Peptide Ligation Research Articles

Glycopeptide ligation via direct aminolysis of selenoester

Direct aminolysis of selenoester in aqueous media was investigated as a glycopeptide ligation strategy. This strategy allows the peptide and glycopeptide ligation to proceed smoothly (even with hindered amino acids) without the need of cysteine residue, N-terminal thiol auxiliary or coupling additive, and to afford the corresponding amide products in excellent yields. No epimerization was observed during ligation reations. In this work, the selenoester of unprotected glycopeptide was readily prepared, and the direct aminolysis of glycopeptide selenoester was successfully applied to synthesize MUC1 mucin sequence efficiently.

On-resin peptide ligation via C-terminus benzyl ester

Here, we report a new approach of on-resin peptide ligation using C-terminal benzyl ester as the stabilized precursor of thioester, which enables both N-terminal elongation and C-terminal peptide ligation on a Rink Amide resin. On-resin native chemical ligation and auxiliary-assisted peptide ligation were successfully achieved. This method is compatible to both protected and unprotected peptide fragments and has potential application in poor water-soluble peptide ligation.

Synthesis of Histidine-Containing Oligopeptides via Histidine-Promoted Peptide Ligation.

Histidine-containing peptides are valuable therapeutic agents for a treatment of neurodegenerative diseases. However, the synthesis of histidine-containing peptides is not trivial due to the potential of imidazole sidechain of histidine to act as a nucleophile if unprotected. A peptide ligation method utilizing the imidazole sidechain of histidine has been developed. The key imidazolate intermediate that acts as an internal acyl transfer catalyst during ligation is generated by deprotonation. Transesterification with amino acids or peptides tethered with C-terminal thioester followed by N→N acyl shifts led to the final ligated products. A range of histidine-containing dipeptides could be synthesized in moderate to good yields via this method without protecting the imidazole sidechain. The protocol was further extended to tripeptide synthesis via a long-range N→N acyl transfer, and tetrapeptide synthesis.

Sortase-Mediated High-Throughput Screening Platform for Directed Enzyme Evolution.

Sortase-catalyzed ligations have emerged as powerful tools for the site-specific ligation of peptides and proteins in material science and biocatalysis. In this work, a directed sortase evolution strategy (SortEvolve) has been developed as a general high-throughput screening (HTS) platform to improve activity of sortase A (application 1) and to perform directed laccase evolution through a semipurification process in 96-well microtiter plate (MTP) (application 2). A semipurification process in polypropylene MTP (PP-MTP) is achieved through the anchor peptide LCI, which acts as adhesion promoter. To validate the SortEvolve screening platform for both applications, three site-saturation mutagenesis (SSM) libraries of sortase A (Sa-SrtA) from Staphylococcus aureus (application 1) and two SSM libraries of the copper efflux oxidase (CueO laccase) from Escherichia coli (application 2) were generated at literature reported positions. After screening and rescreening, an array of Sa-SrtA variants (including the previously reported P94S, D160N, and D165A) and CueO variants (including the previously reported D439A and P444A) were identified. Further recombinant Sa-SrtA variant P94T/D160L/D165Q and CueO variant D439V/P444V were characterized with 22-fold and 103-fold improvements in catalytic efficiency compared with corresponding wild-types, respectively. An important advantage of the SortEvolve screening platform in comparison to many MTP-based screening systems is that the background noise was minimized (decreased 20-fold; application 2) due to the employed semipurification process. In essence, SortEvolve provides a universal surface-functionalized screening platform for sortases and enzymes in which especially background activity can be minimized to enable successful directed evolution campaigns.

Efficient peptide ligation between allyl-protected Asp and Cys followed by palladium-mediated deprotection.

An efficient method for peptide ligation between C-terminal Asp(OAllyl) and N-terminal Cys has been developed. Peptide ligation and removal of the allyl group at the Asp carboxylate side chain proceeded in one pot by adding a small amount of Pd/TPPTS complex. Based on this efficient synthetic method, PEP-19 (61 amino acids), which is highly expressed in Purkinje cells, was synthesized.

The discovery of a freezing-induced peptide ligation during the total chemical synthesis of human interferon-ε.

A counterintuitive freezing-induced peptide ligation was discovered during the total synthesis of human interferon-ε (hIFN-ε) which blocks HIV infection through unique mechanisms. The successful synthesis of hIFN-ε (187 amino acids) in this research laid the foundation for related anti-AIDS drug development. Moreover, alanine mutation based on sequence alignment to solve the maldistribution of the ligation site and freezing-induced dominant conformation that facilitates peptide ligation are expected to be helpful for the synthesis of macrobiomolecules.

Peptide nucleic acid-templated selenocystine-selenoester ligation enables rapid miRNA detection.

The development of a rapid and chemoselective selenocystine-selenoester peptide ligation that operates at nanomolar reactant concentrations has been developed by utilising PNA templation. Kinetic analysis of the templated peptide ligation revealed that the selenocystine-selenoester reaction was 10 times faster than traditional native chemical ligation at cysteine and to our knowledge is the fastest templated ligation reaction reported to date. The efficiency and operational simplicity of this technology is highlighted through the formation of hairpin molecular architectures and in a novel paper-based lateral flow assay for the rapid and sequence specific detection of oligonucleotides, including miRNA in cell lysates.

Thirteen decades of peptide synthesis: key developments in solid phase peptide synthesis and amide bond formation utilized in peptide ligation.

A historical overview of peptide chemistry from T. Curtius to E. Fischer to M. Bergmann and L. Zervas is first presented. Next, the fundamentals of peptide synthesis with a focus on solid phase peptide synthesis by R. B. Merrifield are described. Immobilization strategies to attach the first amino acid to the resin, coupling strategies in stepwise peptide chain elongation, and approaches to synthesize difficult peptide sequences are also shown. A brief comparison between tert-butyloxycarbonyl (Boc)/benzyl (Bzl) strategy and 9-fluorenylmethoxycarbonyl (Fmoc)/tert-butyl (t -Bu) strategy utilized in solid phase peptide synthesis is given with an emphasis on the latter. Finally, the review focuses on the discovery and development of peptide ligation and the latest advances in this field including native amide bond formation strategies, these include the native chemical ligation, α-ketoacid-hydroxylamine ligation, and serine/threonine ligation which are the most commonly used chemoselective ligation methods that provide amide bond at the ligation site. This review provides an overview of the literature concerning the most important advances in the chemical synthesis of proteins and peptides covering the period from 1882 to 2017.

Engineering peptide ligase specificity by proteomic identification of ligation sites.

Enzyme-catalyzed peptide ligation is a powerful tool for site-specific protein bioconjugation, but stringent enzyme–substrate specificity limits its utility. Here, we present an approach for comprehensive characterization of peptide ligase specificity for N termini using proteome-derived peptide libraries. We used this strategy to characterize the ligation efficiency for >25,000 enzyme–substrate pairs in the context of the engineered peptide ligase subtiligase and identified a family of 72 mutant subtiligases with activity toward N-terminal sequences that were previously recalcitrant to modification. We applied these mutants individually for site-specific bioconjugation of purified proteins including antibodies, and in algorithmically selected combinations for sequencing of the cellular N terminome with reduced sequence bias. We also developed a web application to enable algorithmic selection of the most efficient subtiligase variant(s) for bioconjugation to user-defined sequences. These studies provide a new toolbox of enzymes for site-specific protein modification and a general approach for rapidly defining and engineering peptide ligase specificity.

Reciprocal Nucleopeptides as the Ancestral Darwinian Self-Replicator.

Even the simplest organisms are too complex to have spontaneously arisen fully formed, yet precursors to first life must have emerged ab initio from their environment. A watershed event was the appearance of the first entity capable of evolution: the Initial Darwinian Ancestor. Here, we suggest that nucleopeptide reciprocal replicators could have carried out this important role and contend that this is the simplest way to explain extant replication systems in a mathematically consistent way. We propose short nucleic acid templates on which amino-acylated adapters assembled. Spatial localization drives peptide ligation from activated precursors to generate phosphodiester-bond-catalytic peptides. Comprising autocatalytic protein and nucleic acid sequences, this dynamical system links and unifies several previous hypotheses and provides a plausible model for the emergence of DNA and the operational code.

Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis

AbstractA novel and versatile peptide‐based bio‐logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide‐based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND‐INHIBIT), and a complex sequential logic circuit (multi‐input keypad lock). Moreover, a proof‐of‐concept peptide regulatory circuit was developed to analyze the expression profile of cell‐secreted protein biomarkers and trigger cancer‐cell‐specific apoptosis.

Peptide Logic Circuits Based on Chemoenzymatic Ligation for Programmable Cell Apoptosis.

A novel and versatile peptide-based bio-logic system capable of regulating cell function is developed using sortase A (SrtA), a peptide ligation enzyme, as a generic processor. By modular peptide design, we demonstrate that mammalian cells apoptosis can be programmed by peptide-based logic operations, including binary and combination gates (AND, INHIBIT, OR, and AND-INHIBIT), and a complex sequential logic circuit (multi-input keypad lock). Moreover, a proof-of-concept peptide regulatory circuit was developed to analyze the expression profile of cell-secreted protein biomarkers and trigger cancer-cell-specific apoptosis.

Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes.

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester). This task is challenging, with obstacles relating to the preparation and ligation of hydrophobic peptides, as well as the requirement for multiple purification steps due to protecting-group manipulations during the polypeptide assembly process. To address this, our approach uses an easily removable solubilizing tag for the synthesis and ligation of hydrophobic peptides, as well as a more efficient and better-yielding method to remove Cys-protecting groups that uses palladium chemistry (specifically [Pd(allyl)Cl]2 and PdCl2 complexes). The utility of this approach is demonstrated in the syntheses of ubiquitinated H2B at Lys34, phosphorylated H2A at Tyr57 and unmodified H4. Each of these analogs can be prepared in milligram quantities within ∼20-30 d.

Synthesis of Acylborons by Ozonolysis of Alkenylboronates: Preparation of an Enantioenriched Amino Acid Acylboronate

AbstractA concise synthesis of acylborons was achieved by ozonolysis of alkenyl MIDA (N‐methyliminodiacetic acid) boronates. This reaction exhibits excellent functional‐group tolerance and is applicable to various acyl MIDA boronates and potassium acyltrifluroborates (KATs) which could not be synthesized by previous methods. In addition, α‐amino acylborons, which would be essential for peptide ligations, were prepared for the first time. The acylboron of l‐alanine was obtained in high enantiopurity and found to be configurationally stable. Oligopeptide synthesis between the α‐amino KATs and amino acid in dilute aqueous media was studied.

Synthesis of Acylborons by Ozonolysis of Alkenylboronates: Preparation of an Enantioenriched Amino Acid Acylboronate.

A concise synthesis of acylborons was achieved by ozonolysis of alkenyl MIDA (N-methyliminodiacetic acid) boronates. This reaction exhibits excellent functional-group tolerance and is applicable to various acyl MIDA boronates and potassium acyltrifluroborates (KATs) which could not be synthesized by previous methods. In addition, α-amino acylborons, which would be essential for peptide ligations, were prepared for the first time. The acylboron of l-alanine was obtained in high enantiopurity and found to be configurationally stable. Oligopeptide synthesis between the α-amino KATs and amino acid in dilute aqueous media was studied.

A fluorogenic native chemical ligation for assessing the role of distance in peptide-templated peptide ligation

Protein-templated reactions have been used for fragment-based drug discovery as well as for covalent labeling, detection and manipulation of proteins. In spite of the growing interest in protein-templated reactions, little is known about the design criteria. Herein we present a systematic study on the effects of proximity in peptide-templated reactions. To facilitate reaction monitoring at low concentrations we developed a fluorogenic native chemical ligation that is based on the integration of a fluorescence quencher in the thiol leaving group. The reaction system provided up to 39-fold increases of emission from a fluorescein unit. By using templates based on coiled coils as models we investigated the effect of misalignments. The distance-reactivity pattern for remotely aligned peptides was remarkably different to reaction scenarios that involved seamlessly annealed peptides with overhanging functional groups.

Peptide Macrocyclization Assisted by Traceless Turn Inducers Derived from Ugi Peptide Ligation with Cleavable and Resin-Linked Amines.

A multicomponent approach enabling the installation of turn-inducing moieties that facilitate the macrocyclization of short and medium-size oligopeptides is described. The strategy comprises the Ugi ligation of peptide carboxylic acids and isocyanopeptides in the presence of aldehydes and acid or photolabile amines followed by cyclization and cleavage of the backbone N-substituents to render canonical cyclopeptides. Implementing the approach on solid phase with the use of Rink amide resins led to a new class of backbone amide linker strategy.

Recent developments in peptide ligation independent of amino acid side-chain functional group

Chemical synthesis of peptides and proteins has evolved into an indispensable tool for chemical biology. Peptide ligation is a straightforward technique for joining two short peptide fragments together via a native peptide bond to afford a larger natural peptide or protein. However, the junction sites are limited to several specific amino acids because most peptide ligations involve participation of the side-chain functional groups of the junction-site amino acids. To overcome such intrinsic limitations, “general” peptide ligations which do not rely on the side-chain functional group have been developed. This review summarized the recent developments in peptide ligations that are independent of side-chain functional group of ligation-junction-site amino acid.

An orthogonally protected CycloTriVeratrylene (CTV) as a highly pre-organized molecular scaffold for subsequent ligation of different cyclic peptides towards protein mimics

The synthesis of a semi-orthogonally protected CycloTriVeratrilene (CTV) scaffold derivative as well as the sequential introduction of three different peptide loops onto this molecular scaffold via Cu(I)-catalyzed azide alkyne cycloaddition towards a medium-sized protein mimic is described. This approach for the construction of medium-sized protein mimics is illustrated by the synthesis of a paratope mimic of the monoclonal antibody Infliximab (Remicade®) and provides access to a range of highly pre-organized molecular constructs bearing three different peptide segments. This approach may find wide applications for development of protein-protein interaction disruptors as well as synthetic vaccines.

Emerging Roles of Carbonyl Sulfide in Chemical Biology: Sulfide Transporter or Gasotransmitter?

Carbonyl sulfide (COS) is the most prevalent sulfur-containing gas in the Earth's atmosphere, and it plays important roles in the global sulfur cycle. COS has been implicated in origin of life peptide ligation, is the primary energy source for certain bacteria, and has been detected in mammalian systems. Despite this long and intertwined history with terrestrial biology, limited attention has focused on potential roles of COS as a biological mediator. Recent Advances: Although bacterial COS production is well documented, definitive sources of mammalian COS production have not been confirmed. Enzymatic COS consumption in mammals, however, is well documented and occurs primarily by carbonic anhydrase (CA)-mediated conversion to hydrogen sulfide (H2S). COS has been detected in ex vivo mammalian tissue culture, as well as in exhaled breath as a potential biomarker for different disease pathologies, including cystic fibrosis and organ rejection. Recently, chemical tools for COS delivery have emerged and are poised to advance future investigations into the role of COS in different biological contexts. Possible roles of COS as an important biomolecule, gasotransmitter, or sulfide transport intermediate remain to be determined. Key advances in both biological and chemical tools for COS research are needed to further investigate these questions. Further evaluation of the biological roles of COS and disentangling the chemical biology of COS from that of H2S are needed to further elucidate these interactions. Chemical tools for COS delivery and modulation may provide a first avenue of investigative tools to answer many of these questions. Antioxid. Redox Signal. 28, 1516-1532.