Selenocysteine‐Mediated Native Chemical Ligation

Quaderer, Richard; Sewing, Andreas; Hilvert, Donald

doi:10.1002/1522-2675(20010516)84:5<1197::aid-hlca1197>3.0.co;2-#

Cited by 167 publications

(128 citation statements)

References 31 publications

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“…The pMob group can be removed by using a cleavage cocktail of TFA, m-cresol, thioanisole, and trimethylsilyl trifluomethansulfonate (TMSOTf) [42]. Trimethylsilyl bromide has also been used [29]. …”

Section: Synthesis Of Selenocysteine Derivativesmentioning

confidence: 99%

“…In contrast to the genetic approach by Arner, my group and several others have used expressed protein ligation to construct proteins containing selenocysteine semisynthetically [28][29][30][31]. Prior to these efforts, Hilvert had used a total chemical approach to create the first artificial selenoenzyme by activating a serine residue in subtilisin with phenylmethanesulfonyl fluoride and then treating the activated ester with hydrogen selenide to complete the conversion to selenocysteine.…”

Section: Selenium and The Split Personality Of The Genetic Codementioning

confidence: 99%

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Incorporation of Selenocysteine into Proteins Using Peptide Ligation

Hondal

2005

PPL

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Expressed protein ligation has become a frequently used technique to insert non-standard amino acids into proteins. The technique has been adapted to insert selenocysteine residues in place of cysteine residue in proteins, taking advantage of the similarity in the chemistries of sulfur and selenium. This replacement can confer unique structural and catalytic properties to enzymes and proteins. The development of this technique also allows for naturally occurring selenoproteins to be produced semisynthetically. KeywordsSelenocysteine; expressed protein ligation; thioredoxin reductase; semisynthetic Selenium, the mysterious moon-metalThe Swedish chemist, J.J. Berzilius discovered the 34 th element of the periodic table in 1817 and named it after the Greek goddess of the moon, Selene [1]. It is wholly appropriate that he named this element after the moon-goddess, because selenium, like the moon, has remained mysterious to science until the modern eraThe biological role of selenium began to be realized when Schwarz discovered a dietary factor that was necessary to prevent liver necrosis in rats [2]. He called this mysterious nutritional requirement, "Factor 3". He then determined that this dietary factor was the trace element selenium. In the 1970's, Chinese scientists reported that selenium could prevent a childhood cardiomyopathy known as Keshan disease [3], much like vitamin C can prevent the onset of scurvy. Beck and Levander later showed that both influenza and coxsackie viruses accumulate genetic mutations when these viruses have infected animal hosts that are deficient in selenium or vitamin E [4][5]. Keshan disease is caused when coxsackie virus mutates to a more virulent strain in a host deficient in selenium. In the 1980's, it was discovered that selenium is the only trace element that is incorporated directly into proteins as part of the polypeptide chain. [6]. The incorporation of selenium into proteins, known as selenoproteins, is beginning to explain the important role that selenium plays in human health. A number of important examples can be cited. Selenium is vital to the function of the thyroid gland. The key enzyme in the thyroid gland, which converts thyroxin (T4) to triidothyronine (T3), contains selenium [7]. Selenium is absolutely essential to human male fertility because an enzyme in the testes containing selenium protects developing sperm from oxidative injury [8]. There has long been anecdotal evidence that selenium offers † This work was supported by grant GM070742-01 NIH Public Access Selenium and the split personality of the genetic codeMany of the health benefits of selenium can be explained by its incorporation into proteins as the "21 st " amino acid, selenocysteine [12][13]. This amino acid, though rare, is ubiquitous in all of the kingdoms of life being found in eukarya, prokarya, and archea [14][15]. Examples of selenocysteine-containing proteins are found in plants, fungi, mammals, bacteria, and fish [16]. Like the other standard proteinogenic amino acids, selenocysteine i...

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Section: Synthesis Of Selenocysteine Derivativesmentioning

confidence: 99%

Section: Selenium and The Split Personality Of The Genetic Codementioning

confidence: 99%

Incorporation of Selenocysteine into Proteins Using Peptide Ligation

Hondal

2005

PPL

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“…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.…”

Section: Resultsmentioning

confidence: 99%

“…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.…”

Section: Introductionmentioning

confidence: 99%

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

Shimodaira¹,

Iwaoka²

2016

Arkivoc

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Selenocysteine (Sec) derivatives, i.e., Boc-Sec(MBn)-OH (1) and Boc-Sec(MPM)-OH (2), which are useful for chemical synthesis of selenopeptides, were obtained from L-serine in five steps with total yields of 73% and 74%, respectively. The enantiomeric excesses were confirmed to be >99% e.e. by optical resolution using a chiral column on HPLC. On the other hand, for the case of a Fmoc-protected Sec derivative, i.e., FmocSec(MPM)-OH, similar reactions resulted in low yields and partial racemization taking place.

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“…A variety of ligation chemistries has been developed for this purpose (1)(2)(3)(4)(5)(6)(7)(8)(9)(10)(11)(12)(13)(14)(15). Application of the chemical ligation principle (1) has led to practical chemical syntheses of a wide variety of different classes of proteins (16).…”

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confidence: 99%

His₆tag-assisted chemical protein synthesis

Bang

Kent

2005

Proc. Natl. Acad. Sci. U.S.A.

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To make more practical the total chemical synthesis of proteins by the ligation of unprotected peptide building blocks, we have developed a method to facilitate the isolation and handling of intermediate products. The synthetic technique makes use of a His6 tag at the C terminus of the target polypeptide chain, introduced during the synthesis of the C-terminal peptide segment building block. The presence of a His6 tag enables the isolation of peptide or protein products directly from ligation reaction mixtures by Ni-NTA affinity column purification. This simple approach enables facile buffer exchange to alternate reaction conditions and is compatible with direct analytical control by protein MS of the multiple ligation steps involved in protein synthesis. We used syntheses of crambin and a modular tetratricopeptide repeat protein of 17 kDa as models to examine the utility of this affinity purification approach. The results show that His6 tag-assisted chemical protein synthesis is a useful method that substantially reduces handling losses and provides for rapid chemical protein syntheses. (16). Synthetic access to protein molecules has been used to elucidate the molecular basis of protein folding and stability (17), to elucidate the molecular basis of protein function (18), to design and build proteins of novel structure (19), and to determine the molecular structure of proteins by both NMR (20) and x-ray crystallography (21). Chemical protein synthesis has also been used to develop candidate protein therapeutic molecules with improved properties (21,22).Thus far, most research on chemically synthesized protein molecules (16) has been focused on relatively small proteins in the size range of 50 to Ϸ150 aa (i.e., made from two or three peptide segments). This size limitation is the result of two phenomena: (i) practical constraints on the size of the unprotected peptide segment building blocks (23) and (ii) technical challenges in the chemical ligation of more than two or three peptide segments. Even highly optimized stepwise solid-phase peptide synthetic procedures are limited to Ϸ50 amino acid residues for the practical preparation of high-purity unprotected peptides (24). Thus, the chemical synthesis of a protein of typical size (Ϸ300 aa) (25) would require the use of at least six synthetic peptide segments as building blocks.The most effective way of covalently joining unprotected peptide segments to form a protein molecule is native chemical ligation (3,26). Native chemical ligation involves the reaction of a peptide-␣ thioester with a Cys-peptide; reversible thioesterthiol exchange with the N-terminal Cys residue gives a thioesterlinked intermediate that undergoes an irreversible intramolecular rearrangement to give a near-quantitative yield of a single product linked by a native peptide bond at the ligation site. The native chemical ligation reaction is both practical and highly effective. ¶ Each ligation product can be purified by reverse-phase HPLC and characterized with great precision by electrospray M...

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Selenocysteine‐Mediated Native Chemical Ligation

Cited by 167 publications

References 31 publications

Incorporation of Selenocysteine into Proteins Using Peptide Ligation

Incorporation of Selenocysteine into Proteins Using Peptide Ligation

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

His₆tag-assisted chemical protein synthesis

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Selenocysteine‐Mediated Native Chemical Ligation

Cited by 167 publications

References 31 publications

Incorporation of Selenocysteine into Proteins Using Peptide Ligation

Incorporation of Selenocysteine into Proteins Using Peptide Ligation

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

His6tag-assisted chemical protein synthesis

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His₆tag-assisted chemical protein synthesis