Synthesis and Use of a New Bromoacetyl-Derivatized Heterotrifunctional Amino Acid for Conjugation of Cyclic RGD-Containing Peptides Derived from Human Bone Sialoprotein

A new amino acid derivative, N alpha-(tert-butyloxycarbonyl)-N beta-(bromoacetyl)diaminopropionic acid (BBDap), has been synthesized as a reagent for introducing side-chain bromaocetyl groups into any position of a peptide sequence during solid-phase peptide synthesis. By using minor modifications to the protocol of the automated peptide synthesizer and a two-step in situ neutralization procedure, the syntheses of (bromoacetyl)diaminopropionic acid (BDap) in Arg-Gly-Asp-containing peptides from human bone sialoprotein were optimized and completed. Following HPLC purification, the BDap-derivatized peptides were cyclized or/and conjugated to carrier protein or to glass cover slips. In addition, a new procedure for site-specific conjugation of cyclic peptides to protein carriers or to glass was developed. The cell attachment activity of the peptide derivatives and conjugates was tested in cell adhesion assays with human osteoblasts, and the specificity of the binding was confirmed by competition with linear and/or cyclic forms of GRGDS. The results show that conjugates containing the linear and cyclic derivatives of the peptide EPRGDNYR supported cell attachment and spreading in a dose-dependent manner when the peptides were immobilized as described. Cell attachment to the intact bone sialoprotein and to conjugates containing the linear peptides was abolished by competition with linear and cyclic RGD-containing peptides, whereas the attachment to conjugates containing the cyclic peptide was inhibited only partially, and the cell spreading was preserved even in the presence of RGD-peptides.