HPMA as a scaffold for the modular assembly of functional peptide polymers by native chemical ligation

Synthetic peptides are valuable tools in fundamental and applied biomedical research. On one hand, these molecules provide highly efficient access to competitive inhibitors of molecular interactions and enzyme substrates by rational design. On the other hand, peptides may serve as powerful vectors to mediate cellular uptake of molecules that otherwise enter cells only poorly. The coupling of both such functionalities provides access to molecules interfering with molecular processes inside the cell. However, the combination of several functionalities on one synthetic peptide may be compromised by problems associated with the synthesis of long peptides. Native chemical ligation enables the chemoselective coupling of fully deprotected functional building blocks. However, peptide thioesters are still not accessible by standard solid-phase peptide synthesis. Here, we demonstrate the cofunctionalization of a thioester-activated N-hydroxypropyl methacrylamide (HPMA) copolymer (28,500 Da) with the cell-penetrating peptide (CPP) nonaarginine and a bioactive peptide as independent building blocks by native chemical ligation. Nonaarginine was employed as a cell-penetrating peptide (CPP), a fluorescein-labeled analogue of a pro-apoptotic peptide as a biofunctional cargo. Incorporation of the fluorescein label enabled the highly sensitive quantification of the coupling stoichiometry by fluorescence correlation spectroscopy (FCS) using 0.4 pmol/12 ng of labeled construct. A construct only bearing the functional cargo peptide required cellular import by electroporation in order to show activity. In contrast, a construct combining all functionalities was active upon incubation of cells, validating the modular nature of the approach.     

Methods and strategies of peptide ligation.

This review focuses on the concept, methods, and strategies of orthogonal peptide ligation. It updates our previous review in 1999 on the same subject matter in Biopolymers (Peptide Science, 1999, Vol. 51, p. 311). Orthogonal peptide ligation is an amino terminal specific method to couple chemically unprotected peptides or proteins derived from synthetic or biosynthetic sources. Unlike conventional chemical methods, peptide ligation methods do not require coupling reagents or protection schemes, but are achieved through a variable chemoselective capture step and then an invariable intramolecular acyl transfer reaction. It is also a convergent method with the fewest steps. More than a dozen orthogonal ligation methods have been developed based on captures by either imine or thioester chemistries to afford native and unusual amino acids at ligation sites of linear, branched, or cyclic peptides. The ligation strategies for multiple segments including sequential and tandem ligations are also discussed.     

Tandem Peptide Ligation for Synthetic and Natural Biologicals

We describe the concept and methods of peptide ligation and tandem peptide ligation for preparing synthetic and natural biologicals. Peptide ligation is a segment coupling method for free peptides or proteins through an amide bond without the use of a coupling reagent or a protecting group scheme. Because unprotected peptides or proteins prepared from either a chemical or biochemical source are being used as building blocks, the ligation removes the size limitation for peptide and protein synthesis. A key feature of the peptide ligation is that the coupling reaction is orthogonal, i.e. it is specific to a particular alpha-amino terminus (NT). This NT-amino acid-specific feature permits the development of a tandem peptide ligation method employing three unprotected peptide segments containing different NT-amino acids to form consecutively two amide bonds, an Xaa-SPro (thiaproline) and then an Xaa-Cys. This strategy was tested in peptides ranging from 28 to 70 amino acid residues, including analogues of somatostatins and two CC-chemokines MIP-1alpha and MIP-1beta. The thiaproline replacements in these peptides and proteins did not result in altered biological activity. By eliminating the protecting group scheme and coupling reagents, tandem ligation of multiple free peptide segments in aqueous solutions enhances the scope of protein synthesis and may provide a useful approach for preparing protein biologicals and synthetic vaccines.     

An o-nitrobenzyl scaffold for peptide ligation: synthesis and applications

Chemical ligation approaches facilitate the chemoselective assembly of unprotected peptides in aqueous solution. Here, two photolabile auxiliaries are described that enlarge the applicability of native chemical ligation to non-cysteine targets. The auxiliaries, designed to allow reaction with thioester peptides, generate an amide bond between the two initial fragments. The o-nitrobenzyl tertiary benzylamide that is formed at the ligation junction can be transformed into a native amide group under mild photolysis conditions. The veratryl auxiliary was found to be excessively labile during peptide purification and ligation. However, the auxiliary based on the o-nitrobenzyl group shows all the necessary properties for a general application in routine peptide and protein synthesis. In addition, the auxiliary linked to the N-terminus can be efficiently photolyzed, suggesting a new approach for the generation of photocaged amines. Synthesis, solid phase introduction onto peptide chains, ligation properties and photolysis in water are described, and a careful study of compatibility of the method with potentially fragile peptide side chains is reported.     

Chemical ligation and cleavage on solid support facilitate recombinant peptide purification

Recombinant peptide technology offers a promising means alternative to chemical synthesis and natural extraction of peptides. The bottleneck in the process of recombinant peptide production is the paucity of efficient purification protocols to eliminate heterogeneity of the desired preparation. Here, we introduce a combination strategy to facilitate purification of recombinant therapeutic peptide via native chemical ligation and chemical cleavage on a solid support. In this study, one promising therapeutic peptide called for type-2 diabetes, GLP-1(7-37), was prepared with high yield and purity without an expensive HPLC purification. Furthermore, this method is also useful for the preparation of isotopically labeled NMR peptide samples. Hopefully, this strategy combining chemical ligation with chemical cleavage on a solid support will ameliorate the production of important recombinant pharmaceutical peptides.     

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.     

TectoRNP: self‐assembling RNAs with peptide recognition motifs as templates for chemical peptide ligation

TectoRNA, an artificial RNA with self‐assembling ability, has been employed as a structural platform for RNA nanotechnology and RNA synthetic biology. In this study, tectoRNA was applied as a specific template for chemical peptide ligation. On the basis of a self‐assembling tectoRNA, we designed and constructed a template RNA that facilitates peptide ligation depending on controlled dimer formation. Two RNA‐binding peptides were recognized by two peptide‐binding RNA motifs embedded in the template RNA, and chemical ligation was promoted because of the entropic effect of Mg²⁺‐dependent dimerization. In a series of biochemical analyses, we determined the relationship between the structures of the tectoRNA‐based templates and the extent of acceleration in peptide ligation. Copyright © 2012 European Peptide Society and John Wiley & Sons, Ltd.     

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.     

Solvent-induced beta-hairpin to helix conformational transition in a designed peptide

An octapeptide containing a central -Aib-Gly- segment capable of adopting beta-turn conformations compatible with both hairpin (beta(II') or beta(I')) and helical (beta(I)) structures has been designed. The effect of solvent on the conformation of the peptide Boc-Leu-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VIII; Boc: t-butyloxycarbonyl; OMe: methyl ester) has been investigated by NMR and CD spectroscopy. Peptide VIII adopts a well-defined beta-hairpin conformation in solvents capable of hydrogen bonding like (CD(3))(2)SO and CD(3)OH. In solvents that have a lower tendency to interact with backbone peptide groups, like CDCl(3) and CD(3)CN, helical conformations predominate. Nuclear Overhauser effects between the backbone protons and solvent shielding of NH groups involved in cross-strand hydrogen bonding, backbone chemical shifts, and vicinal coupling constants provide further support for the conformational assignments in different solvents. Truncated peptides Boc-Val-Val-Aib-Gly-Leu-Val-Val-OMe (VII), Boc-Val-Val-Aib-Gly-Leu-Val-OMe (VI), and Boc-Val-Aib-Gly-Leu-OMe (IV) were studied in CDCl(3) and (CD(3))(2)SO by 500 MHz (1)H-NMR spectroscopy. Peptides IV and VI show no evidence for hairpin conformation in both the solvents. The three truncated peptides show a well-defined helical conformation in CDCl(3). In (CD(3))(2)SO, peptide VII adopts a beta-hairpin conformation. The results establish that peptides may be designed, which are poised to undergo a dramatic conformational transition.     

Synthesis, isolation and characterization of Plasmodium falciparum antigenic tetrabranched peptide dendrimers obtained by thiazolidine linkages.

Different chemical alternatives were evaluated for obtaining immunogenic polypeptidic macromolecules which could then be used as vaccines. These were based on the ligation reaction between an unprotected immunogenic peptide and an unprotected multifunctional core peptide; polyantigens, designated dendrimers because their form resembles that of dendritic cells, were thus obtained. The antigen-core ligation alternatives, studied by indirect synthesis, were the formation of oxime, hydrazone and thiazolidine linkages, making use of the reaction between a weak base (acting as nucleophile) and an alkyl aldehyde. The other alternative was the formation of a thioether linkage between a sulfydryl and an alkyl halide. Finally, a multiple antigen peptide (MAP) was synthesized by direct synthesis. All reactions were monitored by SEC-HPLC and SDS-PAGE. Dendrimer molecular mass obtained was confirmed by MS MALDI-TOF. Dendrimer purification was first carried out by concentrating crude reaction products with CP-5000 centricons and (using SEC-HPLC) pure tetramers were then obtained. A 20-residue 9376 immunogenic sequence, from Plasmodium falciparum apical merozoite antigen protein (AMA-1), was used to study the best alternative for chemical ligation. It was observed that thiazolidine formation proceeded with greater yield and in less time than the others. A tetramer has been simultaneously synthesized via thiazolidine with the SPf-66 antimalarial vaccine 45-residue monomer, proving the technique's versatility. The 9376 peptide disulfide bound polymer and SPf-66 (as well as their tetrameric thiazolidine dendrimers) were inoculated in rabbits to evaluate their antibody response. It was observed that titers for tetrameric thiazolidine dendrimers were not just greater but were also sustained over time. Western blot for pre-immune and immune sera showed that dendrimer sera recognized specific Plasmodium falciparum proteins as well as disulfide-bound polymers.     

1H-NMR conformational analysis of a high-affinity antigenic 11-residue peptide from the tryptophan synthase beta 2 subunit.

Two synthetic peptides from the beta 2 subunit of tryptophan synthase have been studied by 1H-NMR spectroscopy at 300 MHz. One peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe-Gly-Met-Lys (peptide 11; Ile, isoleucine) is antigenic and binds with a high affinity to a monoclonal antibody that recognizes the native beta 2 subunit. The second peptide, His-Gly-Arg-Val-Gly-Ile-Tyr-Phe (peptide 8) reacts very weakly with the antibody. The 1H-NMR spectra of the two peptides have been assigned from two-dimensional techniques in H2O, 2H2O and (2H6) dimethyl sulfoxide [(2H6)Me2SO]. The structure has been evaluated through analysis of nuclear Overhauser effects, coupling constants, amide-proton exchange rates and their temperature coefficients, and chemical shifts. In aqueous solvent, the C-terminal part of peptide 11 presents some structure centered around residues Phe-Gly-Met. The relationship between the structure found in peptide 11 and its antigenic nature is discussed.     

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.     

Immobilization strategies for small molecule,peptide and protein microarrays

Protein, peptide and small molecule microarrays are valuable tools in biological research. In the last decade, substantial progress has been achieved to make these powerful technologies more reliable and available for researchers. This review describes chemical preparation methods for these microarrays with focus on site‐selective and bioorthogonal immobilization reactions, particularly the Staudinger ligation and the thiol‐ene reaction. In addition, the application of peptide microarrays, which were prepared by Staudinger ligation, to substrate specificity mapping is illustrated. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

Fmoc‐based peptide thioester synthesis with self‐purifying effect: heading to native chemical ligation in parallel formats

The chemical synthesis of proteins has facilitated functional studies of proteins due to the site‐specific incorporation of post‐translational modifications, labels, and non‐proteinogenic amino acids. Moreover, native chemical ligation provides facile access to proteins by chemical means. However, the application of the native chemical ligation reaction in the synthesis of parallel formats such as protein arrays has been complicated because of the often cumbersome and time‐consuming synthesis of the required peptide thioesters. An Fmoc‐based peptide thioester synthesis with self‐purification on the sulfonamide ‘safety‐catch’ linker widens this bottleneck because HPLC purification can be avoided. The method is based on an on‐resin cyclization–thiolysis reaction sequence. A macrocyclization via the N‐terminus of the full‐length peptide followed by a thiolytic C‐terminal ring opening allows selective detachment of the truncation products and the full‐length peptide. A brief overview of the chemical aspects of this method is provided including the optimization steps and the automation process. Furthermore, the application of the cyclization–thiolysis approach combined with the native chemical ligation reaction in the parallel synthesis of a library of 16 SH3‐domain variants of SHO1 in yeast is described, demonstrating the value of this new technique for the chemical synthesis of protein arrays. Copyright © 2013 European Peptide Society and John Wiley & Sons, Ltd.     

Carbopeptides: chemoselective ligation of peptide aldehydes to an aminooxy-functionalized D-galactose template.

Multifunctional, topological template molecules such as linear and cyclic peptides have been used for the attachment of peptide strands to form novel protein models of, for example, 4-alpha-helix bundles. The concept of carbohydrates as templates for de novo design of potential protein models has been previously described and these novel chimeric compounds were termed carbopeptides. Here, a second generation strategy in which carbopeptides are synthesized by chemoselective ligation of a peptide aldehyde to an aminooxy-functionalized alpha-D-galactopyranoside is described. This template was prepared by per-O-acylation of methyl alpha-D-galactopyranoside with N,N-Boc2-aminooxyacetic acid to form a tetra-functionalized template, followed by treatment with TFA-CH2Cl2 to release the aminooxy functionality. The peptide aldehydes Fmoc-Ser-Gly-Gly-H and H-Ala-Leu-Ala-Lys-Leu-Gly-Gly-H were synthesized by a BAL strategy. Four identical copies of peptide aldehyde were smoothly attached to the template by chemoselective ligation to form a 2.1 and a 2.9 kDa carbopeptide, respectively.     

A pH-tunable peptide ligase

A chemical ligation system is reported, in which a highly acidic coiled-coil peptide was used to template two basic peptide fragments and catalyze their condensation, in a pH-tunable fashion, to generate a coiled-coil product. This template showed a high catalytic efficiency (with single turnover) under neutral conditions. Under acidic conditions, however, its catalytic efficiency was reduced by approximately 4500-fold.     

Expanding the Scope of Native Chemical Ligation in Glycopeptide Synthesis

Glycoprotein is one of the important biopolymer in a biological system. In order to understand the complex correlation between the exact oligosaccharide structure of the glycoprotein and its function, preparation of homogeneous glycoprotein is to be essential. For such a purpose, chemical synthesis is one of the most promising methods to obtain homogeneous glycoproteins. Glycopolypeptide, which is a backbone of glycoprotein and an essential intermediate for glycoprotein synthesis, can be obtained through coupling of peptide and glycopeptide segments because straightforward synthesis of such a long glycopolypeptide is still a challenging task. Native chemical ligation (NCL) is one of the powerful methods for the coupling reaction of peptides, however, despite extensive investigation, NCL has site limitation for the coupling. In this context, we discovered NCL at serine site, where is a highly conserved amino acid residue in glycoproteins. This reaction strategy is owed to conversion reaction of cysteine residue to serine residue after conventional NCL. This conversion reaction is consisted of three steps; S-methylation of cysteine, CNBr reaction to afford O-ester linked peptide, and O to N acyl shift to get native peptide linkage with serine residue. During extensive investigation of the strategy, we found new reaction media for CNBr reaction, which is the key reaction in the strategy. This enabled us to synthesize not only N-linked glycopeptides but also O-linked sialyl glycopeptides. Thus we could demonstrate the usefulness of this new glycopeptide ligation strategy. In this short review, we will introduce our newly developed cysteine to serine conversion reaction which will expand the application of NCL in peptide as well as glycopeptide synthesis.     

Structural changes in the C-terminus of Ca2+-bound rat S100B (beta beta) upon binding to a peptide derived from the C-terminal regulatory domain of p53.

S100B(beta beta) is a dimeric Ca2+-binding protein that interacts with p53, inhibits its phosphorylation by protein kinase C (PKC) and promotes disassembly of the p53 tetramer. Likewise, a 22 residue peptide derived from the C-terminal regulatory domain of p53 has been shown to interact with S100B(beta beta) in a Ca2+-dependent manner and inhibits its phosphorylation by PKC. Hence, structural studies of Ca2+-loaded S100B(beta beta) bound to the p53 peptide were initiated to characterize this interaction. Analysis of nuclear Overhauser effect (NOE) correlations, amide proton exchange rates, 3J(NH-H alpha) coupling constants, and chemical shift index data show that, like apo- and Ca2+-bound S100B(beta beta), S100B remains a dimer in the p53 peptide complex, and each subunit has four helices (helix 1, Glu2-Arg20; helix 2, Lys29-Asn38; helix 3, Gln50-Asp61; helix 4, Phe70-Phe87), four loops (loop 1, Glu21-His25; loop 2, Glu39-Glu49; loop 3, Glu62-Gly66; loop 4, Phe88-Glu91), and two beta-strands (beta-strand 1, Lys26-Lys28; beta-strand 2, Glu67-Asp69), which forms a short antiparallel beta-sheet. However, in the presence of the p53 peptide helix 4 is longer by five residues than in apo- or Ca2+-bound S100B(beta beta). Furthermore, the amide proton exchange rates in helix 3 (K55, V56, E58, T59, L60, D61) are significantly slower than those of Ca2+-bound S100B(beta beta). Together, these observations plus intermolecular NOE correlations between the p53 peptide and S100B(beta beta) support the notion that the p53 peptide binds in a region of S100B(beta beta), which includes residues in helix 2, helix 3, loop 2, and the C-terminal loop, and that binding of the p53 peptide interacts with and induces the extension of helix 4.     

Chemoselectively addressable HCan building blocks in peptide synthesis: L-homocanaline derivatives

(S-2-amino-5-(aminooxy)pentanoic acid (L -homocanaline, HCan), a structural analogue of lysine, contains a reactive alkyloxyamine side chain and is therefore considered to react chemoselectively with carbonyl compounds by forming a kinetically stable oxime bond. The chemical synthesis of L -homocanaline starting from protected glutamic acid derivatives is described. Two orthogonally protected homocanaline derivatives were synthesized and their use in standard SPPS procedures was exemplified for the synthesis of a chemoselectively addressable cyclic peptide for use in TASP design. Moreover, the wide range of applications of this unique building block was demonstrated for the chemoselective ligation of an unprotected disaccharide to a HCan containing model peptide resulting in a chimeric glycopeptide structure. © 1998 European Peptide Society and John Wiley & Sons, Ltd.     

Native chemical ligation derived method for recombinant peptide/protein C-terminal amidation

C-terminal amidation is often a requisite structural feature for peptide hormone bio-activity. We report a chemical amidation method that converts peptide/protein thioesters into their corresponding C-terminal amides. The peptide/protein thioester is treated with 1-(2,4-dimethoxyphenyl)-2-mercaptoethyl auxiliary (1b) in a native chemical ligation (NCL) reaction to form an intermediate, which upon removal of the auxiliary with TFA, yields the peptide/protein amide. We have demonstrated the general utility of the approach by successfully converting several synthetic peptide thioesters to peptide amides with high conversion rates. Preliminary results of converting a recombinant peptide thioester to its amide form are also reported.