Schmitt Samantha K, Trebatoski David J, Krutty John D, Xie Angela W, Rollins Benjamin, Murphy William L, Gopalan Padma
Department of Materials Science and Engineering, ‡Department of Biomedical Engineering, §Department of Orthopedics and Rehabilitation, and ∥Department of Chemistry, University of Wisconsin-Madison , Madison, Wisconsin 53706, United States.
Biomacromolecules. 2016 Mar 14;17(3):1040-7. doi: 10.1021/acs.biomac.5b01682. Epub 2016 Feb 15.
Conjugation of biomolecules for stable presentation is an essential step toward reliable chemically defined platforms for cell culture studies. In this work, we describe the formation of a stable and site-specific amide bond via the coupling of a cysteine terminated peptide at low concentration to an azlactone containing copolymer coating. A copolymer of polyethylene glycol methyl ether methacrylate-ran-vinyl azlactone-ran-glycidyl methacrylate P(PEGMEMA-r-VDM-r-GMA) was used to form a thin coating (20-30 nm) on silicon and polycarbonate substrates. The formation and stability of coating-peptide bonds for peptides containing free thiols and amines were quantified by X-ray photoelectron spectroscopy (XPS) after exposure to cell culture conditions. Peptides containing a thiol as the only nucleophile coupled via a thioester bond; however, the bond was labile under cell culture conditions and almost all the bound peptides were displaced from the surface over a period of 2 days. Coupling with N-terminal primary amine peptides resulted in the formation of an amide bond with low efficiency (<20%). In contrast, peptides containing an N-terminal cysteine, which contain both nucleophiles (free thiol and amine) in close proximity, bound with 67% efficiency under neutral pH, and were stable under the same conditions for 2 weeks. Control studies confirm that the stable amide formation was a result of an intramolecular rearrangement through a N-acyl intermediate that resembles native chemical ligation. Through a combination of XPS and cell culture studies, we show that the cysteine terminated peptides undergo a native chemical ligation process at low peptide concentration in aqueous media, short reaction time, and at room temperature resulting in the stable presentation of peptides beyond 2 weeks for cell culture studies.
生物分子的共轭以实现稳定呈现是迈向用于细胞培养研究的可靠化学定义平台的关键一步。在这项工作中，我们描述了通过将低浓度的半胱氨酸末端肽与含氮杂环丙烷的共聚物涂层偶联形成稳定且位点特异性的酰胺键。聚乙二醇甲基醚甲基丙烯酸酯 - 无规 - 乙烯基氮杂环丙烷 - 无规 - 甲基丙烯酸缩水甘油酯共聚物P(PEGMEMA - r - VDM - r - GMA)用于在硅和聚碳酸酯基板上形成薄涂层(20 - 30纳米)。在暴露于细胞培养条件后，通过X射线光电子能谱(XPS)对含游离硫醇和胺的肽的涂层 - 肽键的形成和稳定性进行了定量。仅含硫醇作为亲核试剂的肽通过硫酯键偶联；然而，该键在细胞培养条件下不稳定，并且几乎所有结合的肽在2天内从表面被置换。与N端伯胺肽偶联导致酰胺键形成效率低(<20%)。相比之下，含N端半胱氨酸的肽，其两个亲核试剂(游离硫醇和胺)紧邻，在中性pH下结合效率为67%，并且在相同条件下稳定2周。对照研究证实，稳定的酰胺形成是通过类似于天然化学连接的N - 酰基中间体进行分子内重排的结果。通过结合XPS和细胞培养研究，我们表明半胱氨酸末端肽在水性介质中低肽浓度、短反应时间和室温下经历天然化学连接过程，从而在细胞培养研究中实现肽超过2周的稳定呈现。
