Abstract
Successful strategies for the attachment of oligopeptides to mesoporous silica with pores large enough to load biomolecules should utilize the high surface area of pores to provide an accessible, protective environment. A two-step oligopeptide functionalization strategy is examined here using diazirine-based heterobifunctional linkers. Mesoporous silica nanoparticles (MSNPs) with average pore diameter of ~8 nm and surface area of ~730 m2/g were synthesized and amine-functionalized. Tetrapeptides Gly-Gly-Gly-Gly (GGGG) and Arg-Ser-Ser-Val (RSSV), and a peptide comprised of four copies of RSSV (4RSSV), were covalently attached via their N-terminus to the amine groups on the particle surface by a heterobifunctional linker, sulfo-succinimidyl 6-(4,4′-azipentanamido)hexanoate (sulfo-NHS-LC-diazirine, or SNLD). SNLD consists of an amine-reactive NHS ester group and UV-activable diazirine group, providing precise control over the sequence of attachment steps. Attachment efficiency of RSSV was measured using fluorescein isothiocyanate (FITC)-tagged RSSV (RSSV-FITC). TGA analysis shows similar efficiency (0.29, 0.31 and 0.26 mol peptide/mol amine, respectively) for 4G, RSSV and 4RSSV, suggesting a generalizable method of peptide conjugation. The technique developed here for the conjugation of peptides to MSNPs provides for their attachment in pores and can be translated to selective peptide-based separation and concentration of therapeutics from aqueous process and waste streams.
Highlights
Introduction iationsSynthetic organic functional groups that can mimic the biological specificity of host–guest interactions have been used for analysis, sensing and isolation of different biomolecules, especially in affinity column chromatography [1,2,3,4]
Both MSNPA-RSSV and MSNPA-4RSSV show increased inincreased intensity corresponding toside the arginine side chain stretching vibration, tensity corresponding to the arginine chain stretching vibration, which confirmswhich pepconfirms peptide attachment to the particle surface
Hetero-bifunctional peptide linkers containing a diazirine group provide precise control of the mechanism and orientation during attachment with a high activation wavelength (365 nm), which is more benign to proteins and peptides compared to other linkers that are activated at lower wavelength
Summary
Type-1 attachment of the linker to the particle amine linker Sulfo-NHS-LC-Diazirine (SNLD): (a) Type-1 attachment of the linker to the particle amine group first and attaching to the peptide amine group usingusing the UV-reactive firstusing usingthe theNHS. NHSgroup group and attaching to the peptide amine group the UVreactive diazirine group and (b)attachment. Type-2 attachment of the to the amine peptidegroup aminefirst group first diazirine group and (b) Type-2 of the linker tolinker the peptide using the using the NHS group and attaching to the particle amine group using the UV-reactive diazirine. NHS group and attaching to the particle amine group using the UV-reactive diazirine group
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