SYNTHESIS AND USE OF A NEW BROMOACETYL-DERIVATIZED HETEROTRIFUNCTIONAL AMINO-ACID FOR CONJUGATION OF CYCLIC RGD-CONTAINING PEPTIDES DERIVEDFROM HUMAN BONE SIALOPROTEIN

A new amino acid derivative, loxycarbonyl)-N-beta-(bromoacetyl)diamino propionic acid (BBDap), has been synthesized as a reagent for introduc ing side-chain bromoacetyl groups into any position of a peptide seque nce during solid-phase peptide synthesis. By using minor modifications to the protocol of the automated peptide synthesizer and a two-step i n situ neutralization procedure, the syntheses of (bromoacetyl)diamino propionic acid (BDap) in Arg-Gly-Asp-containing peptides from human bo ne. sialoprotein were optimized and completed. Following HPLC purifica tion, 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 hu man osteoblasts, and the specificity of the binding was confirmed by c ompetition with linear and/or cyclic forms of GRGDS. The results show that conjugates containing the linear and cyclic derivatives of the pe ptide EPRGDNYR supported cell attachment and spreading in a dose-depen dent manner when the peptides were immobilized as described. Cell atta chment to the intact bone sialoprotein and to conjugates containing th e linear peptides was abolished by competition with linear and cyclic RGD-containing peptides, whereas the attachment to conjugates containi ng the cyclic peptide was inhibited only partially, and the cell sprea ding was preserved even in the presence of RGD-peptides.