Electrochemical synthesis of peptide aldehydes via C‒N bond cleavage of cyclic amines.

Peptide aldehydes are crucial biomolecules essential to various biological systems, driving a continuous demand for efficient synthesis methods. Herein, we develop a metal-free, facile, and biocompatible strategy for direct electrochemical synthesis of unnatural peptide aldehydes. This electro-oxidative approach enabled a step- and atom-economical ring-opening via C‒N bond cleavage, allowing for homoproline-specific peptide diversification and expansion of substrate scope to include amides, esters, and cyclic amines of various sizes. The remarkable efficacy of the electro-synthetic protocol set the stage for the efficient modification and assembly of linear and macrocyclic peptides using a concise synthetic sequence with racemization-free conditions. Moreover, the combination of experiments and density functional theory (DFT) calculations indicates that different N-acyl groups play a decisive role in the reaction activity.

Production of constrained L-cyclo-tetrapeptides by epimerization-resistant direct aminolysis.

The synthesis of constrained 12-membered rings is notably difficult. The main challenges result from constraints during the linear peptide cyclization. Attempts to overcome constraints through excessive activation frequently cause peptidyl epimerization, while insufficient activation of the C-terminus hampers cyclization and promotes intermolecular oligomer formation. We present a ß-thiolactone framework that enables the synthesis of cyclo-tetrapeptides via direct aminolysis. This tactic utilizes a mechanism that restricts C-terminal carbonyl rotation while maintaining high reactivity, thereby enabling efficient head-to-tail amidation, reducing oligomerization, and preventing epimerization. A broad range of challenging cyclo-tetrapeptides ( > 20 examples) are synthesized in buffer and exhibits excellent tolerance toward nearly all proteinogenic amino acids. Previously unattainable macrocycles, such as cyclo-L-(Pro-Tyr-Pro-Val), have been produced and identified as µ-opioid receptor (MOR) agonists, with an EC50 value of 2.5 nM. Non-epimerizable direct aminolysis offers a practical solution for constrained peptide cyclization, and the discovery of MOR agonist activity highlights the importance of overcoming synthetic challenges for therapeutic development.

Tetrazine cyclized peptides for one-bead-one-compound library: Synthesis and sequencing.

While most FDA-approved peptide drugs are cyclic, robust cyclization chemistry of peptides and the deconvolution of the cyclic peptide sequences using tandem mass spectrometry render cyclic peptide drug discovery difficult. In this chapter, the protocol for the successful synthesis of tetrazine-linked cyclic peptide library in solid phase, which shows both robust cyclization and easy sequence deconvolution, is described. The protocol for the linearization and cleavage of cyclic peptides from the solid phase by simple UV light irradiation, followed by accurate sequencing using tandem mass spectrometry, is described. We describe the troubleshooting for this dithiol bis-arylation reaction and for the successful cleavage of the aryl cyclic peptide into linear form. This method for efficient solid-phase macrocyclization can be used for the rapid production of loop-based peptides and screening for inhibition of protein-protein interactions, by using the covalent inverse electron-demand Diels Alder reaction to supplement the non-covalent interaction between a protein and its peptide binder, isolating highly selective peptides in the process.

Chemical cyclization of tyrosine-containing peptides via in situ generated triazolinedione peptides.

Tyr-derived cyclic peptide natural products are formed by enzymatic manifolds that oxidatively cross-link embedded phenolic side chains of tyrosine (Tyr) and 4-hydroxyphenylglycine residues during their controlled production. Bioactive Tyr-derived cyclic peptides, such as the arylomycins and vancomycins, continue to motivate the development of enzymatic and chemical strategies for their de novo assembly and modification. However, chemical access to these structurally diverse natural cycles can be challenging and step intensive. Therefore, we developed an oxidative procedure to selectively convert Tyr-containing N4-substituted 1,2,4-triazolidine-3,5-dione peptides (urazole peptides) into stable Tyr-linked cyclic peptides. We show that Tyr-containing urazole peptides are simple to prepare and convert into reactive N4-substituted 1,2,4-triazoline-3,5-dione peptides by oxidation, which then undergo spontaneous cyclization under mildly basic aqueous conditions to form a cross-linkage with the phenol side chain of embedded Tyr residues. Using this approach, we have demonstrated access to over 25 Tyr-linked cyclic peptides (3- to 11-residue cycles) with good tolerance of native residue side chain functionalities. Importantly, this method is simple to perform, and product formation can be quickly confirmed by mass spectrometric and 1H NMR spectroscopic analyses.

Computational Design of Cyclic Peptide Inhibitors of a Bacterial Membrane Lipoprotein Peptidase.

There remains a critical need for new antibiotics against multi-drug-resistant Gram-negative bacteria, a major global threat that continues to impact mortality rates. Lipoprotein signal peptidase II is an essential enzyme in the lipoprotein biosynthetic pathway of Gram-negative bacteria, making it an attractive target for antibacterial drug discovery. Although natural inhibitors of LspA have been identified, such as the cyclic depsipeptide globomycin, poor stability and production difficulties limit their use in a clinical setting. We harness computational design to generate stable de novo cyclic peptide analogues of globomycin. Only 12 peptides needed to be synthesized and tested to yield potent inhibitors, avoiding costly preparation of large libraries and screening campaigns. The most potent analogues showed comparable or better antimicrobial activity than globomycin in microdilution assays against ESKAPE-E pathogens. This work highlights computational design as a general strategy to combat antibiotic resistance.

Synthesis of Membrane-Permeable Macrocyclic Peptides via Imidazopyridinium Grafting.

Macrocyclic peptides (MPs) are a class of compounds that have been shown to be particularly well suited for engaging difficult protein targets. However, their utility is limited by their generally poor cell permeability and bioavailability. Here, we report an efficient solid-phase synthesis of novel MPs by trapping a reversible intramolecular imine linkage with a 2-formyl- or 2-keto-pyridine to create an imidazopyridinium (IP+)-linked ring. This chemistry is useful for the creation of macrocycles of different sizes and geometries, including head-to-side and side-to-side chain configurations. Many of the IP+-linked MPs exhibit far better passive membrane permeability than expected for "beyond Rule of 5" molecules, in some cases exceeding that of much lower molecular weight, traditional drug molecules. We demonstrate that this chemistry is suitable for the creation of libraries of IP+-linked MPs and show that these libraries can be mined for protein ligands.

Novel CCR3-targeted cyclic peptides as potential therapeutic agents for age-related macular degeneration via inhibiting angiogenesis and reducing retinal photoreceptor damage.

The prolonged intravitreal administration of anti-vascular endothelial growth factor (VEGF) drugs is prone to inducing aberrant retinal vascular development and causing damage to retinal neurons. Hence, we have taken an alternative approach by designing and synthesizing a series of cyclic peptides targeting CC motif chemokine receptor 3 (CCR3). Based on the binding mode of the N-terminal region in CCR3 protein to CCL11, we used computer-aided identification of key amino acid sequence, conformational restriction through different cyclization methods, designed and synthesized a series of target cyclic peptides, and screened the preferred compound IB-2 through affinity. IB-2 exhibits excellent anti-angiogenic activity in HRECs. The apoptosis level of 661W cells demonstrated a significant decrease with the escalating concentration of IB-2. This suggests that IB-2 may have a protective effect on photoreceptor cells. In vivo experiments have shown that IB-2 significantly reduces retinal vascular leakage and choroidal neovascularization (CNV) area in a laser-induced mouse model of CNV. These findings indicate the potential of IB-2 as a safe and effective therapeutic agent for AMD, warranting further development.

[Syntheses and Structure-Activity Relationship Studies of Antitumor Bicyclic Hexapeptide RA-VII Analogues].

A series of antitumor bicyclic hexapeptide RA-VII analogues modified at residue 2, 3, or 6 were prepared by the chemical transformation of the hydroxy, methoxy, or carboxy groups or the aromatic rings of natural peptides RA-II, III, V, VII, and X. Analogues with modified side chains or peptide backbones, which cannot be prepared by the chemical transformation of their natural peptides, and newly isolated peptides from Rubia cordifolia roots were synthesized by using protected cycloisodityrosines prepared by the degradation of bis(thioamide) obtained from RA-VII or the diphenyl ether formation of boronodipeptide under the modified Chan-Lam coupling reaction conditions. Studies of the conformational features of the analogues and the newly isolated peptides and their relationships with cytotoxic activities against the HCT-116, HL-60, KATO-III, KB, L1210, MCF-7, and P-388 cell lines revealed the following: the methoxy group at residue 3 is essential for the potent cytotoxic activity; the methyl group at Ala-2 and Ala-4 but not at D-Ala-1 is required to establish the bioactive conformation; the N-methyl group at Tyr-5 is necessary for the peptides to adopt the active conformation preferentially; and the orientation of Tyr-5 and/or Tyr-6 phenyl rings has a significant effect on the cytotoxic activity.

Enhancing anticancer treatment: Development of cRGD-Conjugated F-OH-Evo prodrugs for targeted delivery.

Small molecule drugs sourced from natural products are pivotal for novel therapeutic discoveries. However, their clinical deployment is often impeded by non-specific activity and severe adverse effects. This study focused on 3-fluoro-10-hydroxy-Evodiamine (F-OH-Evo), a potent derivative of Evodiamine, whose development is curtailed due to suboptimal tumor selectivity and heightened cytotoxicity. By harnessing the remarkable stability, specificity, and αvß3 integrin affinity of c(RGDFK), a novel prodrug by conjugating F-OH-Evo with cRGD was synthesized. This innovative prodrug substantially enhanced the tumor-specific targeting of F-OH-Evo and improved the anti-tumor activities. Among them, compound 3c demonstrated the best selective inhibitory activity toward U87 cancer cells in vitro. It selectively enterd U87 cells by binding to αvß3 integrin, releasing the parent molecule under the dual response of ROS and GSH to exert inhibitory activity on topo I. The results highlight the potential of cRGD-conjugated prodrugs in targeted cancer therapy. This approach signifies a significant advancement in developing safer and more effective chemotherapy drugs, emphasizing the role of prodrug strategies in overcoming the limitations of traditional cancer treatments.

Secondary Structure Stabilization of Macrocyclic Antimicrobial Peptides via Cross-Link Swapping.

Lactam cross-links have been employed to stabilize the helical secondary structure and enhance the activity and physiological stability of antimicrobial peptides; however, stabilization of ß-sheets via lactamization has not been observed. In the present study, lactams between the side chains of C- and N-terminal residues have been used to stabilize the ß-sheet conformation in a short ten-residue analogue of chicken angiogenin-4. Designed using a combination of molecular dynamics simulations and Markov state models, the lactam cross-linked peptides are shown to adopt stabilized ß-sheet conformations consistent with simulated structures. Replacement of the peptide side-chain Cys-Cys disulfide by a lactam cross-link enhanced the broad-spectrum antibacterial activity compared to the parent peptide and exhibited greater propensity to induce proinflammatory activity in macrophages. The combination of molecular simulations and conformational and biological analyses of the synthetic peptides provides a useful paradigm for the rational design of therapeutically active peptides with constrained ß-sheet structures.

Computational Design of Novel Cyclic Peptides Endowed with Autophagy-Inhibiting Activity on Cancer Cell Lines.

(1) Autophagy plays a significant role in development and cell proliferation. This process is mainly accomplished by the LC3 protein, which, after maturation, builds the nascent autophagosomes. The inhibition of LC3 maturation results in the interference of autophagy activation. (2) In this study, starting from the structure of a known LC3B binder (LIR2-RavZ peptide), we identified new LC3B ligands by applying an in silico drug design strategy. The most promising peptides were synthesized, biophysically assayed, and biologically evaluated to ascertain their potential antiproliferative activity on five humans cell lines. (3) A cyclic peptide (named Pep6), endowed with high conformational stability (due to the presence of a disulfide bridge), displayed a Kd value on LC3B in the nanomolar range. Assays accomplished on PC3, MCF-7, and A549 cancer cell lines proved that Pep6 exhibited cytotoxic effects comparable to those of the peptide LIR2-RavZ, a reference LC3B ligand. Furthermore, it was ineffective on both normal prostatic epithelium PNT2 and autophagy-defective prostate cancer DU145 cells. (4) Pep6 can be considered a new autophagy inhibitor that can be employed as a pharmacological tool or even as a template for the rational design of new small molecules endowed with autophagy inhibitory activity.

Synthesis of All-Peptide-Based Rotaxane from a Proline-Containing Cyclic Peptide.

Rotaxane cross-linkers enhance the toughness of the resulting rotaxane cross-linked polymers through a stress dispersion effect, which is attributed to the mobility of the interlocked structure. To date, the compositional diversity of rotaxane cross-linkers has been limited, and the poor compatibility of these cross-linkers with peptides and proteins has made their use in such materials challenging. The synthesis of a rotaxane composed of peptides may result in a biodegradable cross-linker that is compatible with peptides and proteins, allowing the fortification of polypeptides and proteins and ultimately leading to the development of innovative materials that possess excellent mechanical properties and biodegradability. However, the chemical synthesis of all-peptide-based rotaxanes has remained elusive because of the absence of strong binding motifs in peptides, which prevents an axial peptide from penetrating a cyclic peptide. Here, we synthesized all-peptide-based rotaxanes using an active template method for proline-containing cyclic peptides. The results of molecular dynamics simulations suggested that cyclic peptides with an expansive inner cavity and carbonyl oxygens oriented toward the center are favorable for rotaxane synthesis. This rotaxane synthesis method is expected to accelerate the synthesis of peptides and proteins with mechanically interlocked structures, potentially leading to the development of peptide- and protein-based materials with unprecedented functionalities.

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.

Large Libraries of Structurally Diverse Macrocycles Suitable for Membrane Permeation.

Macrocycles offer an attractive format for drug development due to their good binding properties and potential to cross cell membranes. To efficiently identify macrocyclic ligands for new targets, methods for the synthesis and screening of large combinatorial libraries of small cyclic peptides were developed, many of them using thiol groups for efficient peptide macrocyclization. However, a weakness of these libraries is that invariant thiol-containing building blocks such as cysteine are used, resulting in a region that does not contribute to library diversity but increases molecule size. Herein, we synthesized a series of structurally diverse thiol-containing elements and used them for the combinatorial synthesis of a 2,688-member library of small, structurally diverse peptidic macrocycles with unprecedented skeletal complexity. We then used this library to discover potent thrombin and plasma kallikrein inhibitors, some also demonstrating favorable membrane permeability. X-ray structure analysis of macrocycle-target complexes showed that the size and shape of the newly developed thiol elements are key for binding. The strategy and library format presented in this work significantly enhance structural diversity by allowing combinatorial modifications to a previously invariant region of peptide macrocycles, which may be broadly applied in the development of membrane permeable therapeutics.

Expansive discovery of chemically diverse structured macrocyclic oligoamides.

Small macrocycles with four or fewer amino acids are among the most potent natural products known, but there is currently no way to systematically generate such compounds. We describe a computational method for identifying ordered macrocycles composed of alpha, beta, gamma, and 17 other amino acid backbone chemistries, which we used to predict 14.9 million closed cycles composed of >42,000 monomer combinations. We chemically synthesized 18 macrocycles predicted to adopt single low-energy states and determined their x-ray or nuclear magnetic resonance structures; 15 of these were very close to the design models. We illustrate the therapeutic potential of these macrocycle designs by developing selective inhibitors of three protein targets of current interest. By opening up a vast space of readily synthesizable drug-like macrocycles, our results should considerably enhance structure-based drug design.

Protocol for photo-controlling the assembly of cyclic peptide nanotubes in solution and inside microfluidic droplets.

In this protocol, we describe how to perform the photo-isomerization of cyclic peptides containing an unsaturated ß-amino acid. This process triggers the formation or disassembly of cyclic peptide nanotubes under appropriate light irradiation. Specifically, we start by describing the solid-phase synthesis of the cyclic peptide component. We also present a technique for performing isomerization studies in solution and how to extend it to microfluidic aqueous droplets. For complete details on the use and execution of this protocol, please refer to Vilela-Picos et al.1.

Synthesis and Structural Revision of the Cyclic Hexapeptide Dimers Antatollamides A and B.

The synthesis and structural revision of the dimerized cyclic hexapeptides antatollamides A (1) and B (2) are reported. These are unique peptides with two proline residues and bicyclic peptides combined by a disulfide bond. Cyclization and disulfide bond formation of the linear peptide led to antatollamide A (1). However, the 1H and 13C NMR spectra of synthetic antatollamide A (1) were not consistent with those of isolated antatollamide A (1). Meanwhile, the NMR spectra of the monomeric cyclic hexapeptide cyclo(Pro-Pro-Phe-dCys-Ile-Val) (3) and the isolated antatollamide A (1) were identified completely. In addition, we found that isolated antatollamide B (2) is cyclo(Pro-Pro-dPhe-dCys-Ile-Val) (4).

Challenges with the Synthesis of a Macrocyclic Thioether Peptide: From Milligram to Multigram Using Solid Phase Peptide Synthesis (SPPS).

We describe an optimization and scale-up of the 45-membered macrocyclic thioether peptide BMS-986189 utilizing solid-phase peptide synthesis (SPPS). Improvements to linear peptide isolation, macrocyclization, and peptide purification were demonstrated to increase the throughput and purification of material on scale and enabled the synthesis and purification of >60 g of target peptide. Taken together, not only these improvements resulted in a 28-fold yield increase from the original SPPS approach, but also the generality of this newly developed SPPS purification sequence has found application in the synthesis and purification of other macrocyclic thioether peptides.

Process Development of a Macrocyclic Peptide Inhibitor of PD-L1.

This article outlines the process development leading to the manufacture of 800 g of BMS-986189, a macrocyclic peptide active pharmaceutical ingredient. Multiple N-methylated unnatural amino acids posed challenges to manufacturing due to the lability of the peptide to cleavage during global side chain deprotection and precipitation steps. These issues were exacerbated upon scale-up, resulting in severe yield loss and necessitating careful impurity identification, understanding the root cause of impurity formation, and process optimization to deliver a scalable synthesis. A systematic study of macrocyclization with its dependence on concentration and pH is presented. In addition, a side chain protected peptide synthesis is discussed where the macrocyclic protected peptide is extremely labile to hydrolysis. A computational study explains the root cause of the increased lability of macrocyclic peptide over linear peptide to hydrolysis. A process solution involving the use of labile protecting groups is discussed. Overall, the article highlights the advancements achieved to enable scalable synthesis of an unusually labile macrocyclic peptide by solid-phase peptide synthesis. The sustainability metric indicates the final preparative chromatography drives a significant fraction of a high process mass intensity (PMI).

Synthesis of Asp-based lactam cyclic peptides using an amide-bonded diaminodiacid to prevent aspartimide formation.

Asp-based lactam cyclic peptides are considered promising drug candidates. However, using Fmoc solid-phase peptide synthesis (Fmoc-SPPS) for these peptides also causes aspartimide formation, resulting in low yields or even failure to obtain the target peptides. Here, we developed a diaminodiacid containing an amide bond as a ß-carboxyl-protecting group for Asp to avoid aspartimide formation. The practicality of this diaminodiacid has been illustrated by the synthesis of lactam cyclic peptide cyclo[Lys9,Asp13] KIIIA7-14 and 1Y.