Surfaces for Immobilization of N-Terminal Cysteine Derivatives via Native Chemical Ligation

This paper presents an improved method for fast, reliable, and quantitative native chemical ligation (NCL - the reaction between an N-terminal cysteine and a thioester to yield an amide bond) at thiophenyl ester-functionalized glass surfaces. For the first time, the degree of surface functionalization has been measured and can be readily controlled by varying the concentration of thiophenyl ester groups on the surface. This methodology facilitates the preparation of tailor-made functionalized glass surfaces for diverse applications. S-Phenyl 11-(chlorodimethylsilyl)undecanethiolate, and the benzyl analogue, are readily prepared from 10-undecanoic acid via thioester formation and platinum-catalyzed hydrosilylation of the terminal alkene functionality. Thioesters covalently bound to glass surfaces were then formed by submerging clean glass in toluene-solutions of the relevant thioester silylchloride (1%) containing the non-nucleophilic base, ethyldiisopropylamine (1%). NCL was explored with a cysteine-lissamine conjugate, and the degree of surface functionalization was quantified by UV/vis absorption spectroscopy, using the lissamine chromophore. NCL at thiophenyl ester surfaces proved fast (half-life less than 10 min) and yielded levels of surface functionalization consistent with a dense monolayer of dimethyloctylsilane (ca. 2.0 molecules/nm(2)). Water contact angles on thiophenyl ester surfaces were found to decrease after NCL reaction with the cysteine-lissamine conjugate, whereas Surfaces treated with the same buffer solution containing an Unreactive alanine-lissamine conjugate showed no significant changes in contact angle, indicating that thioester hydrolysis is not significant during the course of the reaction. NCL with the cysteine-lissamine conjugate at mixed surfaces containing both thiophenyl esters and inert octyl chains showed lower levels of surface functionalization as expected. Plotting the proportion of thiophenyl esters used in the preparation of the substrates against integrated absorption from the surface yielded a linear relationship, demonstrating that NCL on these surfaces occurs in a controllable manner.