Mechanism-Based Design of Quinoline Potassium Acyltrifluoroborates for Rapid Amide-Forming Ligations at Physiological pH

Abstract

Potassium acyltrifluoroborates (KATs) undergo chemoselective amide-forming ligations with hydroxylamines. Under aqueous, acidic conditions these ligations can proceed rapidly, with rate constants of ∼20 M–1 s–1. The requirement for lower pH to obtain the fastest rates, however, limits their use with certain biomolecules and precludes in vivo applications. By mechanistic investigations into the KAT ligation, including kinetic studies, X-ray crystallography, and DFT calculations, we have identified a key role for a proton in accelerating the ligation. We applied this knowledge to the design and synthesis of 8-quinolyl acyltrifluoroborates, a new class of KATs that ligates with hydroxylamines at pH 7.4 with rate constants >4 M–1 s–1. We trace the enhanced rate at physiological pH to unexpectedly high basicity of the 8-quinoline-KATs, which leads to their protonation even under neutral conditions. This proton assists the formation of the key tetrahedral intermediate and activates the leaving groups on the hydroxylamine toward a concerted 1,2-BF3 shift that leads to the amide product. We demonstrate that the fast ligations at pH 7.4 can be carried out with a protein substrate at micromolar concentrations.

An N-Allyl Glyoxylamide Based Bioorthogonal Nitrone Trap

“…The reaction is complementary to other hydroxylamine based chemistries such as the use of N,N-dialkylhydroxylamine reactions with strained alkenes and the reaction of O-functionalized hydroxylamines with acyltrifluoroborates. [30,32] The kinetics of the reaction is first order in glyoxylamide and zero order in hydroxylamine. This facilitates rapid labelling reactions at the low concentrations often encountered under biological conditions.…”

An N-Allylglyoxylamide Based Bioorthogonal Nitrone Trap

“…The reaction is complementary to other hydroxylamine based chemistries such as the use of N,N-dialkylhydroxylamine reactions with strained alkenes and the reaction of O-functionalized hydroxylamines with acyltrifluoroborates. 30,32 The kinetics of the reaction with small molecules is first order in glyoxylamide and catalyst, and zero order in hydroxylamine, although more complex kinetics are present in biological samples.…”

Opportunities for Expanding Encoded Chemical Diversification and Improving Hit Enrichment in mRNA‐Displayed Peptide Libraries

“…The keto‐acid hydroxy acid is one such reaction, but suffers from slow kinetics that may make it less efficient in library generation [147] . Potassium acyltrifluoroborates improve on this aspect, but are synthetically more difficult to access (although this has improved recently) [148] . Nevertheless, both seem well suited to genetic incorporation as a chemical handle in a peptide library for diversification.…”

“… [147] Potassium acyltrifluoroborates improve on this aspect, but are synthetically more difficult to access (although this has improved recently). [148] Nevertheless, both seem well suited to genetic incorporation as a chemical handle in a peptide library for diversification.…”