Thiostrepton maturation involving a deesterification-amidation way to process the C-terminally methylated peptide backbone

Thiopeptides are a class of clinically interesting and highly modified peptide antibiotics. Their biosyntheses share a common paradigm for characteristic core formation but differ in tailoring to afford individual members. Herein we report an unusual deesterification-amidation process in thiostrepton maturation to furnish the terminal amide moiety. TsrB, serving as a carboxylesterase, catalyzes the hydrolysis of the methyl ester intermediate to provide the carboxylate intermediate, which can be converted to the amide product by an amidotransferase, TsrC. These findings revealed a C-terminal methylation of the precursor peptide, which is cryptic in thiostrepton biosynthesis but potentially common in the formation of its homologous series of thiopeptides that vary in the C-terminal form as methyl ester, carboxylate, or amide.     

Synthesis of organogelling, fluoride ion-responsive, cholesteryl-based benzoxazole containing intra- and intermolecular hydrogen-bonding sites

A cholesteryl-based 2-(2′-hydroxyphenyl)benzoxazole (HPB) derivative 3 linked with an amide bond was prepared through an efficient synthetic pathway. The HPB, amide, and cholesteryl groups play important roles in constructing the supramolecular gel structure. UV-vis and fluorescence spectroscopy also showed that HPB and amide groups, which provide intra- and intermolecular hydrogen bonding, respectively, also contribute the recognition of fluoride anions.     

Influence of Ester versus Amide Linkers on the Supramolecular Polymerization Mechanisms of Planar BODIPY Dyes

We report the H‐type supramolecular polymerization of two new hydrophobic BODIPY derivatives equipped with ester and amide linkages. Whereas the ester‐containing BODIPY derivative undergoes an isodesmic supramolecular polymerization in which the monomers are parallel‐oriented, the replacement of the ester by amide groups leads to a highly cooperative self‐assembly process into H‐type aggregates with a rotational displacement of the dye molecules within the stack. The dye organization imposed by simultaneous π–π and hydrogen bonding interactions is the driving force for the cooperative supramolecular polymerization, whereas the absence of additional hydrogen bonds for the ester‐containing moiety does not suffice to induce cooperative phenomena.     

Solid‐Phase Total Synthesis of Yaku'amide B Enabled by Traceless Staudinger Ligation

We report a solid‐phase strategy for total synthesis of the peptidic natural product yaku'amide B ( 1 ), which exhibits antiproliferative activity against various cancer cells. Its linear tridecapeptide sequence bears four β,β‐dialkylated α,β‐dehydroamino acid residues and is capped with an N‐terminal acyl group (NTA) and a C‐terminal amine (CTA). To realize the Fmoc‐based solid‐phase synthesis of this complex structure, we developed new methods for enamide formation, enamide deprotection, and C‐terminal modification. First, traceless Staudinger ligation enabled enamide formation between sterically encumbered alkenyl azides and newly designed phosphinophenol esters. Second, application of Eu(OTf)₃ led to chemoselective removal of the enamide Boc groups without detaching the resin linker. Finally, resin‐cleavage and C‐terminus modification were simultaneously achieved with an ester–amide exchange reaction using CTA and AlMe₃ to deliver 1 in 9.1 % overall yield (24 steps from the resin).     

A Threonine‐Forming Oxazetidine Amino Acid for the Chemical Synthesis of Proteins through KAHA Ligation

α‐Ketoacid‐hydroxylamine (KAHA) ligation allows the coupling of unprotected peptide segments through the chemoselective formation of an amide bond. Currently, the most widely used variant employs a 5‐membered cyclic hydroxylamine that forms a homoserine ester as the primary ligation product. In order to directly form amide‐linked threonine residues at the ligation site, we prepared a new 4‐membered cyclic hydroxylamine building block. This monomer was applied to the synthesis of wild‐type ubiquitin‐conjugating enzyme UbcH5a (146 residues) and Titin protein domain TI I27 (89 residues). Both the resulting UbcH5a and the variant with two homoserine residues showed identical activity to a recombinant variant in a ubiquitination assay.     

Synthesis of macrocyclic bis(phenylbenzoxazole) derivatives via tandem claisen rearrangement and their fluorescence behavior

Novel macrocyclic bis(phenylbenzoxazole) derivatives were easily synthesized from macrocyclic isobutenyl bis(amide‐ether)s by tandem Claisen rearrangement and subsequent intramolecular cyclization of the amide‐phenol intermediates. The position of substitution of the oligoethylene glycol moiety on the phenylamido groups of the macrocycles did not have a large effect on the yields of the bis(benzoxazole)s for the meta and para derivatives. The fluorescence quantum yields of most of the macrocyclic bis(benzoxa‐zole)s were lower than those of the corresponding nonmacrocyclic bis(benzoxazole) model compounds. The quantum yields of the para‐substituted macrocyclic bis(benzoxazole)s were clearly lower than those of the model compounds and decreased with increasing length of the oligoethylene chain.     

Probing intermolecular backbone H-bonding in serine proteinase-protein inhibitor complexes.

BACKGROUND: Intermolecular backbone H-bonding (N-H.O=C) is a common occurrence at the interface of protein-protein complexes. For instance, the amide NH groups of most residues in the binding loop of eglin c, a potent serine proteinase inhibitor from the leech Hirudo medicinalis, are H-bonded to the carbonyl groups of residues in the target enzyme molecules such as chymotrypsin, elastase and subtilisins. We sought to understand the energetic significance of these highly conserved backbone-backbone H-bonds in the enzyme-inhibitor complexes. RESULTS: We synthesized an array of backbone-engineered ester analogs of eglin c using native chemical ligation to yield five inhibitor proteins each containing a single backbone ester bond from P3 to P2' (i.e. -CONH-to -COO-). The structure at the ligation site (P6-P5) is essentially unaltered as shown by a high-resolution analysis of the subtilisin-BPN'-eglin c complex. The free-energy changes (DeltaDeltaGNH-->O) associated with the binding of ester analogs at P3, P1 and P2' with bovine alpha-chymotrypsin, subtilisin Carlsberg and porcine pancreatic elastase range from 0-4.5 kcal/mol. Most markedly, the NH-->O substitution at P2 not only stabilizes the inhibitor but also enhances binding to the enzymes by as much as 500-fold. CONCLUSIONS: Backbone H-bond contributions are context dependent in the enzyme-eglin c complexes. The interplay of rigidity and adaptability of the binding loop of eglin c seems to play a prominent role in defining the binding action.     

Toward the Total Synthesis of Onchidin,a Cytotoxic Cyclic Depsipeptide from a Mollusc

The total synthesis of onchidin ( 1 ), a cytotoxic, C₂‐symmetric cyclic decadepsipeptide from a marine mollusc, according to the published structure, is described. A novel β‐amino acid, (2S,3S)‐3‐amino‐2‐methyl‐7‐octynoic acid (AMO), was efficiently prepared in high yield with high diastereo‐ and enantioselectivity based on a catalytic asymmetric three‐component Mannich‐type reaction with a chiral zirconium catalyst. The formation of sterically unfavorable N‐methyl amide and hindered ester bonds were successfully demonstrated, and final macrocyclization was achieved at a secondary‐amide site. Completion of the synthesis of 1 suggested that a revision of the structure of the natural product is required. Two diastereomers were also synthesized as candidates for the actual structure of onchidin. Furthermore, efficient solid‐phase methods were employed for the combinatorial synthesis of other derivatives to clarify the real structure of onchidin. The solid‐phase assembly of a pentadepsipeptide containing all the building blocks was established followed by dimeric cyclization in solution.     

New organosoluble and thermally stable poly(amide‐imide)s with benzoxazole or benzothiazole pendent groups: synthesis and characterization

Two new benzoxazole or benzothiazole‐containing diimide‐dicarboxylic acid monomers, such as 2‐[3,5‐bis(N‐trimellitimidoyl)phenyl]benzoxazole ( 2 _o ) or 2‐[3,5‐bis(N‐trimellitimidoyl)phenyl]benzothiazole ( 2 _s ) were synthesized from the condensation reaction between 3,5‐diaminobenzoic acid and 2‐aminophenol or 2‐aminothiophenol in polyphosphoric acid (PPA) with subsequent reaction of trimellitic anhydride in the presence of glacial acetic acid, respectively, and two new series of modified aromatic poly(amide‐imide)s were prepared. This preparation was done with pendent benzoxazole or benzothiazole units from the newly synthesized diimide‐dicarboxylic acid and various aromatic diamines by triphenyl phosphite‐activated polycondensation. In addition, the corresponding unsubstituted poly(amide‐imide)s were prepared under identical experimental conditions for comparative purposes. Characterization of polymers was accomplished by inherent viscosity measurements, FT‐IR, UV–visible, ¹H‐NMR spectroscopy and thermogravimetry. The polymers were obtained in quantitative yields with inherent viscosities between 0.39 and 0.81 dl g^?1. The solubilities of modified poly(amide‐imide)s in common organic solvents as well as their thermal stability were enhanced compared to those of the corresponding unmodified poly(amide‐imide)s. The glass transition temperature, 10% weight loss temperature, and char yields at 800°C were, respectively, 7–26°C, 17–46°C and 2–5% higher than those of the unmodified polymers. Copyright © 2010 John Wiley & Sons, Ltd.     

Penicillin acylase-catalyzed peptide synthesis: a chemo-enzymatic route to stereoisomers of 3,6-diphenylpiperazine-2,5-dione

Chiral dipeptides of phenylglycine were synthesized using immobilized Escherichia coli penicillin acylase. The high selectivity of penicillin acylase for l-amino acids as the nucleophile resulted in the efficient acylation of l-phenylglycine by d-phenylglycine amide at pH 9.7 to give d-phenylglycyl-l-phenylglycine in 69% yield. No isomers or tripeptides were formed. The low enantiospecificity of the enzyme for the acyl donor provided the possibility of preparing the corresponding l,l-dipeptides, starting from l-phenylglycine methyl ester as both donor and acceptor at pH 7.5, resulting in a 63% yield of l-phenylglycyl-l-phenylglycine methyl ester. The product precipitated under the reaction conditions; this effectively prevented the formation of oligomers as well as chemical transformation of the product.The dipeptide esters of phenylglycine easily cyclized to diketopiperazines in aqueous methanol. l-Phenylglycyl-l-phenylglycine methyl ester formed l,l-3,6-diphenylpiperazine-2,5-dione (cis); the achiral trans isomer was obtained from d-phenylglycyl-l-phenylglycine methyl ester.     

The Catalytic Mechanism of the Class C Radical S‐Adenosylmethionine Methyltransferase NosN

S ‐Adenosylmethionine (SAM) is one of the most common co‐substrates in enzyme‐catalyzed methylation reactions. Most SAM‐dependent reactions proceed through an S_N2 mechanism, whereas a subset of them involves radical intermediates for methylating non‐nucleophilic substrates. Herein, we report the characterization and mechanistic investigation of NosN, a class C radical SAM methyltransferase involved in the biosynthesis of the thiopeptide antibiotic nosiheptide. We show that, in contrast to all known SAM‐dependent methyltransferases, NosN does not produce S ‐adenosylhomocysteine (SAH) as a co‐product. Instead, NosN converts SAM into 5′‐methylthioadenosine as a direct methyl donor, employing a radical‐based mechanism for methylation and releasing 5′‐thioadenosine as a co‐product. A series of biochemical and computational studies allowed us to propose a comprehensive mechanism for NosN catalysis, which represents a new paradigm for enzyme‐catalyzed methylation reactions.     

The Use of Electrospray Mass Spectrometry to Determine Speciation in a Dynamic Combinatorial Library for Anion Recognition

The composition of a dynamic mixture of similar 2,2′‐bipyridine complexes of iron(II) bearing either an amide (5‐benzylamido‐2,2′‐bipyridine and 5‐(2‐methoxyethane)amido‐2,2′‐bipyridine) or an ester (2,2′‐bipyridine‐5‐carboxylic acid benzylester and 2,2′‐bipyridine‐5‐carboxylic acid 2‐methoxyethane ester) side chain have been evaluated by electrospray mass spectroscopy in acetonitrile. The time taken for the complexes to come to equilibrium appears to be dependent on the counteranion, with chloride causing a rapid redistribution of two preformed heteroleptic complexes (of the order of 1 hour), whereas the time it takes in the presence of tetrafluoroborate salts is in excess of 24 h. Similarly the final distribution of products is dependent on the anion present, with the presence of chloride, and to a lesser extent bromide, preferring three amide‐functionalized ligands, and a slight preference for an appended benzyl over a methoxyethyl group. Furthermore, for the first time, this study shows that the distribution of a dynamic library of metal complexes monitored by ESI‐MS can adapt following the introduction of a different anion, in this case tetrabutylammonium chloride to give the most favoured heteroleptic complex despite the increasing ionic strength of the solution.     

Pathway Complexity Versus Hierarchical Self‐Assembly in N‐Annulated Perylenes: Structural Effects in Seeded Supramolecular Polymerization

Studies were carried out on the hierarchical self‐assembly versus pathway complexity of N‐annulated perylenes 1 – 3 , which differ only in the nature of the linking groups connecting the perylene core and the side alkoxy chains. Despite the structural similarity, compounds 1 and 2 exhibit noticeable differences in their self‐assembly. Whereas 1 forms an off‐pathway aggregate I that converts over time (or by addition of seeds) into the thermodynamic, on‐pathway product, 2 undergoes a hierarchical process in which the kinetically trapped monomer species does not lead to a kinetically controlled supramolecular growth. Finally, compound 3 , which lacks the amide groups, is unable to self‐assemble under identical experimental conditions and highlights the key relevance of the amide groups and their position to govern the self‐assembly pathways.     

Novel and Gram‐Scale Green Synthesis of Flutamide

Isobutyric acid in the presence of cyanuric chloride and N‐methylmorpholine was converted into active ester 3 at 0–5 °C, and it was subsequently treated with 3‐aminobenzotrifluoride 4 at 25 °C to furnish corresponding amide 5. This amide finally, on nitration, produced the desired product flutamide, 2‐methyl‐N‐[4‐nitro‐3‐(trifluoromethyl)phenyl]propionamide 6 in good yield. By‐product 2,4,6‐trihydroxy‐1,3,5‐triazine 7 was converted into the useful starting material cyanuric chloride 1 by refluxing with N,N‐diethylamine and POCl₃.     

Scalable Biosynthesis of the Seaweed Neurochemical,Kainic Acid

Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuropharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six‐steps. Herein we report the discovery and characterization of the concise two‐enzyme biosynthetic pathway to kainic acid from l ‐glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co‐clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α‐ketoglutarate‐dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.     

Scalable Biosynthesis of the Seaweed Neurochemical,Kainic Acid

Kainic acid, the flagship member of the kainoid family of natural neurochemicals, is a widely used neuropharmacological agent that helped unravel the key role of ionotropic glutamate receptors, including the kainate receptor, in the central nervous system. Worldwide shortages of this seaweed natural product in the year 2000 prompted numerous chemical syntheses, including scalable preparations with as few as six‐steps. Herein we report the discovery and characterization of the concise two‐enzyme biosynthetic pathway to kainic acid from l ‐glutamic acid and dimethylallyl pyrophosphate in red macroalgae and show that the biosynthetic genes are co‐clustered in genomes of Digenea simplex and Palmaria palmata. Moreover, we applied a key biosynthetic α‐ketoglutarate‐dependent dioxygenase enzyme in a biotransformation methodology to efficiently construct kainic acid on the gram scale. This study establishes both the feasibility of mining seaweed genomes for their biotechnological prowess.     

Polymerization of 2,5‐diaminoterephthalic acid‐type monomers for the synthesis of polyamides containing ladder unit

To synthesize ladder‐type polyamides by construction of two amide bonds successively, 2,5‐diaminoterephthalic acid derivatives bearing anthranilic acid ester and isatoic anhydride moieties were synthesized and their polymerization was investigated. Polymerization of the methyl ester monomer proceeded in the presence of lithium hexamethyldisilazide (LiHMDS) as a base in tetrahydrofuran (THF). However, mass spectroscopic analysis of the product suggested that not only the bis(trimethylsilyl)amide anion of LiHMDS but also the methoxide anion eliminated at the second amide‐linkage formation reaction decomposed the isatoic anhydride unit of the growing oligomer by nucleophilic attack to disturb the polymerization. To reduce the nucleophilicity of the eliminated anion, methyl ester of the monomer was changed to phenyl ester and its polymerization was studied. The reaction conditions were optimized, and the best result was obtained when the polymerization was conducted in the presence of 1.0 equivalent of LiHMDS without additives in THF at 50 °C. © 2017 Wiley Periodicals, Inc. J. Polym. Sci., Part A: Polym. Chem. 2017 , 55, 2365–2372     

Selective Reductive Removal of Ester and Amide Groups from Arenes and Heteroarenes through Nickel‐Catalyzed C−O and C−N Bond Activation

An inexpensive nickel(II) catalyst and a hydrosilane were used for the efficient reductive defunctionalization of aryl and heteroaryl esters through a decarbonylative pathway. This versatile method could be used for the removal of ester and amide functional groups from various organic molecules. Moreover, a scale‐up experiment and a synthetic application based on the use of a removable carboxylic acid directing group highlight the usefulness of this reaction.     

The Synthesis of Methylated,Phosphorylated, and Phosphonated 3′‐Aminoacyl‐tRNASec Mimics

The twenty first amino acid, selenocysteine (Sec), is the only amino acid that is synthesized on its cognate transfer RNA (tRNA^Sec) in all domains of life. The multistep pathway involves O‐phosphoseryl‐tRNA:selenocysteinyl‐tRNA synthase (SepSecS), an enzyme that catalyzes the terminal chemical reaction during which the phosphoseryl–tRNA^Sec intermediate is converted into selenocysteinyl‐tRNA^Sec. The SepSecS architecture and the mode of tRNA^Sec recognition have been recently determined at atomic resolution. The crystal structure provided valuable insights that gave rise to mechanistic proposals that could not be validated because of the lack of appropriate molecular probes. To further improve our understanding of the mechanism of the biosynthesis of selenocysteine in general and the mechanism of SepSecS in particular, stable tRNA^Sec substrates carrying aminoacyl moieties that mimic particular reaction intermediates are needed. Here, we report on the accurate synthesis of methylated, phosphorylated, and phosphonated serinyl‐derived tRNA^Sec mimics that contain a hydrolysis‐resistant ribose 3′‐amide linkage instead of the natural ester bond. The procedures introduced allow for efficient site‐specific methylation and/or phosphorylation directly on the solid support utilized in the automated RNA synthesis. For the preparation of (S)‐2‐amino‐4‐phosphonobutyric acid–oligoribonucleotide conjugates, a separate solid support was generated. Furthermore, we developed a three‐strand enzymatic ligation protocol to obtain the corresponding full‐length tRNA^Sec derivatives. Finally, we developed an electrophoretic mobility shift assay (EMSA) for rapid, qualitative characterization of the SepSecS‐tRNA interactions. The novel tRNA^Sec mimics are promising candidates for further elucidation of the biosynthesis of selenocysteine by X‐ray crystallography and other biochemical approaches, and could be attractive for similar studies on other tRNA‐dependent enzymes.     

Characterization of a Citrulline 4‐Hydroxylase from Nonribosomal Peptide GE81112 Biosynthesis and Engineering of Its Substrate Specificity for the Chemoenzymatic Synthesis of Enduracididine

The GE81112 tetrapeptides are a small family of unusual nonribosomal peptide congeners with potent inhibitory activity against prokaryotic translation initiation. With the exception of the 3‐hydroxy‐l ‐pipecolic acid unit, little is known about the biosynthetic origins of the non‐proteinogenic amino acid monomers of the natural product family. Here, we elucidate the biogenesis of the 4‐hydroxy‐l ‐citrulline unit and establish the role of an iron‐ and α‐ketoglutarate‐dependent enzyme (Fe/αKG) in the pathway. Homology modelling and sequence alignment analysis further facilitate the rational engineering of this enzyme to become a specific 4‐arginine hydroxylase. We subsequently demonstrate the utility of this engineered enzyme in the synthesis of a dipeptide fragment of the antibiotic enduracidin. This work highlights the value of applying a bioinformatics‐guided approach in the discovery of novel enzymes and engineering of new catalytic activity into existing ones.