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

Abstract

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 (<b>1</b>) obtained from <i>p</i>-arsanilic acid (As­(V)) was shown to readily undergo conjugation to the therapeutic peptide salmon calcitonin (sCT) via bridging of the Cys¹-Cys⁷ disulfide, which was verified by RP-HPLC and MALDI-ToF-MS. Conjugation was shown to proceed rapidly (<i>t</i> < 2 min) <i>in situ</i> 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 <b>1</b> 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, <i>p</i>-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) (PPEGA₄₈₀, DP_<i>n</i> = 10, <i>M</i>_n,NMR = 4900 g·mol^–1, <i><i>Đ</i></i> = 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