Potassium Acyltrifluoroborate (KAT) Ligations are Orthogonal to Thiol‐Michael and SPAAC Reactions: Covalent Dual Immobilization of Proteins onto Synthetic PEG Hydrogels

Abstract

The covalent immobilization of peptides, proteins, and other biomolecules to hydrogels provides a biologically mimicking environment for cell and tissue growth. Bioorthogonal chemical reactions can serve as a tool for this, but the paucity of such reactions and mutual incompatibilities limits the number of distinct molecules that can be introduced. We now report that the potassium acyltrifluoroborate (KAT) amide‐forming ligation is orthogonal to both thiol‐Michael and strain promoted azide alkyne cycloadditions (SPAAC) and the requisite functional groups – KATs and hydroxylamines – are stable and compatible to hydrogel formation, protein modification, and post‐assembly immobilization of biomolecules onto hydrogels. In combination these ligations enables stepwise covalent protein immobilization of multiple BSA‐derivatives onto the hydrogel scaffold regardless of the order of addition.

Post‐Assembly Photomasking of Potassium Acyltrifluoroborates (KATs) for Two‐Photon 3D Patterning of PEG‐Hydrogels

“…[42] By using a slight excess of the KAT PEG building block 7 during hydrogel formation (Scheme 4), covalent functionalization of the hydrogel could be induced through KAT-hydroxylamine ligation at pH 6.0 with a small amount of acyltrifluoroborate groups remaining. The presence of unreacted KATs was established by immersing the gel in hydroxylamine-functionalized fluorophore solutions (50 μM; super-folder green fluorescent protein (sfGFP) [41] 9 or sulforhodamine B [49] 10), leading to visible fluorescence after thorough removal of the excess fluorophore (Scheme 5,A). Treatment of the hydrogels with excess photocage 5 followed by washing and subsequent exposure to hydroxylamines led to no observed fluorescence, as determined by confocal microscopy imaging (Scheme 5,B).…”

Synthesis of Acylboron Compounds

“…Encapsulation of bovine chondrocytes through these hydrogels further showcased the biocompatibility of the acyltrifluoroborate group (Scheme ) . Using the hydrogel as a model system, dual immobilization of proteins inside of the hydrogel was achieved, demonstrating the orthogonality of the KAT ligation toward SPAAC and thiol‐ Michael addition …”

Dynamic Amide Formation by Reversible Nitrone Exchange of Potassium Acyltrifluoroborates (KATs) and Hydroxylamines in Aqueous Conditions

“…Potassium Acyltrifluoroborates (KATs) are a class of bench stable compounds with versatile reactivity, and have recently found various applications in bioconjugation [35][36][37] and material science [38][39][40][41][42] due to their ability to undergo rapid ligations 43,44 with O-substituted hydroxylamines or N-chloro amines under mild and dilute conditions to form amides. The union of O-unsubstituted hydroxylamines and KATs, however, results in a nitrone, 45 which upon further activation can rearrangement to an amide.…”