Chemical Protein Synthesis Research Articles

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.

Development and applications of enzymatic peptide and protein ligation.

Chemical synthesis of complex peptides and proteins continues to play increasingly important roles in industry and academia, where strategies for covalent ligation of two or more peptide fragments to produce longer peptides and proteins in convergent manners have become critical. In recent decades, efficient and site-selective ligation strategies mediated by exploiting the biocatalytic capacity of nature's diverse toolkit (i.e., enzymes) have been widely recognized as a powerful extension of existing chemical strategies. In this review, we present a chronological overview of the development of proteases, transpeptidases, transglutaminases, and ubiquitin ligases. We survey the different properties between the ligation reactions of various enzymes, including the selectivity and efficiency of the reaction, the ligation "scar" left in the product, the type of amide bond formed (natural or isopeptide), the synthetic availability of the reactants, and whether the enzymes are orthogonal to another. This review also describes how the inherent specificity of these enzymes can be exploited for peptide and protein ligation.

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.

Improved one-pot protein synthesis enabled by a more precise assessment of peptide arylthioester reactivity

By investigating 17 peptide arylthioesters that were previously challenging to produce, this study reveals a clear correlation between increased ligation activity and decreased pKa values of their corresponding arylthiols. The observed differences are attributed to variations in thioester bond strength and steric hindrance. These insights have led to the development of an improved one-pot chemical protein synthesis approach that leverages the reactivity differences between peptide arylthioesters with C-terminal Ala-S-Ph(4-NO2) and Ala-S-Ph(2,6-diCH3). This approach eliminates the need for thiol-thioester exchange and additive removal steps while enabling in situ desulfurization, thereby significantly simplifying the protein synthesis process.

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.

Prior disulfide bond-mediated Ser/Thr ligation.

In this work, we developed a novel strategy, prior disulfide bond-mediated Ser/Thr ligation (PD-STL), for the chemical synthesis of peptides and proteins. This approach combines disulfide bond-forming chemistry with Ser/Thr ligation (STL), converting intermolecular STL into intramolecular STL to effectively proceed regardless of concentrations. We demonstrated the effectiveness of PD-STL under high dilution conditions, even for the relatively inert C-terminal proline at the ligation site. Additionally, we applied this method to synthesize the N-terminal cytoplasmic domain (2-104) of caveolin-1 and its Tyr14 phosphorylated form.

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.

Post-translational activation of the C-terminus of polypeptides for the synthesis of peptide thioesters and peptide thioester surrogates.

Semisynthesis using recombinant polypeptides is a powerful approach for the synthesis of proteins having a variety of modifications. Peptide thioesters, of which the peptide C-terminus is activated by a thioester, are utilized for coupling peptide building blocks. Biological methods employing intein have been a center for the C-terminal thioesterification of recombinant polypeptides. Chemical activation has emerged as an alternative methodology for synthesizing peptide thioesters from recombinant polypeptides. Chemical reactions are compatible with various solutions containing organic solvents, chaotropic reagents, or detergents that are generally incompatible with biomolecules such as intein. Despite the potential utility of chemical activation, available methods remain limited. This article introduces the methods for the chemical activation of a peptide C-terminus applied to the chemical synthesis of proteins. By showcasing these methodologies, we aim to accelerate the advancement of new chemical reactions and methodologies and broaden the frontiers for the chemical synthesis of proteins.

Synthesis and Semi-Synthesis of Alpha-Synuclein: Insight into the Chemical Complexity of Synucleinopathies.

The chemical rules governing protein folding have intrigued generations of researchers for decades. With the advent of artificial intelligence (AI), prediction of protein structure has improved tremendously. However, there is still a level of analysis that is only possible through wet laboratory experiments, especially in respect to the investigation of the pathological effect of mutations and posttranslational modifications (PTMs) on proteins of interest. This requires the availability of pure peptides and proteins in sufficient quantities for biophysical, biochemical, and functional studies. In this context, chemical protein synthesis and semi-synthesis are powerful tools in protein research, which help to enlighten the role of protein modification in the physiology and pathology of proteins. A protein of high interest in the field of biomedicine is alpha-synuclein (aSyn), a protein deeply associated with several devastating neurodegenerative disorders such as Parkinson's disease (PD), dementia with Lewy bodies (DLB), or multiple systems atrophy (MSA). Here, we describe several methods and pathways to synthesize native or modified aSyn, and discuss how these approaches enable us to address pathological mechanisms that may open novel perspectives for therapeutic intervention.

Facile Access to Branched Multispecific Proteins.

Approaches that leverage orthogonal chemical reactions to generate protein-protein conjugates have expanded access to bespoke chimeras. Although the literature is replete with examples of the semisynthesis of bispecific proteins, few methods exist for the semisynthesis of protein conjugates of higher complexity (i.e., greater than two-protein fusions). The recent emergence of trispecific cell engagers for immune cell redirection therapies necessitates the development of chemical methods for the construction of trispecific proteins that would otherwise be inaccessible via natural protein synthesis. Here, we demonstrate that 3-bromo-5-methylene pyrrolone (3Br-5MP) can be used to effect the facile chemical synthesis of trispecific peptides and proteins with exquisite control over the addition of each monomer. The multimeric complexes maintain epitope functionality both in human cells and upon immobilization. We anticipate that facile access to trispecific proteins using this 3Br-5MP will have broad utility in basic science research and will quicken the pace of research to establish novel, multimeric immune cell redirection therapies.

Site-Specific Acetylation of the Transcription Factor Protein Max Modulates Its DNA Binding Activity.

Chemical protein synthesis provides a powerful means to prepare novel modified proteins with precision down to the atomic level, enabling an unprecedented opportunity to understand fundamental biological processes. Of particular interest is the process of gene expression, orchestrated through the interactions between transcription factors (TFs) and DNA. Here, we combined chemical protein synthesis and high-throughput screening technology to decipher the role of post-translational modifications (PTMs), e.g., Lys-acetylation on the DNA binding activity of Max TF. We synthesized a focused library of singly, doubly, and triply modified Max variants including site-specifically acetylated and fluorescently tagged analogs. The resulting synthetic analogs were employed to decipher the molecular role of Lys-acetylation on the DNA binding activity and sequence specificity of Max. We provide evidence that the acetylation sites at Lys-31 and Lys-57 significantly inhibit the DNA binding activity of Max. Furthermore, by utilizing high-throughput binding measurements, we assessed the binding activities of the modified Max variants across diverse DNA sequences. Our results indicate that acetylation marks can alter the binding specificities of Max toward certain sequences flanking its consensus binding sites. Our work provides insight into the hidden molecular code of PTM-TFs and DNA interactions, paving the way to interpret gene expression regulation programs.

Chemical synthesis of proteins

Chemical synthesis of proteins

Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S-Benzylidene Thioacetals.

Although solid-phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S-benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V-like (PD-L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.

Selective Peptide Cysteine Manipulation on Demand and Difficult Protein Chemical Synthesis Enabled by Controllable Acidolysis of N,S‐Benzylidene Thioacetals

AbstractAlthough solid‐phase peptide synthesis combining with chemical ligation provides a way to build up customized polypeptides in general, many targets are still presenting challenges for the conventional synthetic process, such as hydrophobic proteins. New methods and strategies are still required to overcome these obstacles. In this study, kinetic studies of Cys/Pen ligation and its acidolysis were performed, from which the fast acidolysis of substituted N,S‐benzylidene thioacetals (NBTs) was discovered. The study demonstrates the potential of NBTs as a promising Cys switchable protection, facilitating the chemical synthesis of peptides and proteins by efficiently disrupting peptide aggregation. The compatibility of NBTs with other commonly adopted Cys protecting groups and their applications in sequential disulfide bond formation were also investigated. The first chemical synthesis of the native human programmed death ligand 1 immunoglobulin V‐like (PD‐L1 IgV) domain was achieved using the NBT strategy, showcasing its potential in difficult protein synthesis.

Recent advances in chemical protein synthesis: method developments and biological applications

Recent advances in chemical protein synthesis: method developments and biological applications

Synthesis of N-Glycosylated Soluble Fas Ligand.

Controlled cell death is essential for the regulation of the immune system and plays a role in pathogen defense. It is often altered in pathogenic conditions such as cancer, viral infections and autoimmune diseases. The Fas receptor and its corresponding membrane-bound ligand (FasL) are part of the extrinsic apoptosis pathway activated in these cases. A soluble form of FasL (sFasL), produced by ectodomain shedding, displays a diverse but still elusive set of non-apoptotic functions and sometimes even serves as a pro-survival factor. To gather more knowledge about the characteristics of this protein and the impact N-glycosylations may have, access to homogeneous posttranslationally modified variants of sFasL is needed. Therefore, we developed a flexible strategy to obtain such homogeneously N-glycosylated variants of sFasL by applying chemical protein synthesis. This strategy can be flexibly combined with enzymatic methods to introduce more complex, site selective glycosylations.

Probing effects of site-specific aspartic acid isomerization on structure and stability of GB1 through chemical protein synthesis.

Chemical modifications of long-lived proteins, such as isomerization and epimerization, have been evoked as prime triggers for protein-damage related diseases. Deamidation of Asn residues, which results in formation of a mixture of l- and d-Asp and isoAsp via an intermediate aspartyl succinimide, can result in the disruption of cellular proteostasis and toxic protein depositions. In contrast to extensive data on the biological prevalence and functional implications of aspartyl succinimide formation, much less is known about the impact of the resulting altered backbone composition on properties of individual proteins at a molecular level. Here, we report the total chemical synthesis, biophysical characterization, and NMR structural analysis of a series of variants of the B1 domain of protein G from Streptococcal bacteria (GB1) in which all possible Asp isomers as well as an aspartyl succinimide were individually incorporated at a defined position in a solvent-exposed loop. Subtle local structural effects were observed; however, these were accompanied by notable differences in thermodynamic folded stability. Surprisingly, the noncanonical backbone connectivity of d-isoAsp led to a variant that exhibited enhanced stability relative to the natural protein.

Azole reagents enabled ligation of peptide acyl pyrazoles for chemical protein synthesis.

Native chemical ligation (NCL) has been playing an increasingly important role in chemical protein synthesis (CPS). More efficient ligation methods that circumvent the requirement of a peptidyl thioester and thiol additive-which allow the following desulfurization or refolding in one pot-are urgently needed for the synthesis of more complex protein targets and in large quantities. Herein, we discover that the weak acyl donor peptidyl N-acyl pyrazole can be activated by azole reagents like 3-methylpyrazole or imidazole to facilitate its ligation directly with an N-terminal cysteine peptide. As it requires no thioester or thiol additive, this ligation strategy can be conveniently combined with metal-free desulfurization (MFD) or oxidative protein folding to allow various one-pot protocols. The utility and generality of the strategy are showcased by the total synthesis of ubiquitin via an N-to-C sequential ligation-MFD strategy, the semi-synthesis of the copper protein azurin, and the efficient assembly of a sulfated hirudin variant and the cyclotide kalata B1, all in a one-pot fashion.