Tuning a Protein‐Labeling Reaction to Achieve Highly Site Selective Lysine Conjugation

Abstract

Activated esters are widely used to label proteins at lysine side chains and N termini. These reagents are useful for labeling virtually any protein, but robust reactivity toward primary amines generally precludes site-selective modification. In a unique case, fluorophenyl esters are shown to preferentially label human kappa antibodies at a single lysine (Lys188) within the light-chain constant domain. Neighboring residues His189 and Asp151 contribute to the accelerated rate of labeling at Lys188 relative to the ≈40 other lysine sites. Enriched Lys188 labeling can be enhanced from 50-70 % to >95 % by any of these approaches: lowering reaction temperature, applying flow chemistry, or mutagenesis of specific residues in the surrounding protein environment. Our results demonstrated that activated esters with fluoro-substituted aromatic leaving groups, including a fluoronaphthyl ester, can be generally useful reagents for site-selective lysine labeling of antibodies and other immunoglobulin-type proteins.

DNAzymes for amine and peptide lysine acylation

“…We then performed in vitro selection experiments with acyl donor oligonucleotides activated in situ from their 5-carboxylic acid (5-CO2H) precursors using two common amide-forming coupling reagents, 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4methylmorpholinium chloride (DMT-MM) 67 or the combination of the water-soluble 1-ethyl-3-(3dimethylaminopropyl)carbodiimide (EDC) and 2,3,5,6-tetrafluorophenol (TFP). [68][69][70][71] Before performing in vitro selection, the resulting DMT and TFP esters (here abbreviated DMTE and TFPE; structures in Figure 1C) were assayed for their uncatalyzed background reactivities, using a DNA splint complementary to the DNA-anchored acyl donor and the simple DNA-anchored amine nucleophile (DNA-C3-NH2; Figure 2A,B). Both DMTE and TFPE led to relatively high uncatalyzed background reactivity, with substantial formation of acylation product; e.g., 34% amide formation in 0.5 min (DMTE) and 15% amide formation in 0.5 min (TFPE), each assessed at pH 7.0.…”

Cysteine-to-lysine transfer antibody fragment conjugation

“…S6 †). 29,30 With the view to tuning down the reactivity of the thioester and hence avoid non-specic reactivity, we turned our attention to the use of alkyl thioesters. MESNa thioester 3 was found to be completely inert upon reaction with native Fab, even at 100 equiv.…”

“…Reagents have been described which offer greater selectivity for certain lysines than conventional reagents by exploiting subtle differences in pK a 's of lysines or local environments. 29,30 We envisaged that an alternative approach to achieve selectivity would be to use proximal cysteine residues as ligating 'hooks', delivering acylating agents specically to certain lysine residues (Fig. 1).…”

Pyrocinchonimides Conjugate to Amine Groups on Proteins via Imide Transfer

“…80,81 The challenge of addressing the combinatorial diversity of lysine-targeted bioconjugates has only recently started to receive a reply. [82][83][84][85] The low DAR values obtained with the Pci reagent 27 suggest that transpyrocinchonimidation may be a viable addition to the NHS alternatives toolkit, particularly in situations where bioconjugate heterogeneity is a persistent problem.…”