title: Peptide Conjugation to a Polymer Coating via Native Chemical Ligation of Azlactones for Cell Culture.
abstract: 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.
authors.affiliation: 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.
authors.forename: Samantha K
authors.initials: SK
authors.lastname: Schmitt
authors.affiliation: 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.
authors.forename: David J
authors.initials: DJ
authors.lastname: Trebatoski
authors.affiliation: 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.
authors.forename: John D
authors.initials: JD
authors.lastname: Krutty
authors.affiliation: 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.
authors.forename: Angela W
authors.initials: AW
authors.lastname: Xie
authors.affiliation: 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.
authors.forename: Benjamin
authors.initials: B
authors.lastname: Rollins
authors.affiliation: 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.
authors.forename: William L
authors.initials: WL
authors.lastname: Murphy
authors.affiliation: 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.
authors.forename: Padma
authors.initials: P
authors.lastname: Gopalan
mesh_terms: D002460:Cell Line; D020099:Coated Materials, Biocompatible / Q000138:chemical synthesis* / Q000494:pharmacology; D003545:Cysteine / Q000737:chemistry; D006801:Humans; D007783:Lactones / Q000737:chemistry; D059630:Mesenchymal Stem Cells / Q000187:drug effects; D010455:Peptides / Q000737:chemistry*; D011076:Polycarboxylate Cement / Q000737:chemistry; D011092:Polyethylene Glycols / Q000138:chemical synthesis / Q000494:pharmacology; D011109:Polymethacrylic Acids / Q000138:chemical synthesis / Q000494:pharmacology; D012828:Silicones / Q000737:chemistry
chemical_list: D020099:Coated Materials, Biocompatible; D007783:Lactones; D010455:Peptides; D011076:Polycarboxylate Cement; D011109:Polymethacrylic Acids; D012828:Silicones; C005506:polycarbonate; D011092:Polyethylene Glycols; D003545:Cysteine
references.citation: Nat Rev Mol Cell Biol. 2013 Aug;14(8):467-73
references.pmid: 23839578
references.citation: Biomaterials. 2012 Oct;33(28):6626-33
references.pmid: 22748769
references.citation: J Biomed Mater Res A. 2012 Feb;100(2):370-9
references.pmid: 22076848
references.citation: Molecules. 2014 Sep 12;19(9):14461-83
references.pmid: 25221869
references.citation: Langmuir. 2013 Mar 19;29(11):3805-12
references.pmid: 23425347
references.citation: Chembiochem. 2012 Aug 13;13(12):1717-24
references.pmid: 22807236
references.citation: J Mater Chem B. 2013 Mar 7;1(9):1349-1360
references.pmid: 32260808
references.citation: Life Sci. 1982 Jul 19;31(3):189-99
references.pmid: 6126794
references.citation: Curr Opin Chem Biol. 2014 Oct;22:70-8
references.pmid: 25285753
references.citation: Biomacromolecules. 2001 Spring;2(1):85-94
references.pmid: 11749159
references.citation: Bioconjug Chem. 2014 Apr 16;25(4):707-17
references.pmid: 24670265
references.citation: Anticancer Agents Med Chem. 2010 Dec;10(10):753-68
references.pmid: 21269250
references.citation: Annu Rev Cell Dev Biol. 1996;12:697-715
references.pmid: 8970741
references.citation: In Vitro. 1982 Jun;18(6):576-84
references.pmid: 7118138
references.citation: Bioconjug Chem. 2011 Jan 19;22(1):4-8
references.pmid: 21128623
references.citation: Annu Rev Chem Biomol Eng. 2012;3:421-44
references.pmid: 22524507
references.citation: Adv Healthc Mater. 2015 Jul 15;4(10):1555-64
references.pmid: 25995154
references.citation: Macromol Biosci. 2015 May;15(5):636-46
references.pmid: 25644402
references.citation: Science. 1994 Nov 4;266(5186):776-9
references.pmid: 7973629
references.citation: Nat Biotechnol. 2010 Jun;28(6):581-3
references.pmid: 20512122
references.citation: Stem Cells Int. 2012;2012:465259
references.pmid: 22448172
references.citation: Biomacromolecules. 2009 Aug 10;10(8):2194-200
references.pmid: 19601644
references.citation: ALTEX. 2003;20(4):275-81
references.pmid: 14671707