-resin with the amino acid side chain protecting groups intact rather than fully cleaved, as is standard.
Note: It is important that any amino acids with reactive side chains are protected to ensure that methacrylamide functionalization only occurs at the primary amine of the N-terminus of the sequence. See Table 1 for amino acids with reactive side groups, and typical protecting groups. Amino acids with protecting groups are incorporated into the sequence during peptide synthesis in the same manner as nonprotected amino acids, and are often available from the same amino acid suppliers.
<table border="0" cellpadding="0" cellspacing="0" width="502">
	<colgroup>
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:144px;">Amino Acid</td>
			<td style="width:163px;">Reactive group</td>
			<td style="width:196px;">Protecting group</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:144px;">Lysine</td>
			<td style="width:163px;">Primary Amine</td>
			<td style="width:196px;">tert-butyloxycarbonyl (Boc)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:144px;">Serine</td>
			<td style="width:163px;">Primary Alcohol</td>
			<td style="width:196px;">tert-butyl (tBu)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:144px;">Threonine</td>
			<td style="width:163px;">Secondary Alcohol</td>
			<td style="width:196px;">tBu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:144px;">Tyrosine</td>
			<td style="width:163px;">Phenol</td>
			<td style="width:196px;">tBu</td>
		</tr>
	</tbody>
</table>
Table 1: Reactive amino acids and typical protecting groups.
<ol>
	<li>Synthesize peptides using standard solid phase peptide synthesis, and store on resin at 4 &#176;C in DMF until ready for use.</li>
	<li>Using a 7 cm B&#252;chner funnel with filter paper and 250 ml&#160;flask, collect the peptide resin from the DMF via filtration.</li>
	<li>If present remove the plastic piece from the lid of the scintillation vial. Transfer the resin to the scintillation vial. Using a disposable pipette, add just enough MA to cover the resin in the scintillation vial.</li>
	<li>Loosely place the cap onto the scintillation vial. Set the microwave to 3 min&#160;on maximum power. Wearing heat resistant gloves, remove the vial from the microwave every 15-20 sec.
	<ol>
		<li>Fully tighten the cap and vortex for 15 sec. Repeat until the solution has been microwaved for the full 3 min.</li>
	</ol>
	</li>
	<li>With the cap loosened, let the peptide solution cool to room temperature. Using a small amount of DMF and a B&#252;chner funnel with filter paper and flask, collect the peptide resin from the vial.</li>
	<li>Transfer the peptide resin to a fresh scintillation vial and cleave and deprotect the peptide.
	<ol>
		<li>Per 0.25 mmol of resin, we use a 2 hr room-temperature reaction with rotation, using a cleavage cocktail of 18.5 ml&#160;trifluoroacetic acid (TFA, 114.02 g/mol) with 0.5 ml&#160;each of triisopropylsilane (TIPS, 158.36 g/mol), 3,6-Dioxa-1,8-octanedithiol (DODT, 182.30 g/mol) and deionized-water (18.02 g/mol). 
		Note: This cocktail is sufficient for most peptides, but will not deprotect 2,2,4,6,7-pentamethyl-dihydrobenzofuran-5-sulfonyl (Pbf)-protected amino acids (commonly used to protect arginine side chains). If the sequence contains any Pbf-protected groups, 0.5 ml&#160;of the TFA should be replaced with 0.5 ml&#160;thioanisole (124.20 g/mol) and cleavage time increased to 4 hr. 
		Note: If a cloudy, crystalline substance forms in the cleavage cocktail, the peptide is likely crashing out of solution and the volume of the cleavage cocktail should be doubled.</li>
	</ol>
	</li>
	<li>Chill 400 ml&#160;anhydrous diethyl ether on ice in a beaker covered with watch glass.</li>
	<li>Precipitate the peptide in 10x excess diethyl ether, and divide the solution evenly amongst four 50 ml&#160;conical tubes. Centrifuge at 3,200 x g for 10 min&#160;to collect the peptide.
	<ol>
		<li>Decant off the ether, and resuspend the peptide in 100 ml&#160;fresh diethyl ether divided between two 50 m&#160;conical tubes. Repeat the centrifugation process, resuspending in 50 ml&#160;of fresh ether twice for a total of 4 ether washes. 
		Note: This removes the chemicals used in the cleavage cocktail and cleaved protecting groups from the solid peptide.</li>
	</ol>
	</li>
	<li>After the last centrifugation step, decant the waste ether and dry the peptide overnight under vacuum.</li>
</ol>
4. Characterization of Peptide Functionalization
<ol>
	<li>Use 50:50 H2O:Acetonitrile (41.05 g/mol) + 0.1% TFA as the solvent for MALDI-ToF analysis of peptide samples. Place a small sample (1 - 2 mg) of the peptide in a 1.5 ml&#160;Eppendorf tube and dissolve the sample in 1 ml&#160;of MALDI solvent.</li>
	<li>Prepare the matrix solution. A commonly employed matrix is &#945;-Cyano-4-hydroxycinnamic acid (CHCA, 189.2 g/mol) as the matrix. Dissolve 10 mg/ml&#160;matrix in MALDI solvent, forming a stock matrix solution. 
	Note: The matrix stock solution can be stored at room temperature for up to one week for additional analyses.</li>
	<li>Combine the peptide and matrix solution in a 1:1 ratio. Spot this combined solution onto three separate locations on the MALDI sample plate, adding 1 &#956;l/spot.
	<ol>
		<li>Dry the spots, either by air drying or using a heat gun. Respot and dry each sample. 
		Note: Respotting produces a more uniform peptide/matrix sample, and assists in obtaining a clear signal. A standard peptide mix should also be combined with the matrix solution in a 1:1 ratio and spotted (only once) onto the MALDI plate.</li>
	</ol>
	</li>
	<li>Collect the MALDI-ToF data. Due to the addition of the methacrylamide group to the N-terminus of the peptide, there should be a 68 g/mol increase in molecular weight above that of the peptide molecular weight alone. 
	Note: Unlike the PEGDM synthesis, peptides cannot be refunctionalized, as upon cleavage the protecting groups on amino acids with reactive side chains are removed, and the selective functionalization of the N-termini can no longer be ensured.
	<ol>
		<li>If the MALDI analysis (Figure 5) indicates the peptide was correctly functionalized and all protecting groups appropriately cleaved, the peptide can be dialyzed (in water, against water) and collected by lyophilization to remove residual contaminants (cleavage cocktail, ether, cleaved protecting groups etc.). The solid peptide should be transferred to a small Eppendorf tube and stored at -20 &#176;C until use.</li>
	</ol>
	</li>
</ol>

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S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ethylene+glycol+methacrylate+phosphate+in+PEG+hydrogels+on+mineralization+and+viability+of+encapsulated+hMSCs.">The effect of ethylene glycol methacrylate phosphate in PEG hydrogels on mineralization and viability of encapsulated hMSCs.</a> Biomaterials. 27, 1377-1386 (2006).</li><li>Hern, D. L., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Incorporation+of+adhesion+peptides+into+nonadhesive+hydrogels+useful+for+tissue+resurfacing.">Incorporation of adhesion peptides into nonadhesive hydrogels useful for tissue resurfacing.</a> J. Biomed. Mater. Res. 39, 266-276 (1998).</li><li>Andreopoulos, F. M., Beckman, E. J., Russell, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Light-induced+tailoring+of+PEG-hydrogel+properties.">Light-induced tailoring of PEG-hydrogel properties.</a> Biomaterials. 19, 1343-1352 (1998).</li><li>Merrill, E. W., Dennison, K. A., Sung, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partitioning+and+Diffusion+of+Solutes+in+Hydrogels+of+Poly(Ethylene+Oxide).">Partitioning and Diffusion of Solutes in Hydrogels of Poly(Ethylene Oxide).</a> Biomaterials. 14, 1117-1126 (1993).</li><li>Engler, A. J., Sen, S., Sweeney, H. L., Discher, D. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+elasticity+directs+stem+cell+lineage+specification.">Matrix elasticity directs stem cell lineage specification.</a> Cell. 126, 677-689 (2006).</li><li>Weber, L. M., Lopez, C. G., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+PEG+hydrogel+crosslinking+density+on+protein+diffusion+and+encapsulated+islet+survival+and+function.">Effects of PEG hydrogel crosslinking density on protein diffusion and encapsulated islet survival and function.</a> J. Biomed. Mater. Res. A. 90A, 720-729 (2009).</li><li>Mellott, M. B., Searcy, K., Pishko, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Release+of+protein+from+highly+cross-linked+hydrogels+of+poly(ethylene+glycol)+diacrylate+fabricated+by+UV+polymerization.">Release of protein from highly cross-linked hydrogels of poly(ethylene glycol) diacrylate fabricated by UV polymerization.</a> Biomaterials. 22, 929-941 (2001).</li><li>Watkins, A. W., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigation+of+molecular+transport+and+distributions+in+poly(ethylene+glycol)+hydrogels+with+confocal+laser+scanning+microscopy.">Investigation of molecular transport and distributions in poly(ethylene glycol) hydrogels with confocal laser scanning microscopy.</a> Macromolecules. 38, 1326-1334 (2005).</li><li>Anseth, K. S., Benoit, D. S. W., Durney, A. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Manipulations+in+hydrogel+degradation+behavior+enhance+osteoblast+function+and+mineralized+tissue+formation.">Manipulations in hydrogel degradation behavior enhance osteoblast function and mineralized tissue formation.</a> Tissue Eng. 12, 1663-1673 (2006).</li><li>Hillwest, J. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Prevention+of+Postoperative+Adhesions+in+the+Rat+by+in-Situ+Photopolymerization+of+Bioresorbable+Hydrogel+Barriers.">Prevention of Postoperative Adhesions in the Rat by in-Situ Photopolymerization of Bioresorbable Hydrogel Barriers.</a> Obstet. Gynecol. 83, 59-64 (1994).</li><li>Sawhney, A. S., Pathak, C. P., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bioerodible+Hydrogels+Based+on+Photopolymerized+Poly(Ethylene+Glycol)-Co-Poly(Alpha-Hydroxy+Acid)+Diacrylate+Macromers.">Bioerodible Hydrogels Based on Photopolymerized Poly(Ethylene Glycol)-Co-Poly(Alpha-Hydroxy Acid) Diacrylate Macromers.</a> Macromolecules. 26, 581-587 (1993).</li><li>Skaalure, S. C., Milligan, I. L., Bryant, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age+impacts+extracellular+matrix+metabolism+in+chondrocytes+encapsulated+in+degradable+hydrogels.">Age impacts extracellular matrix metabolism in chondrocytes encapsulated in degradable hydrogels.</a> Biomed. Mater. 7, 024111-0210 (2012).</li><li>Hoffman, M. D., Benoit, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agonism+of+Wnt-beta-catenin+signalling+promotes+mesenchymal+stem+cell+(MSC)+expansion.">Agonism of Wnt-beta-catenin signalling promotes mesenchymal stem cell (MSC) expansion.</a> J. Tissue. Eng. Regen. Med. , (2013).</li><li>Sawhney, A. S., Pathak, C. P., Vanrensburg, J. J., Dunn, R. C., Hubbell, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Optimization+of+Photopolymerized+Bioerodible+Hydrogel+Properties+for+Adhesion+Prevention.">Optimization of Photopolymerized Bioerodible Hydrogel Properties for Adhesion Prevention.</a> J. Biomed. Mater. Res. 28, 831-838 (1994).</li><li>. Methacrylic Anhydride [Internet] Available from: <a target="_blank" href="https://us.vwr.com/store/catalog/product.jsp?catalog_number=AAAL14357-18">https://us.vwr.com/store/catalog/product.jsp?catalog_number=AAAL14357-18</a> (2013)</li></ol>

The authors declare that they have no competing financial interests.

Poly(ethylene glycol) (PEG) hydrogels are common biomaterials used in regenerative medicine and drug delivery applications1-3. These hydrogels offer significant advantages over other biomaterials. PEG hydrogels are synthetic, offering a high degree of control over engineering properties such as modulus of elasticity and degradation rate compared to their natural biomaterial counterparts1. As they are synthetically derived, PEG has significantly less batch-to-batch variability versus naturally-derived materials4. Owing to the chemical composition of PEG, these hydrogels are highly hydrophilic, resistant to protein adsorption, and biocompatible3. This resistance to protein adsorption allows PEG hydrogels to act as a &#8216;blank slate&#8217;, enabling researchers to interrogate and study specific biological or chemical factors (drugs, biomolecules, cell adhesion peptides, etc.) and the specific roles these factors play in controlling cell and/or tissue behavior.
<img alt="Figure 1" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig1.jpg" width="600px" /> 
<a href="http://www.jove.com/files/ftp_upload/50890/50890fig1highres.jpg" target="_blank">Click here to view larger image</a>.
Figure 1: Examples of poly(ethylene glycol) (PEG) architectures. A) Linear PEG. B) 4-arm PEG with a pentaerythritol core. C) 8-arm PEG with a hexaglycerol core. D) 8-arm PEG with a tripentaerythritol core. n is the number of PEG repeats on each arm. Each repeat has a molecular weight of 44 g/mol, therefore n can be calculated from the overall molecular weight and the structure/arm #.
PEG precursors are available with a variety of architectures and molecular weights (Figure 1). Varying the architecture (arm #) and ethylene glycol repeats (n) of PEG can be used to control properties of hydrogel networks formed from these macromers. Unmodified PEG contains terminal hydroxyl groups which must be replaced with an alternate functionality to facilitate covalent crosslinking via polymerizations, the most commonly employed crosslinking strategy for PEG hydrogels, prior to formation of hydrogel networks. There are a variety of chemical groups which can be incorporated into PEG macromers to facilitate polymerization and network crosslinking (acrylate, methacrylate, vinyl ether, norbornene, etc.). Despite the variety of terminal functionalities available to facilitate crosslinking, there are only two mechanisms by which polymerization can occur: step- and chain-growth (or a mixture of the two, mixed-mode).
<img alt="Figure 2" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig2.jpg" width="600px" /> 
<a href="http://www.jove.com/files/ftp_upload/50890/50890fig2highres.jpg" target="_blank">Click here to view larger image</a>.
Figure 2: Theoretical hydrogel network schematic. A) Traditional chain-growth polymerization results in heterogeneous networks containing dense poly(methacrylate) crosslinking regions and increased network nonidealities such as loops, unreacted precursors, and permanent entanglements. B) Step-growth polymerization results in significantly more homogeneous network structures (not to scale).
Functionalities which crosslink via chain-growth polymerization do not require the presence of an additional crosslinker. However, chain-polymerized hydrogels produce heterogeneous network structures containing dense crosslinking regions (Figure 2A)1. In contrast, step-growth polymerization requires the use of a crosslinker or co-monomer which is reactive with the terminal functional groups of the PEG macromers. As the terminal functional groups on the PEG can only react with the crosslinker and the crosslinker can only react with the terminal functional groups on the PEG, this results in greater network structure homogeneity (Figure 2B)1. Step-growth polymerizations also typically lead to higher conversion of functional groups, decreasing the amount of unreacted precursors and potential for immune/inflammatory responses due to soluble, unincorporated macromers1. Mixed-mode polymerization methods have also been developed that combine both step- and chain-growth polymerization through use of macromers that can both self-react (chain-growth) and react with a crosslinker (step-growth). This produces hydrogels with characteristics of each polymerization mechanism, and can be used to produce more complex, diverse network structures than either step- or chain-growth networks alone1.
While there are a plethora of functional groups which can be used to functionalize PEG and facilitate hydrogel formation, methacrylates and norbornenes are some of the most common chain- and step-growth polymerization moieties, respectively. Both of these functionalities offer excellent spatiotemporal control over network polymerization, and when used to encapsulate cells, these networks support high overall cell survivability5-7. Dimethacrylate functionalized PEG (PEGDM) crosslink via chain polymerization and allows for the incorporation of biomolecules or other factors through co-polymerization with acrylate-, methacrylate-, or similarly-functionalized biomolecules5,6. PEGDM hydrogels have significant advantages over alternate chain-growth polymerization systems such as acrylate functionalized PEG (PEGDA). Using traditional methods, PEGDA can be synthesized more rapidly than PEGDM; however, using microwave-assisted synthesis, PEGDM synthesis is even more efficient. PEGDA is often synthesized in overnight8 or 24-hr9 reactions, but can also be synthesized in four hours at elevated temperatures10. PEGDM is also traditionally synthesized by reacting overnight11 or for 24 hr5, with some methods extending the reaction time to 4 days12. Using the microwave-assisted method demonstrated here, PEGDM can be produced in a 5 min reaction. While PEGDM has slower reaction kinetics than PEGDA13, the crosslinking reaction for PEGDM is still rapid, occurring in minutes, and achieves greater macromer conversion than PEGDA as the increased hydrophobicity of the methacrylate group increases functional group aggregation in solution, thereby increasing the probability of radical transfer and methacrylate conversion14. PEGDM hydrogels are also associated with increased cellular viability and growth as compared to PEGDA hydrogels, likely due to the decreases in reaction rate at any given time, which reduces radical concentration and unreacted macromers present14. Thiol-ene polymerizations such as those using norbornene-functionalized PEG (PEGN) form hydrogels via step-growth polymerization, and require the use of PEGN and a crosslinker that contain an average of greater than two functional groups. Since thiyl radicals react with norbornene carbon-carbon double bonds, multi-thiol containing crosslinkers are commonly used to crosslink PEGN hydrogels, allowing for facile incorporation of peptides with cysteine amino acids functionalities7. While there are numerous other chemistries which react via step-growth polymerization (Michael-addition reactions such as thiol-acrylate15 and thiol-vinyl sulfone16, &#8220;Click&#8221; reactions such as alkyne-azide17 etc.), thiol-norbornene hydrogels are very common, as the strain from the norbornene ring significantly increases the reaction rate and decreases the chance of the norbornene double bond undergoing chain polymerization7.
The decision between methacrylate, norbornene, or alternate functionalization to facilitate hydrogel formation is largely based on the approach. For example, chain-growth polymerized PEGDM networks have been demonstrated as well suited to control cell localization in the development of a tissue-engineered periosteum18,19. Step-growth polymerized PEG networks are better suited for the incorporation of peptide sequences to facilitate enzymatically-responsive hydrogel degradation, due to the ease of incorporation of enzyme substrate sequences using thiol (cysteine) containing peptides and norbornene functionalized macromers20. If the research question will be best addressed by the use of step-growth hydrogels, Fairbanks et al. provides a detailed description of the norbornene functionalization strategy for PEG7. This paper will detail how PEG and peptide sequences can be functionalized (with a methacrylate for PEG, and a methacrylamide for peptides) for chain polymerization reactions.
Traditionally, PEGDM is produced by reacting PEG with methacryloyl chloride and triethylamine in dichloromethane. The reaction is allowed to progress at room temperature overnight11 or for 24 hr5, with some methods extending reaction time to 4 days12 before filtration, precipitation in diethyl ether, and collection. While many variations of this approach exist, all are time-consuming, require a large array of chemical synthesis equipment, and are not environmentally friendly, as they involve the use of relatively large amounts of high-purity reagents and solvent. To circumvent these limitations, Lin-Gibson et al. developed a microwave-assisted, solvent-free method to functionalize PEG with terminal methacrylate groups (Figure 3A)12. In this reaction, the terminal alcohol groups of the PEG react with one of the carbonyl atoms of the methacrylic anhydride to form a carboxyl. This generates the PEGDM product, with methacrylic acid as a side product. This synthesis has many of the characteristic advantages of microwave synthesis, including reduced reaction time and solvent-free synthesis methods21. The microwave synthesis is preferable to the previously discussed methods as it is significantly faster, requires less extensive synthesis equipment (e.g. glassware, reaction plates), and uses less overall reagent and solvent amounts as solvents are only required for product purification/collection and not for synthesis, making it more economical and environmentally friendly.
<img alt="Figure 3" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig3.jpg" width="600px" /> 
<a href="http://www.jove.com/files/ftp_upload/50890/50890fig3highres.jpg" target="_blank">Click here to view larger image</a>.
Figure 3: Functionalization schematics. A) Poly(ethylene glycol) is reacted with 10x molar excess methacrylic anhydride to produce poly(ethylene glycol) methacrylate. B) This same method can be used to functionalize the N-terminus of peptide sequences, forming a methacrylamide functionalized peptide. By performing this procedure prior to cleaving the peptide from the resin, selective functionalization of the N-terminus can be performed as amino acid side groups remain protected. n: number of PEG repeats in the macromer (n=45.5, 227 and 455, respectively, for the 2, 10, and 20 kDa linear PEG used). R1 to RN: amino acid side chains. PG1 to PGN: side chain protecting groups. TFA: trifluoroacetic acid. TIPS: triisopropylsilane. DODT: 3,6-dioxa-1,8-octanedithiol. H2O: water.
The microwave-assisted methacrylation method has been recently adapted by our group to functionalize the N-terminus of peptides with methacrylamide groups (Figure 3B) to facilitate peptide incorporation into a variety of polymers and polymeric networks. In this reaction, the primary amine of the N-terminus of the peptide reacts with the carbonyl atom on the methacrylic anhydride to form an amide. This generates the methacrylamide functionalized peptide, with methacrylic acid produced as a side product. When using this procedure to functionalize the N-terminus of peptide sequences, it is important that amino acids containing reactive side chains (primary amines (lysine), alcohols (serine, threonine), and phenols (tyrosine)) are protected during functionalization, and protecting groups are only cleaved after methacrylamide incorporation.
This article will demonstrate both of these microwave-assisted methods to synthesize PEGDM and functionalize on-resin peptide sequences, highlighting common pitfalls and suggesting troubleshooting methods. In this article, methods to perform analytical chemical techniques commonly employed to assess product functionalization will be detailed, and suggestions and resources for performing more advanced modifications will be given. Typical results will be demonstrated, which include using the synthesized PEGDM to form hydrogel networks, exploiting the formed hydrogels to control release of a model drug, and employing functionalized peptides to facilitate cell-hydrogel interactions. Particular attention will be paid to characterizing hydrogel mesh size and discussing how hydrogel composition can be tuned to affect this underlying physical property, which in turn controls bulk material properties such as stiffness and drug release profile.

<table border="0" cellpadding="0" cellspacing="0" width="619">
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">3,6-Dioxa-1,8-octanedithiol</td>
			<td style="width:115px;">Tokyo Chemical Industry Co, LTD</td>
			<td style="width:119px;">D2649</td>
			<td style="width:167px;">CAS 14970-87-7</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Acetonitrile</td>
			<td style="width:115px;">J.T. Baker</td>
			<td style="width:119px;">UN1648</td>
			<td>CAS 75-05-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Amino Acids</td>
			<td style="width:115px;">AAPPTech</td>
			<td style="width:119px;">Glycine: AFG101</td>
			<td>CAS 29022-11-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:115px;"> </td>
			<td style="width:119px;">Arginine: AFR105</td>
			<td>CAS 154445-77-9</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;"> </td>
			<td style="width:115px;"> </td>
			<td style="width:119px;">Asparagine: AFD105</td>
			<td>CAS 71989-14-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:115px;"> </td>
			<td style="width:119px;">Serine: AFS105</td>
			<td>CAS 71989-33-8</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Anhydrous diethyl ether</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:119px;">UN1155</td>
			<td>CAS 60-29-7</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Citric acid</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">C1857</td>
			<td>CAS 77-92-9</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Deuterated chloroform</td>
			<td style="width:115px;">Cambridge Isotope Laboratories Inc.</td>
			<td style="width:119px;">DLM-7-100</td>
			<td>CAS 865-49-6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dichloromethane</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:119px;">UN1593</td>
			<td>CAS 75-09-2</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Diisopropylethylamine</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">A1181</td>
			<td>CAS 7087-68-5</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Dimethylformamide</td>
			<td style="width:115px;">Fisher Scientific</td>
			<td style="width:119px;">D119-4</td>
			<td>CAS 68-12-2</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">Fmoc-Gly-Wang resin</td>
			<td style="width:115px;">Peptides International</td>
			<td style="width:119px;">RGF-1301-PI</td>
			<td>100-200 mesh size</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Methacrylic anhydride</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">L14357</td>
			<td>CAS 760-93-0</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">N-Methylpyrrolidone</td>
			<td style="width:115px;">VWR</td>
			<td style="width:119px;">BDH1141-4LG</td>
			<td>CAS 872-80-4</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">On-resin peptides</td>
			<td style="width:115px;">Synthesized in-house</td>
			<td style="width:119px;"> </td>
			<td style="width:167px;">On-resin peptides can also be purchased from Peptides International, GenScript, AAPPTec, etc.</td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:216px;">O-Benzotriazole-N,N,N&#8217;,N&#8217;-tetramethyl-uronium-hexafluoro-phosphate</td>
			<td style="width:115px;">AnaSpec Inc</td>
			<td style="width:119px;">510/791-9560</td>
			<td>CAS 94790-37-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Peptide Calibration Standard</td>
			<td style="width:115px;">Care</td>
			<td style="width:119px;">206195</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Piperazine</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">A15019</td>
			<td>CAS 11-85-0</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(ethylene glycol) 2 kDa linear</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">B22181</td>
			<td>CAS 25322-68-3</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Poly(ethylene glycol) 10 kDa linear</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">B21955</td>
			<td></td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">Poly(ethylene glycol) 20 kDa linear</td>
			<td style="width:115px;">Sigma Aldrich</td>
			<td style="width:119px;">81300</td>
			<td style="width:167px;">JenKem Technologies USA is an alternate supplier of linear and multi-arm PEG</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Thioanisole</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">L5464</td>
			<td>CAS 100-68-5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Trifluoroacetic acid</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">A12198</td>
			<td>CAS 76-05-1</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Triisopropylsilane</td>
			<td style="width:115px;">Alfa Aesar</td>
			<td style="width:119px;">L09585</td>
			<td>CAS 6485-79-6</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">&#945;-Cyano-4-hydroxycinnamic acid</td>
			<td style="width:115px;">Tokyo Chemical Industry Co, LTD</td>
			<td style="width:119px;">C1768</td>
			<td>CAS 28166-41-8</td>
		</tr>
	</tbody>
</table>

microwave-assisted functionalization of poly(ethylene glycol) and on-resin peptides for use in chain polymerizations and hydrogel formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Proton nuclear magnetic resonance is one of the most common analytical techniques to assess the efficiency of a chemical reaction, as the area under each spectra peak is proportionate to the relative levels of that proton in the sample, allowing determination of the ratios of product and reactant in the sample. For this reaction, 1H-NMR analysis (Figure 4) can be used to calculate the % functionalization from the observed: theoretical ratio of terminal methacrylate protons (a, b and c) to central PEG protons (d). The PEGDM shown in Figure 4 was 2,000 Da prior to functionalization; therefore n=2,000 Da/(44 Da/PEG repeat) = 45.5, so d=4*(n-1)=178, making the proton: NMR unit ratio 178/102.16=1.74. In this case, peak a cannot be used in assessing % functionalization, as the presence of water in the sample artificially increases the area under the peak. Using peak b, the % functionalization is 1.00*1.74/2*100%=87.1%; using peak c, the % functionalization is 1.08*1.74/2*100%=94.1%. Therefore, the overall % functionalization is 91% and this PEGDM is adequately functionalized for use in hydrogel synthesis. Typically, approximately 90% functionalization is achieved after a single round of methacrylation.
Due to the multitude of proton peaks that arise in 1H-NMR analysis of peptides, peptide functionalization is more easily investigated using MALDI-ToF mass spectrometry. This is demonstrated in Figure 5, where the peptide GKRGDSG was synthesized and subjected to methacrylamide functionalization. A small fraction of the peptide was cleaved for prefunctionalization molecular weight assessment (Figure 5A), which showed the observed molecular weight peak occurring at 676 g/mol, the expected molecular weight of the peptide, indicating correct synthesis of the peptide sequence. The remainder of the peptide underwent methacrylamide functionalization prior to cleavage. As this peptide contains Pbf protected R amino acids, cleavage was performed in a cocktail containing thioanisole for 4 hr. After methacrylamide functionalization, the observed molecular weight peak occurs at 744 g/mol (Figure 5B), the expected weight of the methacrylamide functionalized peptide (676+68 g/mol) and not at the expected molecular weight of the unfunctionalized peptide, indicating correct functionalization.
To demonstrate functionality of both the PEG and the methacrylamide functionalized peptide, PEGDM hydrogels were produced with and without 0.5 mM methacrylamide functionalized GKRGDSG (Figure 6). Hydrogels were produced with 10 wt% linear 10 kDa PEGDM in PBS, with 0.05 wt% Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP) as photoinitiator. The hydrogel precursor solution was injected between two glass slides separated by a glass slide spacer and held together with binder clips. Precursor solution was then exposed to 365 nm UV light at 2 mW/cm2 for 10 min to induce crosslinking, after which 8 mm diameter gels were collected using a cylindrical punch. Gels were rinsed in PBS and allowed to swell for 2 days to ensure equilibrium swelling conditions were achieved16,20 . Human MSCs (passage 3) were grown to 80% confluency and seeded on hydrogels at 15,000 cells/cm2. The cells were allowed to adhere for 48 hr&#160;before being transferred to fresh media containing 0.5 &#181;l/ml&#160;calcein AM and 2 &#181;l/ml&#160;ethidium homodimer (LIVE/DEAD Viability kit from Invitrogen) and imaged under phase contrast and fluorescence at 10X magnification using a Nikon Eclipse Ti 2000. MSCs were unable to adhere to the unfunctionalized PEG hydrogels (Figure 6A), but upon inclusion of the cell adhesion peptide RGD they were able to adhere to and spread on the hydrogel surface (Figure 6B). The LIVE/DEAD images presented do not represent the viability of the seeded MSC population, as MSCs are adhesion-dependent cells that detach from the gel surface upon death and will be removed during the media transfer process, resulting in artificial inflation of seeded cell viability. Rather, the fluorescent images are intended to demarcate between adherent cells and small variations in the hydrogel topology, which can be difficult under phase contrast alone. Interestingly, the nonspread cells seeded on the PEG-only gels stain positive for both calcein AM and ethidium homodimer, indicating that cells were dying at the time of imaging.
One of the many advantages to PEG hydrogels is their highly tunable nature. Modifying the specific makeup of the PEG hydrogel affords researchers a high degree of control over properties such as modulus of elasticity. As illustrated in Figure 7, both PEG molecular weight (Figure 7A) and weight percentage (Figure 7B) are known to control hydrogel mesh size (&#958;) and resultant hydrogel stiffness and release rate of encapsulated drugs. This was demonstrated by producing hydrogels with varying molecular weight PEGDM, and investigating the resultant hydrogel modulus and mesh size (Figure 8). All hydrogels were produced with 10 wt% linear PEGDM in PBS, with 0.05 wt% LAP as photoinitiator. 40 &#181;l&#160;of hydrogel precursor solution was in 1 ml&#160;syringes with the tips cut off, and exposed to 365 nm UV light at 2 mW/cm2 for 10 min to form hydrogels, producing cylindrical geometries approximately 5 mm in diameter and 2 mm in height. Gels were allowed to swell in PBS for 2 days before mechanical testing. Hydrogel modulus was determined using a MTS QT/5 with a 5 N load cell while compressing between 5 and 10% of initial hydrogel height at a rate of 0.1 mm/sec. After mechanical testing was completed, mesh size was determined by measuring hydrogel mass pre-(Ms) and post- (MD) 24 hr&#160;of lyophilization via the Flory-Rehner equation as detailed in the discussion section, with calculations performed in MATLAB. As hypothesized, increasing the molecular weight of the PEG macromer caused an increase in hydrogel mesh size (Figure 8A) and a decrease in hydrogel stiffness (Figure 8B). Hydrogel mesh size and the resultant gel stiffness can also be controlled by altering weight percentage PEG. Hydrogels were produced with varying wt% of linear 10 kDa PEGDM, and hydrogel stiffness and mesh size were determined as previously described (Figure 9). As was illustrated in Figure 7B, increasing wt% PEG causes a significant decrease in mesh size (Figure 9A) and increase in hydrogel stiffness (Figure 9B).
Hydrogels containing encapsulated bovine serum albumin (BSA) were formed using varying molecular weight PEG (2, 10, and 20 kDa). All hydrogels were produced with 10 wt% PEGDM in PBS containing 50 &#181;g/ml&#160;BSA, as described for Figure 6. Gels were incubated in 1 ml&#160;PBS at 37 &#176;C, and transferred to fresh PBS at each time point. Released BSA was quantified using the Bradford Assay from Thermo Scientific. Hydrogel mesh size was determined as described for Figure 8. As illustrated in Figure 7A, BSA release occurs more rapidly from hydrogels formed using higher molecular weight PEGDM, as a result of the larger mesh size within the hydrogel (Figure 10).
<img alt="Figure 4" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig4.jpg" width="500px" /> 
Figure 4. Representative 1H-NMR of 2 kDa linear PEGDM functionalized using the microwave-assisted method.&#160;Percent&#160;functionalization can be calculated based on the observed: theoretical ratio of terminal methacrylate protons (a, b and c) to central PEG protons (d). <a href="https://www.jove.com/files/ftp_upload/50890/50890fig4highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 5" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig5.jpg" width="500px" /> 
Figure 5. Representative MALDI-ToF of peptide GKRGDSG (A) before and (B) after functionalization using the microwave-assisted method. Note that the observed molecular weight peak after functionalization occurs at 744 g/mol, the expected weight of the methacrylamide functionalized peptide (676+68 g/mol) and not at the expected molecular weight of the un-functionalized peptide (676 g/mol). <a href="https://www.jove.com/files/ftp_upload/50890/50890fig5highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 6" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig6.jpg" width="500px" /> 
Figure 6. Representative phase contrast (left) and LIVE/DEAD (green/red) fluorescent images (right) of MSCs cultured on A) PEG gels alone and B) PEG gels containing 0.5 mM methacrylamide functionalized GKRGDSG. MSCs are unable to adhere to and spread on the PEGDA gels alone, but upon incorporation of the cell adhesion peptide RGD, are able to adhere to and spread on the hydrogel surface. <a href="https://www.jove.com/files/ftp_upload/50890/50890fig6highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 7" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig7.jpg" width="500px" /> 
Figure 7. A) PEG molecular weight and B) weight percentage PEG used to form hydrogel networks affects hydrogel mesh size (&#958;) and resultant hydrogel stiffness and release rate of encapsulated drugs. A) Increasing PEG molecular weight (left to right) at constant weight percentage increases hydrogel mesh size, decreasing hydrogel stiffness and increasing the rate of drug release. B) Decreasing the weight percentage of PEG (left to right) used to form hydrogels increases hydrogel mesh size, similarly decreasing hydrogel stiffness and increasing the rate of drug release (not to scale). <a href="https://www.jove.com/files/ftp_upload/50890/50890fig7highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 8" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig8.jpg" width="500px" /> 
Figure 8. A) Mesh size increases and B) hydrogel stiffness decreases with increasing molecular weight of the PEG macromer. n=10, error bars = SEM, *** p&#60;0.001 by one-way ANOVA with Tukey&#39;s HSD post-hoc test. All statistical analyses were performed using Prism 5. <a href="https://www.jove.com/files/ftp_upload/50890/50890fig8highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 9" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig9.jpg" width="500px" /> 
Figure 9. A) Mesh size decreases and B) hydrogel stiffness increases with increasing wt% PEG. n = 9-10, error bars = SEM, ** p&#60;0.01, *** p&#60;0.001 by one-way ANOVA with Tukey&#39;s HSD post-hoc test. <a href="https://www.jove.com/files/ftp_upload/50890/50890fig9highres.jpg" target="_blank">Click here to view larger image</a>.
<img alt="Figure 10" fo:content-width="6in" src="/files/ftp_upload/50890/50890fig10.jpg" width="500px" /> 
Figure 10. Release of encapsulated model drug bovine serum albumin (BSA) from hydrogels formed using 2 kDa, 10 kDa, 20 kDa molecular weight PEG. BSA release occurs more rapidly from hydrogels formed using higher molecular weight PEGDM, as a result of the larger mesh size within the hydrogel. n=6, error bars = SEM. The % BSA released is significantly different (p&#60;0.0001) between all three groups at every time point except t=1 and 2.5 hr&#160;when the release from the 2 and 10 kDa gels are equivalent, by two-way repeat-measures ANOVA with Bonferroni post-hoc test. <a href="https://www.jove.com/files/ftp_upload/50890/50890fig10highres.jpg" target="_blank">Click here to view larger image</a>.

Watch this Scientific Journal Video about Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation at JoVE.com

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

microwave-assisted-functionalization-polyethylene-glycol-on-resin

Research

JoVE Journal

Chemistry

Microwave-assisted Intramolecular Dehydrogenative Diels-Alder Reactions for the Synthesis of Functionalized Naphthalenes/Solvatochromic Dyes

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to the preparation of new organic dyes. Although numerous strategies have been reported to access naphthalene scaffolds, many procedures still present limitations in terms of incorporating functionality, which in turn narrows the range of available substrates. The development of versatile methods for direct access to substituted naphthalenes is therefore highly desirable.
The Diels-Alder (DA) cycloaddition reaction is a powerful and attractive method for the formation of saturated and unsaturated ring systems from readily available starting materials. A new microwave-assisted intramolecular dehydrogenative DA reaction of styrenyl derivatives described herein generates a variety of functionalized cyclopenta[b]naphthalenes that could not be prepared using existing synthetic methods. When compared to conventional heating, microwave irradiation accelerates reaction rates, enhances yields, and limits the formation of undesired byproducts.
The utility of this protocol is further demonstrated by the conversion of a DA cycloadduct into a novel solvatochromic fluorescent dye via a Buchwald-Hartwig palladium-catalyzed cross-coupling reaction. Fluorescence spectroscopy, as an informative and sensitive analytical technique, plays a key role in research fields including environmental science, medicine, pharmacology, and cellular biology. Access to a variety of new organic fluorophores provided by the microwave-assisted dehydrogenative DA reaction allows for further advancement in these fields.

Microwave-assisted intramolecular dehydrogenative Diels-Alder (DA) reactions provide concise access to functionalized cyclopenta[b]naphthalene building blocks. The utility of this methodology is demonstrated by one-step conversion of the dehydrogenative DA cycloadducts into novel solvatochromic fluorescent dyes via Buchwald-Hartwig palladium-catalyzed cross-coupling reactions.

Functionalized naphthalenes have applications in a variety of research fields ranging from the synthesis of natural or biologically active molecules to ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Preparation of Silica Nanoparticles Through Microwave-assisted Acid-catalysis

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle diameters ranging from 30-250 nm by varying the reaction conditions. Through the selection of a microwave compatible solvent, silicic acid precursor, catalyst, and microwave irradiation time, these microwave-assisted methods were capable of overcoming the previously reported shortcomings associated with synthesis of silica nanoparticles using microwave reactors. The siloxane precursor was hydrolyzed using the acid catalyst, HCl. Acetone, a low-tan &#948; solvent, mediates the condensation reactions and has minimal interaction with the electromagnetic field. Condensation reactions begin when the silicic acid precursor couples with the microwave radiation, leading to silica nanoparticle sol formation. The silica nanoparticles were characterized by dynamic light scattering data and scanning electron microscopy, which show the materials&#39; morphology and size to be dependent on the reaction conditions. Microwave-assisted reactions produce silica nanoparticles with roughened textured surfaces that are atypical for silica sols produced by St&#246;ber&#39;s methods, which have smooth surfaces.

Silica nanoparticles were prepared using acid-catalysis of a siloxane precursor and microwave-assisted synthetic techniques resulting in the controlled growth of nanomaterials ranging from 30-250 nm in diameter. The growth dynamics can be controlled by varying the initial silicic acid concentration, time of the reaction, and temperature of reaction.

Microwave-assisted synthetic techniques were used to quickly and reproducibly produce silica nanoparticle sols using an acid catalyst with nanoparticle ...

Ethylene Polymerizations Using Parallel Pressure Reactors and a Kinetic Analysis of Chain Transfer Polymerization

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel &#945;-diimine ethylene polymerization catalyst. Initial polymerizations with the catalyst lead to optimized reaction conditions, including catalyst concentration, ethylene pressure and reaction time. Using gas-uptake data for these reactions, a procedure to calculate the initial rate of propagation (kp) is presented. Using the optimized conditions, the ability of the nickel &#945;-diimine polymerization catalyst to undergo chain transfer with diethylzinc (ZnEt2) during ethylene polymerization was investigated. A procedure to assess the ability of the catalyst to undergo chain transfer (from molecular weight and 13C NMR data), calculate the degree of chain transfer, and calculate chain transfer rates (ke) is presented.

A protocol for the high-throughput analysis of polymerization catalyst, chain transfer polymerizations, polyethylene characterization, and reaction kinetic analysis is presented.

We demonstrate a method for high-throughput catalyst screening using a parallel pressure reactor starting from the initial synthesis of a nickel ...

One-pot Microwave-assisted Conversion of Anomeric Nitrate-esters to Trichloroacetimidates

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate glycosyl donor. Following azido-nitration of a glycal, the product 2-azido-1-nitrate ester can be hydrolyzed under microwave-assisted irradiation. This transformation is usually achieved using strongly nucleophilic reagents and extended reaction times. Microwave irradiation induces hydrolysis, in the absence of reagents, with short reaction times. Following denitration, the intermediate anomeric alcohol is converted, in the same pot, to the corresponding 2-azido-1-trichloroacetimidate.

A 2-azido-1-nitrate-ester can be converted to the corresponding 2-azido-1-trichloroacetimidate in a one-pot procedure. The goal of the manuscript is to demonstrate utility of the microwave reactor in carbohydrate synthesis.

The goal of the following procedure is to provide a demonstration of the one-pot conversion of a 2-azido-1-nitrate-ester to a trichloroacetimidate ...

Surface Functionalization of Metal-Organic Frameworks for Improved Moisture Resistance

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. However, the main issue limiting their applicability is their poor stability in humid conditions. The common methods to overcome this problem involve the formation of strong metal-linker bonds by using highly charged metals, which is limited to a number of structures, the introduction of alkylic groups to the framework by post-synthetic modification (PSM) or chemical vapour deposition (CVD) to enhance overall hydrophobicity of the framework. These last two usually provoke a drastic reduction of the porosity of the material. These strategies do not permit to exploit the properties of the MOF already available and it is imperative to find new methods to enhance the stability of MOFs in water while keeping their properties intact. Herein, we report a novel method to enhance the water stability of MOF crystals featuring Cu2(O2C)4&#160;paddle-wheel units, such as HKUST (where HKUST stands for Hong Kong University of Science &#38; Technology), with the catechols functionalized with alkyl and fluoro-alkyl chains. By taking advantage of the unsaturated metal sites and the catalytic catecholase-like activity of CuII ions, we are able to create robust hydrophobic coatings through the oxidation and subsequent polymerization of the catechol units on the surface of the crystals under anaerobic and water-free conditions without disrupting the underlying structure of the framework. This approach not only affords the material with improved water stability but also provides control over the function of the protective coating, which enables the development of functional coatings for the adsorption and separations of volatile organic compounds. We are confident that this approach could also be extended to other unstable MOFs featuring open metal sites.

Robust functional catechol coatings were produced in one step by direct reaction of the material known as HKUST with synthetic catechols under anaerobic conditions. The formation of homogeneous coatings surrounding the entire crystal is ascribed to the biomimetic catalytic activity of Cu(II) dimers on the external surface of the crystals.

Metal-organic frameworks (MOFs) are a class of porous inorganic materials with promising properties in gas storage and separation, catalysis and sensing. ...

Reducing Willow Wood Fuel Emission by Low Temperature Microwave Assisted Hydrothermal Carbonization

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative environmental impact and slag formation. The selected short rotation coppice (SRC) willow wood has a high ash content (<img alt="Equation 1" src="/files/ftp_upload/58970/58970eq1.jpg" /> = 1.96%) and, therefore, a high content of emission and slag precursors. Therefore, the reduction of minerals from SRC willow wood by low temperature microwave assisted hydrothermal carbonization (MAHC) at 150 &#176;C, 170 &#176;C, and 185 &#176;C is investigated. An advantage of MAHC over conventional reactors is an even temperature conductance in the reaction medium, as microwaves penetrate the whole reactor volume. This allows a better temperature control and a faster cooldown. Therefore, a succession of depolymerization, transformation and repolymerization reactions can be analyzed effectively. In this study, the analysis of the mass loss, ash content and composition, heating values and molar O/C and H/C ratios of the treated and untreated SCR willow wood showed that the mineral content of the MAHC coal was reduced and the heating value increased. The process water showed a decreasing pH and contained furfural and 5-methylfurfural. A process temperature of 170 &#176;C showed the best combination of energy input and ash component reduction. The MAHC allows a better understanding of the hydrothermal carbonization process, while a large-scale industrial application is unlikely because of the high investment costs.

A protocol for the emission precursor depletion from low quality biomass by low temperature microwave assisted hydrothermal carbonization treatment is presented. This protocol includes the microwave parameters and the analysis of the biocoal product and process water.

Biomass is a sustainable fuel, as its CO2 emissions are reintegrated in biomass growth. However, the inorganic precursors in the biomass cause a negative ...

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it efficient in both time and resources and utilizes water as the solvent. 3-Aminocrotononitrile or an appropriate &#945;-cyanoketone is combined with an aryl hydrazine and dissolved in 1 M HCl. The mixture is then heated in a microwave reactor at 150 &#176;C, typically for 10-15 min. The product can be readily obtained by basifying the solution with 10% NaOH and isolating the desired compound with a simple vacuum filtration. The use of water as a solvent in this reaction lends to its ease and utility in production, and this method is easily reproducible with a variety of functional groups. Typical isolated yields range from 70-90%, and reactions can be performed on the milligram to gram scale with little to no change in observed yields. Some of the applications of these molecules and their derivatives include pesticides, anti-malarials, and chemotherapeutics, among many others.

Microwave-Assisted Preparation of 1-Aryl-1H-pyrazole-5-amines

1-Aryl-1H-pyrazole-5-amines are prepared from aryl hydrazines combined with either 3-aminocrotononitrile or an &#945;-cyanoketone in a 1 M HCl solution using a microwave reactor. Most reactions are done in 10-15 minutes and pure product can be obtained via vacuum filtration with typical isolated yields of 70-90%.

A synthetic process for the preparation of a variety of 1-aryl-1H-pyrazole-5-amines was developed. The microwave-mediated nature of this method makes it ...

A Microwave-Assisted Direct Heteroarylation of Ketones Using Transition Metal Catalysis

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal catalysis, the literature on direct heteroarylation is scarce. The presence of heteroatoms such as nitrogen, sulfur and oxygen often make heteroarylation a challenging research field due to catalyst poisoning, product decomposition and the rest. This protocol details a highly efficient direct &#945;-C(sp3) heteroarylation of ketones under microwave irradiation. Key factors for successful heteroarylation include the use of XPhos Palladacycle Gen. 4 Catalyst, excess base to suppress side reactions and the high temperature and pressure achieved in a sealed reaction vial under microwave irradiation. The heteroarylation compounds prepared by this method were fully characterized by proton nuclear magnetic resonance spectroscopy (1H NMR), carbon nuclear magnetic resonance spectroscopy (13C NMR) and high-resolution mass spectrometry (HRMS). This methodology has several advantages over literature precedents including broad substrate scope, rapid reaction time, greener procedure and operational simplicity by eliminating the preparation of intermediates such as silyl enol ether. Possible applications for this protocol include, but are not limited to, diversity-oriented synthesis for the discovery of biologically active small molecules, domino synthesis for the preparation of natural products and ligand development for new transition metal catalytic systems.

Heteroaryl compounds are important molecules utilized in organic synthesis, medicinal and biological chemistry. A microwave-assisted heteroarylation using palladium catalysis provides a rapid and efficient method to attach heteroaryl moieties directly to ketone substrates.

Heteroarylation introduces heteroaryl fragments to organic molecules. Despite the numerous available reactions reported for arylation via transition metal ...

Microwave Synthesis and Characterization of Nickel Hydroxide Nanosheets

Effect of Microwave Synthesis Conditions on the Structure of Nickel Hydroxide Nanosheets

A protocol for rapid, microwave-assisted hydrothermal synthesis of nickel hydroxide nanosheets under mildly acidic conditions is presented, and the effect of reaction temperature and time on the material&#39;s structure is examined. All reaction conditions studied result in aggregates of layered &#945;-Ni(OH)2 nanosheets. The reaction temperature and time strongly influence the structure of the material and product yield. Synthesizing &#945;-Ni(OH)2 at higher temperatures increases the reaction yield, lowers the interlayer spacing, increases crystalline domain size, shifts the frequencies of interlayer anion vibrational modes, and lowers the pore diameter. Longer reaction times increase reaction yields and result in similar crystalline domain sizes. Monitoring the reaction pressure in situ shows that higher pressures are obtained at higher reaction temperatures. This microwave-assisted synthesis route provides a rapid, high-throughput, scalable process that can be applied to the synthesis and production of a variety of transition metal hydroxides used for numerous energy storage, catalysis, sensor, and other applications.

Author Spotlight: A Rapid, Microwave-Assisted Hydrothermal Synthesis Of Nickel Hydroxide Nanosheets 

Nickel hydroxide nanosheets are synthesized by a microwave-assisted hydrothermal reaction. This protocol demonstrates that the reaction temperature and time used for microwave synthesis affects the reaction yield, crystal structure, and local coordination environment.

A protocol for rapid, microwave-assisted hydrothermal synthesis of nickel hydroxide nanosheets under mildly acidic conditions is presented, and the effect ...