Linear Precursor Research Articles

Preparation of poly(oxanorbornene) based single and double-folding polymers via nucleophilic aromatic substitution reaction

A facile approach is introduced to prepare single-chain nanoparticles (SCNPs) utilizing intramolecular crosslinking based on nucleophilic aromatic substitution reaction. The linear polymeric precursor comprising dichlorotriazine (DCT) moiety as a reactive pendant group is synthesized via ring-opening metathesis polymerization (ROMP). The reactive chlorine atoms on DCT motifs are substituted with a dithiol nucleophile in varying degrees to yield individually intramolecularly crosslinked polymer nanoparticles. In addition, double-collapse and modification reactions are exploited through the substitution reaction from the remaining unreacted chlorine atoms. A significant reduction in hydrodynamic volumes of the corresponding SCNPs is observed compared to their linear analogues. The obtained nanoparticles are characterized by size exclusion chromatography (SEC), proton nuclear magnetic resonance spectroscopy (1H NMR), diffusion-ordered spectroscopy (DOSY), dynamic light scattering (DLS), and transmission electron microscopy (TEM) techniques.

Structure of Single-Chain Nanoparticles under Crowding Conditions: A Random Phase Approximation Approach.

The conformation of poly(methyl methacrylate) (PMMA)-based single-chain nanoparticles (SCNPs) and their corresponding linear precursors in the presence of deuterated linear PMMA in deuterated dimethylformamide (DMF) solutions has been studied by small-angle neutron scattering (SANS). The SANS profiles were analyzed in terms of a three-component random phase approximation (RPA) model. The RPA approach described well the scattering profiles in dilute and crowded solutions. Considering all the contributions of the RPA leads to an accurate estimation of the single chain form factor parameters and the Flory-Huggins interaction parameter between PMMA and DMF. The value of the latter in the dilute regime indicates that the precursors and the SCNPs are in good solvent conditions, while in crowding conditions, the polymer becomes less soluble.

Two types of coumarins-specific enzymes complete the last missing steps in pyran- and furanocoumarins biosynthesis

Pyran- and furanocoumarins are key representatives of tetrahydropyrans and tetrahydrofurans, respectively, exhibiting diverse physiological and medical bioactivities. However, the biosynthetic mechanisms for their core structures remain poorly understood. Here we combined multiomics analyses of biosynthetic enzymes in Peucedanum praeruptorum and in vitro functional verification and identified two types of key enzymes critical for pyran and furan ring biosynthesis in plants. These included three distinct P. praeruptorum prenyltransferases (PpPT1–3) responsible for the prenylation of the simple coumarin skeleton 7 into linear or angular precursors, and two novel CYP450 cyclases (PpDC and PpOC) crucial for the cyclization of the linear/angular precursors into either tetrahydropyran or tetrahydrofuran scaffolds. Biochemical analyses of cyclases indicated that acid/base-assisted epoxide ring opening contributed to the enzyme-catalyzed tetrahydropyran and tetrahydrofuran ring refactoring. The possible acid/base-assisted catalytic mechanisms of the identified cyclases were theoretically investigated and assessed using site-specific mutagenesis. We identified two possible acidic amino acids Glu303 in PpDC and Asp301 in PpOC as vital in the catalytic process. This study provides new enzymatic tools in the epoxide formation/epoxide-opening mediated cascade reaction and exemplifies how plants become chemically diverse in terms of enzyme function and catalytic process.

Multicopper Clusters Enable Oxidative Phenol Macrocyclization (OxPM) of Peptides.

The biosynthesis of glycopeptide antibiotics such as vancomycin and other biologically active biaryl-bridged and diaryl ether-linked macrocyclic peptides includes key enzymatic oxidative phenol macrocyclization(s) of linear precursors. However, a simple and step-economical biomimetic version of this transformation remains underdeveloped. Here, we report highly efficient conditions for preparing biaryl-bridged and diaryl ether-linked macrocyclic peptides based on multicopper(II) clusters. The selective syntheses of ring models of vancomycin and the arylomycin cyclic core illustrate the potential of this technology to facilitate the assembly of complex antibiotic macrocyclic peptides, whose syntheses are considered highly challenging. The unprecedented ability of multicopper(II) clusters to chelate tethered diphenols and promote intramolecular over intermolecular coupling reactions demonstrates that copper clusters can catalyze redox transformations that cannot be accessed by smaller metal catalysts.

Tuning the morphology of silicon containing copolymers via macromolecular architecture effect

We demonstrate the successful synthesis of linear and non-linear star block copolymers consisting of the highly segregated polystyrene (PS) and poly(dimethylsiloxane) (PDMS) blocks through sequential anionic polymerization combined with chlorosilane chemistry. To study the macromolecular architecture effect on the adopted morphology star block copolymers with six equivalent diblock arms [(PS-b-PDMS)6] were synthesized and compared with their linear diblock copolymer precursors as well as with the corresponding star block copolymers with smaller arm number (2, 3, 4) but identical molecular characteristics. After verifying the molecular and thermal characteristics, bulk studies were carried out through transmission electron microscopy and small angle X-ray scattering. Well-defined structures with enhanced order and similar domain spacing values, irrespective of the architecture complexity of the samples, were observed. The better order of star block copolymers suggests an alternative way to further reduce the lower limit of repeating period by tuning the architecture which is important for the potential future use of the copolymers for nanotechnology related applications. The findings of the specific study are related to the effects of the increased conformational restriction of the inner block. Herein, we show morphological transformations in the (PS-b-PDMS)6 copolymers having non symmetric volume fraction ratios when compared to their linear diblock precursors and triblock copolymers which has not been observed in previous relevant (PS-b-PDMS)n studies that involved stars with less than six arms. To establish the structure/property relationship as a function of arm number, a series of samples involving the diblock precursor, the triblock, the three-, four- and six- arm star block copolymers was prepared, and a systematic comparison was conducted.

Molecular dynamics of poly(methyl methacrylate) single-chain nanoparticles

Polymer Single-chain nanoparticles (SCNPs) with different cross-linking densities were synthetized from poly (methyl methacrylate-co-(2-acetoacetoxy) ethyl methacrylate) (PMMA-co-PAEMA) random copolymers, and the molecular dynamics of SCNPs was investigated using differential scanning calorimetry (DSC) and broadband dielectric spectroscopy (BDS). The DSC results show that the glass transition temperature (Tg) of SCNPs formed after intra-molecular cross-linking is higher than that of linear precursor, and increases with increasing the cross-linking density. The BDS results reveal that the segment (α) relaxation of SCNPs is significantly inhibited, as evidenced by an increase in the relaxation time and a decrease in the dielectric strength. This is due to the intra-molecular topological constraints which limits the mobility of the polymer segments. Moreover, the distribution width of α relaxation is broader, indicating a more pronounced dynamical heterogeneity. The relaxation time of Johari-Goldstein β (βJG) relaxation increases slightly, neither the dynamics nor the peak width of βJG relaxation is affected by intra-molecular cross-linking.

Preparing cyclic polymers at high concentrations via self-folding cyclization technique by adjusting the contents of hydrophilic units in linear precursors

Cyclic polymers are a class of polymers that feature endless topology, and the synthesis of cyclic polymers has attracted the attention of many researchers. Herein, cyclic polymers were efficiently constructed by self-folding cyclization technique at high concentrations. Linear poly((oligo(ethylene glycol)acrylate)-co-(dodecyl acrylate)) (P(OEGA-co-DDA)) precursors with different ratios of hydrophilic and hydrophobic moieties were synthesized by reversible addition-fragmentation chain transfer (RAFT) polymerization using a bifunctional chain transfer agent with two anthryl end groups. The amphiphilic linear precursors underwent the self-folding process to generate polymeric nanoparticles in water. By irradiating the aqueous solution of the nanoparticles with 365 nm UV light, cyclic polymers were synthesized successfully via coupling of anthryl groups. The effects of the ratios of hydrophilic and hydrophobic moieties in linear P(OEGA-co-DDA) copolymers and polymer concentration on the purity of the obtained cyclic polymers were explored in detail via1H nuclear magnetic resonance (1H NMR), dynamic light scattering (DLS), UV‒visible (vis) analysis, three-detection size exclusion chromatography (TD-SEC) and transmission electron microscopy (TEM). It was found that by adjusting the content of the hydrophilic segments in linear precursors, single chain polymeric nanoparticles (SCPNs) can be generated at high polymer concentrations. Therefore, cyclic polymers with high purity can be constructed efficiently. This method overcomes the limitation of traditional ring-closure method, which is typically conducted in highly dilute conditions, providing an efficient method for the scalable preparation of cyclic polymers.

Genome Mining and Discovery of Imiditides, a Family of RiPPs with a Class-Defining Aspartimide Modification.

Ribosomally synthesized and post-translationally modified peptides (RiPPs) are a large and diverse class of natural products of ribosomal origin. In the past decade, various sophisticated machine-learning-based software packages have been established to discover novel RiPPs that do not resemble the known families. Here, we show that tailoring enzymes that cluster with various RiPP families can serve as effective bioinformatic seeds, providing a complementary approach for novel RiPP discovery. Leveraging the fact that O-methyltransferases homologous to protein isoaspartyl methyltransferases (PIMTs) are associated with lasso peptide, graspetide, and lanthipeptide biosynthetic gene clusters (BGCs), we utilized a C-terminal motif unique to RiPP-associated O-methyltransferases as the search query to discover a novel family of RiPPs, the imiditides. Our genome-mining algorithm reveals a total of 670 imiditide BGCs, distributed across Gram-positive bacterial genomes. In addition, we demonstrate the heterologous production of the founding member of the imiditide family, mNmaAM, encoded in the genome of Nonomuraea maritima. In contrast to other RiPP-associated PIMTs that recognize constrained peptides as substrates, the PIMT homologue in the mNmaAM BGC, NmaM, methylates a specific Asp residue on the linear precursor peptide, NmaA. The methyl ester is then turned into an aspartimide spontaneously. Substrate specificity is achieved by extensive charge-charge interactions between the precursor NmaA and the modifying enzyme NmaM suggested by both experiments and an AlphaFold model prediction. Our study shows that PIMT-mediated aspartimide formation is an emerging backbone modification strategy in the biosynthesis of multiple RiPP families.

Tetradehydro-Diels-Alder Reactions of Flexible Arylalkynes via Folding Inside a Molecular Cage.

The tetradehydro-Diels-Alder (TDDA) reaction is a useful transformation for the rapid assembly of polycyclic scaffolds from simple linear precursors in a single synthetic step. However, the reaction requires careful substrate design and harsh reaction conditions to overcome the inherent entropic cost for this transformation. Herein we report an efficient site-selective TDDA transformation within a self-assembled Pd6L4 cage. Despite the large size, the flexibility of the employed substrates allows for efficient encapsulation within the host cavity. The rate of thermal cyclization of the encapsulated guest was found to be greatly enhanced, and high product selectivity for an unsymmetrical substrate was observed. The efficiency of this system relies on the precise conformational control of the substrate within the confined space of the host cage.

Strategies for preventing gelation in preparation of hyperbranched benzoxazines with high performance

The gelation of linear oligomeric benzoxazine precursors (OBPs) and high-molecular-weight benzoxazine precursors (HMWBPs) have been noticed and addressed by various effective actions, while no similar reports on hyperbranched benzoxazoles have been available. A series of hyperbranched benzoxazines were synthesized by A2 + B3 method using 1,3-bis(3-aminopropyl)tetramethyldisiloxane (BATSi) and 1,1,1-tris(4-hydroxyphenyl)ethane (THPE) and paraformaldehyde, namely HB-TSis, to examine the conditions for preventing gelation. We found that in most cases, increasing the feeding molar ratio of -OH to NH2 group, prolonging the reaction time and increasing the reactant concentration were all favorable for the formation of soluble HB-TSis. Except when the molar ratio of -OH to NH2 was 2:3, a lower reactant concentration was more likely to avoid gel formation. A sufficiently high vacuum degree during drying was reported for the first time as a factor affecting the gelation of polybenzoxazine precursors. In addition, gelation-free HB-TSis exhibited high degrees of branching (DBs) and weight-average molecular weight (Mw) in the range of 0.58–0.69 and 6100–10,100 Da, respectively. Their cured resins, poly(HB-TSi)s, both perform well in terms of thermal stability with high char yield (Yc) varying from 37.9 to 56.2%, and flame retardancy with low the heat release capacity (HRC) varying from 49.9 to 81.9 J/(g K), which are capable of being applied as nonignitable materials.

Robust Chemoenzymatic Synthesis of Keratinimicin Aglycone Analogues Facilitated by the Structure and Selectivity of OxyB.

The emergence of multidrug-resistant pathogens poses a threat to public health and requires new antimicrobial agents. As the archetypal glycopeptide antibiotic (GPA) used against drug-resistant Gram-positive pathogens, vancomycin provides a promising starting point. Peripheral alterations to the vancomycin scaffold have enabled the development of new GPAs. However, modifying the core remains challenging due to the size and complexity of this compound family. The recent successful chemoenzymatic synthesis of vancomycin suggests that such an approach can be broadly applied. Herein, we describe the expansion of chemoenzymatic strategies to encompass type II GPAs bearing all aromatic amino acids through the production of the aglycone analogue of keratinimicin A, a GPA that is 5-fold more potent than vancomycin against Clostridioides difficile. In the course of these studies, we found that the cytochrome P450 enzyme OxyBker boasts both broad substrate tolerance and remarkable selectivity in the formation of the first aryl ether cross-link on the linear peptide precursors. The X-ray crystal structure of OxyBker, determined to 2.8 Å, points to structural features that may contribute to these properties. Our results set the stage for using OxyBker broadly as a biocatalyst toward the chemoenzymatic synthesis of diverse GPA analogues.

Teraryl Braces in Macrocycles: Synthesis and Conformational Landscape Remodeling of Peptides.

The three-dimensional structure of medium-sized cyclic peptides accounts for their biological activity and other important physiochemical properties. Despite significant advances in the past few decades, chemists' ability to fine-tune the structure, in particular, the backbone conformation, of short peptides made of canonical amino acids is still quite limited. Nature has shown that cross-linking the aromatic side chains of linear peptide precursors via enzyme catalysis can generate cyclophane-braced products with unusual structures and diverse activities. However, the biosynthetic path to these natural products is challenging to replicate in the synthetic laboratory using practical chemical modifications of peptides. Herein, we report a broadly applicable strategy to remodel the structure of homodetic peptides by cross-linking the aromatic side chains of Trp, His, and Tyr residues with various aryl linkers. The aryl linkers can be easily installed via copper-catalyzed double heteroatom-arylation reactions of peptides with aryl diiodides. These aromatic side chains and aryl linkers can be combined to form a large variety of assemblies of heteroatom-linked multi-aryl units. The assemblies can serve as tension-bearable multijoint braces to modulate the backbone conformation of peptides as an entry to previously inaccessible conformational space.

Photo-induced synthesis, stereochemistry and antitumor activity of natural cyclopeptide Hikiamide B analogs

Peptide cyclization has been recognized as capable of overcoming the shortcomings of linear peptides such as poor stability, short half-life, low selectivity and low affinity to receptors, and the modification of cyclic peptides to achieve specific activity requirements is receiving increasing attention. Hikiamide B is a cyclic pentadepsipeptide that can induce the expression of peroxisome-proliferator-activated receptor-γ (PPAR-γ). Considering that PPAR-γ can effectively regulate the expression of genes related to tumorigenesis, and is a potential tumor target, in this paper, we have prepared several Hikiamide B analogs by intramolecular photo-induced single-electron-transfer (SET) reaction to find novel antitumor drugs The stereochemistry and the in vitro antitumor activity against HepG-2 and HeLa cells were investigated. We found that the compound with stronger intramolecular hydrogen bonds (compound 2) than other compounds showed more promising antitumor activity, and the cyclic compounds present stronger bioactivity than that of their linear precursors. In vitro studies of apoptosis mechanisms and mitochondrial membrane potential following cyclopeptide 2 treat have shown that the cyclopeptide may induce apoptosis in tumor cells. Finally, in silico ADME analysis has shown the potential of compound 2 for practical application.

Enantioselective Synthesis of Planar Chiral Macrocyclic Metacyclophanes by Pd-Catalyzed C–O Cross-Coupling

Despite the presence of planar chiral elements in natural products and in ligands for asymmetric catalysis, catalytic enantioselective methods for installing planar chirality remain underdeveloped relative to methods for installing central and axial chirality. Here, we report an enantioselective Pd-catalyzed C–O bond-forming macrocyclization to access enantioenriched planar chiral macrocyclic metacyclophanes incorporating 2,3,4-trisubstituted pyridines. A variety of bridging chains and substituents can be incorporated in the metacyclophanes under the same reaction conditions (14 examples, 59–85% yields, 79–92% ee) to generate enantioenriched meta-, metaortho-, and metapara- cyclophanes. The syn-/anti-conformational preferences of metacyclophanes were elucidated by the X-ray structures, 1H NMR analyses, and computational studies. We also found that the two enantiomers of the metacyclophane can be obtained using the same enantiomer of the chiral ligand by starting from two regioisomeric linear precursors. This work provides an entry into the design of enantioselective cross-coupling macrocyclizations to access planar chiral structures for applications in medicinal chemistry and catalysis.

Tactics targeting circular mRNA biosynthesis.

Faced with the development of mRNA technology in the field of medicine and vaccine, circular mRNA (circmRNA) becomes a strong alternative to mRNA for its circular secondary structure and higher stability. At present, the synthesis of circmRNAs has been realized by ligating linear mRNA precursors and is limited by poor efficiency. To solve this challenge, this study started with ribozyme catalysis and enzymatic reaction to explore different circmRNA biosynthesis strategies. In terms of ribozyme method, by screening different group I intron self-splicing system sequences, the sequence from thymidylate synthase (Td) gene of phage T4 showed the highest ligation efficiency. In terms of enzyme method, with the help of 20-bp homologous arm, T4 Rnl 2 was determined as the ligation method with the highest ligation efficiency. By comparing the two ligation methods, the expression level of circmRNA ligated by T4 Rnl 2 was 86% higher than that ligated by Td ribozyme. Based on these ligation methods, the screening results of internal ribosome entry site (IRES) sequences showed that mud crab dicistrovirus IRES was an IRES sequence with high ribosome binding ability and could be widely used in circmRNAs for efficient and stable translation in mammalian cells. These results should provide positive guidance for the industrial production of circmRNAs and the development of mRNA vaccines. Eventually, circmRNAs could widely function in the field of biomedicine.

Cloning and functional verification of PhAEP gene, a key enzyme for biosynthesis of heterophyllin A in Pseudostellaria heterophylla

This paper aimed to study the role of asparagine endopeptidase(AEP) gene in the biosynthesis mechanism of cyclic peptide compounds in Pseudostellaria heterophylla. The transcriptome database of P. heterophylla was systematically mined and screened, and an AEP gene, tentatively named PhAEP, was successfully cloned. The heterologous function verification by Nicotiana benthamiana showed that the expression of the gene played a role in the biosynthesis of heterophyllin A in P. heterophylla. Bioinformatics analysis showed that the cDNA of PhAEP was 1 488 bp in length, encoding 495 amino acids with a molecular weight of 54.72 kDa. The phylogenetic tree showed that the amino acid sequence encoded by PhAEP was highly similar to that of Butelase-1 in Clitoria ternatea, reaching 80%. The sequence homology and cyclase active site analysis revealed that the PhAEP enzyme may specifically hydrolyse the C-terminal Asn/Asp(Asx) site of the core peptide in the HA linear precursor peptide of P. heterophylla, thereby participating in the ring formation of the linear precursor peptide. The results of real-time quantitative polymerase chain reaction(RT-qPCR) showed that the expression level of PhAEP was the highest in fruits, followed by in roots, and the lowest in leaves. The heterophyllin A of P. heterophylla was detected in N. benthamiana that co-expressed PrePhHA and PhAEP genes instantaneously. In this study, the PhAEP gene, a key enzyme in the biosynthesis of heterophyllin A in P. heterophylla, has been successfully cloned, which lays a foundation for further analysis of the molecular mechanism of PhAEP enzyme in the biosynthesis of heterophyllin A in P. heterophylla and has important significance for the study of synthetic biology of cyclic peptide compounds in P. heterophylla.

Exploring the Limits of Cyanobactin Macrocyclase PatGmac: Cyclization of PawS-Derived Peptide Sunflower Trypsin Inhibitor-1 and Cyclotide Kalata B1.

The subtilisin-like macrocyclase PatGmac is produced by the marine cyanobacterium Prochloron didemni. This enzyme is involved in the last step of the biosynthesis of patellamides, a cyanobactin type of ribosomally expressed and post-translationally modified cyclic peptides. PatGmac recognizes, cleaves, and cyclizes precursor peptides after a specific recognition motif comprised of a C-terminal tail with the sequence motif -AYDG. The result is the native macrocyclic patellamide, which has eight amino acid residues. Macrocyclase activity can be exploited by incorporating that motif in other short linear peptide precursors, which then are formed into head-to-tail cyclized peptides. Here, we explore the possibility of using PatGmac in the cyclization of peptides larger than the patellamides, namely, the PawS-derived peptide sunflower trypsin inhibitor-1 (SFTI-1) and the cyclotide kalata B1. These peptides fall under two distinct families of disulfide constrained macrocyclic plant peptides. They are both implicated as scaffolds for drug design due to their structures and unusual stability. We show that PatGmac can be used to efficiently cyclize the 14 amino acid residue long SFTI-1, but less so the 29 amino acid residue long kalata B1.

Toward calix[2]-type macrocycles: Synthesis and structural analysis of cyclic tetraketone and highly strained furanophane

Recent progress in the synthesis of calix[3]pyrrole has proven that cyclic polyketones composed of 3,3-dialkylated pentane-2,4-diones as the repeating unit are promising precursors for strained calix-type macrocycles. Toward the synthesis of calix[2]pyrrole and calix[2]furan, which are the most strained congeners among the possible calix[[Formula: see text]]pyrroles and calix[[Formula: see text]]furans, we report the synthesis of a cyclic dimer of 3,3-dimethylpentane-2,4-dione and its furanophane derivative. These compounds were produced from a linear tetraketone precursor. Single crystal X-ray diffraction analysis revealed that the furan ring in the furanophane was highly strained with deformation angles [Formula: see text] and [Formula: see text] of 3.69[Formula: see text] and 16.00[Formula: see text], respectively. These deformation angles were the largest in the series of 1,4-diketone-linked oligofuran macrocycles. Although these two macrocyclic compounds are likely the most promising precursors for calix[2]-type macrocycles, Paal–Knorr-type pyrrole or furan ring formation did not proceed under standard conditions used for calix[3]pyrrole or calix[3]furan. Theoretical calculations indicated that the second aromatic ring formation on the cyclic tetraketone is a highly uphill process whose activation energy cannot be overcome by standard thermal reactions.

Supramolecular Capsule Catalysis Enables the Exploration of Terpenoid Chemical Space Untapped by Nature.

Terpenes represent the largest and the most diverse class of natural compounds. This is remarkable as the whole variety is accessed from just a handful of highly conserved linear precursors. Modification of the cyclization precursors would enable a dramatic expansion of the accessible chemical space. However, natural enzymes do not enable us to tap into this potential, as they do not tolerate larger deviations from the prototypical substrate structure. Herein we report that supramolecular capsule catalysis enables facile access to diverse and novel terpenoid skeletons that formally can be traced back to C3-phenyl, benzyl, and homoprenyl derivatives of farnesol. Novel skeletons related to the presilphiperfolane core structure, as well as novel neoclovene derivatives were accessed efficiently in only four synthetic steps. Importantly, the products obtained carry functional groups that may be readily derivatized further.

A novel clover-like COFs membrane fabricated via one-step interfacial polymerization for dye/salt separation

Covalent organic frameworks (COFs) membranes have exhibited great applications in wastewater treatment due to their highly ordered porous architecture. Currently, numerous studies have focused on the hexagonal-structured COFs synthesized with linear C2 and C3 precursor. However, the C2 monomer is limited-variety and high-cost. Herein, a novel clover-like structure COFs membrane is successfully fabricated for the first time. Meta-position monomer m-phenylenediamine (MPD) and 1,3,5-triformylbenzene (TFB) are used to synthesize the clover-like COFs membrane by interfacial polymerization (IP) method on the nylon substrate directly. The optimized MPD-TFB/Nylon membrane shows excellent water permeance of 94.4 L m−2 h−1 bar−1, which is higher than hexagonal-structured COFs membrane, in Congo red (CR) and NaCl mixed solution The rejections are 98.6% for CR and 2.9% for NaCl. This work opens a novel route for designing the COFs membrane with clover-like channels, manifesting the usage of linear C2 monomers is unnecessary. Besides, it has been found that the unique clover-like structure can improve the water molecular transport path under the same dye molecules deposition situation compared with hexagonal structure COFs membrane.