Peptide ligation chemistry at selenol amino acids.

Special Issue Review Received: 9 September 2013

Revised: 9 October 2013

Accepted: 10 October 2013

Published online in Wiley Online Library: 28 November 2013

(wileyonlinelibrary.com) DOI 10.1002/psc.2581

Peptide ligation chemistry at selenol amino acids‡ Lara R. Malins,§ Nicholas J. Mitchell§ and Richard J. Payne* The convergent assembly of peptide fragments by native chemical ligation has revolutionized the way in which proteins can be accessed by chemical synthesis. A variation of native chemical ligation involves the reaction of peptides bearing an N-terminal selenocysteine residue with peptide thioesters, which proceeds through the same mechanism as the parent reaction. This transformation was first investigated in 2001 for the installation of selenocysteine into peptides and proteins via ligation chemistry. The recent discovery that selenocysteine residues within peptides can be chemoselectively deselenized without the concomitant desulfurization of cysteine residues has led to renewed interest in ligation chemistry at selenocysteine. This review outlines the use of selenocysteine in ligation chemistry as well as recent investigations of chemoselective ligation–deselenization chemistry at other selenol-derived amino acids that have the potential to greatly expand the number of targets that can be accessed by chemical synthesis. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd. Keywords: native chemical ligation; selenocysteine; peptides; deselenization

Introduction The highly efficient nature of SPPS has provided an extremely reliable platform for the preparation of peptides bearing up to 50 amino acid residues [1,2]. While there are examples of the preparation of large peptides and even small proteins via a single solid-phase synthesis [3–5], this is not always a trivial exercise. The difficulty in accessing large peptides by SPPS is due in major part to the linear nature of the technique, which means that longer syntheses are often plagued by the accumulation of truncated (uncoupled) sequences, unwanted side products, and epimerization. Furthermore, these by-products often accumulate over the iterative coupling and deprotection cycles leading to poor isolated yields and purities of the final products. This inherent size limitation of SPPS has led to the development of chemical ligation methods that serve to assemble large peptides and proteins via the convergent coupling of smaller peptide fragments.

Native Chemical Ligation

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The most widely employed ligation strategy for the assembly of peptides and proteins is native chemical ligation. This methodology, reported by Kent and coworkers in 1994, was first employed in the total chemical synthesis of interleukin-8, a 77-residue cytokine responsible for the proliferation of B cells during an immune response [6]. Over the past two decades, the native chemical ligation transformation has gained widespread attention as an efficient strategy for the convergent ligation of peptides to afford larger peptides and proteins [7–12]. The methodology relies on a chemoselective condensation reaction between a peptide containing an N-terminal cysteine (Cys) residue and another peptide bearing a reactive C-terminal thioester moiety to afford an amide bond (Scheme 1). The proposed reaction mechanism involves a rapid, reversible transthioesterification to generate a thioester intermediate that then undergoes an irreversible S → N acyl shift to generate a native peptide bond [6,7]. Importantly, the reaction proceeds under aqueous conditions at neutral pH and provides the desired ligation product in a rapid and high yielding manner. The scope of native chemical ligation has been

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extensively examined and has proven to be tolerant of most amino acids on the C-terminus of the peptide thioester component [8]. Since its inception almost two decades ago, the method has been successfully employed in the synthesis of hundreds of full-length proteins with and without posttranslational modifications (e.g. glycosylation and phosphorylation) and has been the subject of several reviews [7,10,12–24]. A number of extensions to the native chemical ligation transformation have also been reported in subsequent years including kinetically controlled ligation reactions [25] and expressed protein ligation, whereby one of the two ligation partners is produced recombinantly [11,26–28]. The successful implementation of the methodology in a number of protein syntheses highlights the strength and generality of the technique. Perhaps the only limitation of the native chemical ligation methodology is the requirement for one of the peptide fragments to contain an N-terminal Cys residue (situated at a suitable position within the peptide or protein target) to facilitate the reaction. This caveat initially led to the development of alternate ligation strategies that included the use of N-terminal or side chain thiol auxiliaries [29–40] as well as thiol-free methodologies [41–45]. However, many of these reactions proceed at significantly slower rates compared with native chemical ligation and, as such, are limited in scope, e.g. in the choice of the C-terminal residue of the thioester. In addition, the transformations are often not chemoselective in the presence of other unprotected amines such as the ε-amino side chain of lysine at the pH ranges at which the reactions are conducted. Despite these drawbacks, thiol

* Correspondence to: Richard J. Payne, School of Chemistry, The University of Sydney, Sydney, NSW 2006, Australia. E-mail: [email protected] ‡

§

This article is published in Journal of Peptide Science as part of the Special Issue devoted to contributions presented at the Chemical Protein Synthesis Meeting, April 3-6, 2013, Vienna, edited by Christian Becker (University of Vienna, Austria). These authors contributed equally to this review School of Chemistry, The University of Sydney, Sydney, NSW, 2006, Australia

Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS Biographies Lara R. Malins was born in 1987 in South Carolina, USA. In 2009, she completed her undergraduate studies in Chemistry as a Trustee Scholar at Boston University, where she worked in the laboratory of Associate Professor Scott Schaus on the synthesis of purine-based natural products. She was awarded an International Postgraduate Research Scholarship in 2010 to undertake her PhD at the University of Sydney, Australia, in the group of Associate Professor Richard Payne. She is currently in the final stages of her PhD, which focuses on the development of novel ligation methodologies for the synthesis of complex peptides and glycopeptides. Scheme 1. Proposed mechanism of native chemical ligation.

Dr Nicholas J. Mitchell was born in London, England, in 1981. He received an MChem degree from the University of Southampton in 2004, completing a 6 month industrial placement during his Masters year at Maybridge, Cornwall, as a Synthetic Organic Chemist. In 2005, he joined the group of Dr. Stefan Howorka at University College London to research the chemical modification of oligonucleotides. He was awarded the UCL Graduate Research Scholarship for Cross-Disciplinary Training in 2008 and received his PhD in 2009. Nicholas stayed on at UCL as a Postdoctoral Research Associate under the joint supervision of Profs Alethea B. Tabor and Helen C. Hailes working on MRI and SPECT imaging of liposomal drug and gene delivery. In 2013, he took up a postdoctoral position at the University of Sydney to work with A/Prof. Richard J. Payne on synthetic vaccines and new peptide ligation methodologies. Richard Payne graduated from the University of Canterbury, New Zealand, in 2002. In 2003, he was awarded a Gates Scholarship to undertake his PhD at the Department of Chemistry, University of Cambridge under the supervision of Professor Chris Abell. After his PhD, Richard moved to The Scripps Research Institute under the auspices of a Lindemann Postdoctoral Fellowship where he worked in the laboratory of Professor Chi-Huey Wong. In 2008, he moved to the University of Sydney as a Lecturer of Organic Chemistry and Chemical Biology within the School of Chemistry where he is currently an Associate Professor. Prof. Payne’s research focuses on utilising the tools of synthetic chemistry including peptide chemistry to address problems of biochemical and medicinal significance.

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An approach to expand the application of native chemical ligation to a wider range of targets was first introduced by Yan and Dawson using catalytic desulfurization methods to convert a Cys residue to an alanine (Ala) residue following the ligation event (Scheme 2) [54]. This native chemical ligation–desulfurization protocol allows access to Xaa-Ala ligation sites (where Xaa is any proteinogenic amino acid), which are significantly more abundant (7.8%) in protein sequences compared with Xaa-Cys sites (1.9%). This methodology therefore increases the number of potential targets that can be synthesized by native chemical ligation. In this early work, Raney nickel or Pd/Al2O3 under a hydrogen atmosphere was used for the desulfurization of the Cys residue at the ligation junction [54]. A reported drawback of these metal-based desulfurization methods has been the adsorption of peptides to the metal surfaces, leading to low product recovery [55]. In addition, the metal-based desulfurization methods have been shown to react with methionine (Met) residues (reduced to α-aminobutyric acid) and thiazolidines, commonly used to mask the reactivity of N-terminal Cys residues during the assembly of proteins via iterative ligation reactions. A metal-free desulfurization method (also called metal-free dethiylation) reported by Wan and Danishefsky provided an effective alternative to the

Scheme 2. Native chemical ligation followed by desulfurization of Cys to Ala at the ligation junction.


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auxiliary and thiol-free ligation reactions have been used for the preparation of target post-translationally modified peptides and proteins where native chemical ligation cannot be employed [43,46–53].

Native Chemical Ligation–Desulfurization

MALINS, MITCHELL AND PAYNE traditional reductive desulfurization procedures [56]. The method, based on an early report by Hoffmann et al. [57], utilizes a trialkylphosphine in the presence of a water-soluble radical initiator to facilitate a radical-catalyzed desulfurization. The phosphine of choice in these reactions is usually tris(2-carboxyethyl)phosphine (TCEP) as it is easy to handle and is tolerant of the side chain functionalities of proteinogenic amino acids. Using a water-soluble radical initiator such as 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), TCEP, and a suitable thiol as a hydrogen atom source (e.g. t-BuSH or reduced glutathione [55]) in degassed aqueous media, peptides bearing Cys residues can be efficiently desulfurized to afford Ala-containing peptides in high yields. Notably, the method is orthogonal to Met residues and thiazolidine residues and has been successfully used in the synthesis of numerous proteins and glycoproteins through the native chemical ligation–desulfurization manifold (Scheme 2) [58–64].

Ligation–Desulfurization at Thiol-derived Amino Acids Recently, the repertoire of native chemical ligation has been further expanded through the use of synthetic thiol-derived proteinogenic amino acids (Figure 1) [54,65]. These thiolated derivatives are capable of facilitating ligation reactions in a similar manner to Cys residues in native chemical ligation when incorporated into the N-terminus of peptide fragments. Importantly, these amino acids can be effectively desulfurized via hydrogenation [54] or through the use of the metal-free radical procedure [56]. Over the past 5 years, research aimed at the synthesis and utility of thiolated amino acids in ligation chemistry has been extremely vibrant. To date, synthetic routes to β-thiol phenylalanine 1 [66], β- and γ-thiol valine 2 and 3 [55,67], γ- and δ-thiol lysine 4 and 5 [68–70], β-thiol leucine 6a and 6b [71,72], γ-thiol proline 7a and 7b [73,74], β-thiol threonine 8 [75], β-thiol arginine 9 [76], and β-thiol aspartic acid 10 [77] have been reported by a number of research groups (Figure 1). Very recently, peptide ligation chemistry facilitated by a 2-thiol tryptophan motif has also been reported and is proposed to proceed through a similar mechanism to native chemical ligation [78]. In addition to the use of these functionalized amino acids for the construction of model peptides by ligation–desulfurization chemistry, some of these amino acids have also been used in the chemical synthesis of complex post-translationally modified peptides and proteins [68,71,72,76,77].

The major drawback of using native chemical ligation– desulfurization or ligation–desulfurization at thiol-derived amino acids is that (with the exception of ligation at aspartic acid [77]), the desulfurization reaction is not chemoselective in the presence of other Cys residues in the peptide or protein sequence. While Cys residues are rare, they are often critical to the structure and function of a given protein, and therefore, desulfurization of these residues leads to the formation of a non-native protein, which may possess significantly reduced biological activity. Strategies to keep these structurally important Cys residues intact upon desulfurization have been developed. A key strategy, first reported by Pentelute and Kent, uses side chain acetamidomethyl (Acm)-protecting groups on the Cys sulfhydryl moiety to prevent concomitant desulfurization of important Cys residues [79]. Following the desulfurization step, Acm groups can be removed through the use of silver(I) or mercury(I) salts to afford the native peptide. Although the global protection of Cys residues in the sequence alleviates unwanted desulfurization, side-chain-protected peptides can often exhibit different solubility profiles to unprotected peptides and therefore can be difficult to handle. In addition, this protecting group strategy prohibits the use of expressed peptide and protein fragments in expressed protein ligation applications [11,26–28]. A potential solution to overcome the generally unselective nature of the desulfurization transformation in the presence of unprotected side chain thiols is to instead use selenated amino acids (as Cys surrogates) in peptide ligation chemistry. Ligation chemistry at selenocysteine (Sec) was first reported in 2001 [80–82]; however, this area of research has recently experienced a resurgence, fuelled by a report from Dawson and coworkers, which showed that Sec can be chemoselectively deselenated to generate Ala in the presence of unprotected Cys residues [83]. This review discusses applications of ligation chemistry at selenated amino acids and has been written in response to the renewed interest in this area. The article will highlight the use of ligation chemistry to install Sec into peptides and proteins and will outline recent reports of ligation–deselenization chemistry at Sec and other synthetic selenated amino acid derivatives.

Ligation at Selenocysteine Prior to the 1957 report by Schwarz and Foltz [84], which outlined the biological importance of selenium as an essential trace element in eukaryotic cells, selenium was thought to be generally toxic to biological systems [85]. However, this seminal work,

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Figure 1. Synthetic thiol-derived amino acids for use in ligation–desulfurization chemistry.



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PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS along with a 1973 study that demonstrated that mammalian glutathione peroxidase is a selenoenzyme reliant on the presence of selenium for catalytic activity [86], ignited considerable interest in the biochemistry of selenium. A number of selenoproteins have since been identified, serving a range of important roles in redox signaling, antioxidant defense, oxidoreductions, muscle development and function, and immune responses [85,87,88]. Given the vast array of selenium-dependent biological processes, selenoprotein deficiencies have been correlated to a number of diseases [87], prompting the further study of such compounds. Selenium is incorporated into proteins primarily in the form of selenocysteine (Sec, U) [87–89]. The finding that Sec is encoded by the DNA codon ‘TGA’ and co-translationally incorporated into peptides on the ribosome with the help of a Sec-specific tRNA molecule led to the characterization of Sec as the 21st proteinogenic amino acid [90]. As the selenium analog of Cys, Sec displays a number of similar physicochemical properties to this amino acid (Table 1). There are, however, key differences in pKa, nucleophilicity and the corresponding reactivity of the two amino acids. The combination of unique properties associated with Sec residues is crucial for the function of many selenoproteins, with the Cys mutants of most selenoenzymes characterized by a significant decrease in enzymatic activity [91–93]. Specifically, despite the comparable electronegativities of sulfur (χ = 2.58) and selenium (χ = 2.55), the pKa of Cys is 8.25, while Sec is considerably more acidic, with a pKa of approximately 5.24–5.63 [85,94,95]. Sec is therefore present primarily as the selenolate at physiological pH, whereas Cys exists in its protonated thiol form. Selenolates have also proven to be more nucleophilic than their corresponding thiolates [95], as well as superior leaving groups [91]. Further studies have shown striking differences between the observed redox behavior of Cys and Sec. For instance, the reduction potential of a selenocystine diselenide bridge in an unconstrained glutaredoxin octapeptide was shown to be significantly lower ( 381 mV) than the analogous reduction potential of a Cys disulfide bridge ( 180 mV) [96]. This result is in close agreement with the observed electrode potential for the two-electron reduction of selenocystine at pH 7.0 ( 386 mV) [97]. As a consequence of their highly negative reduction potential, selenols are oxidized much more readily than the corresponding thiols, with oxidation to the diselenide occurring rapidly upon exposure to air [98]. The low redox potential of Sec also ensures preferential oxidation to the diselenide in the presence of Cys. A pair of Sec residues

inserted into a protein sequence in place of native Cys residues can therefore direct and facilitate protein folding, a strategy that was initially employed by Moroder and coworkers in the oxidative folding of Sec analogs of human endothelin-1 [99] and the bee venom toxin, apamin [100,101]. The method has subsequently been applied to a variety of disulfide-rich peptides, including bovine pancreatic trypsin inhibitor (BPTI) [102], Ecballium elaterium trypsin inhibitor II (EETI-II) [103], and a number of conotoxins [104–109] – a group of bioactive peptides derived from the venom of marine snails of the genus Conus. Interest in the structure and function of selenoproteins and the intriguing chemical properties of Sec residues fuelled the first exploration of Sec-mediated ligation chemistry. In 2001, three groups (the laboratories of van der Donk, Raines, and Hilvert) independently reported the extension of native chemical ligation to Sec residues [80–82]. Prior to these discoveries, access to Seccontaining peptides relied primarily on the synthesis of suitably protected Sec amino acid building blocks and incorporation into peptides via standard SPPS methods [85]. The overall logic and mechanism of ligation at Sec is analogous to native chemical ligation at Cys residues and involves the reaction of a C-terminal peptide thioester with a peptide bearing an N-terminal Sec residue (Scheme 3). Initial attack of the selenol moiety onto the thioester acyl donor fragment results in a transesterification reaction generating an intermediate selenoester, which subsequently rearranges via a Se → N acyl shift to afford a new amide bond. Because of the reductive potential of Sec, the products of the ligation reaction usually include the symmetrical diselenide and mixed selenyl sulfide products, the proportion of which is dictated by the amino acid sequence neighboring the Sec residue as well as the nature of the external thiol used to activate the thioester fragment. Because of the unique chemical properties of Sec, including the lower pKa and greater nucleophilicity relative to Cys, reactions at this amino acid exhibit marked differences from standard native chemical ligation reactions. Notably, the enhanced acidity of Sec residues allows reactions to proceed successfully at lower pH (pH 5–6, compared with pH 7–8 for ligations at Cys). This is presumably owing to the large proportion of nucleophilic selenolate ion present in solution, even at slightly acidic pH [80,82]. For instance, at pH 5.0, Raines and coworkers observed

Table 1. Physicochemical properties of cysteine and selenocysteine [85,94–96,110].

Properties

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100 2.58 180 8.25 205

Selenocysteine 115 2.55 381 5.24–5.63 232

Scheme 3. Proposed mechanism of native chemical ligation at selenocysteine.



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Atomic radius (pm) Electronegativity Redox potential (mV) pKa Bond length (pm)

Cysteine

MALINS, MITCHELL AND PAYNE that reaction with Sec was 103-fold faster than ligation with Cys [82]. The authors of this study proposed that the pH-rate profile for Sec and Cys ligations might allow ligations at Sec to proceed chemoselectively in the presence of Cys. In contrast, van der Donk and coworkers noted that reactions at Sec residues were markedly sluggish, requiring relatively high concentrations (3 mM) and 24 h to reach completion, despite the enhanced nucleophilicity of Sec residues [81]. The authors of this report postulated that the rate-determining step of ligation at Sec might be the reduction of the rapidly formed and highly stable diselenide to generate the reactive selenol species capable of facilitating the ligation. This hypothesis was supported by the work of Hilvert and coworkers, which demonstrated that Sec-mediated ligations did not proceed in the absence of a reducing agent, such as TCEP or an exogenous thiol reductant [80]. It is clear that the rate of Sec ligations is therefore dependent on the strength of the reductive conditions used in the ligation. However, the observation that the application of TCEP as a reducing agent in Sec-mediated ligations results in an undesired deselenization pathway [81] has rendered thiol-based additives, for example, mercaptophenylacetic acid (MPAA) [83], the more common choice for these reactions despite their decreased reductive power. Since its inception, Sec-mediated ligation has been employed in the synthesis of a variety of biologically relevant peptide fragments and full-length proteins [111–113]. In 2001, van der Donk

and coworkers prepared a 17-mer corresponding to the C-terminus of ribonucleotide reductase (Scheme 4) [81]. The symmetrical diselenide peptide 11 was prepared from a precursor peptide bearing an N-terminal p-methoxybenzyl (PMB)-protected Sec residue. This was ligated to peptide thioester 12 in denaturing buffer using 4 vol% thiophenol, affording ligation product 13 in 56% yield, which possessed a selenosulfide bridge in place of the naturally occurring disulfide found in ribonucleotide reductase. Hilvert and coworkers have employed Sec ligation chemistry to prepare a selenium-containing derivative of the 58-residue serine protease inhibitor, BPTI [80]. The native protein contains six Cys residues and three disulfides in the folded form. Both the peptide thioester (BPTI-1-37, 14) and N-terminal Seccontaining (C38U-BPTI-38-58, 15) fragments were synthesized by SPPS (Scheme 5). Ligation of these peptides and subsequent oxidation afforded the correctly folded protein 16 in 67% yield, with an intramolecular selenyl sulfide replacing one of the three native disulfides. A novel Sec-mediated ligation was also used to prepare 16-residue cyclic peptides (17 and 18) from bifunctional linear precursor 19 containing a C-terminal thioester and an N-terminal Sec residue (Scheme 6) [114]. In this example, a variety of post-ligation manipulations of the selenol functional group were also carried out, including alkylation with iodoacetamide to afford 20, oxidative elimination in the presence of hydrogen peroxide to generate the corresponding

Scheme 4. Synthesis of a C-terminal fragment of ribonucleotide reductase using a Sec-mediated ligation reaction [81].

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Scheme 5. Synthesis of bovine pancreatic trypsin inhibitor (BPTI) analog via Sec-mediated ligation chemistry [80].



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PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS

Scheme 6. Synthesis of a cyclic peptide via Sec ligation followed by post-ligation elimination and deselenization.

dehydroalanine (Dha) residue in 21, and thiol Michael addition reactions onto 21 to afford a thiol derived side chain (e.g. 22). Finally, using a concept first proposed by Raines and coworkers at Sec [82], and in an analogous fashion to the post-ligation reductive desulfurization of Cys residues [54], reductive deselenization of 17 with Raney nickel was shown to generate cyclic peptide 23 bearing an Ala residue. Danishefsky and coworkers have also demonstrated the application of VA-044-mediated radical deselenization as an alternative, metal-free approach to C–Se bond cleavage in the conversion of Sec to Ala following ligation reactions [56]. The Sec ligation technology has also been implemented in combination with expressed protein ligation to synthesize a Sec analog of ribonuclease A (RNase A) [82]. In this study, a 109-amino-acid recombinant thioester 24 was produced in Escherichia coli and ligated to a short, synthetic peptide 25

bearing an N-terminal Sec residue (Scheme 7). This provided the desired protein, Cys110Sec RNase A (26), albeit in low isolated yield. The resultant semisynthetic RNase A displayed equivalent ribonucleolytic activity to the native protein, suggesting that the Sec-derived protein was correctly folded [82]. The semisynthesis of a Sec-containing variant of azurin, a member of the type 1 blue copper protein family, has also been successfully conducted using a Sec ligation reaction (Scheme 8). This protein is responsible for electron transfer in biological systems and contains a key Cys residue (Cys112), which strongly coordinates the copper ligand. This Cys residue was replaced by a Sec residue in the semisynthetic variant [115]. C-terminally truncated azurin1-111 was expressed as a fusion protein to the N-terminus of the Mycobacterium xenopi (Mxe) intein domain (27). Expressed protein ligation was carried out between

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Scheme 7. Semisynthesis of Cys110Sec RNase A via a Sec ligation.

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MALINS, MITCHELL AND PAYNE

Scheme 8. Semisynthesis of Cys112Sec azurin via expressed Sec ligation.

construct 27 and synthetic peptide 28 corresponding to azurin112-128 with a Sec residue installed at the N-terminus. In the presence of mercaptoethane sulfonate sodium salt (MESNa), which facilitated both intein cleavage and transthioesterification, 27 was converted to protein thioester 29 and subsequently ligated with peptide 28 to afford Cys112Sec azurin. Treatment of the semisynthetic protein with copper(II) generated a copper bound protein 30, which was produced at a concentration of 0.4 mg/L of culture. The Sec mutant protein 30 possessed a similar electronic absorption spectrum and reduction potential to the wild type azurin. The use of Sec in peptide ligation chemistry, together with the possibility of post-ligation side chain functionalization of the selenol moiety, has increased the scope of ligation chemistry to include a range of more functionally diverse peptide and protein targets. However, given the ease of post-ligation conversion of Cys to Ala [54] and the ability to facilitate side chain modifications on this amino acid [116,117], Sec-mediated ligations alone do not offer significant benefits over Cys ligations unless the ultimate goal of the ligation event is to incorporate Sec in the final product (as described earlier). Additionally, Sec amino acid building blocks for incorporation into peptides are considerably less available than the corresponding Cys derivatives and often require multistep syntheses from commercially available precursors [85]. As such, Sec-mediated ligations have not been extensively used for the synthesis of peptides and proteins that do not possess Sec residues in the final product.

Chemoselective Ligation–Deselenization at Selenocysteine

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A groundbreaking discovery that strategically capitalized on the unique chemical properties of Sec was the ability to selectively cleave the side chain C–Se bond of Sec in the presence of unprotected Cys residues, thereby providing a chemoselective approach to Ala ligation junctions [83]. Post-ligation conversions of Cys to Ala and Sec to Ala had previously relied on unselective reduction in the presence of a metal catalyst [54] or a radical-


based protocol (vide supra) [56], both of which effected global conversion to Ala. Access to native Cys-containing peptides via ligation–desulfurization chemistry therefore required the use of side-chain-protecting groups, such as the Cys(Acm) group [79], and subsequent removal following desulfurization. However, in 2010, Dawson and coworkers demonstrated that mild and selective deselenization was possible in the presence of unprotected Cys residues simply by treatment with excess phosphinereducing agent (TCEP) and dithiothreitol (DTT) at ambient temperature [83] – conditions that took advantage of the deselenization by-products previously observed during Sec-mediated ligations in the presence of TCEP (Scheme 9) [81]. The chemoselective deselenization reaction was initially demonstrated on model peptide 31 bearing an intramolecular selenyl sulfide linkage to afford peptide 32 with the Cys residue still intact (Scheme 9A). The applicability of this methodology was subsequently highlighted by the synthesis of a 38 amino acid fragment of the redox enzyme glutaredoxin 3 (Grx3-1–38). Following a Sec ligation to provide the full-length peptide 33 possessing two Cys to Sec mutations at residues 11 and 14 and an Ala to Cys mutation at position 38, treatment with TCEP and DTT in phosphate buffer provided the doubly deselenated product 34 with only trace quantities of the Cys38 desulfurized material detected (Scheme 9B) [83]. Mechanistically, Dawson and coworkers hypothesized that the TCEP-mediated deselenization reaction proceeds via a radical pathway (Scheme 10), in a similar manner to the radical desulfurization of thiols by trialkylphosphines in the presence of UV light or elevated temperatures [118]. It should be noted that Danishefsky and coworkers implicated a similar mechanism in the metal-free desulfurization of Cys residues in the presence of the radical initiator VA-044 [56]. Despite operating under similar pathways, selective radical deselenization of Sec in the presence of Cys is presumably possible because of the mild conditions used to generate the initial selenium-centered radical, which proceeds at room temperature and does not require UV light or a radical initiator. This selective homolysis may therefore be explained by a weaker Se–X bond relative to the S–X bond of Cys. However, it is possible that alternative reaction pathways to that shown in Scheme 10 may be operating.


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PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS

Scheme 9. Chemoselective deselenization in the presence of unprotected Cys residues for (A) a model peptide system and (B) a fragment of Grx3.

Scheme 10. Proposed mechanism for the chemoselective deselenization reaction of Sec-containing peptides with TCEP and DTT.

Ligation–Deselenization at Selenol Amino Acids By providing access to target peptides containing both Cys and Ala ligation junctions without the requirement for side chain protection of native Cys residues, the ligation–chemoselective deselenization protocol at Sec marked a substantial advance and addition to the peptide ligation toolbox [83]. Indeed, this protocol has recently led to the extension of the ligation– deselenization manifold to other proteinogenic amino acid derivatives bearing a selenol moiety, in an analogous manner to the extension of traditional native chemical ligation via incorporation of thiol-derived amino acid building blocks into peptides [65,83,119]. In this case, the concept involves ligation at a selenol-derived amino acid, followed by selective deselenization with excess TCEP to generate a native amino acid at the ligation junction, which proceeds without affecting internal, unprotected Cys residues elsewhere in the peptide sequence (Scheme 11). This strategy has the capacity to greatly expand the scope of chemoselective ligation chemistry by providing access to target proteins that possess Cys residues at impractical ligation sites.

To date, two selenated amino acid derivatives have been synthesized and utilized in peptide ligation chemistry. The first of these amino acids, γ-selenoproline (or 4-selenoproline), was synthesized and utilized in ligation reactions by Danishefsky and coworkers (Scheme 12) [120]. The synthetic route toward a suitable trans-γ-selenoproline building block first involved iodination of Boc-protected hydroxyproline methyl ester 35 under Mitsunobu conditions to afford iodide 36. This was followed by nucleophilic substitution using selenobenzoic acid to provide 37, which was treated with methanolic potassium carbonate to afford diselenide 38. From here, acidolytic cleavage of the Boccarbamate moiety provided unprotected diselenide 39. It should be noted that the authors chose to synthesize the trans-γselenoproline as the thiolated homologue (7) facilitated more facile ligation reactions than the corresponding γ-epimer in a prior study [73]. Diselenide 39 was first incubated with a model peptide thiophenyl thioester at neutral pH in the presence of MPAA as a thiol catalyst and reductant. This provided the ligation product as a mixture of selenyl sulfides (thiophenyl and MPAA) in 92% isolated yield. Having established that ligation chemistry was

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Scheme 11. Chemoselective ligation–deselenization at β-selenol amino acids in the presence of unprotected Cys residues.

MALINS, MITCHELL AND PAYNE possible at γ-selenoproline, the authors next investigated the relative rates of deselenization of peptides bearing this modified amino acid on the N-terminus (Scheme 13). In order to study the efficiency and selectivity of the deselenization reaction, three selenopeptides 40–42 were synthesized. Diselenide peptide 40 was shown to be rapidly deselenized with TCEP in denaturing buffer at pH 5–6 at both low temperature and room temperature to provide 43. The rate of deselenization of peptide 41, possessing a selenyl sulfide linkage (the predominant product when thiophenol is used as the thiol additive in the ligation) was slightly slower, requiring first the addition of DTT as a reductant and subsequent treatment with TCEP to afford compound 43. These conditions are similar to those reported by Dawson and coworkers for selective deselenization of Sec in the presence of unprotected Cys residues [83] and were also capable of facilitating selective deselenization of peptide 42, bearing an internal, unprotected γ-thiol proline residue, to provide peptide 44 in excellent yield. Having established conditions for the ligation and deselenization reactions at trans-γ-selenoproline, the authors next incorporated protected diselenide building block 38 into model peptide 45, which was reacted with a range of peptide thioesters to gauge the scope of the ligation reaction (Table 2). Ligation reactions proved to be rapid at peptide thioesters bearing C-terminal Gly and Ala residues, reaching completion after 4.5 h (entries 1 and 2, Table 2). Following deselenization, which could be achieved in a one-pot fashion following completion of the ligation reaction, native peptides were produced in excellent yields. Ligation reactions of 45 with peptide thioesters containing C-terminal Phe and Val residues were slower, requiring 10–12 h to reach completion. Nonetheless, ligation products were isolated in excellent yields following deselenization (entries 3 and 4, Table 2). It should be noted that ligation at Val

thioesters is also sluggish for native chemical ligation facilitated by Cys residues [8] as well as for ligations facilitated by the γ-thiol proline analog [73,120]. Reaction with a peptide thioester bearing a C-terminal proline residue did not afford the desired ligation product despite extended reaction times (entry 5, Table 1). The second selenated amino acid to be investigated in the context of peptide ligation–deselenization chemistry is β-selenophenylalanine [121]. The synthesis of protected building block 46 for direct incorporation into SPPS was proposed from Garner’s aldehyde 47 (Scheme 14). This chiral precursor has also been proposed as a general starting material to access several thiol- and selenol-amino acid derivatives and has since been employed for the preparation of β-thiol Arg [76]. Synthesis began with the Grignard addition of phenyl magnesium bromide into the aldehyde of 47 providing 48 in 80% yield as a mixture of diastereoisomers (2 : 3 syn : anti). Activation of the alcohol as the mesylate followed by treatment with potassium selenocyanate provided 49 as only the syn-diastereoisomer. The stereochemical outcome of this reaction was proposed to be a result of the sterically hindered nature of the syn-mesylate, and as a result, only the anti-mesylate was competent in the SN2 inversion. From here, acidic cleavage of the hemiaminal protecting group followed by oxidation with pyridinium dichromate yielded carboxylic acid 51 in 57% yield. Finally, reduction of the selenocyanate functionality of 51 with sodium borohydride and subsequent protection afforded the corresponding PMB protected β-selenophenylalanine building block 46. Selenol-derived building block 46 was subsequently incorporated into model peptides 52 and 53 via Fmoc-strategy SPPS (Scheme 15) [121]. Following assembly and cleavage from the resin with a TFA-based cocktail, the PMB selenyl ether remained intact in peptides 54 and 55. This group could be

Scheme 12. Synthesis of trans-γ-selenoproline building blocks 38 and 39.

72

Scheme 13. Efficiency of deselenization of trans-γ-selenoproline-containing peptides.



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PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS Table 2. Scope of ligation–deselenization chemistry at trans-γselenoproline.

Entry 1 2 3 4 5

Thioester (X =) G A F V P

Reaction time (h)

Yield (%)

4.5 4.5 10 12 24

84 88 80 66 0

removed by re-treatment with TFA in the presence of 2,2′dithiobis(5-nitropyridine) (DTNP) [122], which provided symmetrical diselenides 52 and 53. Ligation reactions between 52 and 53 and a variety of C-terminal peptide thioester coupling partners (Ac-LYRANX-S(CH2)2CO2Et, X = G, A, M, F, V) were investigated to gauge the scope of ligation at β-selenophenylalanine. Optimized ligation conditions involved the use of ligation buffer (6 M Gn.HCl/0.1 M Na2HPO4, 5 mM with respect to the selenopeptide) in the presence of 200 mM MPAA, which served as both a thiol catalyst and a mild reductant. Ligations

were performed at room temperature at a final pH of 7.0–7.3, a slight modification of the conditions employed by Dawson and coworkers for ligations at Sec [83]. Under these conditions, ligation of 52 with a peptide thioester bearing a C-terminal Gly residue proceeded to completion in less than 24 h to provide the ligation product as a mixture of the diselenide dimer and the selenyl-MPAA sulfide adduct in 56% yield (entry 1, Table 3). Ligation reactions of 52 with peptide thioesters bearing C-terminal Ala, Met, and Phe residues also proceeded to completion within 24 h, affording the desired ligation products (as a mixture of the diselenide and corresponding selenyl sulfide adduct) in 51–53% isolated yields (entries 2–4, Table 3). However, ligation with a peptide thioester bearing a sterically encumbered Val residue led to poor conversion to the desired ligation product even after 96 h (21% yield, entry 5, Table 3). The lengthy reaction times were proposed to be a result of the low steady-state concentration of free selenol in solution caused by the highly negative reduction potential of the diselenide bond in the starting peptide together with the use of a relatively mild reductant (MPAA) in the ligation reaction [83]. However, effects to this extent were not observed in the ligation studies at γ-selenoproline, which suggests that subtle changes in the reaction conditions can result in dramatic effects on the reaction rate. Alternatively, it is possible that reduction potential differences between the selenol-derived amino acids accounts for these differences in reactivity. Importantly, ligation reactions at β-selenophenylalanine were also shown to be chemoselective in the presence of the ε-amino side chain of lysine residues. This was demonstrated by the successful ligation of peptide 53 bearing an internal lysine residue with peptide thioesters containing C-terminal Gly and Phe residues (entries 6 and 7, Table 3). Although the reactions at β-selenophenylalanine provided satisfactory yields of the corresponding ligation products, purification of the ligation reactions was complicated by the generation of both symmetrical diselenide and selenyl-MPAA adducts. In addition, attempted deselenization of the purified ligation product mixture led to the production of significant quantities

Scheme 14. Synthesis of β-selenophenylalanine building block 46 from Garner’s aldehyde 47.

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73

Scheme 15. Synthesis of model peptides 52 and 53 bearing an N-terminal β-selenophenylalanine residue.

MALINS, MITCHELL AND PAYNE Table 3. Ligation–deselenization reactions of β-selenophenylalanine-containing peptides 52 and 53 with a range of peptide thioesters.

Entry

1 2 3 4 5 6 7

Selenopeptide (Z =)

Thioester (X =)

S (52) S (52) S (52) S (52) S (52) K (53) K (53)

Isolated yield (%)

Isolated yield (%)

Ligation

One-pot ligation–deselenization

56 53 51 53 21 52 54

35a,c 38a,c 47b 43b 21 nd nd

G A M F V G F

Ligation conditions: buffer (6 M Gn.HCl/0.1 M Na2HPO4, 5 mM with respect to selenopeptide), 200 mM MPAA, pH 7.0–7.3, rt, 24 h. One-pot deselenization conditions: TCEP (42 eq) and DTT (4.9 eq) added to ligation mixture. nd, not determined. a t = 48 h. b t = 24 h. c Additional TCEP (42 eq) and DTT (4.9 eq) were added after 24 h.

of radical hydroxylation by-products. In order to facilitate a more operationally simple ligation–deselenization reaction at β-selenophenylalanine, the authors chose to explore a onepot ligation–deselenization protocol. Ligation reactions between selenopeptide 52 and a range of C-terminal thioesters were carried out using the same conditions described earlier (entries 1–5, Table 3). These crude reactions were directly subjected to deselenization with DTT (4.9 eq) and TCEP (42 eq) (Table 3). The one-pot deselenization reactions were relatively slow, requiring 24–48 h to reach completion, and in the case of reaction with the model Gly (entry 1) and Ala (entry 2) thioesters, additional dosing with DTT and TCEP was required. As suggested by Dawson and

coworkers in the deselenization of Sec [83] and by Brik and coworkers in their exploration of one-pot ligation radical desulfurization reactions at Cys [64,123], the sluggish rate of deselenization may be attributed to the large excess (200 mM) of MPAA present in the ligation buffer, which may act as a competitive radical scavenger. Nonetheless, the one-pot ligation–deselenization protocol provided the target peptide products in 21–47% yield over the two steps with the hydroxylation pathway substantially suppressed relative to the stepwise protocol (Table 3) [121]. The chemoselectivity of the one-pot ligation–deselenization reaction in the presence of unprotected Cys residues was also investigated. Specifically, selenopeptide 52 and selenopeptide

Table 4. Ligation–deselenization reactions of β-selenophenylalanine-containing peptides 52 and 56 with a range of peptide thioesters.

Entry

1 2 3

Selenopeptide (Z =) C S S

Thioester

Isolated yield (%) One-pot ligation–deselenization

LYRANG LYRCNG LYRCNM

52 61 48

74

Ligation conditions: buffer (6 M Gn.HCl/0.1 M Na2HPO4, 5 mM with respect to selenopeptide), 200 mM MPAA, pH 7.0–7.3, rt, 48 h. One-pot deselenization conditions: TCEP (42 eq) and DTT (4.9 eq) added to ligation mixture, 48 h.



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PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS 56 (bearing an internal Cys residue) were reacted with peptide thioesters with and without unprotected Cys residues (entries 1–3, Table 4). Ligation reactions were slower than those described in Table 3, requiring 48 h to reach completion. This was owing to the formation of a stable intramolecular selenyl-sulfide bond (entry 1, Table 4) and unproductive thioester formation with the unprotected Cys residues (entries 2 and 3, Table 4). However, the one-pot ligation–deselenization reaction provided the desired peptides bearing a native Phe residue at the ligation junction in 48–61% yield over the two steps. Perhaps most importantly, the deselenization step was completely chemoselective, leaving the internal, unprotected Cys residues untouched.

Summary and Outlook Inspired by native chemical ligation at Cys, peptide ligation chemistry at selenocysteine (Sec) has provided an efficient and robust means to incorporate Sec residues into peptides and proteins. Importantly, the methodology enables the preparation of both native and non-natural Sec-containing peptides and proteins that can be difficult to access via recombinant technologies. The recent finding that Sec residues can be deselenized to afford native Ala residues in the presence of unprotected Cys residues has greatly expanded the utility of Sec ligation chemistry for the assembly of large polypeptides and proteins without the need to implement protecting groups. Importantly, a similar chemoselective desulfurization cannot be effected following native chemical ligation at Cys or (for the most part) after a ligation reaction at a thiol-derived amino acid. The recent extension of this concept to ligation–chemoselective deselenization chemistry at selenol-derived proline and phenylalanine has further expanded the repertoire of peptide ligation chemistry and the targets that can now be assembled using this technology. It is anticipated that synthetic routes to additional selenol-derived proteinogenic amino acids will be developed in the coming years, which will serve to expand the utility of selenol ligation– chemoselective deselenization chemistry for the chemical synthesis of proteins.

References

J. Pept. Sci. 2014; 20: 64–77



75

1 Merrifield RB. Solid phase peptide synthesis. I. The synthesis of a tetrapeptide. J. Am. Chem. Soc. 1963; 85: 2149–2154. 2 Barany G, Merrifield RB. Solid-phase peptide synthesis. The Peptides, Vol. 2. Academic Press: New York, 1979; 1–284. 3 Gutte B, Merrifield RB. The total synthesis of an enzyme with ribonuclease A activity. J. Am. Chem. Soc. 1969; 91: 501–502. 4 Kent SBH. Chemical synthesis of peptides and proteins. Annu. Rev. Biochem. 1988; 57: 957–989. 5 Clark-Lewis I, Aebersold R, Ziltener H, Schrader JW, Hood LE, Kent SBH. Automated chemical synthesis of a protein growth factor for hematopoietic cells, interleukin-3. Science 1986; 231: 134–139. 6 Dawson PE, Muir TW, Clark-Lewis I, Kent SBH. Synthesis of proteins by native chemical ligation. Science 1994; 266: 776–779. 7 Dawson PE, Kent SB. Synthesis of native proteins by chemical ligation. Annu. Rev. Biochem. 2000; 69: 923–960. 8 Hackeng TM, Griffin JH, Dawson PE. Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 10068–10073. 9 Dirksen A, Dawson PE. Expanding the scope of chemoselective peptide ligations in chemical biology. Curr. Opin. Chem. Biol. 2008; 12: 760–766. 10 Kent SB. Total chemical synthesis of proteins. Chem. Soc. Rev. 2009; 38: 338–351.

11 Muir TW. Semisynthesis of proteins by expressed protein ligation. Annu. Rev. Biochem. 2003; 72: 249–289. 12 Hackenberger CPR, Schwarzer D. Chemoselective ligation and modification strategies for peptides and proteins. Angew. Chem. Int. Ed. 2008; 47: 10030–10074. 13 Kent S. Total chemical synthesis of enzymes. J. Pept. Sci. 2003; 9: 574–593. 14 Yeo DSY, Srinivasan R, Chen GYJ, Yao SQ. Expanded utility of the native chemical ligation reaction. Chem.-Eur. J. 2004; 10: 4664–4672. 15 Nilsson BL, Soellner MB, Raines RT. Chemical synthesis of proteins. Annu. Rev. Biophys. Biomolec. Struct. 2005; 34: 91–118. 16 Haase C, Seitz O. Extending the scope of native chemical peptide coupling. Angew. Chem. Int. Ed. 2008; 47: 1553–1556. 17 Dirksen A, Dawson PE. Expanding the scope of chemoselective peptide ligations in chemical biology. Curr. Opin. Chem. Biol. 2008; 12: 760–766. 18 Lemieux GA, Bertozzi CR. Chemoselective ligation reactions with proteins, oligosaccharides and cells. Trends Biotechnol. 1998; 16: 506–513. 19 Tam JP, Xu J, Eom KD. Methods and strategies of peptide ligation. Biopolymers 2001; 60: 194–205. 20 Davis BG. Synthesis of glycoproteins. Chem. Rev. 2002; 102: 579–601. 21 Gamblin DP, Scanlan EM, Davis BG. Glycoprotein synthesis: an update. Chem. Rev. 2009; 109: 131–163. 22 Payne RJ, Wong CH. Advances in chemical ligation strategies for the synthesis of glycopeptides and glycoproteins. Chem. Commun. 2010; 46: 21–43. 23 Macmillan D. Evolving strategies for protein synthesis converge on native chemical ligation. Angew. Chem. Int. Ed. 2006; 45: 7668–7672. 24 Hemantha HP, Narendra N, Sureshbabu VV. Total chemical synthesis of polypeptides and proteins: chemistry of ligation techniques and beyond. Tetrahedron 2012; 68: 9491–9537. 25 Bang D, Pentelute BL, Kent SB. Kinetically controlled ligation for the convergent chemical synthesis of proteins. Angew. Chem. Int. Ed. 2006; 45: 3985–3988. 26 Muir TW, Sondhi D, Cole PA. Expressed protein ligation: a general method for protein engineering. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 6705–6710. 27 Severinov K, Muir TW. Expressed protein ligation, a novel method for studying protein-protein interactions in transcription. J. Biol. Chem. 1998; 273: 16205–16209. 28 Muralidharan V, Muir TW. Protein ligation: an enabling technology for the biophysical analysis of proteins. Nat. Methods 2006; 3: 429–438. 29 Botti P, Carrasco MR, Kent SBH. Native chemical ligation using removable N-α-(1-phenyl-2-mercaptoethyl) auxiliaries. Tetrahedron Lett. 2001; 42: 1831–1833. 30 Offer J, Dawson PE. N-α-2-mercaptobenzylamine-assisted chemical ligation. Org. Lett. 2000; 2: 23–26. 31 Offer J, Boddy CNC, Dawson PE. Extending synthetic access to proteins with a removable acyl transfer auxiliary. J. Am. Chem. Soc. 2002; 124: 4642–4646. 32 Macmillan D, Anderson DW. Rapid synthesis of acyl transfer auxiliaries for cysteine-free native glycopeptide ligation. Org. Lett. 2004; 6: 4659–4662. 33 Wu B, Chen JH, Warren JD, Chen G, Hua ZH, Danishefsky SJ. Building complex glycopeptides: development of a cysteine-free native chemical ligation protocol. Angew. Chem. Int. Ed. 2006; 45: 4116–4125. 34 Offer J. Native chemical ligation with N-α acyl transfer auxiliaries. Biopolymers 2010; 94: 530–541. 35 Brik A, Yang YY, Ficht S, Wong CH. Sugar-assisted glycopeptide ligation. J. Am. Chem. Soc. 2006; 128: 5626–5627. 36 Ficht S, Payne RJ, Brik A, Wong CH. Second-generation sugar-assisted ligation: a method for the synthesis of cysteine-containing glycopeptides. Angew. Chem. Int. Ed. 2007; 46: 5975–5979. 37 Payne RJ, Ficht S, Tang S, Brik A, Yang YY, Case DA, Wong CH. Extended sugar-assisted glycopeptide ligations: development, scope and applications. J. Am. Chem. Soc. 2007; 129: 13527–13536. 38 Yang YY, Ficht S, Brik A, Wong CH. Sugar-assisted glycoprotein synthesis. J. Am. Chem. Soc. 2007; 129: 7690–7701. 39 Bennett CS, Dean SM, Payne RJ, Ficht S, Brik A, Wong CH. Sugarassisted glycopeptide ligation with complex oligosaccharides: scope and limitations. J. Am. Chem. Soc. 2008; 130: 11945–11952. 40 Lutsky MY, Nepomniaschiy N, Brik A. Peptide ligation via side-chain auxiliary. Chem. Commun. 2008; 10: 1229–1231.

MALINS, MITCHELL AND PAYNE

76

41 Aimoto S. Polypeptide synthesis by the thioester method. Biopolymers 1999; 51: 247–265. 42 Chen G, Wan Q, Tan Z, Kan C, Hua Z, Ranganathan K, Danishefsky SJ. Development of efficient methods for accomplishing cysteine-free peptide and glycopeptide coupling. Angew. Chem. Int. Ed. 2007; 119: 7527–7531. 43 Payne RJ, Ficht S, Greenberg WA, Wong CH. Cysteine-free peptide and glycopeptide ligation by direct aminolysis. Angew. Chem. Int. Ed. 2008; 47: 4411–4415. 44 Thomas GL, Payne RJ. Phosphate-assisted peptide ligation. Chem. Commun. 2009: 4260–4262. 45 Thomas GL, Hsieh YSY, Chun CKY, Cai ZL, Reimers JR, Payne RJ. Peptide ligations accelerated by N-terminal aspartate and glutamate residues. Org. Lett. 2011; 13: 4770–4773. 46 Hojo H, Haginoya E, Matsumoto Y, Nakahara Y, Nabeshima K, Toole BP, Watanabe Y. The first synthesis of peptide thioester carrying N-linked core pentasaccharide through modified Fmoc thioester preparation: synthesis of an N-glycosylated Ig domain of emmprin. Tetrahedron Lett. 2003; 44: 2961–2964. 47 Hojo H, Matsumoto Y, Nakahara Y, Ito E, Suzuki Y, Suzuki M, Suzuki A, Nakahara Y. Chemical synthesis of 23 kDa glycoprotein by repetitive segment condensation: a synthesis of MUC2 basal motif carrying multiple O-GalNAc moieties. J. Am. Chem. Soc. 2005; 127: 13720–13725. 48 Hojo H, Murasawa Y, Katayama H, Ohira T, Nakahara Y, Nakahara Y. Application of a novel thioesterification reaction to the synthesis of chemokine CCL27 by the modified thioester method. Org. Biomol. Chem. 2008; 6: 1808–1813. 49 Katayama H, Hojo H, Shimizu I, Nakahara Y, Nakahara Y. Chemical synthesis of mouse pro-opiomelanocortin(1–74) by azido-protected glycopeptide ligation via the thioester method. Org. Biomol. Chem. 2010; 8: 1966–1972. 50 Yuan Y, Chen J, Wan Q, Tan ZP, Chen G, Kan C, Danishefsky SJ. Toward homogeneous erythropoietin: fine tuning of the C-terminal acyl donor in the chemical synthesis of the Cys(29)-Gly(77) glycopeptide domain. J. Am. Chem. Soc. 2009; 131: 5432–5437. 51 Tan ZP, Shang SY, Halkina T, Yuan Y, Danishefsky SJ. Toward homogeneous erythropoietin: non-NCL-based chemical synthesis of the Gln(78)-Arg(166) glycopeptide domain. J. Am. Chem. Soc. 2009; 131: 5424–5431. 52 Thompson RE, Jolliffe KA, Payne RJ. Total synthesis of microcin B17 via a fragment condensation approach. Org. Lett. 2011; 13(4): 680–683. 53 Wilkinson BL, Chun CKY, Payne RJ. Synthesis of MUC1 glycopeptide thioesters and ligation via direct aminolysis. Pept. Sci. 2011; 18: 147–154. 54 Yan LZ, Dawson PE. Synthesis of peptides and proteins without cysteine residues by native chemical ligation combined with desulfurization. J. Am. Chem. Soc. 2001; 123: 526–533. 55 Haase C, Rohde H, Seitz O. Native chemical ligation at valine. Angew. Chem. Int. Ed. 2008; 47: 6807–6810. 56 Wan Q, Danishefsky SJ. Free-radical-based, specific desulfurization of cysteine: a powerful advance in the synthesis of polypeptides and glycopolypeptides. Angew. Chem. Int. Ed. 2007; 46: 9248–9252. 57 Hoffmann FW, Ess RJ, Simmons TC, Hanzel RS. The desulfurization of mercaptans with trialkyl phosphites. J. Am. Chem. Soc. 1956; 78: 6414–6414. 58 Brailsford JA, Danishefsky SJ. Probing the stability of nonglycosylated wild-type erythropoietin protein via reiterative alanine ligations. Proc. Natl. Acad. Sci. U. S. A. 2012; 109: 7196–7201. 59 Wilkinson BL, Stone RS, Capicciotti CJ, Thaysen-Andersen M, Matthews JM, Packer NH, Ben RN, Payne RJ. Total synthesis of homogeneous antifreeze glycopeptides and glycoproteins. Angew. Chem. Int. Ed. 2012; 51: 3606–3610. 60 Wang P, Dong S, Brailsford JA, Iyer K, Townsend SD, Zhang Q, Hendrickson RC, Shieh J, Moore MAS, Danishefsky SJ. At last: erythropoietin as a single glycoform. Angew. Chem. Int. Ed. 2012; 51: 11576–11584. 61 Murakami M, Okamoto R, Izumi M, Kajihara Y. Chemical synthesis of an erythropoietin glycoform containing a complex-type disialyloligosaccharide. Angew. Chem. Int. Ed. 2012; 51: 3567–3572. 62 Sakamoto I, Tezuka K, Fukae K, Ishii K, Taduru K, Maeda M, Ouchi M, Yoshida K, Nambu Y, Igarashi J, Hayashi N, Tsuji T, Kajihara Y. Chemical synthesis of homogeneous human glycosyl-interferon-β that exhibits potent antitumor activity in vivo. J. Am. Chem. Soc. 2012; 134: 5428–5431.


63 Liu SH, Pentelute BL, Kent SBH. Convergent chemical synthesis of 24,38,83 ] human erythropoietin. Angew. Chem. Int. Ed. 2012; [lysine 51: 993–999. 64 Siman P, Blatt O, Moyal T, Danieli T, Lebendiker M, Lashuel HA, Friedler A, Brik A. Chemical synthesis and expression of the HIV-1 Rev protein. ChemBioChem 2011; 12: 1097–1104. 65 Dawson PE. Native chemical ligation combined with desulfurization and deselenization: a general strategy for chemical protein synthesis. Isr. J. Chem. 2011; 51: 862–867. 66 Crich D, Banerjee A. Native chemical ligation at phenylalanine. J. Am. Chem. Soc. 2007; 129: 10064–10065. 67 Chen J, Wan Q, Yuan Y, Zhu JL, Danishefsky SJ. Native chemical ligation at valine: a contribution to peptide and glycopeptide synthesis. Angew. Chem. Int. Ed. 2008; 47: 8521–8524. 68 Ajish Kumar KS, Haj-Yahya M, Olschewski D, Lashuel HA, Brik A. Highly efficient and chemoselective peptide ubiquitylation. Angew. Chem. Int. Ed. 2009; 48: 8090–8094. 69 Yang RL, Pasunooti KK, Li FP, Liu XW, Liu CF. Dual native chemical ligation at lysine. J. Am. Chem. Soc. 2009; 131: 13592–13593. 70 El Oualid F, Merkx R, Ekkebus R, Hameed DS, Smit JJ, de Jong A, Hilkmann H, Sixma TK, Ovaa H. Chemical synthesis of ubiquitin, ubiquitin-based probes, and diubiquitin. Angew. Chem. Int. Ed. 2010; 49: 10149–10153. 71 Harpaz Z, Siman P, Kumar KSA, Brik A. Protein synthesis assisted by native chemical ligation at leucine. ChemBioChem 2010; 11: 1232–1235. 72 Tan ZP, Shang SY, Danishefsky SJ. Insights into the finer issues of native chemical ligation: an approach to cascade ligations. Angew. Chem. Int. Ed. 2010; 49: 9500–9503. 73 Shang SY, Tan ZP, Dong SW, Danishefsky SJ. An advance in proline ligation. J. Am. Chem. Soc. 2011; 133: 10784–10786. 74 Ding H, Shigenaga A, Sato K, Morishita K, Otaka A. Dual kinetically controlled native chemical ligation using a combination of sulfanylproline and sulfanylethylanilide peptide. Org. Lett. 2011; 13: 5588–5591. 75 Chen J, Wang P, Zhu JL, Wan Q, Danishefsky SJ. A program for ligation at threonine sites: application to the controlled total synthesis of glycopeptides. Tetrahedron 2010; 66: 2277–2283. 76 Malins LR, Cergol KM, Payne RJ. Peptide ligation-desulfurization chemistry at arginine. ChemBioChem 2013; 14: 559–563. 77 Thompson RE, Chan B, Radom L, Jolliffe KA, Payne RJ. Chemoselective peptide ligation-desulfurization at aspartate. Angew. Chem. Int. Ed. 2013; 14: 559–563. 78 Malins LR, Cergol KM, Payne RJ. Chemoselective sulfenylation and peptide ligation at tryptophan. Chem. Sci. 2014; 5(1): 260–266. 79 Pentelute BL, Kent SBH. Selective desulfurization of cysteine in the presence of Cys(Acm) in polypeptides obtained by native chemical ligation. Org. Lett. 2007; 9: 687–690. 80 Quaderer R, Sewing A, Hilvert D. Selenocysteine-mediated native chemical ligation. Helv. Chim. Acta 2001; 84: 1197–1206. 81 Gieselman MD, Xie L, van Der Donk WA. Synthesis of a selenocysteine-containing peptide by native chemical ligation. Org. Lett. 2001; 3: 1331–1334. 82 Hondal RJ, Nilsson BL, Raines RT. Selenocysteine in native chemical ligation and expressed protein ligation. J. Am. Chem. Soc. 2001; 123: 5140–5141. 83 Metanis N, Keinan E, Dawson PE. Traceless ligation of cysteine peptides using selective deselenization. Angew. Chem. Int. Ed. 2010; 49: 7049–7053. 84 Schwarz K, Foltz CM. Selenium as an integral part of factor 3 against dietary necrotic liver degeneration. J. Am. Chem. Soc. 1957; 79: 3292–3293. 85 Muttenthaler M, Alewood PF. Selenopeptide chemistry. J. Pept. Sci. 2008; 14: 1223–1239. 86 Flohe L, Guenzler WA, Schock HH. Glutathione peroxidase: a selenoenzyme. FEBS Lett. 1973; 32: 132–134. 87 Lu J, Holmgren A. Selenoproteins. J. Biol. Chem. 2009; 284: 723–727. 88 Johansson L, Gafvelin G, Arner ESJ. Selenocysteine in proteins— properties and biotechnological use. Biochim. Biophys. Acta, Gen. Subj. 2005; 1726: 1–13. 89 Cone JE, Martin DRR, Davis JN, Stadtman TC. Chemical characterization of the selenoprotein component of clostridial glycine reductase: identification of selenocysteine as the organoselenium moiety. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2659–2663.


J. Pept. Sci. 2014; 20: 64–77

PEPTIDE LIGATION CHEMISTRY AT SELENOL AMINO ACIDS 90 Bock A, Forchhammer K, Heider J, Leinfelder W, Sawers G, Veprek B, Zinoni F. Selenocysteine: the 21st amino acid. Mol. Microbiol. 1991; 5: 515–520. 91 Zhong L, Arnér ESJ, Holmgren A. Structure and mechanism of mammalian thioredoxin reductase: the active site is a redox-active selenolthiol/ selenenylsulfide formed from the conserved cysteine-selenocysteine sequence. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5854–5859. 92 Lee SR, Bar Noy S, Kwon J, Levine RL, Stadtman TC, Rhee SG. Mammalian thioredoxin reductase: oxidation of the C-terminal cysteine/ selenocysteine active site forms a thioselenide, and replacement of selenium with sulfur markedly reduces catalytic activity. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 2521–2526. 93 Axley MJ, Böck A, Stadtman TC. Catalytic properties of an Escherichia coli formate dehydrogenase mutant in which sulfur replaces selenium. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8450–8454. 94 Arnold AP, Tan KS, Rabenstein DL. Nuclear magnetic resonance studies of the solution chemistry of metal complexes. 23. Complexation of methylmercury by selenohydryl-containing amino acids and related molecules. Inorg. Chem. 1986; 25: 2433–2437. 95 Huber R, Criddle RS. Comparison of the chemical properties of selenocysteine and selenocystine with their sulfur analogs. Arch. Biochem. Biophys. 1967; 122: 164–173. 96 Besse D, Siedler F, Diercks T, Kessler H, Moroder L. The redox potential of selenocystine in unconstrained cyclic peptides. Angew. Chem. Int. Ed. 1997; 36: 883–885. 97 Nauser T, Dockheer S, Kissner R, Koppenol WH. Catalysis of electron transfer by selenocysteine. Biochemistry 2006; 45: 6038–6043. 98 Guenther WHH. Methods in selenium chemistry. III. Reduction of diselenides with dithiothreitol. J. Org. Chem. 1967; 32: 3931–3934. 99 Pegoraro S, Fiori S, Rudolph-Böhner S, Watanabe TX, Moroder L. Isomorphous replacement of cystine with selenocystine in endothelin: oxidative refolding, biological and conformational properties of [Sec3, Sec11,Nle7]-endothelin-1. J. Mol. Biol. 1998; 284: 779–792. 100 Pegoraro S, Fiori S, Cramer J, Rudolph-Böhner S, Moroder L. The disulfide-coupled folding pathway of apamin as derived from diselenide-quenched analogs and intermediates. Protein Sci. 1999; 8: 1605–1613. 101 Fiori S, Pegoraro S, Rudolph-Böhner S, Cramer J, Moroder L. Synthesis and conformational analysis of apamin analogues with natural and non-natural cystine/selenocystine connectivities. Biopolymers 2000; 53: 550–564. 102 Metanis N, Hilvert D. Strategic use of non-native diselenide bridges to steer oxidative protein folding. Angew. Chem. Int. Ed. 2012; 51: 5585–5588. 103 Walewska A, Jaśkiewicz A, Bulaj G, Rolka K. Selenopeptide analogs of EETI-II retain potent trypsin inhibitory activities. Chem. Biol. Drug Des. 2011; 77: 93–97. 104 Armishaw CJ, Daly NL, Nevin ST, Adams DJ, Craik DJ, Alewood PF. α-Selenoconotoxins, a new class of potent α7 neuronal nicotinic receptor antagonists. J. Biol. Chem. 2006; 281: 14136–14143. 105 Walewska A, Zhang M-M, Skalicky JJ, Yoshikami D, Olivera BM, Bulaj G. Integrated oxidative folding of cysteine/selenocysteine containing peptides: improving chemical synthesis of conotoxins. Angew. Chem. Int. Ed. 2009; 48: 2221–2224.

106 Muttenthaler M, Nevin ST, Grishin AA, Ngo ST, Choy PT, Daly NL, Hu S-H, Armishaw CJ, Wang C-IA, Lewis RJ, Martin JL, Noakes PG, Craik DJ, Adams DJ, Alewood PF. Solving the α-conotoxin folding problem: efficient selenium-directed on-resin generation of more potent and stable nicotinic acetylcholine receptor antagonists. J. Am. Chem. Soc. 2010; 132: 3514–3522. 107 Gowd KH, Yarotskyy V, Elmslie KS, Skalicky JJ, Olivera BM, Bulaj G. Site-specific effects of diselenide bridges on the oxidative folding of a cystine knot peptide, ω-selenoconotoxin GVIA. Biochemistry 2010; 49: 2741–2752. 108 de Araujo AD, Callaghan B, Nevin ST, Daly NL, Craik DJ, Moretta M, Hopping G, Christie MJ, Adams DJ, Alewood PF. Total synthesis of the analgesic conotoxin MrVIB through selenocysteine-assisted folding. Angew. Chem. Int. Ed. 2011; 50: 6527–6529. 109 Steiner AM, Woycechowsky KJ, Olivera BM, Bulaj G. Reagentless oxidative folding of disulfide-rich peptides catalyzed by an intramolecular diselenide. Angew. Chem. Int. Ed. 2012; 51: 5580–5584. 110 Clark KM, van der Donk WA, Lu Y. Chapter 5 expressed protein ligation for metalloprotein design and engineering. Methods Enzymol. 2009; 462: 97–115. 111 Hondal RJ, Raines RT. Semisynthesis of proteins containing selenocysteine. Methods Enzymol. 2002; 347: 70–83. 112 Hondal RJ. Incorporation of selenocysteine into proteins using peptide ligation. Protein Pept. Lett. 2005; 12: 757–764. 113 Gieselman MD, Zhu YT, Zhou H, Galonic D, van der Donk WA. Selenocysteine derivatives for chemoselective ligations. ChemBioChem 2002; 3: 709–716. 114 Quaderer R, Hilvert D. Selenocysteine-mediated backbone cyclization of unprotected peptides followed by alkylation, oxidative elimination or reduction of the selenol. Chem. Commun. 2002: 2620–2621. 115 Berry SM, Gieselman MD, Nilges MJ, van der Donk WA, Lu Y. An engineered azurin variant containing a selenocysteine copper ligand. J. Am. Chem. Soc. 2002; 124: 2084–2085. 116 Chalker JM, Bernardes GJL, Lin YA, Davis BG. Chemical modification of proteins at cysteine: opportunities in chemistry and biology. Chem. Asian J. 2009; 4: 630–640. 117 Chalker JM, Bernardes GJL, Davis BG. A “tag-and-modify” approach to site-selective protein modification. Acc. Chem. Res. 2011; 44: 730–741. 118 Walling C, Basedow OH, Savas ES. Some extensions of the reaction of trivalent phosphorus derivatives with alkoxy and thiyl radicals; a new synthesis of thioesters. J. Am. Chem. Soc. 1960; 82: 2181–2184. 119 Wong CTT, Tung CL, Li X. Synthetic cysteine surrogates used in native chemical ligation. Mol. Biosyst. 2013; 9: 826–833. 120 Townsend SD, Tan Z, Dong S, Shang S, Brailsford JA, Danishefsky SJ. Advances in proline ligation. J. Am. Chem. Soc. 2012; 134: 3912–3916. 121 Malins LR, Payne RJ. Synthesis and utility of β-selenol-phenylalanine for native chemical ligation-deselenization chemistry. Org. Lett. 2012; 14: 3142–3145. 122 Harris KM, Flemer S, Hondal RJ. Studies on deprotection of cysteine and selenocysteine side-chain protecting groups. J. Pept. Sci. 2007; 13: 81–93. 123 Moyal T, Hemantha HP, Siman P, Refua M, Brik A. Highly efficient one-pot ligation and desulfurization. Chem. Sci. 2013; 4: 2496–2501.

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Templated native chemical ligation: peptide chemistry beyond protein synthesis.

Native chemical ligation: a boon to peptide chemistry.

Non-natural phenolic amino acids Synthesis and application in peptide chemistry.

One-pot peptide ligation-desulfurization at glutamate.

Radiation chemistry of amino acids and peptides in aqueous solutions.

Peptide modification by incorporation ofα-trifluoromethyl substituted amino acids.

Structural alterations of amino acids at the level of aminoacyl-tRNAs: transformation of dicarboxylic amino acids.

Use of tetrabutylammonium salts of amino acids in peptide synthesis.

Trifluoroethanethiol: an additive for efficient one-pot peptide ligation-desulfurization chemistry.

Peptidomimetics via modifications of amino acids and peptide bonds.

Peptide modification by introduction ofα-trifluoromethyl substituted amino acids.

Solid phase peptide synthesis utilizing 9-fluorenylmethoxycarbonyl amino acids.

Protein-templated peptide ligation.

Histones: at the crossroads of peptide and protein chemistry.

Templated DNA ligation with thiol chemistry.

Excited state chemistry of aromatic amino acids and related peptides. I. Tyrosine.

Bioinorganic chemistry of silver: its interactions with amino acids and peptides.

Structural alterations of amino acids at the level of aminoacyl-tRNAs: identification of the transformation products of dicarboxylic amino acids.

Nucleic acids--chemistry and applications.

Lysyl dipeptide and tripeptide model systems for racemization studies in amino acid and peptide chemistry.

Transformation of some hydroxy amino acids to other amino acids.

Application of hexafluoroacetone as protecting and activating reagent in amino acid and peptide chemistry.

[Use of tosylaminocarbonyl group as an amino protecting group in peptide chemistry].

Peptide ligation from alkoxyamine based radical addition.