Facile synthetic procedure for omega, primary amine functionalization directly in water for subsequent fluorescent labeling and potential bioconjugation of RAFT-synthesized (co)polymers.
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.
York
Adam W
AW
Department of Polymer Science and Department of Chemistry and Biochemistry, The University of Southern Mississippi, Hattiesburg, Mississippi 39406, USA.
Scales
Charles W
CW
Huang
Faqing
F
McCormick
Charles L
CL
D000588:Amines / Q000737:chemistry*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D008689:Methacrylates / Q000138:chemical synthesis / Q000737:chemistry; D009555:Ninhydrin / Q000737:chemistry; D014867:Water / Q000737:chemistry
C115825:6-(fluorescein-5-carboxamido)hexanoic acid succimidyl ester; D000588:Amines; D005452:Fluoresceins; D005456:Fluorescent Dyes; D008689:Methacrylates; C523539:poly(N-(2-hydroxypropyl) methacrylamide-b-N-(3-(dimethylamino)propyl) methacrylamide); D014867:Water; D009555:Ninhydrin