PEGylation and Dimerization of Expressed Proteins under Near Equimolar Conditions with Potassium 2-Pyridyl Acyltrifluoroborates

Abstract

The covalent conjugation of large, functionalized molecules remains a frontier in synthetic chemistry, as it requires rapid, chemoselective reactions. The potassium acyltrifluoroborate (KAT)–hydroxylamine amide-forming ligation shows promise for conjugations of biomolecules under aqueous, acidic conditions, but the variants reported to date are not suited to ligations at micromolar concentrations. We now report that 2-pyridyl KATs display significantly enhanced ligation kinetics over their aryl counterparts. Following their facile, one-step incorporation onto the termini of polyethylene glycol (PEG) chains, we show that 2-pyridyl KATs can be applied to the construction of protein–polymer conjugates in excellent (>95%) yield. Four distinct expressed, folded proteins equipped with a hydroxylamine could be PEGylated with 2–20 kDa 2-pyridyl mPEG KATs in high yield and with near-equimolar amounts of coupling partners. Furthermore, the use of a bis 2-pyridyl PEG KAT enables the covalent homodimerization of proteins with good conversion. The 2-pyridyl KAT ligation offers an effective alternative to conventional protein–polymer conjugation by operating under aqueous acidic conditions well suited for the handling of folded proteins.

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).…”

“…[36 -38] The KAT reaction has shown faster second order rate constants than most ligation techniques, albeit requiring acidic conditions. [39] To date, the KAT reaction has been used for both the assembly of small molecules and peptides, [40] as well as for the conjugation of larger molecules including PEGylation, homodimerization of proteins, [41] and the formation of hydrogels. [42] We envisioned that addition of a photocontrollable component to this process would expand the repertoire of its applications.…”

Chemoselective Derivatization of Folded Synthetic Insulin Variants with Potassium Acyltrifluoroborates (KATs)

“…[17] More recently, we reported the PEGylation and dimerization of expressed proteins using KAT ligation; in this case the hydroxylamines were attached to a surface exposed cysteine using a labeling reagent. [18] In this report, we document the preparation of a synthetic, folded protein bearing a protected ornithine hydroxylamine, which following facile deprotection with UV light undergoes conjugation with KAT reagents (Scheme 1,b). Advances in chemical protein synthesis make it competitive to expression for the production of small proteins (less than 200 residues) and allows the facile incorporation of non-canonical residues.…”

Chemoselective 18F-incorporation into pyridyl acyltrifluoroborates for rapid radiolabelling of peptides and proteins at room temperature

“…18 The Bode group successfully used this method for the modification of a synthetic GLP1 analogue 19 and for the two-step modification of recombinant proteins. 20 In order to harness the advantages of the KAT ligation for radiochemistry applications, we sought to develop a new 18 F-labelling prosthetic group based on this chemistry.…”

“…To further extend this method towards larger biomolecules, we modified sfGFP(S147C) 20 with a maleimide functionality bearing the hydroxylamine group at the exposed cysteine residue (Scheme 4). Due to the low concentrations of the protein used, small amounts of [ 18 F]FPAT (50-100 MBq) were used respectively to avoid interference of the cold [ 19 F]FPAT.…”