Peptide Synthesis for Hydrogel Biomaterials

• Brian Harrity, Material Science, University of Delaware

• April Kloxin, MSEG/BCE, University of Delaware

Solid-phase peptide synthesis (SPPS) enables researchers to build peptides with exact amino acid sequences and functional side chains. This research project concentrated on understanding and performing Fmoc-based SPPS which builds peptide chains through sequential addition on solid resin beads. The Fmoc protecting group is removed by piperidine before the next amino acid is attached on the resulting amine through a coupling step involving Oxyma and N,N‘-Diisopropylcarbodiimide (DIC). The process relies on protected amino acid side chains to prevent any unwanted chemical reactions. The completed peptide sequence is detached from the resin using a trifluoroacetic acid (TFA) cocktail solution which simultaneously removes any side chain protection groups. The cleaved peptide is then precipitated in cold diethyl ether to remove any excess TFA and is centrifuged to isolate the crude peptide. The resulting peptide is purified using high-performance liquid chromatography (HPLC) and characterized by liquid chromatography–mass spectrometry (LC-MS) to verify the correct product and assess its purity. The peptides I have synthesized through this method serve important roles in hydrogel biomaterials used for cell culture and tissue engineering applications. When incorporated, different sequences can have various impacts on the hydrogel’s environment. Some promote cell adhesion, such as RDG (Arg-Gly-Asp), which binds to integrins enabling cells to attach, grow, and function normally. Others, like the GFOGER sequence found in collagen, mimic proteins found in natural tissue environments, helping to recreate the conditions that would be present for that type of tissue. Certain sequences, such as GPQGIWGQ, can also be designed to degrade in response to enzymes secreted by cells, allowing the hydrogel to change over time, mimicking how real tissues act in the human body. These peptide-functionalized hydrogels are an important part in the creation of tunable microenvironments that can mimic both healthy and diseased tissue providing an effective system for studying cellular responses.