Selenomethionine as an Expressible Handle for Bioconjugations

Abstract

<b>Site-selective chemical protein ligation reactions are enabling tools for chemical biology. Herein, we employ a physical organic study to refine the selenomethionine (SeM) benzylation as a practical protein bioconjugation strategy. 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, selectively labile to glutathione and embodies a broadly tuneable reactivity profile. Guided by a mechanistic analysis of the reaction, a 4-bromomethylphenylacetyl linker is identified for efficient conjugations of complex organic molecules to SeM containing proteins. This optimized benzyl linker exhibits a rate constant of 3x10<sup>-1</sup> M<sup>-1</sup>s<sup>-1</sup>, facilitating efficient conjugation at micromolar concentrations. The selenonium conjugate is further advanced through a linker that can be selectively photo-locked or reductively cleaved on demand. This tool-kit of selenonium forming reagents have broad potential in the development of chemically enhanced proteins.</b>

Peptide and Protein Cysteine Modification by Virtue of Highly Chemo-, Regio- and Stereoselective Hydrosulfuration of Ynamide

“…Reaction conditions: 1) peptide 4l (1 mM) and ynamide 2t (5 mM) were incubated in 0.1 M PB, pH 8, at 37 °C for 1 h. 2), azide 2u (2 mM), CuSO 4 (0.1 mM), ligand (bathophenanthroline disulfonic acid disodium salt, 0.5 mM) and sodium ascorbate (5 mM) were added to the reaction, which was incubated at 37 °C for 30 min. 6513.5 Da) (c) The conversion of BSA modi cation is 99%, no BSA (10) was observed after 12 h. (d) Tratsuzumab (12, 0.5 mg/mL) was incubated with 10 equiv TCEP in 0.1 M PB (pH 8), 37 ºC for 40 min followed by adding 200 equiv of ynamide (2n) for 20 h. Deconvoluted MS spectra of partly reduced tratsuzumab labeled with 2n. LC, light chain; HC, heavy chain; subscripts, number of labeling.…”

“…[3] Consequently, chemical modi cations of peptides and proteins have made enormous impact for the developments of biotherapeutics [4] as well as for basic biological studies. [5] Despite the progress in the chemoselective bioconjugation of previously unexplored amino acid side chains, including serine (Ser), [6] selenocysteine (Sec), [7] histidine (His), [8] methionine (Met), [9] selenomethionine (Sem), [10] tyrosine (Tyr) [11] and tryptophan (Trp), [12] nevertheless lysine (Lys) [13] and cysteine (Cys) [14] are still the preferred residues for chemical protein modi cation due to their unique nucleophilicity. [15] While Lys is highly common in protein (make up ~6% of human proteins sequences [16] ), many of which are solvent exposed, Cys is one of the least frequent amino acids in proteins (~ 1.7%), [17] especially those in the reduced form on the protein surfaces, which are readily accessible for chemical modi cations.…”

In vivo, in vitro and in silico: an open space for the development of microbe‐based applications of synthetic biology

“…However, again, much of the corresponding works are more proofs of concept than ready to become, biotechnology‐adapted synthetic microbes growing in industrial fermenters. More limited approaches have already been used to produce synthetic variants of proteins, for example, using analogues of amino acids such as selenomethionine for bioconjugations (Flood et al ., 2021) or variants of norleucine, known to replace methionine in proteins in E. coli for a very long time (Moroder and Budisa, 2010; van Eldijk and van Hest, 2018). Nevertheless, a creation somewhat similar in its concept to that of E. chlori was based on growth of cells on tryptophan analogues.…”

Highly Chemo-, Regio- and Stereoselective Cysteine Modification of Peptides and Proteins with Ynamides

“…[2][3] In addition, chemists have developed a variety of chemical tools to mimic the function of some protein PTMs [4][5][6][7] , and chemical modifications of peptides and proteins have made enormous impact for the developments of biotherapeutics [8][9][10] as well as for basic biological studies [11][12][13][14] . Despite the progress in the chemoselective bioconjugation of previously unexplored amino acid side chains, including serine (Ser) 15 , selenocysteine (Sec) [16][17] , histidine (His) 18 , methionine (Met) [19][20][21][22] , selenomethionine (Sem) 23 , tyrosine (Tyr) [24][25][26][27][28] and tryptophan (Trp), [29][30][31] lysine (Lys) [32][33][34][35][36] and cysteine (Cys) [37][38][39][40][41] are still the preferred residues for chemical protein modification due to their unique nucleophilicity [42][43] . While Lys is highly common in protein sequences (~6% human proteins sequences 44 ), many of which are solvent exposed, Cys is one of the least frequent amino acids in proteins (~ 1.7%)...…”