Improved Electrochemical Peptide Synthesis Enabled by Electron-Rich Triaryl Phosphines.

While remarkable progress has been made in the development of peptide medicines, many problems related to peptide synthesis remain unresolved. Previously, we reported electrochemical peptide synthesis using a phosphine as a potentially recyclable coupling reagent. However, there was room for improvement from the point of view of reaction efficiency, especially in the carboxylic acid activation step and the peptide bond formation step. To overcome these challenges, we searched for the optimal phosphine. Among phosphines with various electronic properties, we found that electron-rich triaryl phosphines improved the reaction efficiency. Consequently, we successfully performed electrochemical peptide synthesis on sterically hindered and valuable amino acids. We also synthesized oligopeptides that were challenging with our previous method. Finally, we examined the effect of substituents on the phosphine cations, and gained some insights into reactivity, which will aid researchers designing reactions involving phosphine cations.

Mono-ADP-ribosylation of peptides: an overview of synthetic and chemoenzymatic methodologies.

Adenosine diphosphate (ADP)-ribosylation is a ubiquitous post-translational modification that regulates vital biological processes like histone reorganization and DNA-damage repair through the modification of various amino acid residues. Due to advances in mass-spectrometry, the collection of long-known ADP-ribose (ADPr) acceptor sites, e.g. arginine, cysteine and glutamic acid, has been expanded with serine, tyrosine and histidine, among others. Well-defined ADPr-peptides are valuable tools for investigating the exact structures, mechanisms of action and interaction partners of the different flavors of this modification. This review provides a comprehensive overview of synthetic and chemoenzymatic methodologies that enabled the construction of peptides mono-ADP-ribosylated on various amino acids, and close mimetics thereof.

Synthesis, derivatization, and conformational scanning of peptides containing N-Aminoglycine.

N-alkylated glycine residues are the main constituent of peptoids and peptoid-peptide hybrids that are employed across the biomedical and materials sciences. While the impact of backbone N-alkylation on peptide conformation has been extensively studied, less is known about the effect of N-amination on the secondary structure propensity of glycine. Here, we describe a convenient protocol for the incorporation of N-aminoglycine into host peptides on solid support. Amide-to-hydrazide substitution also affords a nucleophilic handle for further derivatization of the backbone. To demonstrate the utility of late-stage hydrazide modification, we synthesized and evaluated the stability of polyproline II helix and ß-hairpin model systems harboring N-aminoglycine derivatives. The described procedures provide facile entry into peptidomimetic libraries for conformational scanning.

Designing and synthesizing peptide-based quorum sensing modulators.

Quorum sensing (QS) is a density-dependent bacterial communication system that uses small molecules as regulatory modulators. Synthetic changes to these molecules can up-or-down-regulate this system, leading to control of phenotypes, like competence and virulence factor production, that have implications in human health. In this chapter, a methodology for library design and screening of synthetic autoinducing peptides (AIPs) to uncover QS SARs is delineated. Additionally, procedures for the synthesis, purification and analysis of linear and cyclic AIPs are detailed. This includes solutions for potential synthetic challenges including diketopiperazine formation when using N-methyl amino acids and cyclization of peptides containing N-terminal cysteine residues. These procedures have and are currently being applied to develop potent QS modulators in Streptococcus pneumoniae, Bacillus cereus, Streptococcus gordonii and Lactiplantibacillus plantarum.

1,3,5-Triazine as Branching Connector for the Construction of Novel Antimicrobial Peptide Dendrimers: Synthesis and Biological Characterization.

Peptides displaying antimicrobial properties are being regarded as useful tools to evade and combat antimicrobial resistance, a major public health challenge. Here we have addressed dendrimers, attractive molecules in pharmaceutical innovation and development displaying broad biological activity. Triazine-based dendrimers were fully synthesized in the solid phase, and their antimicrobial activity and some insights into their mechanisms of action were explored. Triazine is present in a large number of compounds with highly diverse biological targets with broad biological activities and could be an excellent branching unit to accommodate peptides. Our results show that the novel peptide dendrimers synthesized have remarkable antimicrobial activity against Gram-negative bacteria (E. coli and P. aeruginosa) and suggest that they may be useful in neutralizing the effect of efflux machinery on resistance.

Synthesis and Biological Studies of New Temporin A Analogs Containing Unnatural Amino Acids in Position 7.

(1) Background: Antimicrobial resistance is growing at an extreme pace and has proven to be an urgent topic, for research into alternative treatments. Such a prospective possibility is hidden in antimicrobial peptides because of their low to no toxicity, effectiveness at low concentrations, and most importantly their ability to be used for multiple treatments. This work was focused on the study of the effect of the modification in position 7 of Temporin A on its biological activity; (2) Methods: The targeted peptides were synthesized using Fmoc/Ot-Bu SPPS. The antibacterial activity of the analogs was determined using the broth microdilution method and disk-diffusion method. In vitro tests were performed to determine the cytotoxicity, phototoxicity, and antiproliferative activity of the peptide analogs on a panel of tumor and normal cell lines; (3) Results: All analogs except DTCit showed good antibacterial activity, with DTDab having the best activity according to the disk-diffusion method. However, DTCit had an acceptable cytotoxicity, combined with good selectivity against the test MCF-7 cell line; (4) Conclusions: The obtained results revealed the importance of the basicity and length of the side chain at position 7 in the Temporin A sequence for both tested activities.

Synthesis and anticancer properties of novel dolastatin 10 analogs featuring five-membered heterocyclic rings with a linkable group at the C-terminus.

Dolastatin 10 (Dol-10), a natural marine-source pentapeptide, is a powerful antimitotic agent regarded as one of the most potent anticancer compounds found to date. Dol-10 however, lacks chemical conjugation capabilities, which restricts the feasibility of its application in targeted drug therapy. This limitation has spurred the prospect that chemical structure of the parent molecule might allow conjugation of the derivatives to drug carriers such as antibodies. By first employing docking studies, we designed and prepared a series of novel Dol-10 analogs with a modified C-terminus, preserving high potency of the parent compound while enhancing conjugation capability. The modifications involved the introduction of a methyleneamine functionality at position 4 of the 1,3-thiazole ring, along with the substitution of the thiazole ring with a 1,2,3-triazole moiety, furnished with methylenehydroxy, carboxy, methyleneamine, and N(Me)-methyleneamine tethering functionalities at position 4. Among the synthesized pentapeptides, DA-1 exhibited the highest potency in prostate cancer (PC-3) cells, eliciting apoptosis (IC50 0.2 ± 0.1 nm) and cell cycle arrest at the mitotic stage after at least 6 days of culture. This delayed response suggests the accumulation of cellular stress or significant physiological alterations that profoundly impact the cell cycle. We believe that these novel Dol-10 derivates represent a new and straightforward route for the development of C-terminus modified Dol-10-based microtubule inhibitors, thereby advancing targeted anticancer therapy.

The crystal structure of the ammonium salt of 2-aminomalonic acid.

The salt ammonium 2-aminomalonate (systematic name: ammonium 2-azaniumylpropanedioate), NH4+·C3H4NO4-, was synthesized in diethyl ether from the starting materials malonic acid, ammonia and bromine. The salt was recrystallized from water as colourless blocks. In the solid state, intramolecular medium-strong N-H...O, weak C-H...O and weak C-H...N hydrogen bonds build a three-dimensional network.

Structure-Activity Relationship of Synthetic Linear KTS-Peptides Containing Meta-Aminobenzoic Acid as Antagonists of α1ß1 Integrin with Anti-Angiogenic and Melanoma Anti-Tumor Activities.

To develop peptide drugs targeting integrin receptors, synthetic peptide ligands endowed with well-defined selective binding motifs are necessary. The snake venom KTS-containing disintegrins, which selectively block collagen α1ß1 integrin, were used as lead compounds for the synthesis and structure-activity relationship of a series of linear peptides containing the KTS-pharmacophore and alternating natural amino acids and 3-aminobenzoic acid (MABA). To ensure a better stiffness and metabolic stability, one, two and three MABA residues, were introduced around the KTS pharmacophore motif. Molecular dynamics simulations determined that the solution conformation of MABA peptide 4 is more compact, underwent larger conformational changes until convergence, and spent most of the time in a single cluster. The peptides' binding affinity has been characterized by an enzyme linked immunosorbent assay in which the most potent peptide 4 inhibited with IC50 of 324 ± 8 µM and 550 ± 45 µM the binding of GST-α1-A domain to collagen IV fragment CB3, and the cell adhesion to collagen IV using α1-overexpressor cells, respectively. Docking studies and MM-GBSA calculations confirmed that peptide 4 binds a smaller region of the integrin near the collagen-binding site and penetrated deeper into the binding site near Trp1. Peptide 4 inhibited tube formation by endothelial cell migration in the Matrigel angiogenesis in vitro assay. Peptide 4 was acutely tolerated by mice, showed stability in human serum, decreased tumor volume and angiogenesis, and significantly increased the survival of mice injected with B16 melanoma cells. These findings propose that MABA-peptide 4 can further serve as an α1ß1-integrin antagonist lead compound for further drug optimization in angiogenesis and cancer therapy.

Synthesis of the Antimicrobial Peptide Murepavadin Using Novel Coupling Agents.

The problem of antimicrobial resistance is becoming a daunting challenge for human society and healthcare systems around the world. Hence, there is a constant need to develop new antibiotics to fight resistant bacteria, among other important social and economic measures. In this regard, murepavadin is a cyclic antibacterial peptide in development. The synthesis of murepavadin was undertaken in order to optimize the preparative protocol and scale-up, in particular, the use of new activation reagents. In our hands, classical approaches using carbodiimide/hydroxybenzotriazole rendered low yields. The use of novel carbodiimide and reagents based on OxymaPure® and Oxy-B is discussed together with the proper use of chromatographic conditions for the adequate characterization of peptide crudes. Higher yields and purities were obtained. Finally, the antimicrobial activity of different synthetic batches was tested in three Pseudomonas aeruginosa strains, including highly resistant ones. All murepavadin batches yielded the same highly active MIC values and proved that the chiral integrity of the molecule was preserved throughout the whole synthetic procedure.

Amino acid analogues provide multiple plausible pathways to prebiotic peptides.

Prebiotic peptide synthesis has consistently been a prominent topic within the field of the origin of life. While research predominantly centres on the 20 classical amino acids, the synthesis process encounters significant thermodynamic barriers. Consequently, amino acid analogues are being explored as potential building blocks for prebiotic peptide synthesis. This review delves into the pathway of polypeptide formation, identifying specific amino acid analogues that might have existed on early Earth, potentially participating in peptide synthesis and chemical evolution. Moreover, considering the complexity and variability of the environment on early Earth, we propose the plausibility of coevolution between amino acids and their analogues.

A Type of Stable Amides Behaves as Acyl Transfer Reagents upon Visible-Light Irradiation through Self-Aromatization.

Organic molecules with light-modifiable reactivity are important in many fields because they can serve as the "switch" for light to trigger chemical processes. Herein, we disclose a new type of stable non-twisted amides, the reactivity of which can be turned on by light as acyl transfer reagents. Upon photo-activation, these amides react with various nucleophiles including amines, phenols, hydroxide, thiols, boronic acids, and alkynes either under metal-free or metal-catalysis conditions. This reactivity hinges on the design and synthesis of a photo-activatable reagent (7-nitro-5,6-dihydrophenanthridine), which undergoes self-aromatization enabled by an internal oxidant under light. This masked acyl donor group is anticipated to be useful in scenarios where light is preferred to trigger a chemical process.

A stability-indicating method development and validation for the determination of related substances in novel synthetic decapeptide by HPLC.

In the present scenario, peptide is an emerging field of research having vast therapeutic applications. Diverse impurities may rise from various stages of the synthesis process and storage of the peptides. Because these contaminants may have an impact on the therapeutic safety and effectiveness of peptides in their approaching applications, they must be identified and carefully monitored. Considering the pharmaceutical importance of the extent of peptides, we were motivated to synthesize a decapeptide and establish a novel gradient reversed-phase high-performance liquid chromatography (RP-HPLC) method for its analysis along with efficient separation of its six related impurities. Different buffers, organic modifiers, and columns were used in the tests for good separation of these impurities. To establish a stability-indicating method, a stress study was also conducted. The International Conference on Harmonization (ICH) guidelines have been followed for validation of the developed analytical method. The validated method revealed sufficient accuracy, specificity, linearity, robustness, precision, and high sensitivity for its intended use. The proposed method could be appropriate for routine analysis and stability assessment of the decapeptide, which might be useful for further scientific investigation.

Macromolecular tool box to elucidate CLAVATA3/Embryo Surrounding Region-Related-RLK binding, signaling and downstream effects.

Plant peptides communicate by binding to a large family of receptor-like kinases (RLKs) and they share a conserved binding mechanism, which may account for their promiscuous interaction with several RLKs. In order to understand the in vivo binding specificity of CLAVATA3/Embryo Surrounding Region-Related peptide family, we have developed a novel set of CLAVATA 3 (CLV3) based peptide tools. After carefully evaluating the CLE peptide binding characteristics, using solid phase synthesis process, we have modified the CLV3 peptide and attached a fluorophore and a photoactivable side group. We observed that the labeled CLV3 shows binding specificity within CLAVATA1 clade of RLKs while avoiding the distantly-related PEP RECEPTOR clade, thus resolving the contradictory results obtained previously by many in vitro methods. Furthermore, we observed that the RLK-bound CLV3 undergoes clathrin-mediated endocytosis and gets trafficked to vacuole via ARA7-labeled endosomes. Additionally, modifying CLV3 for light-controlled activation enabled spatial and temporal control over CLE signalling. Hence, our CLV3 macromolecular toolbox can be used to study rapid cell specific down-stream effects. Given the conserved binding properties, in the future our toolbox can also be used as a template to modify other CLE peptides.

Synthesis and structural optimization of oncolytic peptide LTX-315.

Oncolytic peptides represented potential novel candidates for anticancer treatments especially drug-resistant cancer cell lines. One of the most promising and extensively studied is LTX-315, which is considered as the first in class oncolytic peptide and has entered phase I/II clinical trials. Nevertheless, the shortcomings including poor proteolytic stability, moderate anticancer durability and high synthesis costs may hinder the widespread clinical applications of LTX-315. In order to reduce the synthesis costs, as well as develop derivatives possessing both high protease-stability and durable anticancer efficiency, twenty LTX-315-based derived-peptides were designed and efficiently synthesized. Especially, through solid-phase S-alkylation, as well as the optimized peptide cleavage condition, the derived peptides could be prepared with drastically reduced synthesis cost. The in vitro anticancer efficiency, serum stability, anticancer durability, anti-migration activity, and hemolysis effect were systematically investigated. It was found that derived peptide MS-13 exhibited comparable anticancer efficiency and durability to those of LTX-315. Strikingly, the D-type peptide MS-20, which is the enantiomer of MS-13, was demonstrated to possess significantly high proteolytic stability and sustained anticancer durability. In general, the cost-effective synthesis and stability-guided structural optimizations were conducted on LTX-315, affording the highly hydrolysis resistant MS-20 which possessed durable anticancer activity. Meanwhile, this study also provided a reliable reference for the future optimization of anticancer peptides through the solid-phase S-alkylation and L-type to D-type amino acid substitutions.

An Introductory Guide to Protease Sensitive Linker Design Using Matrix Metalloproteinase 13 as an Example.

Proteases play a crucial role, not only in physiological, but also in pathological processes, such as cancer, inflammation, arthritis, Alzheimer's, and infections, to name but a few. Their ability to cleave peptides can be harnessed for a broad range of biotechnological purposes. To do this efficiently, it is essential to find an amino acid sequence that meets the necessary requirements, including interdependent factors like specificity, selectivity, cleavage kinetics, or synthetic accessibility. Cleavage sequences from natural substrates of the protease may not be optimal in terms of specificity and selectivity, which is why these frequently require arduous and sometimes unsuccessful optimization such as by iterative exchange of single amino acids. Hence, here we describe the systematic design of protease sensitive linkers (PSLs)─peptide sequences specifically cleaved by a target protease─guided by the mass spectrometry based determination of target protease specific cleavage sites from a proteome-based peptide library. It includes a procedure for identifying bespoke PSL sequences, their optimization, synthesis, and validation and introduces a program that can indicate potential cleavage sites by hundreds of enzymes in any arbitrary amino acid sequence. Thereby, we provide an introduction to PSL design, illustrated by the example of matrix metalloproteinase 13 (MMP13). This introduction can serve as a guide and help to greatly accelerate the development and use of protease-sensitive linkers in diverse applications.

d-type peptides based fluorescent probes for "turn on" sensing of heparin.

Developing "turn on" fluorescent probes was desirable for the detection of the effective anticoagulant agent heparin in clinical applications. Through combining the aggregation induced emission (AIE) fluorogen tetraphenylethene (TPE) and heparin specific binding peptide AG73, the promising "turn on" fluorescent probe TPE-1 has been developed. Nevertheless, although TPE-1 could achieve the sensitive and selective detection of heparin, the low proteolytic stability and undesirable poor solubility may limit its widespread applications. In this study, seven TPE-1 derived fluorescent probes were rationally designed, efficiently synthesized and evaluated. The stability and water solubility were systematically estimated. Especially, to achieve real-time monitoring of proteolytic stability, the novel Abz/Dnp-based "turn on" probes that employ the internally quenched fluorescent (IQF) mechanism were designed and synthesized. Moreover, the detection ability of synthetic fluorescent probes for heparin were systematically evaluated. Importantly, the performance of d-type peptide fluorescent probe XH-6 indicated that d-type amino acid substitutions could significantly improve the proteolytic stability without compromising its ability of heparin sensing, and attaching solubilizing tag 2-(2-aminoethoxy) ethoxy) acid (AEEA) could greatly enhance the solubility. Collectively, this study not only established practical strategies to improve both the water solubility and proteolytic stability of "turn on" fluorescent probes for heparin sensing, but also provided valuable references for the subsequent development of enzymatic hydrolysis-resistant d-type peptides based fluorescent probes.

Chemical and Chemoenzymatic Synthesis of Peptide and Protein Therapeutics Conjugated with Human N-Glycans.

Glycosylation is one of the most ubiquitous post-translational modifications. It affects the structure and function of peptides/proteins and consequently has a significant impact on various biological events. However, the structural complexity and heterogeneity of glycopeptides/proteins caused by the diversity of glycan structures and glycosylation sites complicates the detailed elucidation of glycan function and hampers their clinical applications. To address these challenges, chemical and/or enzyme-assisted synthesis methods have been developed to realize glycopeptides/proteins with well-defined glycan morphologies. In particular, N-glycans are expected to be useful for improving the solubility, in vivo half-life and aggregation of bioactive peptides/proteins that have had limited clinical applications so far due to their short duration of action in the blood and unsuitable physicochemical properties. Chemical glycosylation performed in a post-synthetic procedure can be used to facilitate the development of glycopeptide/protein analogues or mimetics that are superior to the original molecules in terms of physicochemical and pharmacokinetic properties. N-glycans are used to modify targets because they are highly biodegradable and biocompatible and have structures that already exist in the human body. On the practical side, from a quality control perspective, close attention should be paid to their structural homogeneity when they are to be applied to pharmaceuticals.

Optimization of peptide synthesis time and sustainability using novel eco-friendly binary solvent systems with induction heating on an automated peptide synthesizer.

On December 12th, 2023, the European Commission took regulatory action to amend Annex XVII of REACH, imposing restrictions on the use of N,N-dimethylformamide (DMF) within the EU market owing to its high toxicity. Historically, DMF has been widely considered the gold standard for solid-phase peptide synthesis (SPPS). Being urgent to propose alternative solvents, we tested the suitability of non-hazardous neat and mixed solvents. Notably, binary solvent mixtures containing dimethyl sulfoxide as one of the solvent partners demonstrated high efficacy in solubilizing reagents while maintaining the desired swelling characteristics of common resins. A series of binary solvent mixtures were tested in automated SPPS, both at room temperature and high temperature, employing the PurePep® Chorus synthesizer, which enabled controlled induction heating between 25 and 90°C with oscillation mixing. The performances were assessed in challenging peptide sequences, i.e., ACP (65-74), and in longer and aggregating sequences like SARS-CoV-2 RBM (436-507) and ß-amyloid (1-42). Furthermore, as part of the proposed sustainable approach to minimize the utilization of hazardous solvents, we coupled the novel PurePep EasyClean catch-and-release purification technology. This work, addressing regulatory compliance, emphasizes the crucial role of green chemistry in advancing safer and more environmentally friendly practices in SPPS.

Facile Peptide Macrocyclization and Multifunctionalization via Cyclen Installation.

Cyclen-peptide bioconjugates are usually prepared in multiple steps that require individual preparation and purification of the cyclic peptide and hydrophilic cyclen derivatives. An efficient strategy is discovered for peptide cyclization and functionalization toward lanthanide probe via three components intermolecular crosslinking on solid-phase peptide synthesis with high conversion yield. Multifunctionality can be conferred by introducing different modular parts or/and metal ions on the cyclen-embedded cyclopeptide. As a proof-of-concept, a luminescent Eu3+ complex and a Gd3+-based contrasting agent for in vitro optical imaging and in vivo magnetic resonance imaging, respectively, are demonstrated through utilizing this preparation of cyclen-embedded cyclic arginylglycylaspartic acid (RGD) peptide.