title: Comparison of hydrazone heterobifunctional cross-linking agents for reversible conjugation of thiol-containing chemistry.
abstract: Reversible covalent conjugation chemistries that allow site- and condition-specific coupling and uncoupling reactions are attractive components in nanotechnologies, bioconjugation methods, imaging, and drug delivery systems. Here, we compare three heterobifunctional cross-linkers, containing both thiol- and amine-reactive chemistries, to form pH-labile hydrazones with hydrazide derivatives of the known and often published water-soluble polymer, poly[N-(2-hydroxypropyl methacrylamide)] (pHPMA), while subsequently coupling thiol-containing molecules to the cross-linker via maleimide addition. Two novel cross-linkers were prepared from the popular heterobifunctional cross-linking agent, succinimidyl-4-(N-maleimidomethyl) cyclohexane-1-carboxylate (SMCC), modified to contain either terminal aldehyde groups (i.e., 1-(N-3-propanal)-4-(N-maleimidomethyl) cyclohexane carboxamide, PMCA) or methylketone groups (i.e., 1-(N-3-butanone)-4-(N-maleimidomethyl) cyclohexane carboxamide, BMCA). A third cross-linking agent was the commercially available N-4-acetylphenyl maleimide (APM). PMCA and BMCA exhibited excellent reactivity toward hydrazide-derivatized pHPMA with essentially complete hydrazone conjugation to polymer reactive sites, while APM coupled only ∼60% of available reactive sites on the polymer despite a 3-fold molar excess relative to polymer hydrazide groups. All polymer hydrazone conjugates bearing these bifunctional agents were then further reacted with thiol-modified tetramethylrhodamine dye, confirming cross-linker maleimide reactivity after initial hydrazone polymer conjugation. Incubation of dye-labeled polymer conjugates in phosphate buffered saline at 37 °C showed that hydrazone coupling resulting from APM exhibited the greatest difference in stability between pH 7.4 and 5.0, with hydrolysis and dye release increased at pH 5.0 over a 24 h incubation period. Polymer conjugates bearing hydrazones formed from cross-linker BMCA exhibited intermediate stability with hydrolysis much greater at pH 5.0 at early time points, but hydrolysis at pH 7.4 was significant after 5 h. Hydrazones formed with the PMCA cross-linker showed no difference in release rates at pH 7.4 and 5.0.
authors.affiliation: Department of Chemistry, Colorado State University, Fort Collins, Colorado 80523-1872, USA.
authors.forename: R James
authors.initials: RJ
authors.lastname: Christie
authors.forename: Diana J
authors.initials: DJ
authors.lastname: Anderson
authors.forename: David W
authors.initials: DW
authors.lastname: Grainger
mesh_terms: D003432:Cross-Linking Reagents / Q000737:chemistry*; D015195:Drug Design; D006835:Hydrazones / Q000737:chemistry*; D006863:Hydrogen-Ion Concentration; D008689:Methacrylates / Q000737:chemistry; D008823:Micelles; D012235:Rhodamines / Q000737:chemistry; D013438:Sulfhydryl Compounds / Q000737:chemistry*
chemical_list: C114557:5-carboxytetramethylrhodamine succinimidyl ester; D003432:Cross-Linking Reagents; D006835:Hydrazones; D008689:Methacrylates; D008823:Micelles; D012235:Rhodamines; D013438:Sulfhydryl Compounds; C447720:methacryloylglycylglycine-4-nitrophenyl ester