Library Of Macrocycles Research Articles

Solid-Phase Synthesis of Diverse Macrocycles by Regiospecific 2-Pyridone Formation: Scope and Applications.

This study introduces a novel solid-phase macrocyclization method generating 2-pyridone rings. This method relies on the intramolecular condensation between secondary and tertiary dimethoxy-propionic amide units. This selective reaction leads to the formation of a single well-defined regioisomer. The method demonstrates remarkable efficiency in producing diverse peptidic and nonpeptidic bioactive targets, paving the way for the development of innovative macrocycle libraries featuring the 2-pyridone unit.

Triply Adaptive Libraries of Dynamic Covalent Macrocycles: Switching between Sorted and Unsorted States.

Dynamic noncovalent and covalent chemistries have enabled the constitutional modulation of chemical entities within chemical dynamic systems. The switching between order and disorder, i.e., self-sorted and unsorted states of constitutional dynamic libraries, remains challenging. Herein, we study the adaptive behaviors of a dynamic library of imine macrocycles generated from dialdehydes and diamines, seeking ways to exert control over sorting and unsorting processes. The distribution of constituents in the present library of dynamic macrocycles is modulated in response to internal and external effectors (e.g., time, metal cations, and chemical fuels), resulting in the transient amplification of self-sorted constituents in out-of-equilibrium states. The present study showcases higher complexity in systems subject to multiple adaptation through the dynamic interconversion between singularity/order and diversity/disorder.

Bulking Up the Bay-Position Substituents Enables Enhanced Selectivity of Cs-Symmetric Boron Subphthalocyanine-Subnaphthalocyanine Hybrids.

The precise synthesis of subporphyrinoid hybrids with π-expanded topologies and unique material properties plays a promising role in the design of functional macrocycles. Easy, selective, and controllable routes to boron subphthalocyanine-subnaphthalocyanine hybrids, Bsub(Pc3-p-Ncp)s, are desirable for this purpose yet synthetically challenging due to random mixtures of Cs-, C3v-, and, in some cases, C1-symmetric compounds that form during traditional statistical mixed cyclotrimerizations. Herein, we addressed this issue by developing a sterically driven mixed cyclotrimerization with enhanced selectivity for the targeted Cs-symmetric hybrid and complete suppression of sterically crowded macrocyclic byproducts. This process, coupled with a rationally designed precursor bearing bulky phenyl substituents, enabled the synthesis and characterization of bay-position phenylated Ph2-(Rp)8Bsub(Pc2-Nc1) hybrids with halogens (Rp = Cl or F) in their peripheral isoindole rings. Reaction selectivity ranged between 59 and 72% with remarkable yields, significantly higher than that of conventional mixed cyclotrimerizations. These findings were augmented by theoretical calculations on precursor Lewis basicity as guiding principles into hybrid macrocycle formation. Additionally, the incorporation of unfused phenyl groups and halogen atoms into the hybrid framework resulted in fine-tuned optical, structural, electronic, and electrochemical properties. This straightforward approach achieved improved selectivity and controlled narrowing of the product distribution, affording the efficient synthesis of structurally sophisticated Bsub(Pc2-Nc1) hybrids. This then expands the library of 3-dimensional π-extended macrocycles for use in a range of applications, such as in optoelectronic devices with precisely tailored optical properties.

Promiscuity of Omphalotin A Biosynthetic Enzymes Allows de novo Production of Non-Natural Multiply Backbone N-Methylated Peptide Macrocycles in Yeast.

Multiple backbone N-methylation and macrocyclization improve the proteolytic stability and oral availability of therapeutic peptides. Chemical synthesis of such peptides is challenging, in particular for the generation of peptide libraries for screening purposes. Enzymatic backbone N-methylation and macrocyclization occur as part of both non-ribosomal and ribosomal peptide biosynthesis, exemplified by the fungal natural products cyclosporin A and omphalotin A, respectively. Omphalotin A, a 9fold backbone N-methylated dodecamer isolated from the agaricomycete Omphalotus olearius, can be produced in Pichia pastoris by coexpression of the ophMA and ophP genes coding for the peptide precursor protein harbouring an autocatalytic peptide α-N-methyltransferase domain, and a peptide macrocyclase, respectively. Since both OphMA and OphP were previously shown to be relatively promiscuous in terms of peptide substrates, we expressed mutant versions of ophMA, encoding OphMA variants with altered core peptide sequences, along with wildtype ophP and assessed the production of the respective peptide macrocycles by the platform by high-performance liquid chromatography, coupled with tandem mass spectrometry (HPLC-MS/MS). Our results demonstrate the successful production of fifteen non-natural omphalotin-derived macrocycles, containing polar, aromatic and charged residues, and, thus, suggest that the system may be used as biotechnological platform to generate libraries of non-natural multiply backbone N-methylated peptide macrocycles.

Employing unnatural promiscuity of sortase to construct peptide macrocycle libraries for ligand discovery

We report a sortase-based macrocyclization strategy in which low-reactive electrophiles are introduced into the SrtA recognition sequence (LPXTG) to enable the construction of phage-displayed peptide macrocycle libraries for ligand discovery.

Display‐Selektion eines Foldamer‐Peptid‐Makrozyklus‐Hybriden

AbstractDie Erweiterung der chemischen Vielfalt von Peptid‐Makrozyklenbibliotheken für die Display‐Selektion ist wünschenswert, um ihr Potenzial zur Bindung an biomolekulare Ziele zu verbessern. Wir haben nun eine erhebliche Erweiterung durch die Einbeziehung eines großen, aromatischen helikalen Foldamers umgesetzt. Es wurde ein helikales aromatisches Foldamer identifiziert, welches eine Flexizym‐vermittelte tRNA‐Acylierung durchläuft und in der Lage ist, die ribosomale Translation mit Ausbeuten durchzuführen, die ausreichend hoch sind, um eine mRNA‐Display‐Selektion von makrozyklischen‐Foldamer‐Peptid‐Hybriden zu vollziehen. Ein hybrider Makrozyklus mit nanomolarer Affinität an C‐lobe der E6AP HECT‐Domäne wurde ausgewählt, der eine stark konvergente Peptidsequenz aufwies. Eine Kristallstruktur und Moleküldynamik‐Simulationen haben gezeigt, dass sowohl das Peptid als auch das Foldamer in einer faszinierenden, reziproken Heftklammer‐artigen Art helikal sind. Die starke Konvergenz der Reste könnte durch ihre Beteiligung an spezifischen Wechselwirkungen mit dem Zielprotein erklärt werden. Das Foldamer stabilisiert die Peptidhelix durch Heftklammern und durch Kontakte mit wesentlichen Resten. Diese Ergebnisse stellen insgesamt eine erhebliche Erweiterung des chemischen Raums dar, der für die Display‐Selektion zur Verfügung steht, und unterstreichen die möglichen Vorteile des Einfügens eines aromatischen Foldamers in einen Peptidmakrozyklus zum Zwecke der Proteinerkennung.

Display Selection of a Hybrid Foldamer-Peptide Macrocycle.

Expanding the chemical diversity of peptide macrocycle libraries for display selection is desirable to improve their potential to bind biomolecular targets. We now have implemented a considerable expansion through a large aromatic helical foldamer inclusion. A foldamer was first identified that undergoes flexizyme-mediated tRNA acylation and that is capable of initiating ribosomal translation with yields sufficiently high to perform an mRNA display selection of macrocyclic foldamer-peptide hybrids. A hybrid macrocyclic nanomolar binder to the C-lobe of the E6AP HECT domain was selected that showed a highly converged peptide sequence. A crystal structure and molecular dynamics simulations revealed that both the peptide and foldamer are helical in an intriguing reciprocal stapling fashion. The strong residue convergence could be rationalized based on their involvement in specific interactions with the target protein. The foldamer stabilizes the peptide helix through stapling and through contacts with key residues. These results altogether represent a significant extension of the chemical space amenable to display selection and highlight possible benefits of inserting an aromatic foldamer into a peptide macrocycle for the purpose of protein recognition.

De Novo Sequencing of Synthetic Bis-cysteine Peptide Macrocycles Enabled by "Chemical Linearization" of Compound Mixtures.

A "chemical linearization" approach was applied to synthetic peptide macrocycles to enable their de novo sequencing from mixtures using nanoliquid chromatography-tandem mass spectrometry (nLC-MS/MS). This approach─previously applied to individual macrocycles but not to mixtures─involves cleavage of the peptide backbone at a defined position to give a product capable of generating sequence-determining fragment ions. Here, we first established the compatibility of "chemical linearization" by Edman degradation with a prominent macrocycle scaffold based on bis-Cys peptides cross-linked with the m-xylene linker, which are of major significance in therapeutics discovery. Then, using macrocycle libraries of known sequence composition, the ability to recover accurate de novo assignments to linearized products was critically tested using performance metrics unique to mixtures. Significantly, we show that linearized macrocycles can be sequenced with lower recall compared to linear peptides but with similar accuracy, which establishes the potential of using "chemical linearization" with synthetic libraries and selection procedures that yield compound mixtures. Sodiated precursor ions were identified as a significant source of high-scoring but inaccurate assignments, with potential implications for improving automated de novo sequencing more generally.

Pushing the Boundaries of Diversity: Innovative Design and Synthesis of Hydrazone-Based Dynamic Combinatorial Libraries of Macrocycles

Pushing the Boundaries of Diversity: Innovative Design and Synthesis of Hydrazone-Based Dynamic Combinatorial Libraries of Macrocycles

Adapting antibacterial display to identify serum-active macrocyclic peptide antibiotics.

The lack of available treatments for many antimicrobial-resistant infections highlights the critical need for antibiotic discovery innovation. Peptides are an underappreciated antibiotic scaffold because they often suffer from proteolytic instability and toxicity toward human cells, making in vivo use challenging. To investigate sequence factors related to serum activity, we adapt an antibacterial display technology to screen a library of peptide macrocycles for antibacterial potential directly in human serum. We identify dozens of new macrocyclic peptide antibiotic sequences and find that serum activity within our library is influenced by peptide length, cationic charge, and the number of disulfide bonds present. Interestingly, an optimized version of our most active lead peptide permeates the outer membrane of Gram-negative bacteria without strong inner-membrane disruption and kills bacteria slowly while causing cell elongation. This contrasts with traditional cationic antimicrobial peptides, which kill rapidly via lysis of both bacterial membranes. Notably, this optimized variant is not toxic to mammalian cells and retains its function in vivo, suggesting therapeutic promise. Our results support the use of more physiologically relevant conditions when screening peptides for antimicrobial activity which retain in vivo functionality.

Macrocyclizing DNA-Linked Peptides via Three-Component Cyclization and Photoinduced Chemistry.

While DNA-encoded macrocyclic libraries have gained substantial attention and several hit compounds have been identified from DNA-encoded library technology, efficient on-DNA macrocyclic methods are also required to construct DNA-linked libraries with a high degree of cyclization and DNA integrity. In this paper, we reported a set of on-DNA methodologies, including the use of an OPA-mediated three-component cyclization with native handles of amino acids and photoredox chemistries. These chemistries proceed smoothly under mild conditions in good to excellent conversions, successfully generating novel isoindole, isoindoline, indazolone, and bicyclic scaffolds.

Synthesis of a DNA-Encoded Macrocyclic Library Utilizing Intramolecular Benzimidazole Formation.

Macrocycles occupy chemical space "beyond the rule of five". They bridge traditional bioactive small molecule drugs and macromolecules and have the potential to modulate challenging targets such as PPI or proteases. Here we report an on-DNA macrocyclization reaction utilizing intramolecular benzimidazole formation. A 129-million-member macrocyclic library composed of a privileged benzimidazole core, a dipeptide sequence (natural or non-natural), and linkers of varying length and flexibility was designed and synthesized.

Solid Phase Synthesis of Fluorosulfate Containing Macrocycles for Chemoproteomic Workflows

AbstractMacrocyclic peptides are attractive for chemoproteomic applications due to their modular synthesis and potential for high target selectivity. We describe a solid phase synthesis method for the efficient generation of libraries of small macrocycles that contain an electrophile and alkyne handle. The modular synthesis produces libraries that can be directly screened using simple SDS‐PAGE readouts and then optimal lead molecules applied to proteomic analysis. We generated a library of 480 macrocyclic peptides containing the weakly reactive fluorosulfate (OSF) electrophile. Initial screening of a subset of the library containing each of the various diversity elements identified initial molecules of interest. The corresponding positional and confirmational isomers were then screened to select molecules that showed specific protein labeling patterns that were dependent on the probe structure. The most promising hits were applied to standard chemoproteomic workflows to identify protein targets. Our results demonstrate the feasibility of rapid, on‐resin synthesis of diverse macrocyclic electrophiles to generate new classes of covalent ligands.

Photoresponsive Dissipative Macrocycles Using Visible-Light-Switchable Azobenzenes.

Visible light can be used to shift dynamic covalent imine assemblies out of equilibrium. We studied a fluorinated azobenzene building block that reliably undergoes geometric isomerism upon irradiation. The building block was used in combination with two different amines, ethylenediamine and R,R-1,2-diaminocyclohexane, to create a library of imine macrocycles. Whereas the simple amine can be used to access a polymeric state and a defined bowl-shaped macrocycle, the chiral amine gives access to a rich network of macrocycles that undergo both isomerisation as well as interconversion between different macrocyclic species, thereby allowing for control over the number of monomers involved in the cyclo-oligomerization; 1 H- and 19 F-DOSY NMR, MALDI-MS measurements, and UV/Vis spectroscopy were used to study the processes.

Crystallization- and Metal-Driven Selection of Discrete Macrocycles/Cages and Their Metallosupramolecular Polymers from Dynamic Systemic Networks

Reversible imine- and metal-coordination reactions are dynamic enough to produce complex libraries of macrocycles, cages, and supramolecular polymers in solution, from which amplification effects have been identified in solution or during crystallization in response to ligand- and metal-driven selection modes. Crystallization-driven selection can lead to the amplification of unexpected metallosupramolecular architectures. The addition of Ag+ triggered the change of the optimal components, so that the crystallization process showed different ligand preferences than in solution. The most packed constituents are amplified in the solid state, taking into account the optimal coordination of metal ions together with non-specific non-covalent interactions between the macrocycle packed in dimers or trimers in the solid state.

Synthesis and direct assay of large macrocycle diversities by combinatorial late-stage modification at picomole scale

Macrocycles have excellent potential as therapeutics due to their ability to bind challenging targets. However, generating macrocycles against new targets is hindered by a lack of large macrocycle libraries for high-throughput screening. To overcome this, we herein established a combinatorial approach by tethering a myriad of chemical fragments to peripheral groups of structurally diverse macrocyclic scaffolds in a combinatorial fashion, all at a picomole scale in nanoliter volumes using acoustic droplet ejection technology. In a proof-of-concept, we generate a target-tailored library of 19,968 macrocycles by conjugating 104 carboxylic-acid fragments to 192 macrocyclic scaffolds. The high reaction efficiency and small number of side products of the acylation reactions allowed direct assay without purification and thus a large throughput. In screens, we identify nanomolar inhibitors against thrombin (Ki = 44 ± 1 nM) and the MDM2:p53 protein-protein interaction (Kd MDM2 = 43 ± 18 nM). The increased efficiency of macrocycle synthesis and screening and general applicability of this approach unlocks possibilities for generating leads against any protein target.

Aza-Oxa-Triazole Based Macrocycles with Tunable Properties: Design, Synthesis, and Bioactivity.

A modular platform for the synthesis of tunable aza-oxa-based macrocycles was established. Modulations in the backbone and the side-chain functional groups have been rendered to achieve the tunable property. These aza-oxa-based macrocycles can also differ in the number of heteroatoms in the backbone and the ring size of the macrocycles. For the proof of concept, a library of macrocycles was synthesized with various hanging functional groups, different combinations of heteroatoms, and ring sizes in the range of 17–27 atoms and was characterized by NMR and mass spectrometry. In light of the importance of the copper-catalyzed azide-alkyne cycloaddition (CuAAC) reaction and the significance of triazole groups for various applications, we employed the click-reaction-based macrocyclization. The competence of the synthesized macrocycles in various biomedical applications was proven by studying the interactions with the serum albumin proteins; bovine serum albumin and human serum albumin. It was observed that some candidates, based on their hanging functional groups and specific backbone atoms, could interact well with the protein, thus improving the bioactive properties. On the whole, this work is a proof-of-concept to explore the backbone- and side-chain-tunable macrocycle for different properties and applications.

Noninvasive and Individual-Centered Monitoring of Uric Acid for Precaution of Hyperuricemia via Optical Supramolecular Sensing.

Characterized by an excessively increased uric acid (UA) level in serum, hyperuricemia induces gout and also poses a great threat to renal and cardiovascular systems. It is urgent and meaningful to perform early warning by noninvasive diagnosis, thus conducing to blockage of disease aggravation. Here, guanidinocalix[5]arene (GC5A) is successfully identified from the self‐built macrocyclic library to specifically monitor UA from urine by the indicator displacement assay. UA is strongly bound to GC5A at micromolar‐level, while simultaneously excluding fluorescein (Fl) from the GC5A·Fl complex in the “switch‐on” mode. This method successfully differentiates patients with hyperuricemia from volunteers except for those with kidney dysfunction and targets a volunteer at high risk of hyperuricemia. In order to meet the trend from hospital‐centered to individual‐centered testing, visual detection of UA is studied through a smartphone equipped with a color‐scanning feature, whose adaptability and feasibility are demonstrated in sensing UA from authentic urine, leading to a promising method in family‐centered healthcare style. A high‐throughput and visual detection method is provided here for alarming hyperuricemic by noninvasive diagnosis.

Design, synthesis and bioactive properties of a class of macrocycles with tunable functional groups and ring size

The design and synthesis of a versatile class of macrocycles with tunable functional groups and ring size are unfolded. Herein, a synthetic strategy is reported to furnish a new class of macrocycles in multi-gram scale in a two-step reaction. The total time taken for synthesizing a macrocycle is 1.5 h. Dithiocarbamates, an important functional group in biomedical and material sciences, is strategically incorporated in the macrocyclic backbone without metal for the first time. It is noteworthy that when state-of-the-art macrocycle synthesis is in millimolar concentration, this work employs the reaction in molar concentration (0.2–0.4 M). As proof-of-principle, a library of macrocycles was synthesized, varying the functional groups and ring size. The physicochemical properties of macrocycles revealed their druggable nature and are affirmed by protein (serum albumin) interaction study theoretically and experimentally. Diverse functional groups and ring sizes of macrocycles brought about twenty-five-fold difference in binding constant with the model protein.

Synthesis and Screening of a DNA-Encoded Library of Non-Peptidic Macrocycles.

There is considerable interest in the development of libraries of non‐peptidic macrocycles as a source of ligands for difficult targets. We report here the solid‐phase synthesis of a DNA‐encoded library of several hundred thousand thioether‐linked macrocycles. The library was designed to be highly diverse with respect to backbone scaffold diversity and to minimize the number of amide N−H bonds, which compromise cell permeability. The utility of the library as a source of protein ligands is demonstrated through the isolation of compounds that bind Streptavidin, a model target, with high affinity.