Template assembled synthetic proteins: Condensation of a multifunctional peptide to a topological template via chemoselective ligation

A chemoselective ligation via oxime bond formation is used for the chemical synthesis of template assembled peptides according to the TASP (Template Assembled Synthetic Proteins) approach. Aminooxyacetylation of the multifunctional partial sequence Lys- Arg- Asp- Ser of lactoferrin and subsequent condensation in aqueous solution with a topological template containing four selectively addressable aldehyde functions as attachment sites gives readily access to the TASP molecule.     

Multivalent Design of Apoptosis‐Inducing Bid‐BH3 Peptide–Oligosaccharides Boosts the Intracellular Activity at Identical Overall Peptide Concentrations

Multivalent peptide–oligosaccharide conjugates were prepared and used to investigate the multivalency effect concerning the activity of Bid‐BH3 peptides in live cells. Dextran oligosaccharides were carboxyethylated selectively in the 2‐position of the carbohydrate units and activated for the ligation of N‐terminally cysteinylated peptides. Ligation through maleimide coupling was found to be superior to the native chemical ligation protocol. Monomeric Bid‐BH3 peptides were virtually inactive, whereas pentameric peptide conjugates induced apoptosis up to 20‐fold stronger at identical peptide concentrations. Comparison of lowly multivalent and highly multivalent peptide dextrans proved a multivalency effect in life cells which was specific for the BH3 peptide sequence.     

A cassette ligation strategy with thioether replacement of three Gly-Gly peptide bonds: total chemical synthesis of the 101 residue protein early pregnancy factor [psi(CH(2)S)28-29,56-57,76-77

The 101 residue protein "early pregnancy factor" (EPF), also known as human chaperonin 10, was synthesized from four functionalized, but unprotected, peptide segments by a sequential thioether ligation strategy. The approach exploits the differential reactivity of a peptide-NHCH(2)CH(2)SH thiolate with XCH(2)CO-peptides, where X = Cl or I/Br. Initial model studies with short functionalized (but unprotected) peptides showed a significantly faster reaction of a peptide-NHCH(2)CH(2)SH thiolate with a BrCH(2)CO-peptide than with a ClCH(2)CO-peptide, where thiolate displacement of the halide leads to chemoselective formation of a thioether surrogate for the Gly-Gly peptide bond. This rate difference was used as the basis of a novel sequential ligation approach to the synthesis of large polypeptide chains. Thus, ligation of a model bifunctional N(alpha)-chloroacetyl, C-terminal thiolated peptide with a second N(alpha)-bromoacetyl peptide demonstrated chemoselective bromide displacement by the thiol group. Further investigations showed that the relatively unreactive N(alpha)-chloroacetyl peptides could be "activated" by halide exchange using saturated KI solutions to yield the highly reactive N(alpha)-iodoacetyl peptides. These findings were used to formulate a sequential thioether ligation strategy for the synthesis of EPF, a 101 amino acid protein containing three Gly-Gly sites approximately equidistantly spaced within the peptide chain. Four peptide segments or "cassettes" comprising the EPF protein sequence (BrAc-[EPF 78-101] 12, ClAc-[EPF 58-75]-[NHCH(2)CH(2)SH] 13, ClAc-[EPF 30-55]-[NHCH(2)CH(2)SH] 14, and Ac-[EPF 1-27]-[NHCH(2)CH(2)SH] 15) of EPF were synthesized in high yield and purity using Boc SPPS chemistry. In the stepwise sequential ligation strategy, reaction of peptides 12 and 13 was followed by conversion of the N-terminal chloroacetyl functional group to an iodoacetyl, thus activating the product peptide for further ligation with peptide 14. The process of ligation followed by iodoacetyl activation was repeated to yield an analogue of EPF (EPF psi(CH(2)S)(28)(-)(29,56)(-)(57,76)(-)(77)) 19 in 19% overall yield.     

Design and synthesis of alpha Gal-conjugated peptide T20 as novel antiviral agent for HIV-immunotargeting

An efficient chemo-enzymatic synthesis of alpha Gal-conjugated peptide T20 as novel HIV-immuno-targeting agent is described. The synthesis involves chemo-enzymatic preparation of maleimide-functionalized alpha Gal epitope and its chemoselective ligation with the peptide T20. The title compound contains two functional domains: the trisaccharide alpha Gal epitope that binds to human natural anti-Gal antibodies and the 36-amino acid gp41 peptide (T20) that recognizes the gp41 N-terminal ectodomain of the HIV envelope. Biological assays demonstrated that the synthetic conjugate could readily bind to natural anti-Gal antibodies (both IgG and IgM type) in normal human serum and exhibited potent anti-HIV activity even in the absence of human antibodies and complement system. The experimental data suggest that the synthetic alpha Gal-T20 might be valuable for in vivo HIV-immuno-targeting via antibody-mediated cytotoxicity and/or antibody-dependent, complement-mediated lysis of HIV particles and HIV-infected cells, thus providing an additional dimension of HIV intervention.     

Development of highly pure α-helical lipoglycopeptides as self-adjuvanting vaccines

The incorporation of lipid moieties into synthetic peptide vaccines has been demonstrated to self-adjuvant otherwise poorly immunogenic peptides, whereas carbohydrates have emerged to be advantageous carriers for assembling these peptides. With the advent of an efficient native chemical ligation method, which is compatible with both peptides and carbohydrates, we have developed highly pure self-adjuvanting tetravalent group A streptococcal vaccine candidates assembled on carbohydrate templates. The utility of chemoselective ligation has overcome difficulties in the synthesis and purification of branched high molecular weight peptides. Circular dichroism measurements provided the evidence of α-helix formation of the assembled peptide epitopes, which may have impact on their immunogenicity.     

Singlet Oxygen‐Induced Furan Oxidation for Site‐Specific and Chemoselective Peptide Ligation

A novel chemoselective ligation methodology has been developed for the facile construction of peptide‐based fluorescent probes. Furan‐containing peptides were activated by singlet oxygen and covalently engaged by nitrogen nucleophiles to yield stable conjugates. Singlet oxygen was compatible with sensitive amino acid residues within the peptides and a range of fluorophores, bearing different functionalities, were successfully incorporated, illustrating the broad scope of the developed strategy.     

Single-nucleotide-specific PNA-peptide ligation on synthetic and PCR DNA templates

DNA-directed chemical synthesis has matured into a useful tool with applications such as fabrication of defined (nano)molecular architectures, evolution of amplifiable small-molecule libraries, and nucleic acid detection. Most commonly, chemical methods were used to join oligonucleotides under the control of a DNA or RNA template. The full potential of chemical ligation reactions can be uncovered when nonnatural oligonucleotide analogues that can provide new opportunities such as increased stability, DNA affinity, hybridization selectivity, and/or ease and accuracy of detection are employed. It is shown that peptide nucleic acid (PNA) conjugates, nonionic biostable DNA analogues, allowed the fashioning of highly chemoselective and sequence-selective peptide ligation methods. In particular, PNA-mediated native chemical ligations proceed with sequence selectivities and ligation rates that reach those of ligase-catalyzed oligodeoxynucleotide reactions. Usually, sequence-specific ligations can only be achieved by employing short-length probes, which show DNA affinities that are too low to allow stable binding to target segments in large, double-stranded DNA. It is demonstrated that the PNA-based ligation chemistry allowed the development of a homogeneous system in which rapid single-base mutation analyses can be performed even on double-stranded PCR DNA templates.     

New isocysteine building blocks and chemoselective peptide ligation

Boc-, Fmoc- and Cbz-protected isocysteine building blocks were prepared by a concise three-step procedure starting from thiomalic acid. The use of Boc/Trt-protected isocysteine provided convenient access to isocysteinyl peptides that allow the chemoselective ligation of unprotected peptide fragments in water. The pH-dependency of the isocysteine-mediated ligation was compared with that of cysteine-mediated native chemical ligation.     

Template assembled cyclopeptides as multimeric system for integrin targeting and endocytosis

The alpha(V)beta(3) integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. c[-RGDfV-] peptide represents a selective alpha(V)beta(3) integrin ligand that has been extensively used for research, therapy, and diagnosis of neoangiogenesis. We report here the modular synthesis and biological characterization of template assembled cyclopeptides as a multimeric system for targeting and endocytosis of cells expressing alpha(V)beta(3) integrin. c[-RGDfK-] was cleanly assembled in a multivalent mode by chemoselective oxime bond formation to a cyclodecapeptides template labeled by different reporter groups. Binding propensity to the alpha(V)beta(3) receptor and the associated good uptake property displayed by the multivalent molecules demonstrated the interest in the RAFT molecule to design new multimeric system with hitherto unreported properties. These compounds offer an interesting perspective for the reevaluation of integrins as angiogenesis regulators (Hynes, R. O. Nature Med. 2003, 9, 918-921) as well as for the design of more sophisticated systems such as molecular conjugate vectors.     

Design and synthesis of a bimodal target-specific contrast agent for angiogenesis

[structure: see text] A bimodal target-specific contrast agent based on a cyclic peptide containing the target-specific NGR sequence, gadolinium(III) diethylenetriaminepentaacetic acid (Gd(III)DTPA), and Oregon Green 488 (OG488) suitable for both MR imaging and optical imaging of angiogenesis is developed. The synthetic strategy for this target-specific contrast agent exploits the use of highly efficient, chemoselective reactions, such as native chemical ligation, and gives a straightforward approach for double labeling of peptides in general.     

Dynamic Expression of DNA Complexation with Self‐assembled Biomolecular Clusters

We report herein the implementation of a dynamic covalent chemistry approach to the generation of multivalent clusters for DNA recognition. We show that biomolecular clusters can be expressed in situ by a programmed self‐assembly process using chemoselective ligations. The cationic clusters are shown, by fluorescence displacement assay, gel electrophoresis and isothermal titration calorimetry, to effectively complex DNA through multivalent interactions. The reversibility of the ligation was exploited to demonstrate that template effects occur, whereby DNA imposes component selection in order to favor the most active DNA‐binding clusters. Furthermore, we show that a chemical effector can be used to trigger DNA release through component exchange reactions.     

Peptide recognition via hierarchical imprinting

Silica particles containing immobilised peptidic templates have been used for the generation of hierarchically imprinted polymers. The pores of the silica mould were filled with a mixture of monomers/initiator and polymerised, followed by dissolution of the silica template. This method leaves behind imprinted polymers with binding sites located at the surface, which are capable of recognising larger molecules with the same immobilised epitope. All the products resulting from solid-phase synthesis of peptides were characterised by elemental analysis, FT-IR spectroscopy and fluorescence microscopy. The hierarchically imprinted polymers generated from these products were characterised by elemental analysis, FT-IR spectroscopy, fluorescence microscopy, scanning electron microscopy (SEM) and nitrogen adsorption, providing evidence concerning the reproducibility of each step. The chromatographic properties of the materials have been investigated and the advantages of the immobilisation method have been proven. The materials exhibit selectivity for their templates and other structurally related dipeptides. Furthermore, the polymers proved to be capable of recognising larger peptides containing the immobilised sequence.     

Peptide/protein-polymer conjugates: synthetic strategies and design concepts

This feature article provides a compilation of tools available for preparing well-defined peptide/protein-polymer conjugates, which are defined as hybrid constructs combining (i) a defined number of peptide/protein segments with uniform chain lengths and defined monomer sequences (primary structure) with (ii) a defined number of synthetic polymer chains. The first section describes methods for post-translational, or direct, introduction of chemoselective handles onto natural or synthetic peptides/proteins. Addressed topics include the residue- and/or site-specific modification of peptides/proteins at Arg, Asp, Cys, Gln, Glu, Gly, His, Lys, Met, Phe, Ser, Thr, Trp, Tyr and Val residues and methods for producing peptides/proteins containing non-canonical amino acids by peptide synthesis and protein engineering. In the second section, methods for introducing chemoselective groups onto the side-chain or chain-end of synthetic polymers produced by radical, anionic, cationic, metathesis and ring-opening polymerization are described. The final section discusses convergent and divergent strategies for covalently assembling polymers and peptides/proteins. An overview of the use of chemoselective reactions such as Heck, Sonogashira and Suzuki coupling, Diels-Alder cycloaddition, Click chemistry, Staudinger ligation, Michael's addition, reductive alkylation and oxime/hydrazone chemistry for the convergent synthesis of peptide/protein-polymer conjugates is given. Divergent approaches for preparing peptide/protein-polymer conjugates which are discussed include peptide synthesis from synthetic polymer supports, polymerization from peptide/protein macroinitiators or chain transfer agents and the polymerization of peptide side-chain monomers.     

Extending synthetic access to proteins with a removable acyl transfer auxiliary

A chemo- and regioselective auxiliary-mediated peptide ligation has been developed that is effective under nonidealized conditions for the synthesis of proteins. This general amide bond ligation utilizes a removable auxiliary that is analogous to the role of cysteine in native chemical ligation, combining chemoselective thioester exchange with efficient regioselective intramolecular acyl transfer. Acid lability and improved ligation efficiency were introduced into the 2-mercaptobenzyl auxiliary by increasing the electron density of the aromatic ring. The 62 amino acid SH3 domain from alpha-spectrin was synthesized using the auxiliary-mediated ligation at a Lys-Gly sequence. The auxiliary was removed with TFA and scavengers from the ligated product. This methodology enables unprotected peptides to be coupled at noncysteine ligation sites expanding the scope of protein synthesis and semisynthesis.     

Multimeric cyclic RGD peptides as potential tools for tumor targeting: solid-phase peptide synthesis and chemoselective oxime ligation

The alpha v beta 3 integrin receptor plays an important role in human metastasis and tumor-induced angiogenesis. Targeting this receptor may provide information about the receptor status of the tumor and enable specific therapeutic planning. Solid-phase peptide synthesis of multimeric cyclo(-RGDfE-)-peptides is described, which offer the possibility of enhanced integrin targeting due to polyvalency effects. These peptides contain an aminooxy group for versatile chemoselective oxime ligation. Conjugation with para-trimethylstannylbenzaldehyde results in a precursor for radioiododestannylation, which would allow them to be used as potential tools for targeting and imaging alpha v beta 3-expressing tumor cells. The conjugates were obtained in good yield without the need of a protection strategy and under mild conditions.     

Chemoselective Ligation and Modification Strategies for Peptides and Proteins

The investigation of biological processes by chemical methods, commonly referred to as chemical biology, often requires chemical access to biologically relevant macromolecules such as peptides and proteins. Building upon solid‐phase peptide synthesis, investigations have focused on the development of chemoselective ligation and modification strategies to link synthetic peptides or other functional units to larger synthetic and biologically relevant macromolecules. This Review summarizes recent developments in the field of chemoselective ligation and modification strategies and illustrates their application, with examples ranging from the total synthesis of proteins to the semisynthesis of naturally modified proteins.     

Identification of by-products from an orthogonal peptide ligation by oxime bonds using mass spectrometry and tandem mass spectrometry

Synthetic proteins with unusual architecture are obtained through chemoselective ligation, a method based on the condensation of unprotected peptides under mild aqueous conditions. The last step of a new procedure leading to a tri-branched conjugate consists of the chemoselective ligation reaction between an (aminooxy)acetyl peptide and a peptide aldehyde resulting from a first ligation via an oxime bond. In order to optimize the reaction conditions, electrospray ionization mass spectrometry combined with liquid chromatography and tandem mass spectrometry has been used. In addition to the target tri-branched conjugate, two other conjugates were characterized allowing documentation of transoximation reactions in peptide chemistry. A fourth conjugate was identified as a side product appearing after the first ligation. Data obtained by low-energy collision-induced dissociation led to a rapid and reliable identification of impurities observed in the (aminooxy)acetyl peptide despite a previous high performance liquid chromatography (HPLC) purification. This highlights the great reactivity of the aminooxy group towards carbonyl-containing compounds.     

Peptide and glycopeptide dendrimer apple trees as enzyme models and for biomedical applications

Solid phase peptide synthesis (SPPS) provides peptides with a dendritic topology when diamino acids are introduced in the sequences. Peptide dendrimers with one to three amino acids between branches can be prepared with up to 38 amino acids (MW ~ 5,000 Da). Larger peptide dendrimers (MW ~ 30,000) were obtained by a multivalent chloroacetyl cysteine (ClAc) ligation. Structural studies of peptide dendrimers by CD, FT-IR, NMR and molecular dynamics reveal molten globule states containing up to 50% of α-helix. Esterase and aldolase peptide dendrimers displaying dendritic effects and enzyme kinetics (k(cat)/k(uncat) ~ 10(5)) were designed or discovered by screening large combinatorial libraries. Strong ligands for Pseudomonas aeruginosa lectins LecA and LecB able to inhibit biofilm formation were obtained with glycopeptide dendrimers. Efficient ligands for cobalamin, cytotoxic colchicine conjugates and antimicrobial peptide dendrimers were also developed showing the versatility of dendritic peptides. Complementing the multivalency, the amino acid composition of the dendrimers strongly influenced the catalytic or biological activity obtained demonstrating the importance of the "apple tree" configuration for protein-like function in peptide dendrimers.     

Glycoconjugate and oligosaccharide mimetics by chemoselective ligation

Different approaches for the synthesis of neoglycoconjugates, exploiting chemoselective ligation procedures, are reported. Based on the reaction of the carbonyl group of ketones or aldehydes (including the anomeric centre of the sugar) with strong nucleophiles such as hydroxylamino- or hydrazino-derivatives, or on the addition of sulfidryl groups to a variety of electrophiles, these procedures afford bioactive neo-glycoconjugates avoiding protection–deprotection and activation steps. Conjugation of two copies of the C-saccharide analogue of the T_n epitope with the immunogenic peptide OVA^(327–339) by way of chemoselective ligation, afforded a neo-glycopeptide capable of inducing in vivo B- and T-cell response to tumours exposing T_n epitope. To cite this article: F. Peri et al., C. R. Chimie 6 (2003).     

A Tripeptide Approach to the Solid-Phase Synthesis of Peptide Thioacids and N-Glycopeptides

A general and robust method for the incorporation of aspartates with a thioacid side chain into peptides has been developed. Pseudoproline tripeptides served as building blocks for the efficient fluorenylmethyloxycarbonyl (Fmoc) solid-phase synthesis of thioacid-containing peptides. These peptides were readily converted to complex N-glycopeptides by using a fast and chemoselective one-pot deprotection/ligation procedure. Furthermore, a novel side reaction that can lead to site-selective peptide cleavage using thioacids (CUT) was discovered and studied in detail.