RESUMEN
Thiofunctional polymers are the established standard for the coating and biofunctionalization of gold nanoparticles (AuNPs). However, the nucleophilic and oxidative character of thiols provokes polymeric crosslinking and significantly limits the chemical possibilities to introduce biological functions. Thioethers represent a chemically more stable potential alternative to thiols that would offer easier functionalization, yet a few studies in the literature report inconclusive data regarding the efficacy of thioethers to stabilize AuNPs in comparison to thiols. A systematic comparison is presented of mono- versus multivalent thiol- and thioether-functional polymers, poly(ethylene glycol) versus side chain functional poly(glycidol) (PG) and it is shown that coating of AuNPs with multivalent thioether-functional PG leads to superior colloidal stability, even under physiological conditions and after freeze-drying and resuspension, as compared to thiol analogs at comparable polymer surface coverages. In addition, it is shown that a wide range of functional groups can be introduced in these polymers. Using diazirine functionalization as example, it is demonstrated that proteins can be covalently immobilized, and that conjugation of antibodies via this strategy enables efficient targeting and laser-irradiation induced killing of cells.
RESUMEN
The carbonyl reductase from Candida parapsilosis (CPCR2) is an industrially attractive biocatalyst for producing chiral alcohols from ketones. The homodimeric enzyme has a broad substrate spectrum and an excellent stereoselectivity, but is rapidly inactivated at aqueous-organic interfaces. The latter limits CPCR2's application in biphasic reaction media. Reengineering the protein surface of CPCR2 yielded a variant CPCR2-(A275N, L276Q) with 1.5-fold increased activity, 1.5-fold higher interfacial stability (cyclohexane/buffer system), and increased thermal resistance (ΔT50=+2.7 °C). Site-directed and site-saturation mutagenesis studies discovered that position 275 mainly influences stability and position 276 governs activity. After single site-saturation of position 275, amino acid exchanges to asparagine and threonine were discovered to be stabilizing. Interestingly, both positions are located at the dimer interface and close to the active site and computational analysis identified an inter-subunit hydrogen bond formation at position 275 to be responsible for stabilization. Finally, the variant CPCR2-(A275S, L276Q) was found by simultaneous site-saturation of positions 275 and 276. CPCR2-(A275S, L276Q) has compared to wtCPCR2 a 1.4-fold increased activity, a 1.5-fold higher interfacial stability, and improved thermal resistance (ΔT50=+5.2 °C).