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

Abstract

Synthetic folded insulin variants containing an ornithine‐hydroxylamine residue are readily modified in aqueous buffers by amide‐forming ligations with potassium acyltrifluoroborates (KATs). The synthetic insulin analogs were prepared by Fmoc‐SPPS, α‐ketoacid‐hydroxylamine (KAHA) ligation, and a prosthetic C‐peptide that delivers the correct disulfide pattern and allows facile incorporation at the B0 position of Glargine M2 of a new ornithine hydroxylamine protected with a photolabile group. The folded insulin is readily modified by photo‐deprotection followed by amide‐forming KAT ligation to give insulin variants labeled with dyes, lipids, and PEGs, as well as the formation of a covalent dimer.

Recent Advances in the Synthesis of Acylboranes and Their Widening Applicability

“…The previously described method using hydroxylamines could nevertheless be used by the same group to PEGylate and dimerize expressed and already folded proteins. 21 Hydroxylamine-equipped proteins were readily ligated with 2-pyridyl KATs bearing a PEG-linker. 21a The use of 2-pyridyl KATs was essential as they were shown to be significantly more susceptible toward ligation than other aryl KATs.…”

“…The previously described method using hydroxylamines could nevertheless be used by the same group to PEGylate and dimerize expressed and already folded proteins . Hydroxylamine-equipped proteins were readily ligated with 2-pyridyl KATs bearing a PEG-linker .…”

Recent Advances in the Chemical Synthesis of Insulin and Related Peptides

“…An additional element of this versatile approach was the use of a base-labile arginine solubilizing handle to enhance A-chain aqueous solubility. The Bode research group subsequently integrated their KAHA and KAT (potassium acyltrifluoroborate) ligation methods to yield a photoprotected hydroxylamine amino acid [23]. Through this synthetic intermediate a series of site-specific glargine-like insulin conjugates were further prepared.…”

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.…”