Selenomethionine as an expressible handle for bioconjugations

Abstract

Site-selective chemical bioconjugation reactions are enabling tools for the chemical biologist. Guided by a careful study of the selenomethionine (SeM) benzylation, we have refined the reaction to meet the requirements of practical protein bioconjugation. SeM is readily introduced through auxotrophic expression and exhibits unique nucleophilic properties that allow it to be selectively modified even in the presence of cysteine. The resulting benzylselenonium adduct is stable at physiological pH, is selectively labile to glutathione, and embodies a broadly tunable cleavage profile. Specifically, a 4-bromomethylphenylacetyl (BrMePAA) linker has been applied for efficient conjugation of complex organic molecules to SeM-containing proteins. This expansion of the bioconjugation toolkit has broad potential in the development of chemically enhanced proteins.

Keywords: bioconjugation; protein chemistry; selenomethionine.

Fig. 1.

The selenomethionine benzylation.

Fig. 2.

(A) The selenomethionine benzylation reaction conditions. (B) pH dependence of SeM benzylation (2.5 mM peptide 1, 5 mM BnBr, 30% MeCN, 5% DMSO, 1 mM Boc-Tyr-OH as internal standard). Some error bars are smaller than point markers.

Fig. 3.

(A) The selenomethionine benzylation reaction conditions. (B) Hammett correlation of the selenomethionine benzylation [the Hammett constant for p-CH₂NEt₃⁺ was estimated from p-CH₂N(Me)₃⁺]. (C) Conversion of 1 to the corresponding selenonium species of representative linkers. Shown are BrMePAA (Green), benzamide (blue), and quaternary ammonium linker (red) [1 mM 1, 2 mM R-BnBr, 1 mM Boc-Tyr-OH (internal standard), 10 mM ascorbic acid, 40 mM MES pH 6, 30% MeCN, 5% DMSO in water]. Rate constants were derived by fitting rate conversion experiments to a second-order integrated rate law.

Fig. 4.

(A) Benzyl-selenonium stability in plasma mimic (1 mM peptide, in PBS pH 7.4, monitored by HPLC). (B) Benzyl selenonium stability in cytosol mimic (1 mM peptide, in PBS pH 7.4, with 7.5 mM GSH, monitored by HPLC).

Fig. 5.

(A) Modification of SpA (PDB: 1BDD) protein 3 with tags varying pH (0.15 mM 3; 0.2 mM tag 4, 5, or 6; 20 mM MES; 10 mM ascorbic acid). (B) HPLC chromatogram of reaction of 3 and 5 (0.15 mM 3, 0.2 mM tag 6, 20 mM MES buffer pH 5). Deconvolution was performed manually from centroid collapsed peaks between m/z 600 and 1,250.

Fig. 6.

(A) Tev-GATE-16 (PDB: 4CO7) 7 surface, SeM residues shown in blue, Se highlighted in orange, and the Cys residue shown in green. (B) Labeling conditions. (C) Mass spectrum of starting material and labeled product.

Fig. 7.

(A) His-MBP-Tev (PDB: 1ANF) 9 surface, SeM residues shown in blue, Se highlighted in orange. (B) Labeling conditions. (C) Mass spectrum of starting material and labeled product.

Fig. 8.

(A) Tev-GATE-16 (PDB: 4CO7) 7 surface, SeM residues shown in blue, Se atom highlighted in orange, and the Cys residue shown in green. (B) Mass spectrum of starting material and labeled product.