Facile synthesis of a chitosan hybrid of a laminin-related peptide and itsantimetastatic effect in mice

Laminin, a cell adhesion protein, consists of three peptide chains (alpha-1 , beta-1 and gamma-1). The beta-1 chain contains a Tyr-Ile-Gly-Ser-Arg (YIG SR) sequence that; has been found to inhibit experimental metastasis in mic e. We have prepared a hybrid of a water-soluble chitosan and a laminin-rela ted peptide, and have examined its inhibitory effect on experimental metast asis in mice. A laminin-related peptide, acetyl-Tyr-Ile-Gly-Ser-Arg-beta Ala-OH (Ac-YIGSR beta A-OH), was prepared by a solid-phase method. Ac-YIGSR beta A-OH was t hen reacted with a water-soluble chitosan. beta Ala is a spacer and was pla ced to avoid racemization of the Arg residue when the peptide was coupled w ith chitosan. Although chitosan has amino groups, they did not react with t he peptide. Four methods were tried to achieve a coupling reaction, the dip henylphosphoryl azide method, the diisopropylcarbodiimide/1-hydroxybenzotri azole method, the water-soluble carbodiimide (WSC), and the 2-(1H-benzotria zole-1-yl)-1,1,3,3-tetramethyluronium tetrafluoroborate (TBTU) method, but all four methods were unsuccessful. Therefore, a small spacer, tert-butylox ycarbonyl-Gly, was intercalated in chitosan, by the TBTU method, to facilit ate its coupling with the peptide. After removal of the protecting group, t he Gly-chitosan was coupled with Ac-YIGSR beta A-OH by the water-soluble ca rbodiimide method to give Ac-YIGSR beta AG-chitosan. Conjugation of the peptide with the larger chitosan molecule did not reduce the inhibitory effect of the peptide on experimental metastasis in mice, i t actually potentiated the antimetastatic effect, demonstrating that chitos an may be effective as a drug carrier for peptides.