Total chemical synthesis of the B1 domain of protein L from Peptostreptococcus magnus

Reported here is a native chemical ligation strategy for the total chemical synthesis of the B1 domain of protein L. A synthetic construct of this 76 amino acid protein domain was prepared by the chemoselective ligation of two unprotected polypeptide fragments, one containing an N-terminal cysteine residue and one containing a C-terminal thioester moiety. The polypeptide fragments utilized in the ligation reaction were readily prepared by stepwise solid phase peptide synthesis (SPPS) methods for Boc-chemistry. The milligram quantities of protein required for conventional biophysical studies were readily accessible using the synthetic protocol described here. The folding properties of the synthetic protein L construct were also determined and found to be very similar to those of a similar wild-type protein L constructs prepared by recombinant-DNA methods. This work facilitates future unnatural amino acid mutagenesis experiments on this model protein system to further dissect the molecular basis of its folding and stability.     

Native chemical ligation of hydrophobic [corrected] peptides in lipid bilayer systems

The covalent modification of water-insoluble membrane polypeptides incorporated into lipid bilayers by native chemical ligation is described. The key feature of this strategy is the use of cubic lipidic phase (CLP) matrixes as reaction media. The CLP-matrix consists of a lipid bilayer into which hydrophobic polypeptides and folded membrane proteins can be inserted and two unbounded aqueous channels that give the aqueous phase access to both sides of an infinite lipid bilayer and thus ensure that modification of solvent-exposed sites is independent of the topology of membrane incorporation. The enzymatic removal of an N-terminal proteolytic cleavage sequence from the membrane polypeptide exposes an N-terminal cysteine residue. Subsequently, a C-terminal thioester peptide is joined to the N-terminus of the polypeptide by a native chemical ligation reaction. By use of this approach, incorporation of a variety of molecular tools, such as spectroscopic probes, unnatural amino acids, and molecular markers into membrane proteins that cannot be easily solubilized in detergent or denaturant solutions, may be achieved.     

Protein semisynthesis and expressed protein ligation: chasing a protein's tail

The adaptation of native chemical ligation to protein semisynthesis has become a powerful way to address problems in the analysis of protein structure and function. In particular, the exploitation of nature's inteins in expressed protein ligation is now a standard approach in the study of proteins. Site-specific incorporation of unnatural amino acids, biophysical probes and post-translational modifications in proteins have led to new insights into enzyme mechanisms, protein folding, ion channel function, translation and signaling.     

Lanthanide-binding tags with unnatural amino acids: sensitizing Tb3+ and Eu3+ luminescence at longer wavelengths

Lanthanide-binding tags (LBTs) are small, genetically encoded, versatile protein fusion partners that selectively bind lanthanide ions with high affinity. The LBT motif features a strategically positioned tryptophan residue that sensitizes Tb3+ luminescence upon excitation at 280 nm. Herein, we describe the preparation of new LBT peptides that incorporate unnatural amino acids in place of tryptophan, and which sensitize both Tb3+ and Eu3+ luminescence at lower energies. We also report the semisynthesis of proteins tagged with these new LBTs using native chemical ligation. This expands the scope of LBTs and will enable their wider use in luminescence applications.     

Thiocarbamate‐linked peptides by chemoselective peptide ligation

Peptide chemical ligation chemistries, which allow the chemoselective coupling of unprotected peptide fragments, are useful tools for synthesizing native polypeptides or unnatural peptide‐based macromolecules. We show here that the phenylthiocarbonyl group can be easily introduced into peptides on α or ε amino groups using phenylthiochloroformate and standard solid‐phase method. It reacts chemoselectively with cysteinyl peptides to give an alkylthiocarbamate bond. S,N‐shift of the alkylaminocarbonyl group from the Cys side chain to the α‐amino group did not occur. The method was used for linking two peptide chains through their N‐termini, for the synthesis of a cyclic peptide or for the synthesis of di‐ or tetravalent multiple antigenic peptides (MAPs). Thiocarbamate ligation is thus complementary to thioether, thioester or disulfide ligation methods. Copyright © 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis of an O‐acyl isopeptide by using native chemical ligation in an aqueous solvent system

O‐Acyl isopeptides, in which the N‐acyl linkage on the hydroxyamino acid residue (e.g. Ser and Thr) is replaced by an O‐acyl linkage, generally suppress unfavorable aggregation properties derived from the corresponding parent peptides. Here, we report the synthesis of an O‐acyl isopeptide of 34‐mer pyroGlu‐ADan (2), a component of amyloid deposits in hereditary familial Danish dementia, by using native chemical ligation. Native chemical ligation of pyroGlu¹‐ADan(1‐21)‐SCH₂CH₂SO₃^?Na⁺ (3) and Cys²²‐O‐acyl isopeptide (4), in which the amino group of the Ser²⁹ residue at the isopeptide moiety was protected by an allyloxycarbonyl group, proceeded well in an aqueous solvent to yield a ligated O‐acyl isopeptide (5). Subsequent disulfide bond formation and deprotection of the allyloxycarbonyl group followed by HPLC purification gave 2 with a reasonable overall yield. 2 was converted to the parent peptide 1 via an O‐to‐N acyl migration reaction. The sequential method, namely (i) native chemical ligation of the O‐acyl isopeptide, (ii) HPLC purification as the O‐acyl isopeptide form, and (iii) O‐to‐N acyl migration into the desired polypeptide, would be helpful to solve problems with HPLC purification of hydrophobic polypeptides in the process of chemical protein synthesis. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Chemical synthesis and biotinylation of the thrombospondin domain TSR2

The type 1 repeat domain from thrombospondin has potent antiangiogenic activity and a structurally interesting fold, making it an attractive target for protein engineering. Chemical synthesis is an attractive approach for studying protein domains because it enables the use of unnatural amino acids for site‐specific labeling and detailed structure‐function analysis. Here, we demonstrate the first total chemical synthesis of the thrombospondin type 1 repeat domain by native chemical ligation. In addition to the natural domain, five sites for side chain modification were evaluated and two were found to be compatible with oxidative folding. Several challenges were encountered during peptide synthesis due to the functional complexity of the domain. These challenges were overcome by the use of new solid supports, scavengers, and the testing of multiple ligation sites. We also describe an unusual sequence‐specific protecting group migration observed during cleavage resulting in +90 Da and +194 Da adducts. Synthetic access to this domain enables the synthesis of a number of variants that can be used to further our understanding of the biochemical interaction network of thrombospondin and provide insight into the structure and function of this important antitumorogenic protein domain.     

Direct production of proteins with N-terminal cysteine for site-specific conjugation

Proteins with N-terminal cysteine can undergo native chemical ligation and are useful for site-specific N-terminal labeling or protein semisynthesis. Recombinant production of these has usually been by site-specific cleavage of a precursor fusion protein at an internal cysteine residue. Here we describe a simpler route to producing these proteins. Overexpression in E. coli of several proteins containing cysteine as the second amino acid residue yielded products in which the initiating methionine residue had been completely cleaved by endogenous methionine aminopeptidase. While secondary modification of the terminal cysteine was a complicating factor, conditions were identified to eliminate or minimize this problem. Recombinant proteins produced in this way were suitable for site-specific modification of the amino terminus via native chemical ligation technology, as demonstrated by conjugation of a thioester-containing derivative of fluorescein to one such protein. The ability to directly produce proteins with N-terminal cysteine should simplify the application of native chemical ligation technology to recombinant proteins and make the technique more amenable to researchers with limited expertise in protein chemistry.     

Synthesis of lucifensin by native chemical ligation and characteristics of its isomer having different disulfide bridge pattern

The antimicrobial 40‐amino‐acid‐peptide lucifensin was synthesized by native chemical ligation (NCL) using N‐acylbenzimidazolinone (Nbz) as a linker group. NCL is a method in which a peptide bond between two discreet peptide chains is created. This method has been applied to the synthesis of long peptides and proteins when solid‐phase synthesis is imcompatible. Two models of ligation were developed: [15 + 25] Ala‐Cys and [19 + 21] His‐Cys. The [19 + 21] His‐Cys method gives lower yield because of the lower stability of 18‐peptide‐His‐Nbz‐CONH₂ peptide, as suggested by density functional theory calculation. Acetamidomethyl‐deprotection and subsequent oxidation of the ligated linear lucifensin gave a mixture of lucifensin isomers, which differed in the location of their disulfide bridges only. The dominant isomer showed unnatural pairing of cysteines [C1?6], [C3?5], and [C2?4], which limits its ability to form α‐helical structure. The activity of isomeric lucifensin toward Bacillus subtilis, Staphylococcus aureus, and Micrococcus luteus was lower than that of the natural lucifensin. The desired product native lucifensin was prepared from this isomer using a one‐pot reduction with dithiotreitol and subsequent air oxidation in slightly alkaline medium. Copyright © 2014 European Peptide Society and John Wiley & Sons, Ltd.     

Synthesis and use of a pseudo-cysteine for native chemical ligation.

The process of native chemical ligation (NCL) is well described in the literature. An N-terminal cysteine-containing peptide reacts with a C-terminal thioester-containing peptide to yield a native amide bond after transesterification and acyl transfer. An N-terminal cysteine is required as both the N-terminal amino function and the sidechain thiol participate in the ligation reaction. In certain circumstances it is desirable, or even imperative, that the N-terminal region of a peptidic reaction partner remain unmodified, for Instance for the retention of biological activity after ligation. This work discusses the synthesis of a pseudo-N-terminal cysteine building block for incorporation into peptides using standard methods of solid phase synthesis. Upon deprotection, this building block affords a de facto N-terminal cysteine positioned on an amino acid sidechain. which is capable of undergoing native chemical ligation with a thioester. The syntheses of several peptides and structures containing this motif are detailed, their reactions discussed. and further applications of this technology proposed.     

Ultrafiltration,a useful method for isolation of intermediates in native chemical ligation exemplified with the total synthesis of Sortase AΔN59

In this paper, ultrafiltration was employed to facilitate the isolation of intermediates in native chemical ligation. Depending on the molecular weight cutoff of the membrane used, molecules with different sizes could be purified, separated, or concentrated by the ultrafiltration process. Total chemical synthesis of the polypeptide chain of the enzyme Sortase A_ΔN59 was used as an example of the application of ultrafiltration in chemical protein synthesis. Sortase A is a ligase that catalyzes transpeptidation reactions between proteins that have C‐terminal LPXTG recognition sequence and Gly5‐ on the peptidoglycan of bacterial cell walls [3]. Ultrafiltration technique facilitated synthesis of Sortase A_ΔN59 and was a promising tool in isolation of intermediates in native chemical ligation. Copyright © 2015 European Peptide Society and John Wiley & Sons, Ltd.     

An amalgamation of solid phase peptide synthesis and ribosomal peptide synthesis

Expressed protein ligation (EPL) is a protein semisynthesis technique that allows the site-specific introduction of unnatural amino acids and biophysical probes into proteins. In the present study, we illustrate the utility of the approach through the generation of two semisynthetic proteins bearing spectroscopic probes. Dihydrofolate reductase containing a single (13)C probe in an active site loop was generated through the ligation of a synthetic peptide-alpha-thioester to a recombinantly generated fragment containing an N-terminal Cys. Similarly, c-Crk-II was assembled by the sequential ligation of three recombinant polypeptide building blocks, allowing the incorporation of (15)N isotopes in the central domain of the protein. These examples showcase the scope of the protein ligation strategy for selective introduction of isotopic labels into proteins, and the protocols described will be of value to those interested in using EPL on other systems.     

Synthesis of full length PB1-F2 influenza A virus proteins from 'Spanish flu' and 'bird flu'.

The proapoptotic influenza A virus PB1-F2 protein contributes to viral pathogenicity and is present in most human and avian isolates. Previous synthetic protocols have been improved to provide a synthetic full length H1N1 type PB1-F2 protein that is encoded by the 'Spanish flu' isolate and an equivalent protein from an avian host that is representative of a highly pathogenic H5N1 'bird flu' isolate, termed SF2 and BF2, respectively. Full length SF2, different mutants of BF2 and a number of fragments of these peptides have been synthesized by either the standard solid-phase peptide synthesis method or by native chemical ligation of unprotected N- and C-terminal peptide fragments. For SF2 chemical ligation made use of the histidine and the cysteine residues located in positions 41 and 42 of the native sequence, respectively, to afford a highly efficient synthesis of SF2 compared to the standard SPPS elongation method. By-product formation at the aspartic acid residue in position 23 was prevented by specific modifications of the SPPS protocol. As the native sequence of BF2 does not contain a cysteine residue two different mutants of BF2 (Y42C) and BF2 (S47C) with appropriate cysteine exchanges were produced. In addition to the full length molecules, fragments of the native sequences were synthesized for comparison of their physical characteristics with those from the H1N1 human isolate A/Puerto Rico/8/34 (H1N1). All peptides were analyzed by mass spectrometry, (1)H NMR spectroscopy, and SDS-PAGE. The protocols allow the synthesis of significant amounts of PB1-F2 and its related peptides. Copyright (c) 2008 European Peptide Society and John Wiley & Sons, Ltd.     

Engineering an unnatural Nalpha-anchored disulfide into BPTI by total chemical synthesis: structural and functional consequences

A disulfide-engineered analogue of bovine pancreatic trypsin inhibitor (BPTI), ((N(alpha)-(CH2)2S-)Gly38)BPTI, has been prepared using a thioester-mediated auxiliary functional group chemical ligation of a N(alpha)-ethanethiol-containing peptide segment with a peptide-alphaCOSR segment. In this study, Nalpha-(ethanethiol)Gly38 replaces the native Cys38, providing the sulfhydryl group required for ligation and folding. Comparisons between ((Nalpha-(CH2)2SH)Gly38)BPTI, synthetic native BPTI and reference BPTI purchased from Sigma were made using mass spectroscopy, enzyme inhibitor association constant determination (K(a)) and 1H-nuclear magnetic resonance total correlated spectroscopy (1H-NMR TOCSY) measurements. The K(a) value for ((Nalpha-(CH2)2SH)Gly38)BPTI was approximately 20-fold lower than synthetic and reference BPTI, which was attributed to perturbations in the binding loop of the protein (near Cys14). This hypothesis was confirmed by two-dimensional (2D) 1H-NMR TOCSY experiments. The data reported here demonstrate that total chemical synthesis by auxiliary functional group chemical ligation is a practical method for the synthesis of a novel class of biologically active protein analogues containing additional functional groups linked to the protein backbone.     

The use of 5''-phospho-2 deoxyribocytidylylriboadenosine as a facile route to chemical aminoacylation of tRNA.

Methodology is described for the synthesis and chemical aminoacylation of the hybrid dinucleotide 5'-phospho-2'-deoxyribocytidylylriboadenosine (pdCpA). Ligation of aminoacylated pdCpA to a truncated amber suppressor tRNACUA (-CA) using T4 RNA ligase generates an aminoacylated suppressor tRNA which can be used for site-specific incorporation of unnatural amino acids into proteins. Both the ligation and in vitro suppression efficiencies are the same when either pCpA or pdCpA is used. The use of deoxycytidine simplifies the chemistry involved in the synthesis of the dinucleotide pCpA. In addition, these results demonstrate that ribocytidine is not required for recognition of the aminoacylated tRNA during protein synthesis.     

Orthogonal ligation strategies for peptide and protein

This review focuses on the concept, criteria, and methods of an orthogonal amide ligating strategy suitable for syntheses of peptides, peptide mimetics, and proteins. Utilizing unprotected peptides or proteins derived from chemical or biosynthetic sources, this ligation strategy has been shown to be general and exceptionally mild. Its orthogonality in ligating two unprotected segments with free N-terminal (NT)-amines at a specific NT-amine is achieved through a chemoselective capture step and then an intramolecular acyl transfer reaction. Both coupling reagents for enthalpic activation and protection schemes therefore become unnecessary. More than a dozen orthogonal ligation methods based on either imine or thioester captures have been developed to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. Because unprotected peptides and proteins of different sizes and forms can be obtained from either chemical or recombinant sources, orthogonal ligation removes the size limitation imposed on the chemical synthesis of a protein with a native or non-native structure. Furthermore, by using building blocks from biosynthetic sources, orthogonal ligation provides a unifying operational concept for both total and semisynthesis of peptides and proteins.     

Native chemical ligation in dimethylformamide can be performed chemoselectively without racemization

Native chemical ligation of unprotected peptides in organic solvents has been previously reported as a fast, efficient, and suitable method for coupling of hydrophobic peptides. However, it has not been determined whether the reaction can be carried out without possible side reactions or racemization. Here, we present a study on the chemoselectivity of this method by model reactions designed to test the reactivity of Arg and Lys side chains as well as that of α‐amino groups. A possible racemization of the C‐terminal amino acid of the N‐terminal peptide was also investigated. The results show that ligation in organic solvents can be conducted chemoselectively without side reactions with other nucleophilic groups. Furthermore, no racemization of the C‐terminal amino acid was observed if both educts were added simultaneously. Thus, native chemical ligation can be performed either in aqueous buffer systems or in organic solvents paving the way for the synthesis of larger hydrophobic peptides and/or membrane proteins. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

A new scaffold for amide ligation.

Highly chemoselective amide forming ligation reactions have facilitated the synthetic access to proteins and other amide-linked bioconjugates. In order to generalize this approach, a N(alpha)-2-phenyl ethanethiol scaffold has been developed to promote S to N acyl transfer in a manner analogous to native chemical ligation with N-terminal cysteine residues. Analysis of scaffold-mediated ligation reactions in aqueous solution indicate that the ligation rate at Xaa-Gly junctions is sufficient for the synthesis of large polypeptides. In addition, it was found that the ligation rate is independent of the stereocenter in the scaffold and S- to N-acyl transfer is rate limiting. These studies indicate that the N(alpha)-2-phenyl ethanethiol scaffold is a good candidate for the development of a ligation chemistry for the formation of Xaa-Gly peptides and other unhindered amides.     

Chemical synthesis of circular proteins

Circular proteins, once thought to be rare, are now commonly found in plants. Their chemical synthesis, once thought to be difficult, is now readily achievable. The enabling methodology is largely due to the advances in entropic chemical ligation to overcome the entropy barrier in coupling the N- and C-terminal ends of large peptide segments for either intermolecular ligation or intramolecular ligation in end-to-end cyclization. Key elements of an entropic chemical ligation consist of a chemoselective capture step merging the N and C termini as a covalently linked O/S-ester intermediate to permit the subsequent step of an intramolecular O/S-N acyl shift to form an amide. Many ligation methods exploit the supernucleophilicity of a thiol side chain at the N terminus for the capture reaction, which makes cysteine-rich peptides ideal candidates for the entropy-driven macrocyclization. Advances in desulfurization and modification of the thiol-containing amino acids at the ligation sites to other amino acids add extra dimensions to the entropy-driven ligation methods. This minireview describes recent advances of entropy-driven ligation to prepare circular proteins with or without a cysteinyl side chain.