Organic Arsenicals As Efficient and Highly Specific Linkers for Protein/Peptide-Polymer Conjugation

摘要

The entropy-driven affinity of trivalent (in) organic arsenicals for closely spaced dithiols has been exploited to develop a novel route to peptide/protein-polymer conjugation. A trivalent arsenous acid (As(Ⅲ)) derivative (1) obtained from p-arsanilic acid (As(Ⅴ)) was shown to readily undergo conjugation to the therapeutic peptide salmon calcitonin (sCT) via bridging of the Cys~1-Cys~7 disulfide, which was verified by RP-HPLC and MALDI-ToF-MS. Conjugation was shown to proceed rapidly (t ＜ 2 min) in situ and stoichiometrically through sequential reduction-conjugation protocols, therefore exhibiting conjugation efficiencies equivalent to those reported for the current leading disulfide-bond targeting strategies. Furthermore, using bovine serum albumin as a model protein, the trivalent organic arsenical 1 was found to demonstrate enhanced specificity for disulfide-bond bridging in the presence of free cysteine residues relative to established maleimide functional reagents. This specificity represents a shift toward potential orthogonality, by clearly distinguishing between the reactivity of mono- and disulfide-derived (vicinal or neighbors-through-space) dithiols. Finally, p-arsanilic acid was transformed into an initiator for aqueous single electron-transfer living radical polymerization, allowing the synthesis of hydrophilic arsenic-functional polymers which were shown to exhibit negligible cytotoxicity relative to a small molecule organic arsenical, and an unfunctionalized polymer control. Poly(poly[ethylene glycol] methyl ether acrylate) (PPEGA_(480), DP_n = 10, M_(n,NMR) = 4900 g· mol~(-1), D = 1.07) possessing a pentavalent arsenic acid (As(Ⅴ)) α-chain end was transformed into trivalent As(Ⅲ) post-polymerization via initial reduction by biological reducing agent glutathione (GSH), followed by binding of GSH. Conjugation of the resulting As(Ⅲ)-functional polymer to sCT was realized within 35 min as indicated by RP-HPLC and verified later by thermodynamically driven release of sCT, from the conjugate, in the presence of strong chelating reagent ethanedithiol.