Facile Synthetic Procedure for ω, Primary Amine Functionalization Directly in Water for Subsequent Fluorescent Labeling and Potential Bioconjugation of RAFT-Synthesized (Co)Polymers

Abstract

We describe a facile method to amine functionalize and subsequently fluorescently label polymethacrylamides synthesized via reversible addition-fragmentation chain transfer (RAFT) polymerization. RAFT-generated poly(N-(2-hydroxypropyl) methacrylamide-b-N-[3-(dimethylamino)propyl] methacrylamide) (poly(HPMA-b-DMAPMA)), a water soluble biocompatible polymer, is first converted to a polymeric thiol and functionalized with a primary amine through a disulfide exchange reaction with cystamine and subsequently reacted with the amine-functionalized fluorescent dye, 6-(fluorescein-5-carboxamido)hexanoic acid, succinimidyl ester (5-SFX). Poly(HPMA258-b-DMAPMA13) (Mn = 39 700 g/mol, Mw/Mn = 1.06), previously synthesized by RAFT polymerization, was used to demonstrate this facile labeling method. The problem with labeling the omega-terminal chain end of a RAFT-synthesized polymethacrylamide is that the reduced end yields a tertiary thiol with low reactivity. The key to labeling poly(HPMA-b-DMAPMA) is to first reduce the dithioester chain end with a strong reducing agent such as NaBH4, and then functionalize the tertiary polymeric thiol with a primary amine through a disulfide exchange reaction with dihydrochloride cystamine. We show that the disulfide exchange reaction is efficient and that the amine-functionalized poly(HPMA-b-DMAPMA) can be easily labeled with the fluorescent dye, 5-SFX. This concept is proven by using a ninhydrin assay to detect primary amines and UV-vis spectroscopy to measure the degree of conjugation.

177Lu-labeled HPMA copolymers utilizing cathepsin B and S cleavable linkers: Synthesis, characterization and preliminary in vivo investigation in a pancreatic cancer model

“…[26] A ninhydrin solution was prepared by dissolving 5.61 mmole of ninhydrin in 25 mL ethylene glycol. A SnCl 2 solution was made by dissolving 0.177 mmole of SnCl 2 .2H 2 O in 25 mL of 0.2 M citrate buffer.…”

Modification of RAFT‐polymers via thiol‐ene reactions: A general route to functional polymers and new architectures

“…4,27,30,[73][74][75][76] To ensure the presence of trithiocarbonate or dithiobenzoate RAFT end-groups on the generated polymers, termination was minimized by selecting a low ratio of the polymerization initiator to the RAFT agent concentrations in the feed solutions (([Initiator] 0 / [RAFT] 0 ), i.e., 1/5 (theoretical functionality…”

“…au) or V. Bulmus (E-mail: vbulmus@unsw.edu.au) been the removal of the RAFT end-group by reduction or aminolysis 5,6,48,52-59 yielding functional polymers 27,60,61 and polymers with thiol end-groups that can be further modified. 54,56,[62][63][64] This aminolysis approach has a potential serious drawback, as the resultant thiol is susceptible to further oxidation to disulfide leading to bimodal polymer populations composed of thiol and disulfide functional polymers.…”

Anwendung von Poly(ethylenglycol) beim Wirkstoff‐Transport: Vorteile, Nachteile und Alternativen

“…B. Atom-Transfer-Radikal-Polymerisation (ATRP) oder Additions-Fragmentierungs-Kettenübertragungs(RAFT)-Polyme-risation) synthetisiert werden, [209][210][211] die zu niedrigen PDIWerten und verschiedenen definierten Endgruppen führen, die weitere chemische Modifizierungen ermöglichen. [212] Zur Beurteilung der Polymerstabilität wurde die Degradation bei Erhitzen untersucht. [213] Wie bei den Vinylpolymeren wurde ein Abbau festgestellt, der jedoch unter physiologischen Bedingungen nicht stattfindet.…”