Selenocysteine in Native Chemical Ligation and Expressed Protein Ligation

Abstract

L-Selenocysteine (Sec or U) has been called the "21st amino acid". 1 Like the twenty common amino acids, selenocysteine is inserted during the translation of mRNA and has its own tRNA Sec and codon, UGA. This codon also serves as the opal stop codon. Decoding a UGA codon as one for selenocysteine requires a special structure in the 3′ untranslated region of the mRNA called a selenocysteine insertion sequence (SECIS) element. Because eukaryotic and prokaryotic cells use a different SECIS element to decode UGA as selenocysteine, the production of eukaryotic selenocysteine-containing proteins in prokaryotes is problematic. 2 Here, we describe a general semisynthetic route to proteins containing selenocysteine. 3,4 In "native chemical ligation", the thiolate of an N-terminal cysteine residue in one peptide attacks a C-terminal thioester in another peptide to produce, ultimately, an amide bond between the two peptides (Scheme 1). 5 "Expressed protein ligation" is an extension in which the C-terminal thioester is produced by using recombinant DNA (rDNA) technology. 6 We reasoned that selenocysteine, like cysteine, could effect both native chemical ligation and expressed protein ligation, and thereby provide a means to incorporate selenocysteine into proteins.We used AcGlySCH 2 C(O)NHCH 3 as a model thioester to test the feasibility of using selenocysteine in native chemical ligation. 7 Reaction with cystine ((CysOH) 2 ) in the presence of the reducing agent tris-(2-carboxyethyl)phosphine (TCEP) produced AcGlyCysOH, as well as some (AcGlyCysOH) 2 . When selenocystine ((SecOH) 2 ) was used in the same reaction, the product was (AcGlySecOH) 2 . 8 A selenolate (RSe -) is more nucleophilic than is its analogous thiolate (RS -). 9 Moreover, the pK a of a selenol (RSeH) is lower than that of its analogous thiol (RSH). 9a,10 These properties suggested to us that native chemical ligation with selenocysteine could be more rapid than with cysteine, especially at low pH. To test this hypothesis, we used the chromogenic thioester AcGly-SC 6 H 4 -p-NO 2 (1; Scheme 2) to determine the rate of native chemical ligation as a function of pH. 11 The resulting pH-rate profile is shown in Figure 1. Reaction with selenocysteine is 10 3 -fold faster than with cysteine at pH 5.0. Thus, native chemical ligation with selenocysteine can be chemoselective. 12 Having demonstrated the effectiveness of selenocysteine in native chemical ligation, we next set out to explore its utility in expressed protein ligation. As a model protein, we chose ribonuclease A (RNase A; EC 3.1.27.5; Figure 2), which has been the object of much seminal work in protein chemistry. 14 RNase A has 8 cysteine residues that form 4 disulfide bonds in the native For other means to incorporate selenocysteine into semisynthetic and synthetic proteins, see: (a) Wu, Z. P.; Hilvert, D. J. Am. Chem. Soc. 1989, 111, 4513-4514. (b) Fiori, S.; Pegoraro, S.; Rudolph-Bohner, S.; Cramer, J.; Moroder, L. Biopolymers 2000, 53, 550-564. (4) For a means to produce selenopeptide libraries o...

Studies on deprotection of cysteine and selenocysteine side‐chain protecting groups

“…Fmoc-selenocysteine(Mob)-OH was synthesized as had been reported previously by us [18,19]. All peptides were synthesized with a Burrell Model 75 wrist-action shaker (Pittsburgh, PA).…”

Diels–Alder Ligation of Peptides and Proteins

“…Raines et al have showed that the concept of NCL can also be extended to include selenocysteines. [37] The main disadvantage of NCL is the necessity of a cysteine residue at the ligation site.…”

Improved synthetic routes to the selenocysteine derivatives useful for Boc-based peptide synthesis with benzylic protection on the selenium atom

“…Since an excess amount of Sec derivatives are necessary for high-yield synthesis of selenopeptides, the improved routes reported here will be valuable for promoting the research that utilizes various selenopeptides as selenoenzyme models 5 or for other purposes. [12][13][14][15][16] On the other hand, for the case of Fmoc-protected Sec derivatives, it was found that a similar route through an iodide intermediate 14 (and tosylate 12) and subsequent selenation with a selenolate nucleophile (RSe -) does not work well, resulting in a low yield and partial racemization taking place. However, the racemization problem can be overcome by repeated recrystallization, though the yield decreases.…”

“…The spectral data of 13 were identical to the literature. 36 The purity was confirmed by 14. Under nitrogen atmosphere, triphenyl phosphine (124 mg, 0.47 mmol) and imidazole (40 mg, 0.58 mmol) were dissolved in dry dichloromethane (3 mL).…”

“…12,13 Long selenopeptides that possess a couple of Sec residues along the chain have also been synthesized by application of selenocysteine-based native chemical ligation (Sec-NCL). [14][15][16] To synthesize selenopeptides in a flask, particular Sec derivatives have to be utilized. (Figure 1), which have Boc protection on the amino group and MBn or MPM protection on the selenium atom, are useful for Boc-based selenopeptide synthesis as the MBn and MPM groups in these targets can be deprotected under satisfactory conditions in the final step of selenopeptide synthesis.…”