Experimental studies on the role of intermolecular forces in mediating specific peptide-protein interactions have largely focused on the importance of peptide side chain identity and sequence rather than contributions from backbone hydrogen bonding. In developing peptidomimetic self-assembling hydrogel biomaterials with backbone ester inclusions to enhance degradability, we sought to understand the cellular response to a depsipeptide analog of the cell-adhesive RGD peptide motif. Using both solution- and solid-phase methods, we synthesized Fmoc-F-R-Glc-D, the glycolic acid (Glc)-containing side chain analog of Fmoc-FRGD, a short peptide known to self-assemble to form fibrous supramolecular structures that entrap and gel water. Fmoc-F-R-Glc-D was found also to self-assemble despite the reduced capability for supramolecular stabilization through hydrogen bonding.<br/>To assess the biological effect of the backbone ester modification, we opted to assess cell attachment and spreading in a simplified 2-D system in which RGD or R-Glc-D molecules were built on glass functionalized with an amine-terminated poly(ethylene glycol) (PEG, M<sub>n</sub> = 2000). NIH-3T3 cells were allowed to attach and spread on (depsi)peptide-PEG surfaces for 5 hours in serum-free conditions. Fluorescent staining of cell nuclei and actin filaments show clearly that cells attach at high number and spread with a high degree of cytoskeletal organization on RGD surfaces, but remain rounded on negative control surfaces (PEG only and RGE surfaces). Cells on R-Glc-D surfaces are morphologically similar to those on negative control surfaces; indeed, area and perimeter length measurements from micrographs are statistically greater only for cells on RGD surfaces. These results suggest that hydrogen bonding mediated by the R-to-G amide bond in RGD is crucial for functional integrin binding. Future work will involve quantification of depsipeptide-integrin binding affinity and kinetics using cell-free surface plasmon resonance-based techniques and biochemical assessment of upregulation of intracellular signaling molecules in the integrin signaling pathway.

Examining the importance of backbone hydrogen bonding in the RGD-integrin interaction: Consequences for engineering degradable cell-adhesive biomaterials

249th National Meeting (2015)

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