Chemical Ligation Research Articles

Identification and synthesis of a long-chain antimicrobial peptide from the venom of the Liocheles australasiae scorpion.

Scorpion venom contains linear peptides without disulfide bonds in addition to peptides with disulfide bonds. Many such linear peptides have an amphiphilic α-helical structure, often with antimicrobial activity and can be classified into three groups based on their molecular size. Among them, long-chain antimicrobial peptides consisting of more than 40 residues have not been thoroughly studied due to the difficulty of synthesizing them. We have previously reported a transcriptome analysis of the venom gland of Liocheles australasiae that revealed precursor sequences of long-chain antimicrobial peptides. In the study reported here, we identified the mature structure of one such long-chain antimicrobial peptide, LaCT1, which we synthesized using chemical ligation to confirm its structure and evaluate its biological activities. The result showed that LaCT1 exhibited significant antimicrobial activity. In addition, we identified its partial peptides consisting of an N- or C-terminal region, which may be generated by enzymatic cleavage in the venom. Among them, only the peptide containing the N-terminal half region was active. LaCT1 also not only showed insecticidal activity but also synergistically enhanced the effects of another insecticidal peptide identified in L. australasiae venom as well. These results provide insights into the role of antimicrobial peptides in scorpion venom.

Kinetic and Mechanistic Studies of Native Chemical Ligation with Phenyl α-Selenoester Peptides

Kinetic and Mechanistic Studies of Native Chemical Ligation with Phenyl α-Selenoester Peptides

Coiled Coil Peptide Tiles (CCPTs): Expanding the Peptide Building Block Design with Multivalent Peptide Macrocycles.

Owing to their synthetic accessibility and protein-mimetic features, peptides represent an attractive biomolecular building block for the fabrication of artificial biomimetic materials with emergent properties and functions. Here, we expand the peptide building block design space through unveiling the design, synthesis, and characterization of novel, multivalent peptide macrocycles (96mers), termed coiled coil peptide tiles (CCPTs). CCPTs comprise multiple orthogonal coiled coil peptide domains that are separated by flexible linkers. The constraints, imposed by cyclization, confer CCPTs with the ability to direct programmable, multidirectional interactions between coiled coil-forming "edge" domains of CCPTs and their free peptide binding partners. These fully synthetic constructs are assembled using a convergent synthetic strategy via a combination of native chemical ligation and Sortase A-mediated cyclization. Circular dichroism (CD) studies reveal the increased helical stability associated with cyclization and subsequent coiled coil formation along the CCPT edges. Size-exclusion chromatography (SEC), analytical high-performance liquid chromatography (HPLC), and fluorescence quenching assays provide a comprehensive biophysical characterization of various assembled CCPT complexes and confirm the orthogonal colocalization between coiled coil domains within CCPTs and their designed on-target free peptide partners. Lastly, we employ molecular dynamics (MD) simulations, which provide molecular-level insights into experimental results, as a supporting method for understanding the structural dynamics of CCPTs and their complexes. MD analysis of the simulated CCPT architectures reveals the rigidification and expansion of CCPTs upon complexation, i.e., coiled coil formation with their designed binding partners, and provides insights for guiding the designs of future generations of CCPTs. The addition of CCPTs into the repertoire of coiled coil-based building blocks has the potential for expanding the coiled coil assembly landscape by unlocking new topologies having designable intermolecular interfaces.

Selective Activation of Peptide‐Thioester Precursors for Templated Native Chemical Ligations

AbstractChemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid‐phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C‐terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield‐sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi‐segment templated NCL approach permits the one‐pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of the full‐length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one‐pot approach removes the need for intermediate purification. We report the synthesis of two E. coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

An Enabling Peptide Ligation Induced by Thiol-Salicylaldehyde Ester for Chemical Protein Synthesis.

Chemical protein synthesis by amide-forming ligation of two unprotected peptide segments offers an effective strategy for the preparation of protein derivatives that are not accessible through bioengineering approaches. Herein, an unprecedented chemical ligation between peptides with C-terminal 2-mercaptobenzaldehyde (thiol-salicylaldehyde, TSAL) esters and peptides bearing N-terminal cysteine/penicillamine is reported. Reactive peptide TSAL esters can be obtained from peptide hydrazides in an operationally simple and highly effective manner. This chemoselective peptide ligation enables the rapid production of N,S-benzylidene acetal intermediates, which can readily be converted into native amide bonds even at sterically hindered junctions. In addition, the current method can be applied compatibly in concert with other types of ligations and subsequent desulfurization chemistry, thereby facilitating convergent protein synthesis. The effectiveness of this new method is also showcased by the total synthesis of proteins ubiquitin and hyalomin-3 (Hyal-3), the efficient synthesis of protein ubiquitin-fold modifier 1 (UFM1) via a C-to-N sequential TSAL ester-induced ligation strategy, and the chemical synthesis of protein Mtb CM through a combined strategy of Ser/Thr ligation and TSAL ester-induced ligations.

Promotion of RNF168-Mediated Nucleosomal H2A Ubiquitylation by Structurally Defined K63-Polyubiquitylated Linker Histone H1.

The chemical synthesis of histones with homogeneous modifications is a powerful approach for quantitatively deciphering the functional crosstalk between different post-translational modifications (PTMs). In this study, we developed an expedient site-specific (poly)ubiquitylation strategy (CAEPL, Cysteine Aminoethylation coupled with Enzymatic Protein Ligation), which integrates the Cys-aminoethylation reaction with the process of ubiquitin-activating enzyme UBA1-assisted native chemical ligation. Using this strategy, we successfully prepared monoubiquitylated and K63-linked di- and tri-ubiquitylated linker histone H1.0 proteins, which were incorporated into individual chromatosomes. Quantitative biochemical analysis of different RNF168 constructs on H1 ubiquitylated chromatosomes with different ubiquitin chain lengths demonstrated that K63-linked polyubiquitylated H1.0 could directly stimulate RNF168 ubiquitylation activity by enhancing the affinity between RNF168 and the chromatosome. Subsequent cryo-EM structural analysis of the RNF168/UbcH5c-Ub/H1.0-K63-Ub3 chromatosome complex revealed the potential recruitment orientation between RNF168 UDM1 domain and K63-linked ubiquitin chain on H1.0. Finally, we explored the impact of H1.0 ubiquitylation on RNF168 activity in the context of asymmetric H1.0-K63-Ub3 di-nucleosome substrate, revealing a comparable stimulation effect of both the inter- and intra-nucleosomal crosstalk. Overall, our study highlights the significance of access to structurally defined polyubiquitylated H1.0 by the CAEPL strategy, enabling in-depth mechanistic investigations of in-trans PTM crosstalk between linker histone H1.0 and core histone H2A ubiquitylation.

BracketMaker: Visualization and optimization of chemical protein synthesis.

Chemical protein synthesis (CPS), in which custom peptide segments of ~20-60 aa are produced by solid-phase peptide synthesis and then stitched together through sequential ligation reactions, is an increasingly popular technique. The workflow of CPS is often depicted with a "bracket" style diagram detailing the starting segments and the order of all ligation, desulfurization, and/or deprotection steps to obtain the product protein. Brackets are invaluable tools for comparing multiple possible synthetic approaches and serve as blueprints throughout a synthesis. Drawing CPS brackets by hand or in standard graphics software, however, is a painstaking and error-prone process. Furthermore, the CPS field lacks a standard bracket format, making side-by-side comparisons difficult. To address these problems, we developed BracketMaker, an open-source Python program with built-in graphic user interface (GUI) for the rapid creation and analysis of CPS brackets. BracketMaker contains a custom graphics engine which converts a text string (a protein sequence annotated with reaction steps, introduced herein as a standardized format for brackets) into a high-quality vector or PNG image. To aid with new syntheses, BracketMaker's "AutoBracket" tool automatically performs retrosynthetic analysis on a set of segments to draft and rank all possible ligation orders using standard native chemical ligation, protection, and desulfurization techniques. AutoBracket, in conjunction with an improved version of our previously reported Automated Ligator (Aligator) program, provides a pipeline to rapidly develop synthesis plans for a given protein sequence. We demonstrate the application of both programs to develop a blueprint for 65 proteins of the minimal Escherichia coli ribosome.

Rapid and large-scale glycopeptide enrichment strategy based on chemical ligation.

Protein glycosylation, the most universal post-translational modification, is thought to play a crucial role in regulating multiple essential cellular processes. However, the low abundance of glycoproteins and the heterogeneity of glycans complicate their comprehensive analysis. Here, we develop a rapid and large-scale glycopeptide enrichment strategy via bioorthogonal ligation and trypsin cleavage. The enrichment process is performed in one tube to minimize sample loss and time costs. This method combines convenience and practicality, identifying over 900 O-GlcNAc sites from a 500μg sample. Surprisingly, it allows simultaneous identification of N-glycosites, O-GlcNAc sites, O-GalNAc sites and N-glycans via a two-step enzymatic release strategy. Combined with quantitative analysis, it reveals the distinct O-GlcNAcylation patterns in different compartments during oxidative stress. In summary, our study offers a convenient and robust tool for glycoproteome and glycome profiling, facilitating in-depth analysis to elucidate the biological functions of glycosylation.

Access to capped RNAs by chemical ligation.

A distinctive feature of eukaryotic mRNAs is the presence of a cap structure at the 5' end. The typical cap consists of 7-methylguanosine linked to the first transcribed nucleotide through a 5',5'-triphosphate bridge. It plays a key role in many processes in eukaryotic cells, including splicing, intracellular transport, initiation of translation and turnover. Synthetic capped oligonucleotides have served as useful tools for elucidating these physiological processes. In addition, cap mimics with artificial modifications are of interest for the design of mRNA-based therapeutics and vaccines. While the short cap mimics can be obtained by chemical synthesis, the preparation of capped analogs of mRNA length is still challenging and requires templated enzymatic ligation of synthetic RNA fragments. To increase the availability of capped mRNA analogs, we present here a practical and non-templated approach based on the use of click ligation resulting in RNAs bearing a single triazole linkage within the oligo-phosphate backbone. Capped RNA fragments with up to 81 nucleotides in length have thus been obtained in nanomolar yields and are in demand for biochemical, spectroscopic or structural studies.

Epimerization-Free Native Chemical Ligation Using β-Lactone for the Synthesis of Polypeptides

Epimerization-Free Native Chemical Ligation Using β-Lactone for the Synthesis of Polypeptides

Rapid Production of Native and Mirror-Image Tumor Necrosis Factor-α Enabled by Automated Flow Peptide Synthesis Technology.

Tumor necrosis factor-α (TNF-α) plays a central role in immune response regulation. Because elevated TNF-α production is correlated with a range of diseases, inhibiting the interaction of this protein with its native receptors has been thoroughly explored as a therapeutic avenue. Despite advancements in the development of TNF-α inhibitors, concerns remain regarding immunogenicity and loss of activity in vivo. To facilitate the discovery of stable and less immunogenic therapeutic modalities, we applied a single-shot automated fast-flow peptide synthesis (AFPS) strategy to produce full-length TNF-α, resulting in a complex reaction mixture. Leveraging the ability of AFPS to generate long peptides with high purity, we combined this technology with native chemical ligation (NCL). An NCL reaction using two fragments readily produced by AFPS afforded synthetic L- and D-TNF-α in milligram quantities (up to 5.5 mg, ∼28% yield). Following the oxidative folding of synthetic TNF-α using established conditions, higher molecular weight species were generated. Through high-throughput screening of refolding conditions, functional synthetic L- and mirror-image D-TNF-α were obtained, exhibiting characteristics analogous to those of the recombinant TNF-α. Overall, this approach can serve as a general protocol for accessing proteins that are intractable by modern protein synthesis methods, therefore, streamlining the development of novel therapeutics.

Facile incorporation of non-canonical heme ligands in myoglobin through chemical protein synthesis

The incorporation of non-canonical amino acids (ncAAs) into the metal coordination environments of proteins has endowed metalloproteins with enhanced properties and novel activities, particularly in hemoproteins. In this work, we disclose a scalable synthetic strategy that enables the production of myoglobin (Mb) variants with non-canonical heme ligands, i.e., HoCys and f4Tyr. The ncAA-containing Mb* variants (with H64V/V68A mutations) were obtained through two consecutive native chemical ligations and a subsequent desulfurization step, with overall isolated yield up to 28.6 % in over 10-milligram scales. After refolding and heme b cofactor reconstitution, the synthetic Mb* variants showed typical electronic absorption bands. When subjected to the catalysis of the cyclopropanation of styrene, both synthetic variants, however, were not as competent as the His-ligated Mb*. We envisioned that the synthetic method reported herein would be useful for incorporating a variety of ncAAs with diverse structures and properties into Mb for varied purposes.

Selective Activation of Peptide-Thioester Precursors for Templated Native Chemical Ligations.

Chemical protein synthesis enables access to proteins that would otherwise be difficult or impossible to obtain with traditional means such as recombinant expression. Chemoselective ligations provide the ability to join peptide segments prepared by solid-phase peptide synthesis. While native chemical ligation (NCL) is widely used, it is limited by the need for C-terminal thioesters with suitable reaction kinetics, properly placed native Cys or thiolated derivatives, and peptide segment solubility at low mM concentrations. Moreover, repetitive purifications to isolate ligated products are often yield-sapping, hampering efficiency and progress. In this work, we demonstrate the use of Controlled Activation of Peptides for Templated NCL (CAPTN). This traceless multi-segment templated NCL approach permits the one-pot synthesis of proteins by harnessing selective thioester activation and orthogonal conjugation chemistries to favor formation of full-length ligated product while minimizing side reactions. Importantly, CAPTN provides kinetic enhancements allowing ligations at sterically hindered junctions and low peptide concentrations. Additionally, this one-pot approach removes the need for intermediate purification. We report the synthesis of two E.coli ribosomal subunits S16 and S17 enabled by the chemical tools described herein. We anticipate that CAPTN will expedite the synthesis of valuable proteins and expand on templated approaches for chemical protein synthesis.

Accessing Therapeutically-Relevant Multifunctional Antisense Oligonucleotide Conjugates Using Native Chemical Ligation.

Antisense oligonucleotide (ASO) therapies hold significant promise in the realm of molecular medicine. By precisely targeting RNA molecules, ASOs offer an approach to modulate gene expression and protein production, making them valuable tools for treating a wide range of genetic and acquired diseases. As the precise intracellular targeting and delivery of ASOs is challenging, strategies for preparing ASO-ligand conjugates are in exceedingly high demand. This work leverages the utility of native chemical ligation to conjugate ASOs with therapeutically relevant chemical modifications including locked nucleic acids and phosphorothioate backbone modifications to peptides and sugars via a stable amide linkage. A suite of post-ligation functionalizations through modification of the cysteine ligation handle are highlighted, including chemoselective radical desulfurization, lipidation, and alkylation with a range of valuable handles (e.g. alkyne, biotin, and radionuclide chelating ligands), affording multifunctional constructs for further applications in biology and medicine. Application of the methodology to a clinically-relevant triantennary-GalNAc ASO conjugate and validation of its binding and functional activity underpins the applicability of the technique to oligonucleotide-based therapeutics.

Mix-and-Read Digital MicroRNA Analysis Based on Flow Cytometric Counting of Target-Clicked Nanobead Dimer.

A one-step, enzyme-free, and highly sensitive digital microRNA (miRNA) assay is rationally devised based on flow cytometric counting of target miRNA-clicked nanobead dimers via a facile mix-and-read manner. In this strategy, highly efficient miRNA-sandwiched click chemical ligation of two DNA probes may remarkably stabilize and boost the dimer formation between two kinds of fluorescence-coded nanobeads, and the number of as-produced bead dimers will be target dose-responsive, particularly when the trace number of miRNA is much less than that of employed nanobeads. Finally, each fluorescence-coded bead dimer can be easily identified and digitally counted by a powerful flow cytometer (FCM) and accordingly, the amount of target miRNA can be accurately quantified in a digital way. This new digital miRNA assay can be accomplished with a facile mix-and-read operation just by simply mixing the target miRNA with two kinds of preprepared DNA probe-functionalized nanobeads, which do not require any nucleic acid amplification, purification, and complex operation procedures. In spite of the extremely simple one-step operation, benefiting from the low-background but high target-mediated click ligation efficiency, and the powerfully digital statistical capability of FCM, this strategy achieves high sensitivity with a quite low detection limit of 5.2 fM target miRNA as well as high specificity and good generality for miRNA analysis, pioneering a new direction for fabricating digital bioassays.

Uncovering a Latent Bioactive Interleukin-6 Glycoform.

A bioinspired semisynthesis of human-interleukin-6 bearing N-glycan at Asn143 (143glycosyl-IL-6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide-backbone activation. 143Glycosyl-IL-6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6-polypeptide (1-141-position) with an intentionally inserted cysteine at 142-position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two-disulfide bonds (43-49 and 72-82). Utilizing the successfully formed free-142-cysteine, sequential conversions using cyanation of 142-cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1-141)-thioester. However, the resultant polypeptide-thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two-disulfide bonds of polypeptide (1-141)-thioester, an unprecedented hydrophilic N-glycan tag was inserted at the resultant cysteine thiols. The N-glycan tags greatly stabilized polypeptide-thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl-IL-6 polypeptide (183-amino acids). Removal of four N-glycan tags and immediate one-pot in vitro folding protocol efficiently produced the folded 143glycosyl-IL-6. The folded 143glycosyl-IL-6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl-IL-6.

Uncovering a Latent Bioactive Interleukin‐6 Glycoform

A bioinspired semisynthesis of human‐interleukin‐6 bearing N‐glycan at Asn143 (143glycosyl‐IL‐6) was performed by intentional glycosylation effects and protein folding chemistry for regioselective peptide‐backbone activation. 143Glycosyl‐IL‐6 is a genetically coded cytokine, but isolation was difficult owing to a tiny amount. IL6‐polypeptide (1‐141‐position) with an intentionally inserted cysteine at 142‐position was expressed in E. coli. The expressed polypeptide was treated with a chemical folding process to make a specific helices bundle conformation through native two‐disulfide bonds (43–49 and 72–82). Utilizing the successfully formed free‐142‐cysteine, sequential conversions using cyanation of 142‐cysteine, hydrazinolysis, and thioesterification created a long polypeptide (1–141)‐thioester. However, the resultant polypeptide‐thioester caused considerable aggregation owing to a highly hydrophobic peptide sequence. After the reduction of two‐disulfide bonds of polypeptide (1–141)‐thioester, an unprecedented hydrophilic N‐glycan tag was inserted at the resultant cysteine thiols. The N‐glycan tags greatly stabilized polypeptide‐thioester. The subsequent native chemical ligation and desulfurization successfully gave a whole 143glycosyl‐IL‐6 polypeptide (183‐amino acids). Removal of four N‐glycan tags and immediate one‐pot in vitro folding protocol efficiently produced the folded 143glycosyl‐IL‐6. The folded 143glycosyl‐IL‐6 exhibited potent cell proliferation activity. The combined studies with molecular dynamics simulation, semisynthesis, and bioassays predict the bioactive conformation of latent 143glycosyl‐IL‐6.

Chemical Synthesis of Interleukin-6 for Mirror-Image Screening.

Interleukin-6 (IL-6), a multifunctional cytokine, is an attractive therapeutic target for immunological and inflammatory diseases. We investigated the chemical synthesis of IL-6 and its enantiomer (d-IL-6) using a sequential N-to-C native chemical ligation strategy from six peptide segments. Solubilizing Trt-K10 tags improved the intermediate solubility and served as protecting groups during the metal-free desulfurization to facilitate the synthesis of full-length IL-6 protein. Synthetic l-IL-6 and recombinant IL-6 exhibited nearly identical structural and binding properties. The symmetrical binding property of d-IL-6 was also demonstrated by functional analysis using IL-6-binding peptides. The resulting functional d-IL-6 was employed to screen a phage-displayed antibody fragment library, leading to the identification of several d-IL-6-binding single-domain antibodies. This work will contribute to the development of novel, potent IL-6 inhibitors without the adverse effects of undesired immune activation.

Chemical Synthesis of Human Proteoforms and Application in Biomedicine.

Limited understanding of human proteoforms with complex posttranslational modifications and the underlying mechanisms poses a major obstacle to research on human health and disease. This Outlook discusses opportunities and challenges of de novo chemical protein synthesis in human proteoform studies. Our analysis suggests that to develop a comprehensive, robust, and cost-effective methodology for chemical synthesis of various human proteoforms, new chemistries of the following types need to be developed: (1) easy-to-use peptide ligation chemistries allowing more efficient de novo synthesis of protein structural domains, (2) robust temporary structural support strategies for ligation and folding of challenging targets, and (3) efficient transpeptidative protein domain-domain ligation methods for multidomain proteins. Our analysis also indicates that accurate chemical synthesis of human proteoforms can be applied to the following aspects of biomedical research: (1) dissection and reconstitution of the proteoform interaction networks, (2) structural mechanism elucidation and functional analysis of human proteoform complexes, and (3) development and evaluation of drugs targeting human proteoforms. Overall, we suggest that through integrating chemical protein synthesis with in vivo functional analysis, mechanistic biochemistry, and drug development, synthetic chemistry would play a pivotal role in human proteoform research and facilitate the development of precision diagnostics and therapeutics.

Enhanced native chemical ligation by peptide conjugation in trifluoroacetic acid.

Chemical ligation of peptides is increasingly used to generate proteins not readily accessible by recombinant approaches. However, a robust method to ligate "difficult" peptides remains to be developed. Here, we report an enhanced native chemical ligation strategy mediated by peptide conjugation in trifluoroacetic acid (TFA). The conjugation between a carboxyl-terminal peptide thiosalicylaldehyde thioester and a 1,3-dithiol-containing peptide in TFA proceeds rapidly to form a thioacetal-linked intermediate, which is readily converted into the desired native amide bond product through simple postligation treatment. The effectiveness and practicality of the method was demonstrated by the successful synthesis of several challenging proteins, including the SARS-CoV-2 transmembrane Envelope (E) protein and nanobodies. Because of the ability of TFA to dissolve virtually all peptides and prevent the formation of unreactive peptide structures, the method is expected to open new opportunities for synthesizing all families of proteins, particularly those with aggregable or colloidal peptide segments.