Peptide Ligation Research Articles

The use of tyrosinases in a chemoenzymatic cascade as a peptide ligation strategy.

Peptides play many key roles in biological systems and numerous methods have been developed to generate both natural and unnatural peptides. However, straightforward, reliable coupling methods that can be achieved under mild reactions conditions are still sought after. In this work, a new N-terminal tyrosine-containing peptide ligation method with aldehydes, utilising a Pictet-Spengler reaction is described. In a key step, tyrosinase enzymes have been used to convert l-tyrosine to l-3,4-dihydroxyphenyl alanine (l-DOPA) residues, generating suitable functionality for the Pictet-Spengler coupling. This new chemoenzymatic coupling strategy can be used for fluorescent-tagging and peptide ligation purposes.

Convergent synthesis of proteins using peptide-aminothiazoline.

Sequential peptide coupling plays a central role in chemical protein synthesis. This paper describes a new peptide derivative, peptide-aminothiazoline (At), whereof the C-terminus is functionalized with 2-aminothiazoline. Peptide-At streamlined the sequential peptide ligation in a one-pot manner and demonstrated the convergent synthesis of a circular protein and homogeneous glycoproteins.

Total synthesis of interleukin-2 via a tunable backbone modification strategy.

Chemical synthesis of hydrophobic proteins presents a formidable task as they are often difficultly achieved via peptide synthesis, purification, and peptide ligation. Thus, peptide solubilizing strategies are needed to integrate with peptide ligation to achieve protein total synthesis. Herein, we report a tunable backbone modification strategy, taking advantage of the tunable stability of the Cys/Pen ligation intermediate, which allows for readily introducing a solubilizing tag for both peptide purification and ligation processes. The effectiveness of this strategy was demonstrated by the chemical synthesis of interleukin-2.

Modular Synthesis of Unnatural Peptides via Rh(III)-Catalyzed Diastereoselective Three-Component Carboamidation Reaction.

Herein we report a modular peptide ligation methodology that couples dioxazolones, arylboronic acids, and acrylamides to construct amide bonds in a diastereoselective manner under mild conditions, facilitated by Rh(III) catalysis. By converting the C-terminus of one peptide into a dioxazolone and the N-terminus of a second peptide into an acrylamide, the two pieces can be bridged by an arylboronic acid to construct unnatural phenylalanine, tyrosine, and tryptophan residues at the junction point with diastereoselectivity for their corresponding d-stereocenters. The reaction exhibits excellent functional group tolerance with a large substrate scope and is compatible with a wide array of protected amino acid residues that are utilized in Fmoc solid phase peptide synthesis. The methodology is applied to the synthesis of six diastereomeric proteasome inhibitor analogs, as well as the ligation of two 10-mer oligopeptides to construct a 21-mer polypeptide with an unnatural phenylalanine residue at the center.

Cyclic Peptide Screening Methods for Preclinical Drug Discovery.

Cyclic peptides are among the most diverse architectures for current drug discovery efforts. Their size, stability, and ease of synthesis provide attractive scaffolds to engage and modulate some of the most challenging targets, including protein-protein interactions and those considered to be "undruggable". With a variety of sophisticated screening technologies to produce libraries of cyclic peptides, including phage display, mRNA display, split intein circular ligation of peptides, and in silico screening, a new era of cyclic peptide drug discovery is at the forefront of modern medicine. In this perspective, we begin by discussing cyclic peptides approved for clinical use in the past two decades. Particular focus is placed around synthetic chemistries to generate de novo libraries of cyclic peptides and novel methods to screen them. The perspective culminates with future prospects for generating cyclic peptides as viable therapeutic options and discusses the advantages and disadvantages currently being faced with bringing them to market.

Repetitive Thiazolidine Deprotection Using a Thioester‐Compatible Aldehyde Scavenger for One‐Pot Multiple Peptide Ligation**

Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N′,N′-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

Repetitive Thiazolidine Deprotection Using a Thioester-Compatible Aldehyde Scavenger for One-Pot Multiple Peptide Ligation.

Strategies for one-pot peptide ligation enable chemists to access synthetic proteins at a high yield in a short time. Herein, we report a novel one-pot multi-segments ligation strategy using N-terminal thiazolidine (Thz) peptide and a newly designed formaldehyde scavenger. Among the designed 2-aminobenzamide-based aldehyde scavengers, 2-amino-5-methoxy-N',N'-dimethylbenzohydrazide (AMDBH) can remarkably convert Thz into unprotected cysteine at pH 4.0. Furthermore, AMDBH degrades Thz at a considerably low rate at pH 7.5, and thioester degradation caused by this scavenger is negligible. As a result, we have developed an efficient one-pot peptide ligation strategy by simply repetitively changing the pH with AMDBH. Finally, we synthesize mono-ubiquitinated histone H2A.Z (209 amino acids) via AMDBH-mediated one-pot four-segment peptide ligation in good yield.

Rhodium-Catalyzed Ligation of Peptides with Olefins

Rhodium-Catalyzed Ligation of Peptides with Olefins

Superfast desulfurization for protein chemical synthesis and modification

Superfast desulfurization for protein chemical synthesis and modification

Ligation Embedding Aggregation Disruptor Strategy Enables the Chemical Synthesis of PD-1 Immunoglobulin and Extracellular Domains.

Chemical synthesis of proteins with aggregable or colloidal peptide segments presents a formidable task, as such peptides prove to be difficult for both solid-phase peptide synthesis and peptide ligation. To address this issue, we have developed ligation embedding aggregation disruptor (LEAD) as an effective strategy for the chemical synthesis of difficult-to-obtain proteins. The N,O/S-benzylidene acetals generated from Ser/Thr ligation and Cys/Pen ligation are found to effectively disrupt peptide aggregation, and they can be carried for sequential ligations toward protein synthesis. The effectiveness and generality of this strategy have been demonstrated with total syntheses of programmed cell death protein 1 immunoglobulin like V-type domain and extracellular domain.

Versatile Synthesis of Multivalent Porphyrin-Peptide Conjugates by Direct Porphyrin Construction on Resin.

Multifunctional porphyrin–peptide conjugates with different propensities for self‐assembly into various supramolecular nanoarchitectures play important roles in advanced materials and biomedical research. However, preparing prefunctionalized core porphyrins by traditional low‐yielding statistical synthesis and purifying them after peptide ligation through many rounds of HPLC purification is tedious and unsustainable. Herein, we report a novel integrated solid‐phase synthetic protocol for the construction of porphyrin moieties from simple aldehydes and dipyrromethanes on resin‐bound peptides directly to form mono‐, cis/trans‐di‐, and trivalent porphyrin–peptide conjugates in a highly efficient and controllable manner; moreover, only single final‐stage HPLC purification of the products is needed. This efficient strategy enables the rapid, greener, and substrate‐controlled diversity‐oriented synthesis of multivalent porphyrin–(long) peptide conjugate libraries for multifarious biological and materials applications.

Versatile Synthesis of Multivalent Porphyrin–Peptide Conjugates by Direct Porphyrin Construction on Resin

AbstractMultifunctional porphyrin–peptide conjugates with different propensities for self‐assembly into various supramolecular nanoarchitectures play important roles in advanced materials and biomedical research. However, preparing prefunctionalized core porphyrins by traditional low‐yielding statistical synthesis and purifying them after peptide ligation through many rounds of HPLC purification is tedious and unsustainable. Herein, we report a novel integrated solid‐phase synthetic protocol for the construction of porphyrin moieties from simple aldehydes and dipyrromethanes on resin‐bound peptides directly to form mono‐, cis/trans‐di‐, and trivalent porphyrin–peptide conjugates in a highly efficient and controllable manner; moreover, only single final‐stage HPLC purification of the products is needed. This efficient strategy enables the rapid, greener, and substrate‐controlled diversity‐oriented synthesis of multivalent porphyrin–(long) peptide conjugate libraries for multifarious biological and materials applications.

PIII‐Directed Late‐Stage Ligation and Macrocyclization of Peptides with Olefins by Rhodium Catalysis

AbstractTransition metal‐catalyzed C−H activation is a step‐economical strategy for peptide functionalization. Herein, we report the method of late‐stage peptide ligation and macrocyclization through rhodium‐catalyzed alkylation of tryptophan residues at the C7 position. This method utilizes a N‐PtBu2 directing group and tolerates various peptide and alkene substrates. Utilizing internal olefins, this study represents the first example of site‐selective peptide C−H alkylation through deconjugative isomerization. Furthermore, our method provides access to peptide macrocycles with unique Trp(C7)‐alkyl crosslinks and potent cytotoxicity towards cancer cells.

PIII -Directed Late-Stage Ligation and Macrocyclization of Peptides with Olefins by Rhodium Catalysis.

Transition metal-catalyzed C-H activation is a step-economical strategy for peptide functionalization. Herein, we report the method of late-stage peptide ligation and macrocyclization through rhodium-catalyzed alkylation of tryptophan residues at the C7 position. This method utilizes a N-Pt Bu2 directing group and tolerates various peptide and alkene substrates. Utilizing internal olefins, this study represents the first example of site-selective peptide C-H alkylation through deconjugative isomerization. Furthermore, our method provides access to peptide macrocycles with unique Trp(C7)-alkyl crosslinks and potent cytotoxicity towards cancer cells.

Prebiotic Catalytic Peptide Ligation Yields Proteinogenic Peptides by Intramolecular Amide Catalyzed Hydrolysis Facilitating Regioselective Lysine Ligation in Neutral Water.

The prebiotic origin of catalyst-controlled peptide synthesis is fundamental to understanding the emergence of life. Building on our recent discovery that thiols catalyze the ligation of amino acids, amides, and peptides with amidonitriles in neutral water, we demonstrate the outcome of ligation depends on pH and that high pKa primary thiols are the ideal catalysts. While the most rapid thiol catalyzed peptide ligation occurs at pH 8.5–9, the most selective peptide ligation, that tolerates all proteinogenic side chains, occurs at pH 7. We have also identified the highly selective mechanism by which the intermediate peptidyl amidines undergo hydrolysis to α-peptides while demonstrating that the hydrolysis of amidines with nonproteinogenic structures, such as β- and γ-peptides, displays poor selectivity. Notably, this discovery enables the highly α-selective protecting-group-free ligation of lysine peptides at neutral pH while leaving the functional ε-amine side chain intact.

In Situ Assembly of Transmembrane Proteins from Expressed and Synthetic Components in Giant Unilamellar Vesicles.

Reconstituting functional transmembrane (TM) proteins into model membranes is challenging due to the difficulty of expressing hydrophobic TM domains, which often require stabilizing detergents that can perturb protein structure and function. Recent model systems solve this problem by linking the soluble domains of membrane proteins to lipids, using noncovalent conjugation. Herein, we test an alternative solution involving the in vitro assembly of TM proteins from synthetic TM domains and expressed soluble domains using chemoselective peptide ligation. We developed an intein mediated ligation strategy to semisynthesize single-pass TM proteins in synthetic giant unilamellar vesicle (GUV) membranes by covalently attaching soluble protein domains to a synthetic TM polypeptide, avoiding the requirement for detergent. We show that the extracellular domain of programmed cell death protein 1, a mammalian immune checkpoint receptor, retains its ligand-binding function at a membrane interface after ligation to a synthetic TM peptide in GUVs, facilitating the study of receptor-ligand interactions.

Relative configuration of Cys-Pro ester peptides in thioester formation.

A peptide containing a cysteinyl prolyl ester (CPE) moiety at the C-terminus (CPE peptide) was transformed into a diketopiperazine (DKP) thioester via an intramolecular N-S acyl shift reaction and was then used for peptide ligation. The difference in reactivity between the CPE peptide stereoisomers was examined. In reactions of the CPE peptides that contained L-Cys-L-Pro or D-Cys-D-Pro, the desired DKP thioester was formed at the preceding amino acid residue. On the other hand, in reactions of the CPE peptides that contained D-Cys-L-Pro or L-Cys-D-Pro, a thiolactone was formed at the C-terminal prolyl ester, and the ligation occurred at the C-terminal Pro residue. Using this reaction, it was possible to efficiently prepare a cyclic peptide.

Head-to-tail cyclization of side chain-protected linear peptides to recapitulate genetically-encoded cyclized peptides.

Genetically‐encoded cyclic peptide libraries allow rapid in vivo screens for inhibitors of any target protein of interest. In particular, the Split Intein Circular Ligation of Protein and Peptides (SICLOPPS) system exploits spontaneous protein splicing of inteins to produce intracellular cyclic peptides. A previous SICLOPPS screen against Aurora B kinase, which plays a critical role during chromosome segregation, identified several candidate inhibitors that we sought to recapitulate by chemical synthesis. We describe the syntheses of cyclic peptide hits and analogs via solution‐phase macrocyclization of side chain‐protected linear peptides obtained from standard solid‐phase peptide synthesis. Cyclic peptide targets, including cyclo‐[CTWAR], were designed to match both the variable portions and conserved cysteine residue of their genetically‐encoded counterparts. Synthetic products were characterized by tandem high‐resolution mass spectrometry to analyze a combination of exact mass, isotopic pattern, and collisional dissociation‐induced fragmentation pattern. The latter analyses facilitated the distinction between targets and oligomeric side products, and served to confirm peptidic sequences in a manner that can be readily extended to analyses of complex biological samples. This alternative chemical synthesis approach for cyclic peptides allows cost‐effective validation and facile chemical elaboration of hit candidates from SICLOPPS screens.

Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs).

Chemical synthesis of proteins with poor solubility presents a challenging task. The existing solubilizing tag strategies are not suitable for the expressed protein segment. To address this issue, we report herein that solubilizing tags could be introduced at the side chain of the peptide and C-terminal peptide salicylaldehyde esters via a disulfide linker. Such reducible solubilizing tags (RSTs) are compatible with peptide salicylaldehyde ester-mediated Ser/Thr ligation and Cys/Pen ligation for purifying and ligating peptides with poor solubility. This strategy features operational simplicity and readily accessible materials. Both the protein 2B4 cytoplasmic tail and FCER1G protein have been successfully synthesized via this strategy. Of particular note, the RST strategy could be used for solubilizing the expressed protein segment for protein semi-synthesis of the HMGB1 protein.

Diselenide-selenoester ligation in the chemical synthesis of proteins.

Peptides and proteins represent an important class of biomolecules responsible for a plethora of structural and functional roles in vivo. Following their translation on the ribosome, the majority of eukaryotic proteins are post-translationally modified, leading to a proteome that is much larger than the number of genes present in a given organism. In order to understand the functional role of a given protein modification, it is necessary to access peptides and proteins bearing homogeneous and site-specific modifications. Accordingly, there has been significant research effort centered on the development of peptide ligation methodologies for the chemical synthesis of modified proteins. In this chapter we outline the discovery and development of a contemporary methodology called the diselenide-selenoester ligation (DSL) that enables the rapid and efficient fusion of peptide fragments to generate synthetic proteins. The practical aspects of using DSL for the preparation of chemically modified peptides and proteins in the laboratory is described. In addition, recent advances in the application of the methodology are outlined, exemplified by the synthesis and biological evaluation of a number of complex protein targets.