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.     

Western blot analysis of Src kinase assays using peptide substrates ligated to a carrier protein

We have applied intein-mediated peptide ligation (IPL) to the use of peptide substrates for kinase assays and subsequent Western blot analysis. IPL allows for the efficient ligation of a synthetic peptide with an N-terminal cysteine residue to an intein-generated carrier protein containing a cysteine reactive C-terminal thioester through a native peptide bond. A distinct advantage of this procedure is that each carrier protein molecule ligates only one peptide, ensuring that the ligation product forms a sharp band on sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). We demonstrate the effectiveness of this approach by mutational analysis of peptide substrates derived from human cyclin-dependent kinase, Cdc2, which contains a phosphorylation site of human c-Src protein tyrosine kinase.     

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.     

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.     

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.     

A NEW “NATIVE LIGATION” PROCEDURE FOR PEPTIDE-OLIGONUCLEOTIDE CONJUGATION

“Native ligation”, a powerful method of joining peptide fragments, has been applied successfully to peptide-oligonucleotide conjugation. Novel reagents are described for the solid-phase synthesis of peptide N-terminal thioesters and 5′-cysteinyl oligonucleotides suitable for ligation reactions.     

Studies of ion channels using expressed protein ligation

Expressed protein ligation (EPL) is a semisynthetic technique for the chemoselective ligation of a synthetic peptide to a recombinant peptide that results in a native peptide bond at the ligation site. EPL therefore allows us to engineer proteins with chemically defined, site-specific modifications. While EPL has been used mainly in investigations of soluble proteins, in recent years it has been increasingly used in investigations of integral membrane proteins. These include studies on the KcsA K(+) channel, the non-selective cation channel NaK, and the porin OmpF. These studies are discussed in this review.     

Solid phase synthesis of peptide-selenoesters

The synthesis of proteins by native chemical ligation greatly enhances the application of chemistry to complex molecules such as proteins. The essential building blocks for this approach traditionally have been peptide-thioester segments that are linked chemoselectively in consecutive reactions. By using peptide selenoesters instead of thioesters, the ligation rate can be significantly accelerated permitting couplings at difficult sites and potentially enabling new ligation strategies. To facilitate the routine synthesis of selenoester peptides, a general and straightforward procedure has been developed that generates a suitably functionalized resin from which the desired selenoester peptide can be readily synthesized. This simple approach utilizes readily available and cheap chemical agents and enables production of peptide selenoesters of excellent quality in short time and with high recovery. In addition, the stability of peptide selenoesters was examined under different native chemical ligation conditions and compared to thioesters. Selenoesters are slightly more reactive and more susceptible to hydrolysis and aminolysis than thioesters but sufficiently stable under mildly acidic conditions (pH 6.5). Under these conditions, rapid selenoester-mediated ligation is kinetically favoured.     

Autocrine IL-12 is involved in dendritic cell modulation via CD40 ligation.

Ligation of CD40 on dendritic cells (DC) triggers production of IL-12. Using an adoptive transfer model we have previously shown that rIL-12 acts directly on DC to enhance presentation of an otherwise poorly immunogenic tumor peptide. Using the same experimental model, we now describe a similar adjuvanticity of CD40 ligation on peptide presentation by DC. We also explore the possibility that the IL-12 resulting from CD40 ligation directly affects the APC function of DC, mediating or contributing to the adjuvant effect of CD40 ligation. CD40 engagement in vitro and rIL-12 at concentrations in the range induced by CD40 ligation were equally effective in priming DC for presentation of the tumor peptide in vivo. Remarkably, the copresence in vitro of neutralizing Ab to IL-12, but not to TNF-alpha, IL-1beta, or IFN-gamma, ablated the enhancing effect of CD40 engagement on the APC function of DC. These data suggest a major role for autocrine IL-12 in DC modulation via CD40 ligation.     

Peptide-Templated Nucleic Acid Ligation

Abstract Short oligonucleotide and peptide replicators have been described. To determine whether cross-replication could have occurred between such systems, we have attempted to show that peptides can specifically template the ligation of nucleic acids. A complex between a 35-mer anti-Rev RNA aptamer and a 17-mer arginine-rich motif (ARM) peptide from the HIV-1 Rev protein served as a model system. Aptamer half-molecules were activated for ligation via two activation chemistries, representing two distinct kinetic possibilities for early replicators. Cyanogen bromide activation was transient relative to oligonucleotides that terminated with a 5′-iodine and a 3′phosphorothioate, respectively. The Rev ARM specifically enhanced the degree or rate of ligation by both methods: there was a 10-fold increase in the production of full-length aptamer in the presence of cyanogen bromide and a 5.9- to 7.6-fold enhancement in the rate of ligation for stably activated aptamer half-molecules. These results support the possibility that life could have originated with peptide replicators and transitioned to nucleic acid replicators or that peptide and nucleic acid replicators could have been interdependent.     

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.     

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.     

Tandem ligation at X-Cys and Gly-Gly positions via an orthogonally protected auxiliary group

4,5-dimethoxy-2-mercaptobenzylamine (Dmmb) has been protected by acetamidomethyl (Acm) and incorporated into a peptide thioester for use in tandem native chemical ligation. Upon ligation between the thioester and a Cys-peptide, Acm was removed from Dmmb using silver acetate, and a second ligation reaction was done at the Dmmb position. Dmmb removal using TFMSA-TFA effected overall tandem ligation at X-Cys and Gly-Gly.     

Methionine ligation strategy in the biomimetic synthesis of parathyroid hormones

In biological systems, both proteolysis and aminolysis of amide bonds produce activated intermediates through acyl transfer reactions either inter- or intramolecularly. Protein splicing is an illustrative example that proceeds through a series of catalyzed acyl transfer reactions and culminates at an O- or S-acyl intermediate. This intermediate leads to an uncatalyzed acyl migration to form an amide bond in the spliced product. A ligation method mimicking the uncatalyzed final steps in protein splicing has been developed utilizing the acyl transfer amide-bond feature for the blockwise coupling of unprotected, free peptide segments at methionine (Met). The latent thiol moiety of Met can be exploited using homocysteine at the α-amino terminal position of a free peptide for transthioesterification with another free peptide containing an α-thioester to give an S-acyl intermediate. A subsequent, proximity-driven S- to N-acyl migration of this acyl intermediate spontaneously rearranges to form a homocysteinyl amide bond. S-methylation with excess p-nitrobenezensulfonate yields Met at the ligation site. The methionine ligation is selective and orthogonal, and is usually completed within 4 h when performed at slightly basic pH and under strongly reductive conditions. No side reactions due to acylation were observed with any other α-amines of both peptide segments as seen in the synthesis of parathyroid hormone peptides. Furthermore, cyclic peptide can also be obtained through the same strategy by placing both homocysteine at the amino terminus and the thioester at the carboxyl terminus in an unprotected peptide precursor. These biomimetic ligation strategies hold promise for engineering novel peptides and proteins. © 1998 John Wiley & Sons, Inc. Biopoly 46: 319–327, 1998     

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.     

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.     

Synthesis of proteins by native chemical ligation using Fmoc-based chemistry

C-terminal peptide alpha-thioesters are valuable intermediates in the synthesis/semisynthesis of proteins by native chemical ligation. They are prepared either by solid-phase peptide synthesis (SPPS) or biosynthetically by protein splicing techniques. The present paper reviews the different methods available for the chemical synthesis of peptide alpha-thioesters using Fmoc-based SPPS.     

Making Ends Meet: Microwave-Accelerated Synthesis of Cyclic and Disulfide Rich Proteins Via In Situ Thioesterification and Native Chemical Ligation

The development of synthetic methodologies for cyclic peptides is driven by the discovery of cyclic peptide drug scaffolds such as the plant-derived cyclotides, sunflower trypsin inhibitor 1 (SFTI-1) and the development of cyclized conotoxins. Currently, the native chemical ligation reaction between an N-terminal cysteine and C-terminal thioester group remains the most robust method to obtain a head-to-tail cyclized peptide. Peptidyl thioesters are effectively generated by Boc SPPS. However, their generation is challenging using Fmoc SPPS because thioester linkers are not stable to repeated piperidine exposure during deprotection. Herein we describe a Fmoc-based protocol for synthesizing cyclic peptides adapted for microwave assisted solid phase peptide synthesis. The protocol relies on the linker Di-Fmoc-3,4-diaminobenzoic acid, and we demonstrate the use of Gly, Ser, Arg and Ile as C-terminal amino acids (using HBTU and HATU as coupling reagents). Following synthesis, an N-acylurea moiety is generated at the C-terminal of the peptide; the resin bound acylurea peptide is then deprotected and cleaved from the resin. The fully deprotected peptide undergoes thiolysis in aqueous buffer, generating the thioester in situ. Ultimately, the head-to-tail cyclized peptide is obtained via native chemical ligation. Two naturally occurring cyclic peptides, the prototypical Möbius cyclotide kalata B1 and SFTI-1 were synthesized efficiently, avoiding potential branching at the diamino linker, using the optimized protocol. In addition, we demonstrate the possibility to use the approach for the synthesis of long and synthetically challenging linear sequences, by the ligation of two truncated fragments of a 50-residue long plant defensin.     

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.     

Expressed protein ligation. Method and applications.

The introduction of noncanonical amino acids and biophysical probes into peptides and proteins, and total or segmental isotopic labelling has the potential to greatly aid the determination of protein structure, function and protein-protein interactions. To obtain a peptide as large as possible by solid-phase peptide synthesis, native chemical ligation was introduced to enable synthesis of proteins of up to 120 amino acids in length. After the discovery of inteins, with their self-splicing properties and their application in protein synthesis, the semisynthetic methodology, expressed protein ligation, was developed to circumvent size limitation problems. Today, diverse expression vectors are available that allow the production of N- and C-terminal fragments that are needed for ligation to produce large amounts and high purity protein(s) (protein alpha-thioesters and peptides or proteins with N-terminal Cys). Unfortunately, expressed protein ligation is still limited mainly by the requirement of a Cys residue. Of course, additional Cys residues can be introduced into the sequence by site directed mutagenesis or synthesis, however, those mutations may disturb protein structure and function. Recently, alternative ligation approaches have been developed that do not require Cys residues. Accordingly, it is theoretically possible to obtain each modified protein using ligation strategies.