Facile chemoselective synthesis of dehydroalanine-containing peptides

Useful methodology is described for the synthesis of dehydroalanine residues (II) within peptides. The unnatural amino acid (Se)-phenylselenocysteine (I) can be incorporated into growing peptide chains via standard peptide synthesis procedures. Subsequent oxidative elimination affords a dehydroalanine at the desired position. The oxidation conditions are mild and tolerate functionalities commonly found in peptides, including variously protected cysteine residues. To illustrate its utility, cyclic lanthionines have been synthesized by this method.     

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.     

Water-driven chemoselective reaction of squarate derivatives with amino acids and peptides

Here, we report a new class of highly chemoselective reactions between squarate derivatives and the amino acid cysteine or unprotected peptides with a N-terminus cysteine that proceed most efficiently in entirely aqueous solution at neutral pH. Kinetic and structural studies reveal that the presence of hydrogen bonding in water is primarily responsible for both the high yield and fast rate of the reaction.     

Tandem ligation of multipartite peptides with cell-permeable activity

To prepare multipartite peptides with several functional cargoes including a cell-permeable sequence or transportant for intracellular delivery, tandem ligation of peptides is a convenient convergent approach with the fewest synthetic steps. It links three or four unprotected segments forming two or more regiospecific bonds consecutively without a deprotection step. This paper describes a tandem ligation strategy to prepare multipartite peptides with normal and branched architectures carrying a novel transportant peptide that is rich in arginine and proline to permit their cargoes to be translocated across membranes to affect their biological functions in cytoplasm. Our strategy consists of three ligation methods specific for amino terminal cysteine (Cys), serine/threonine (Ser/Thr), and N(alpha)-chloroacetylated amine to afford Xaa-Cys, Xaa-OPro (oxaproline) and Xaa-psiGly (pseudoglycine) at the ligation sites, respectively. Assembly of single-chain peptides from three different segments was achieved by the tandem Cys/OPro ligation to form two amide bonds, an Xaa-Cys and then an Xaa-OPro. Assembly of two- and three-chain peptides with branched architectures from four different segments was accomplished by tandem Cys/psiGly/OPro ligation. These NT-specific tandem ligation strategies were successful in generating cell-permeable multipartite peptides with one-, two-, and three-chain architectures, ranging in size from 52 to 75 residues and without the need of a protection or deprotection step. In addition, our results show that there is considerable flexibility in architectural design to obtain cell-permeable multipartite peptides containing a transportant sequence.     

Construction of diverse peptide structural architectures via chemoselective peptide ligation

Herein, we report the development of a facile synthetic strategy for constructing diverse peptide structural architectures via chemoselective peptide ligation. The key advancement involved is to utilize the benzofuran moiety as the peptide salicylaldehyde ester surrogate, and Dap–Ser/Lys–Ser dipeptide as the hydroxyl amino functionality, which could be successfully introduced at the side chain of peptides enabling peptide ligation. With this method, the side chain-to-side chain cyclic peptide, branched/bridged peptides, tailed cyclic peptides and multi-cyclic peptides have been designed and successfully synthesized with native peptidic linkages at the ligation sites. This strategy has provided an alternative strategic opportunity for synthetic peptide development. It also serves as an inspiration for the structural design of PPI inhibitors with new modalities.

Methods of introducing peptide salicylaldehyde esters and hydroxyl amine functionality into the peptide side chain have been developed. Diverse peptide structural motifs were constructed via ligation with native amide linkages at the ligation sites.     

Harnessing chemoselective imine ligation for tethering bioactive molecules to platinum(IV) prodrugs

Platinum(II) anticancer drugs are among the most effective and often used chemotherapeutic drugs. In recent years, there has been increasing interest in exploiting inert platinum(IV) scaffolds as a prodrug strategy to mitigate the limitations of platinum(II) anticancer complexes. In this prodrug strategy, the axial ligands are released concomitantly upon intracellular reduction to the active platinum(II) congener, offering the possibility of conjugating bioactive co-drugs which may synergistically enhance cytotoxicity on cancer cells. Existing techniques of tethering bioactive molecules to the axial positions of platinum(IV) prodrugs suffer from limited scope, poor yields and low reliability. This report explores the applications of current chemoselective ligation chemistries to platinum(IV) anticancer complexes with the aim of addressing the aforementioned limitations. Here, we describe the synthesis of a platinum(IV) complex bearing an aromatic aldehyde functionality and explored the scope of imine ligation with various hydrazide and aminooxy functionalized substrates. As a proof of concept, we tethered a six sequence long peptide mimetic (AMVSEF) of the anti-inflammatory protein, ANXA1.     

Staudinger-phosphonite reactions for the chemoselective transformation of azido-containing peptides and proteins

Site-specific functionalization of proteins by bioorthogonal modification offers a convenient pathway to create, modify, and study biologically active biopolymers. In this paper the Staudinger reaction of aryl-phosphonites for the chemoselective functionalization of azido-peptides and proteins was probed. Different water-soluble phosphonites with oligoethylene substituents were synthesized and reacted with unprotected azido-containing peptides in aqueous systems at room temperature in high conversions. Finally, the Staudinger-phosphonite reaction was successfully applied to the site-specific modification of the protein calmodulin.     

A "traceless" Staudinger ligation for the chemoselective synthesis of amide bonds

[reaction: see text] Here we report a novel modification of our previously reported "Staudinger ligation" that generates an amide bond from an azide and a specifically functionalized phosphine. This method for the selective formation of an amide bond, which does not require the orthogonal protection of distal functional groups, should find general utility in synthetic and biological chemistry.     

A new bioorthogonal cross-linker with alkyne and hydrazide end groups for chemoselective ligation. Application to antibody labelling

We describe here the synthesis of the first bioorthogonal cross-linking reagent based on aminocaproic acid core with a hydrazide function at one end to react with glycoproteins and an alkyne group at the other end for Cu(I)-catalyzed click chemistry to azide-derivatized probes. As an application, this cross-linker was used to orthogonally conjugate profluorescent 3-azido-7-hydroxycoumarin to immunoglobulin G (IgG). An immunoassay showed that IgG was mostly not affected by the Cu(I)-catalyzed click chemistry conditions. Successful conjugations and retained immunoreactivity demonstrate the potential of this new bioorthogonal cross-linker in chemoselective ligation.     

Diels-Alder ligation of peptides and proteins

The development of the Diels-Alder cycloaddition as a new method for the site-specific chemoselective ligation of peptides and proteins under mild conditions is reported. Peptides equipped with a 2,4-hexadienyl ester and an N-terminal maleimide react in aqueous media to give cycloadducts in high yields and depending on the amino acid sequence with high stereoselectivity. Except for the cysteine SH group the transformation is compatible with all amino acid side chain functional groups. For ligation to proteins the hexadienyl group was attached to avidin and streptavidin noncovalently by means of complex formation with a biotinylated peptide or by covalent attachment of a hexadienyl ester-containing label to lysine side chains incorporated into the proteins. Site-specific attachment of the hexadienyl unit into a Rab protein was achieved by means of expressed protein ligation followed by protection of the generated cysteine SH by means of Ellman's reagent. The protein reacted with different maleimido-modified peptides under mild conditions to give the fully functional cycloadducts in high yield. The results demonstrate that the Diels-Alder ligation offers an advantageous and technically straightforward new opportunity for the site-specific equipment of peptides and proteins with further functional groups and labels. It proceeds under very mild conditions and is compatible with most functional groups found in proteins. Its combination with other ligation methods, in particular expressed protein ligation is feasible.     

Dehydropeptides from orthogonal ligation of unprotected peptides

A facile method has been developed to synthesize linear and cyclic dehydropeptides from unprotected peptide precursors. This method exploits an N-terminal Cys for a Cys-thioester ligation to generate an unprotected peptide and as a precursor for conversion to DeltaAla by beta-elimination under mild conditions.     

Efficient conjugation of peptides to oligonucleotides by "native ligation"

A new strategy has been developed for conjugation of peptides to oligonucleotides. The method is based on the "native ligation" of an N-terminal thioester-functionalized peptide to a 5'-cysteinyl oligonucleotide. Two new reagents were synthesized for use in solid-phase peptide and oligonucleotide synthesis, respectively. Pentafluorophenyl S-benzylthiosuccinate was used in the final coupling step in standard Fmoc-based solid-phase peptide assembly. Deprotection with trifluoracetic acid generated in solution peptides substituted with an N-terminal S-benzylthiosuccinyl moiety. O-trans-4-(N-alpha-Fmoc-S-tert-butylsulfenyl-L-cysteinyl)aminoc yclohe xyl O-2-cyanoethyl-N,N-diisopropylphosphoramidite was used in the final coupling step in standard phosphoramidite solid-phase oligonucleotide assembly. Deprotection with aqueous ammonia solution generated in solution 5'-S-tert-butylsulfenyl-L-cysteinyl functionalized oligonucleotides. Functionalized peptides and oligonucleotides were used without purification in native ligation conjugation reactions in aqueous/organic solution using tris-(2-carboxyethyl)phosphine to remove the tert-butylsulfenyl group in situ and thiophenol as a conjugation enhancer. A range of peptide-oligonucleotide conjugates were prepared by this route and purified by reversed-phase HPLC.     

Postsynthetic modification of peptides via chemoselective N-alkylation of their side chains

A chemoselective, mild, and versatile method for performing postsynthetic modifications of peptide sequences is described. It requires only activated molecular sieves in the presence of an alkyl halide in order to N-alkylate lysine side chains. This reaction is fully compatible with most of the peptide functionalities, discriminates the reactivity of differently protected lysines, and proceeds in good yield. The mild conditions employed were further proved by performing the N-alkylation of a peptide containing a disulfide bridge.     

Staudinger ligation of peptides at non-glycyl residues

The Staudinger ligation provides a means to form an amide bond between a phosphinothioester and azide. This reaction holds promise for the ligation of peptides en route to the total chemical synthesis of proteins. (Diphenylphosphino)methanethiol is the most efficacious of known reagents for mediating the Staudinger ligation of peptides, providing high (> 90%) isolated yields for equimolar couplings in which a glycine residue is at the nascent junction. Surprisingly, the yields are lower (< 50%) for non-glycyl couplings due to an aza-Wittig reaction that diverts the reaction toward a phosphonamide byproduct. Here, the partitioning of the reaction toward Staudinger ligation (and away from the aza-Wittig reaction) is shown to increase with increasing electron density on phosphorus. This electron density can be tuned either by installing functional groups on the phenyl substituents of (diphenylphosphino)methanethiol or by changing the polarity of the solvent. Installing p-methoxy groups and using a solvent of low polarity (such as toluene or dioxane) provide especially high (> 80%) isolated yields for the ligation of two non-glycyl residues. These conditions retain the high chemoselectivity of the reaction and do not lead to a substantial change in reaction rate. The traceless Staudinger ligation is now poised to enable the iterative ligation of peptides with little regard for their sequence, as well as the synthesis of amide bonds for other purposes.     

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.     

Asymmetric synthesis of enantiopure isoxazolidinone monomers for the synthesis of β-oligopeptides by chemoselective amide ligation

The design and general synthesis of enantiopure isoxazolidinone monomers as precursors for the preparation of enantiopure N-terminal hydroxylamine-β³-oligopeptides, which may be used as reaction partners with α-ketoacids in the decarboxylative amide ligation reaction, is described.     

Spatially addressable chemoselective C-terminal ligation of an intein fusion protein from a complex mixture to a hydrazine-terminated surface

Protein immobilization on surfaces is useful in many areas of research, including biological characterization, antibody purification, and clinical diagnostics. A critical limitation in the development of protein microarrays and heterogeneous protein-based assays is the enormous amount of work and associated costs in the purification of proteins prior to their immobilization onto a surface. Methods to address this problem would simplify the development of interfacial diagnostics that use a protein as the recognition element. Herein, we describe an approach for the facile, site-specific immobilization of proteins on a surface without any preprocessing or sample purification steps that ligates an intein fusion protein at its C-terminus by reaction with a hydrazine group presented by a surface. Furthermore, we demonstrate that this methodology can directly immobilize a protein directly from cell lysate onto a protein-resistant surface. This methodology is also compatible with soft lithography and inkjet printing so that one or more proteins can be patterned on a surface without the need for purification.     

Aziridine-mediated ligation and site-specific modification of unprotected peptides

A synthesis of aziridine-containing peptides via the Cu(II)-promoted coupling of unprotected peptide thioacids and N-H aziridine-2-carbonyl peptides is reported. The unique reactivity of the resulting N-acylated aziridine-2-carbonyl peptides facilitates their subsequent regioselective and stereoselective nucleophilic ring-opening to give unprotected peptides that are specifically modified at the ligation site. The aziridine-mediated peptide ligation concept is exemplified using H(2)O as the nucleophile, producing a Xaa-Thr linkage (where Xaa can be an epimerizable and hindered amino acid). The overall process is compatible with a variety of unprotected amino acid functionality, most notably the N-terminal and Lys side chain amines.     

Alternating chemical ligation reactivity of S-acyl peptides explained with theory and computations

Previously discovered alternating reactivity of S-acyl di-, tri-, and tetrapeptide in internal chemical ligation reactions is rationalised using conformational search, virtual screening methods and quantum chemical calculations. Conformational preorganisation is shown to be the major controller of reactivity, with hydrogen bonding providing additional stabilisation for the tetrapeptide structure.