Context‐Dependence of the Reactivity of Cysteine and Lysine Residues

Abstract

The S‐alkylation of Cys residues with a maleimide and the Nϵ‐acylation of Lys residues with an N‐hydroxysuccinimide (NHS) ester are common methods for bioconjugation. Using Cys and Lys derivatives as proxies, we assessed differences in reactivity depending on the position of Cys or Lys in a protein sequence. We find that Cys position is exploitable to improve site‐selectivity in maleimide‐based modifications. Reactivity decreases substantially in the order N‐terminal>in‐chain>C‐terminal Cys due to modulation of sulfhydryl pKa by the α‐ammonium and carboxylate groups at the termini. A lower pKa value yields a larger fraction thiolate, which promotes selectivity while somewhat decreasing thiolate nucleophilicity in accord with βnormalnnormalunormalc =0.41. Lowering pH and salt concentration enhances selectivity still further. In contrast, differences in the reactivity of Lys towards an NHS ester were modest due to an appreciable decrease in amino group nucleophilicity with a lower pKa of its conjugate acid. Hence, site‐selective Lys modification protocols will require electrophiles other than NHS esters.

A Membrane‐Associated Light‐Harvesting Model is Enabled by Functionalized Assemblies of Gene‐Doubled TMV Proteins

“…In previous work, we have used 1% or close to 100% dye labeling of TMV constructs at engineered cysteine residues to measure both isolated and networked dye excitation properties. [ 9,33 ] An intermediate modification level was used in this case to avoid potential off‐target modification with the NHS ester dye [ 34 ] while ensuring nearly every dTMV disk would contain at least one dye for visualization. A construct without the reactive lysine residue, dTMV‐S123‐S123′C, showed no modification with AF647 under these conditions (Figure S8a, Supporting Information).…”

Chemoselective Methionine Labelling of Recombinant Trastuzumab Shows High In Vitro and In Vivo Tumour Targeting

“…Despite this, the application of conventional conjugation techniques that enable labelling of solvent‐accessible lysine [16] and cysteine [17] residues have been the most widely investigated for generating antibody conjugates. Cysteine residues, in particular, offer distinct advantages over the labelling of lysine residues due to the lower occurrence of cysteine within the backbone of proteins as well as the ability to target cysteine residues specifically due to changes in their reactivity depending on their protein environment [18] . As cysteine remains the only proteinogenic amino acid able to be reliably targeted for site‐specific protein labelling, [17] significant effort has been made to diversify labelling techniques to allow alternate residues to be addressed [7,19] .…”

Phenylpropynones as Selective Disulfide Rebridging Bioconjugation Reagents

“…We checked this reactivity in buffer by incubating reagents 1 – 9 with the hydrochloride salt of N -acetyl- O -methyl-lysine 12 (Table ). A slow aza-Michael reactivity is expected at physiological pH, due to the protonated state of amines in buffer . At 40 °C, phenylpropynones 1 – 5 nonetheless showed full conversion to the expected aza-Michael adducts, although phenylpropynone 4 unexpectedly also showed some more complex reactivity (see the Supporting Information).…”

“…A slow aza-Michael reactivity is expected at physiological pH, due to the protonated state of amines in buffer. 11 At 40 °C, phenylpropynones 1−5 nonetheless showed full conversion to the expected aza-Michael adducts, although phenylpropynone 4 unexpectedly also showed some more complex reactivity (see the Supporting Information). Propynamide 6 did not show any reactivity toward amine 12 in buffer.…”

“…The reagents that showed promising thiol−thiol coupling reactivity in an organic solvent (Scheme 2) were studied in model bioconjugation trials in an aqueous buffer. Reagents 1− 6, 8, and 9 were incubated for 24 h at 40 °C with 2.0 equiv of N-acetyl-O-methyl-cysteine (11) in a 1:1 mixture of acetonitrile and a 0.1 M aqueous phosphate buffer solution (pH 8.0), and the reaction outcomes were compared (Table 1). To our delight, most alkynes gave the expected dithioacetal product (DTA) as the major reaction product within 24 h. In a small kinetic study, phenylpropynones 1−3 showed some marked differences (see the Supporting Information).…”