All posts in Raw-Journal Peptide Science

A considerable quantity of an alkylation by-product is observed when using 3,6-dioxa-1,8-octanedithiol as a scavenger during acidic release of peptides containing the thioether amino acid methionine from the solid support. Adjustment of the cleavage conditions by replacement of 3,6-dioxa-1,8-octanedithiol with ethane dithiol or by using methionine sulfoxide as an alternative to methionine resulted in no such impurity. The by-product was detectable by liquid chromatography and mass spectrometry and characterised by NMR spectroscopy of an isolated model peptide. It could be effectively removed in a separate post cleavage step by treatment with dilute aqueous acid at 37 °C. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

We outline a novel alkylation when using the dithiol 3,6-dioxa-1,8-octanedithiol (DODT) as a scavenger in TFA-mediated peptide cleavage from the resin. Peptides containing unprotected methionine were extensively modified by a DODT-derived alkylating agent affording a previously unreported by-product. Conditions for the prevention or subsequent removal of the by-product are outlined.

Ubiquitin and ubiquitin-like proteins such as SUMO represent important and abundant post-translational modifications involved in many cellular processes. These modifiers are reversibly attached via an isopeptide bond to lysine side chains of their target proteins by the action of specific E1, E2, and E3 enzymes. A significant challenge in studying ubiquitylation and SUMOylation is the frequently encountered inability to access desired conjugates at a defined position of the target protein and in homogenous form by using enzymatic preparation. In recent years, several chemical conjugation approaches have been developed to overcome this limitation. In this study, we aimed to selectively SUMOylate a 189-amino acid fragment of human RanGAP1 (amino acids 398–587) at the position of Lys524 by applying two recently reported approaches based on the Cu(I)-catalyzed alkyne-azide cycloaddition. Because of low yields observed for the incorporation of an unnatural amino acid with an azide moiety by the tRNA suppression technology, this route was abandoned. However, installing a single cysteine at position 524 and its selective alkylation was successful to introduce the azide group. The triazole-linked SUMO1**RanGAP1 conjugate could be obtained in good yields, purified, and was shown to specifically interact with RanBP2/Ubc9. Thus, we expand the scope of proteins accessible to chemical conjugation with ubiquitin-like proteins and underline the importance of having alternative approaches to do so. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

A chemical SUMO1-RanGAP1 conjugate was generated via the copper-catalyzed alkyne-azide cycloaddition (CuAAC). The triazole-linked analog was compared to the native isopeptide-linked conjugate and showed similar binding to form the multiprotein complex with RanBP2 and Ubc9. Our study illustrates advantages and disadvantages of two different routes for SUMO conjugation by CuAAC and further underlines the potential of chemical conjugates to investigate the biochemistry of ubiquitin and ubiquitin-like post-translational modifiers.

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.

This article provides a comprehensive review of ligation chemistry at selenol amino acids. Peptide ligation chemistry at selenocysteine is initially outlined as an effective means to generate selenocysteine-containing peptides by chemical synthesis or semisynthesis. The recent finding that selenocysteine residues can be chemoselectively deselenized to afford alanine residues following a ligation reaction has greatly expanded the utility of selenol ligation chemistry to access targets bearing internal, unprotected cysteine residues. This concept has recently been further exploited in ligation–chemoselective deselenization chemistry at proline and phenylalanine and is also discussed.

Protein total chemical synthesis enables the atom-by-atom control of the protein structure and therefore has a great potential for studying protein function. Native chemical ligation of C-terminal peptide thioesters with N-terminal cysteinyl peptides and related methodologies are central to the field of protein total synthesis. Consequently, methods enabling the facile synthesis of peptide thioesters using Fmoc-SPPS are of great value. Herein, we provide a detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

A detailed protocol for the preparation of bis(2-sulfanylethyl)amino polystyrene resin as a starting point for the synthesis of C-terminal bis(2-sulfanylethyl)amido peptides and of peptide thioesters derived from 3-mercaptopropionic acid is described.

The R5 peptide is derived from silaffin peptides naturally occurring in the diatom Cylindrotheca fusiformis and exhibits outstanding activity in silica precipitation. Because of its ability to cause silicification under mild conditions, several biotechnological applications based on R5-mediated biomimetic silica formation have already been reported. Yet a more detailed understanding of the R5 peptide and its intrinsic silica precipitation activity will help the rational design of R5 peptide variants as efficient agents for defined silica precipitation. The herein presented analysis of the relationship between the R5 amino acid sequence and its activity in silica precipitation emphasizes the essential role of the lysine residues in mediating silica polycondensation. Furthermore, a tetra amino acid motif (RRIL) has to be present within the R5 sequence, but in contrast to previous reports, we demonstrate that localization of the RRIL motif shows minor impact on silica precipitation activity but rather on morphology of the resulting silica material. The amino acid sequence of silaffin peptides is a well-balanced arrangement in terms of charges, functional groups and distances. The impact of this pattern of charges and functionalities was highlighted by the disturbed morphology of silica spheres resulting from R5 variants with scrambled sequences. A detailed understanding of the highly evolved silaffin sequence(s) will contribute to unravel the intriguing process of silica biomineralization in diatoms. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

A functional analysis of the R5 peptide highlights the characteristics of its amino acid sequence essential for silica precipitating activity. The RRIL amino acids are important for peptide self-assembly and lysine residues mediate silica formation. Overall, the well-balanced arrangement of charges and functionalities in the amino acid sequence of R5 turns out to be perfectly evolved for efficient silica precipitation.

The ligation of peptide hydrazides at a Gly site carrying a removal auxiliary was found to be an efficient process. This technology was successfully used for the synthesis of ubiquitin C-terminal conjugates. Recombinant Ub(1–75)-NHNH₂ was prepared through the hydrozinolysis of the Ub(1–75)-intein fusion protein. It was ligated with a glycine derivative modified with an acid-sensitive thiol auxiliary. The final acid treatment produced the desired bioactive ubiquitin conjugates in practical quantities. Thus, the method described here extends the protocols of expressed protein ligation. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.

Native chemical ligation of a recombinant peptide hydrazide with a Gly-peptide carrying a removal auxiliary was examined and used to synthesize ubiquitin C-terminal conjugates.