title: Conjugation of Methotrexate-Amino Derivatives to Macromolecules through Carboxylate Moieties Is Superior Over Conventional Linkage to Amino Residues: Chemical, Cell-Free and In Vitro Characterizations.
abstract: In this study, we examined the possibility of introducing methotrexate (MTX) to the carboxylate rather than to the ε-amino side chains of proteins. We found that MTX-amino compounds covalently linked to the carboxylate moieties of macromolecules, undergo unusual peptide-bond cleavage, with the release of the MTX amino derivatives from the conjugates. This event takes place at an accelerated rate under acidic conditions, and at a slower rate at physiological pH values. The glutamate portion of MTX is responsible for this behavior, with little or no contribution of the p-aminobenzoate-pteridine ring that is linked to the α-amino side chain of the glutamate. Carboxylate-linked Fmoc-Glu-γ-CONH-(CH2)6-NH2 undergoes hydrolysis in a nearly indistinguishable fashion. A free α carboxylate moiety is essential for this effect. Carboxylate linked Fmoc-glutamic-amide-γ-CONH-(CH2)6-NH2 undergoes no hydrolysis under acidic conditions. Based on these findings, we engineered a cysteine specific MTX containing reagent. Its linkage to bovine serum albumin (BSA) yielded a conjugate with profound antiproliferative efficacy in a MTX-sensitive glioma cell line. In conclusion, carboxylate linked MTX-amino derivatives in particular, and carboxylate linked R-α-GLU-γ amino compounds in general are equipped with'built-in chemical machinery' that releases them under mild acidic conditions.
authors.affiliation: BBB-group, The Joseph Sagol Neuroscience Center, Sheba Medical Center, Tel Hashomer, Ramat Gan, 52621, Israel.
authors.forename: Itzik
authors.initials: I
authors.lastname: Cooper
authors.affiliation: Department of Organic Chemistry, The Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Mati
authors.initials: M
authors.lastname: Fridkin
authors.affiliation: Department of Biomolecular Sciences, The Weizmann Institute of Science, Rehovot, 76100, Israel.
authors.forename: Yoram
authors.initials: Y
authors.lastname: Shechter
mesh_terms: D000818:Animals; D000970:Antineoplastic Agents / Q000737:chemistry / Q000494:pharmacology; D002264:Carboxylic Acids / Q000737:chemistry*; D002417:Cattle; D045744:Cell Line, Tumor; D003545:Cysteine / Q000737:chemistry; D005910:Glioma / Q000473:pathology; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D008727:Methotrexate / Q000737:chemistry* / Q000494:pharmacology; D010455:Peptides / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry; D012710:Serum Albumin, Bovine / Q000737:chemistry*
chemical_list: D000970:Antineoplastic Agents; D002264:Carboxylic Acids; D010455:Peptides; D012710:Serum Albumin, Bovine; D011092:Polyethylene Glycols; D003545:Cysteine; D008727:Methotrexate