Use of Native Chemical Ligation as a Tool for Polymer Construction over Solid Substrates

摘要

We previously showed [1] that polypeptides can be rapidly immobilized on glass substrates by means of thiol catalyzed native chemical ligation (NCL) [2]. The aim of the present contribution is to build long unordered molecules on glass surfaces for biosensors application. Linear ωamino-PEG thioesters of 10 kDa (PEG = polyethylene glycol) were to be attached on glass surfaces and to be extended to 20 kDa oligomers. NCL proved to be a useful method to attach PEG-thioestcr over cysteine modified substrate. In this study, surface plasmon resonance (SPR) has been used to follow attachement of these PEG to the substrate. Native chemical ligation reaction rates of 5 and 10 kDa a-Boc-PEGω-thioester were monitored and compared. Using the same strategy 20 kDa oligomers were created using successive NCL. For this purpose, first α-Boc-Cys(Boc)-PEG-ω-thioester was synthesized and coupled to cysteine modified glass surfaces by NCL. A second PEG unit was then attached by the same procedure after acid deprotection of the Boc group of the first BocCys(Boc)-PEG unit. In order to measure the length of the attached constructions, a-biotinyl PEG (10kDa)ω-thioester modified surfaces were probed by atomic force microscopy (AFM). AFM cantilevers were coated with biotinyl BSA and then with avidin. PEG length was measured by pulling perpendicularly and the pulling force was recorded.