Secondary Amide Bonds Research Articles

Discovery of (±)-Penindolenes Reveals an Unusual Indole Ring Cleavage Pathway Catalyzed by P450 Monooxygenase.

(±)-Penindolenes A-D (1-4), the first representatives of indole terpenoids featuring a γ-lactam skeleton, were isolated from the mangrove-derived endophytic fungus Penicillium brocae MA-231. Our bioactivity tests revealed their potent antimicrobial and acetylcholinesterase inhibitory activities. The biosynthetic reactions by the five enzymes PbaABCDE leading to γ-lactam ring formation were identified with heterologous expression and in vitro enzymatic assays. Remarkably, the cytochrome P450 monooxygenase PbaB and its homolog in Aspergillus oryzae catalyzed the 2,3-cleavage of the indole ring to generate two keto groups in 1. This is the first example of the oxidative cleavage of indole by a P450 monooxygenase. In addition, rare secondary amide bond formation by the glutamine synthetase-like enzyme PbaD was reported. These findings will contribute to the engineered biosynthesis of unnatural, bioactive indole terpenoids.

Discovery of (±)‐Penindolenes Reveals an Unusual Indole Ring Cleavage Pathway Catalyzed by P450 Monooxygenase

Abstract(±)‐Penindolenes A−D (1–4), the first representatives of indole terpenoids featuring a γ‐lactam skeleton, were isolated from the mangrove‐derived endophytic fungus Penicillium brocae MA‐231. Our bioactivity tests revealed their potent antimicrobial and acetylcholinesterase inhibitory activities. The biosynthetic reactions by the five enzymes PbaABCDE leading to γ‐lactam ring formation were identified with heterologous expression and in vitro enzymatic assays. Remarkably, the cytochrome P450 monooxygenase PbaB and its homolog in Aspergillus oryzae catalyzed the 2,3‐cleavage of the indole ring to generate two keto groups in 1. This is the first example of the oxidative cleavage of indole by a P450 monooxygenase. In addition, rare secondary amide bond formation by the glutamine synthetase‐like enzyme PbaD was reported. These findings will contribute to the engineered biosynthesis of unnatural, bioactive indole terpenoids.

Tertiary amide bond formation by an engineered asparaginyl ligase.

Transpeptidases are powerful tools for site-specific protein modification, enabling the production of tailored biologics to investigate protein function and aiding the development of next-generation therapeutics and diagnostics. Although protein labelling at the N- or C-terminus is readily accomplished using a range of established transpeptidases, these reactions are generally limited to forming products that are linked by a standard (secondary) amide bond. Here we show that, unlike other widely used transpeptidases, an engineered asparaginyl ligase is able to efficiently synthesise tertiary amide bonds by accepting diverse secondary amine nucleophiles. These reactions proceed efficiently under mild conditions (near-neutral pH) and allow the optimal recognition elements for asparaginyl ligases (P1 Asn and P2'' Leu) to be preserved. Certain products, particularly proline-containing products, were found to be protected from recognition by the enzyme, allowing for straightforward sequential labelling of proteins. Additionally, incorporation of 4-azidoproline enables one-pot dual labelling directly at the ligation junction. These capabilities further expand the chemical diversity of asparaginyl ligase-catalysed reactions and provide an alternative approach for straightforward, successive modification of protein substrates.

Optimization of Pharmacokinetic and In Vitro Safety Profile of a Series of Pyridine Diamide Indirect AMPK Activators.

A set of focused analogues have been generated around a lead indirect adenosine monophosphate-activated kinase (AMPK) activator to improve the rat clearance of the molecule. Analogues were focused on inhibiting amide hydrolysis by the strategic placement of substituents that increased the steric environment about the secondary amide bond between 4-aminopiperidine and pyridine-5-carboxylic acid. It was found that placing substituents at position 3 of the piperidine ring and position 4 of the pyridine could all improve clearance without significantly impacting on-target potency. Notably, trans-3-fluoropiperidine 32 reduced rat clearance from above liver blood flow to 19 mL/min/kg and improved the hERG profile by attenuating the basicity of the piperidine moiety. Oral dosing of 32 activated AMPK in mouse liver and after 2 weeks of dosing improved glucose handling in a db/db mouse model of Type II diabetes as well as lowering fasted glucose and insulin levels.

Conformational Properties of Aromatic Amides Bearing Imidazole Ring and Acid-Induced Trans–Cis Amide Switching

Aromatic amides bearing secondary amide bond exist in trans conformation both in the crystal and in solution, whereas the conformation of the N-methylated derivatives is cis in the crystal and predominantly cis in various solvents. The cis conformational preference of N-alkylated benzanilide provides access to aromatic foldamers such as oligo(N-alkyl-p-benzamide)s, which adopt dynamic helical structures. Here, the conformational properties of imidazole-substituted amide in the crystal and in solution were examined. Imidazole-substituted amides 2a and 4a existed mainly in the cis conformation in solution. The ratio of the cis conformer of N-methyl-N-(1-methyl-1H-imidazol-4-yl)benzamide (4a) was smaller than that of N,1-dimethyl-N-phenyl-1H-imidazole-2-carboxamide (2a) or N-methylbenzanilide, but the introduction of a substituent strongly affected the conformer ratio. Compounds 6a and 7a bearing an electron-withdrawing group on the imidazole ring existed predominantly in trans form. On the other hand, the introduction of an electron-withdrawing group on the phenyl ring or a bulky substituent on the amide nitrogen of 4a increased the ratio of cis conformer. Further, the major conformer of N-alkylated N-imidazolylamides was switched from cis to trans by the addition of acid. These results suggest that imidazole-substituted amides might be applicable as conformational switches in aromatic foldamers to enable environment-dependent structural change.

Oxidative Damage of Aliphatic Amino Acid Residues by the Environmental Pollutant NO3·: Impact of Water on the Reactivity.

The rate of oxidative damage of aliphatic amino acids and dipeptides by the environmental pollutant nitrate radical (NO3·) in an aqueous acidic environment was studied by laser flash photolysis. The reactivity dropped by a factor of about four for amino acid residues with secondary amide bonds and by a factor of up to nearly 20 for amino acid residues with tertiary amide bonds, compared with that in acetonitrile. According to density functional theory studies, the lower reactivity is due to protonation of the amide moiety, whereas in neutral water, hydrogen bonding with the amide should have little impact on the absolute reaction rate compared with that in acetonitrile. This finding can be rationalized by the high reactivity and broad reaction pattern of NO3·. Although hydrogen bonding involving the amide group raises the energies associated with some electron transfer processes, alternative low-energy pathways remain available so that the overall reaction rate is barely affected. The undiminished high reactivity of NO3· toward aliphatic amino acid residues in a neutral aqueous environment highlights the health-damaging potential of exposure to the combined air pollutants nitrogen dioxide (NO2·) and ozone (O3).

Ammonolytic Hydrogenation of Secondary Amides: An Efficient Method for the Recycling of Long-Chain Polyamides

Long-chain polyamides (LCPAs) are depolymerized by ammonolysis of the secondary amide bonds into building blocks for new polymer synthesis. Research on the ammonolysis of PA is quite limited and often proposes harsh reaction conditions with homogeneous catalysts. Here, we describe a heterogeneous catalytic system based on Nb2O5. Reactions were performed at a relatively low temperature of 200 °C in cyclopentyl methyl ether (CPME) as a green solvent and with limited addition of NH3 and H2. The ammonolysis of secondary amides was extensively studied, initially with N-hexylhexanamide as a model compound. Since ammonolysis is an equilibrium reaction, it was eventually coupled to a hydrogenation process, with addition of a RuWOx/MgAl2O4 hydrogenation catalyst, in order to achieve complete depolymerization. Industrial LCPA samples were successfully and completely depolymerized to α,ω-diamine monomers and oligomers, resulting in product distributions of 62% primary amines and 36% secondary amines. The catalytic system was proven to be very robust against a variety of contaminants, e.g., fillers, other plastics, and additives. Only sulfur-containing compounds poison the Ru-catalyst and have to be removed completely.

Selective Modification of Peptides through Cleavage of a Secondary Amide Bond

Selective Modification of Peptides through Cleavage of a Secondary Amide Bond

Development of Androgen-Antagonistic Coumarinamides with a Unique Aromatic Folded Pharmacophore.

First-generation nonsteroidal androgen receptor (AR) antagonists, such as flutamide (2a) and bicalutamide (3), are effective for most prostate cancer patients, but resistance often appears after several years due to the mutation of AR. Second-generation AR antagonists are effective against some of these castration-resistant prostate cancers, but their structural variety is still limited. In this study, we designed and synthesized 4-methyl-7-(N-alkyl-arylcarboxamido)coumarins as AR antagonist candidates and evaluated their growth-inhibitory activity toward androgen-dependent SC-3 cells. Coumarinamides with a secondary amide bond did not show inhibitory activity, but their N-methylated derivatives exhibited AR-antagonistic activity. Especially, 19b and 31b were more potent than the lead compound 7b, which was comparable to hydroxyflutamide (2b). Conformational analysis showed that the inactive coumarinamides with a secondary amide bond have an extended structure with a trans-amide bond, while the active N-methylated coumarinamides have a folded structure with a cis-amide bond, in which the two aromatic rings are placed face-to-face. Docking study suggested that this folded structure is important for binding to AR. Selected coumarinamide derivatives showed AR-antagonistic activity toward LNCaP cells with T877A AR, and they had weak progesterone receptor (PR)-antagonistic activity. The folded coumarinamide structure appears to be a unique pharmacophore, different from those of conventional AR antagonists.

Enzymatic Hydrolysis of Tertiary Amide Bonds by anti Nucleophilic Attack and Protonation.

The molecular mechanisms conferring high resistance of planar tertiary amide bonds to hydrolysis by most enzymes have remained elusive. To provide a chemical explanation to this unresolved puzzle, UB3LYP calculations were performed on an active site model of Xaa-Pro peptidases. The calculated reaction mechanism demonstrates that biocatalysts capable of tertiary amide bond hydrolysis capitalize on anti nucleophilic attack and protonation of the amide nitrogen, in contrast to the traditional syn displayed by amidases and proteases acting on secondary amide bonds.

Polymerization of PPTA in Ionic Liquid/Cosolvent Mixtures

1-Octyl-3-methylimidazolium chloride ([C8MIM][Cl]) shows the greatest potential to replace N-methylpyrrolidone/CaCl2 as solvent for the synthesis of poly(p-phenylene terephthalamide) (PPTA), a high-strength material when spun into a fiber. Coordinating anions are crucial to prevent precipitation of the polymer during synthesis. Additionally, the imidazolium cations play an essential role in gelation of the reaction mixture and enable the polycondensation reaction to continue in a gel state. Analysis of Kamlet–Taft parameters and Gutmann donor and acceptor numbers of ionic liquids and various solvents showed that balanced interactions between the secondary amide bonds of the polymer, anion, cation, and solvent are required to form a network of hydrogen bond interactions throughout the reaction mixture. This hypothesis was supported by the fact that tri-n-butyl phosphate (TBP), containing similar Kamlet–Taft and Gutmann solvent parameters as N-methylpyrrolidone, was able to produce PPTA with an inherent vis...

Microbial transglutaminase displays broad acyl-acceptor substrate specificity

The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts with the side chain of a lysine, forming a secondary amide bond. Long alkylamines carrying diverse chemical entities can substitute for lysine as acyl-acceptor substrates, to link molecules of interest onto peptides or proteins. Here, we explore short and chemically varied acyl-acceptor substrates, to better understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys, and Trp--but not Ile--also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group--particularly if aromatic--was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acyl-acceptor substrates, providing a robust route to minimally modified, "clickable" peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and nonnatural acyl-acceptor substrates, which broadens the scope for modification of Gln-containing peptides and proteins.

3-Mercaptopropionic acid capped ZnSe quantum dot-cytochrome P450 3A4 enzyme biotransducer for 17β-estradiol

A 3-mercaptopropionic acid capped ZnSe quantum dot/cytochrome P450 3A4 enzyme electrochemical biotransducer for 17β-estradiol (endocrine disrupting compound) is presented. The biotransducer combines both the electric and chemical properties of 3-mercaptopropionic acid capped ZnSe quantum dots as well as redox and binding affinity properties of the cytochrome P450 3A4 enzyme to steroids. 3-mercaptopropionic acid capped ZnSe quantum dots were conjugated to gold electrode, previously modified with self-assembled l-cysteamine. Cytochrome P450 3A4 enzyme was then conjugated onto the modified gold electrode for 3h. A Fourier transform infrared spectra of this biotransducer showed a band at 2956cm−1, confirming the formation of a secondary amide bond from free carboxylic groups on the ZnSe quantum dots surface and amine groups on the cytochrome P450 3A4 enzyme. The biotransducer showed proportional increase in current with increasing concentration of 17β-estradiol in a phosphate buffer under aerobic conditions. A high sensitivity (1.08×10−5AμM−1) and a low Michaelis–Menten constant (Kmapp=0.15mM) obtained for this biotransducer confirms that the cytochrome P450 3A4 was immobilized in a biocompatible microenvironment, retained its catalytic properties and exhibited high enzymatic activity towards 17β-estradiol. The detection limit was 1.03×10−10molL−1.

New Fluorinated Rhodamines for Optical Microscopy and Nanoscopy

New photostable rhodamine dyes represented by the compounds 1 a-r and 3-5 are proposed as efficient fluorescent markers with unique combination of structural features. Unlike rhodamines with monoalkylated nitrogen atoms, N',N-bis(2,2,2-trifluoroethyl) derivatives 1 e, 1 i, 1 j, 3-H and 5 were found to undergo sulfonation of the xanthene fragment at the positions 4' and 5'. Two fluorine atoms were introduced into the positions 2' and 7' of the 3',6'-diaminoxanthene fragment in compounds 1 a-d, 1 i-l and 1 m-r. The new rhodamine dyes may be excited with λ=488 or 514 nm light; most of them emit light at λ=512-554 nm (compounds 1 q and 1r at λ=576 and 589 nm in methanol, respectively) and have high fluorescence quantum yields in solution (up to 98 %), relatively long excited-state lifetimes (>3 ns) and are resistant against photobleaching, especially at high laser intensities, as is usually applied in confocal microscopy. Sulfonation of the xanthene fragment with 30 % SO3 in H2SO4 is compatible with the secondary amide bond (rhodamine-CON(Me)CH2CH2COOH) formed with MeNHCH2CH2COOCH3 to providing the sterically unhindered carboxylic group required for further (bio)conjugation reactions. After creating the amino reactive sites, the modified derivatives may be used as fluorescent markers and labels for (bio)molecules in optical microscopy and nanoscopy with very-high light intensities. Further, the new rhodamine dyes are able to pass the plasma membrane of living cells, introducing them as potential labels for recent live-cell-tag approaches. We exemplify the excellent performance of the fluorinated rhodamines in optical microscopy by fluorescence correlation spectroscopy (FCS) and stimulated emission depletion (STED) nanoscopy experiments.

Total Synthesis of Cyclosporine: Access to N-Methylated Peptides via Isonitrile Coupling Reactions

Recent developments in the use of isonitriles to furnish secondary and tertiary amide bond formations have been applied to a novel total synthesis of the important cyclic polypeptide cyclosporine A. Specifically, the disclosed synthetic route demonstrates the utility of microwave-mediated carboxylic acid isonitrile couplings, thioacid isonitrile couplings at ambient temperature, and isonitrile-mediated couplings of carboxylic acids and thioacids with amines to form challenging amide bonds.

An efficient route to VEGF-like peptide porphyrin conjugates via microwave-assisted ‘click-chemistry’

Synthetic cyclopeptides, and particularly those derived from VEGF sequence, present considerable interest for the development of nanodevices devoted to tumour imaging or targeting. In order to provide selective peptide-targeted tetrapyrrolic structures, we designed two meso-porphyrin derivatives anchored to a 17-residue-long cyclopeptide, potent antagonist of VEGF receptors, via a flexible tetraethylene glycol chain. Anchoring was achieved by two different strategies: a classical secondary amide bond formation and microwave-assisted Cu(I)-catalyzed azide-alkyne 1,3-dipolar cycloaddition (‘click-chemistry’). These compounds appear to be promising candidates for applications in PDT.

Interaction Energies and Dynamics of Acid−Base Pairs Isolated in Cavitands

The use of capsules and cavitands in physical organic chemistry is briefly reviewed, and their application to the study of salt bridges is introduced. Carboxylate/ammonium ion pairs are generated within an environment that more or less surrounds the functional groups within a synthetic fixed introverted solvent sphere. This is provided by cavitands that fold around amines and present them with a carboxylic acid function. Both organic and water-soluble versions were prepared, and their equilibrium affinities with quinuclidine bases were determined by NMR methods. The association constants range from approximately 10(3) M(-1) in water to more than 10(5) M(-1) in organic solvents. Studies of nitrogen inversion and tumbling of [2.2.2]-diazabicyclooctane within the introverted acids also illustrate the strength of the acid-base interactions. The barriers to in-out exchange of several amine guests were determined to be in the range from 15 to 24 kcal mol(-1). Some parallels with enzymes are drawn: the receptor folds around the guest species; presents them with inwardly directed functionality; and provides a generally hydrophobic environment and a periphery of secondary amide bonds.

Kinetics and Equilibria of Cis/Trans Isomerization of Backbone Amide Bonds in Peptoids

The biological activities of N-substituted glycine oligomers (peptoids) have been the subject of extensive research. As compared to peptides, both the cis and trans conformations of the backbone amide bonds of peptoids can be significantly populated. Thus, peptoids are mixtures of configurational isomers, with the number of isomers increasing by a factor of 2 with each additional monomer residue. Here we report the results of a study of the kinetics and equilibria of cis/trans isomerization of the amide bonds of N-acetylated peptoid monomers, dipeptoids, and tripeptoids by NMR spectroscopy. Resonance intensities indicate the cis conformation of the backbone amide bonds of the peptoids studied is more populated than is generally the case for the analogous secondary amide bond to proline residues in acyclic peptides. Rate constants were measured by inversion-magnetization transfer techniques over a range of temperatures, and activation parameters were derived from the temperature dependence of the rate constants. The rate of cis/trans isomerization by rotation around the amide bonds in the peptoids studied is generally slower than that around amide bonds to proline residues and takes place on the NMR inversion-magnetization transfer time scale only by rotation around the amide bond to the C-terminal peptoid residue.

Mass spectrometric identification of pyroglutamic acid in peptides following selective hydrolysis

The formation of a pyroglutamyl residue by enzymatic cyclization of an amino terminus glutamine residue is a common posttranslational modification [1–3]. Several biologically active peptides, most notably the thyrotropinreleasing hormone $(TRH)^1$ and gonadotropin-releasing hormone (GnRH), were among the first examples where the N-terminal modification was characterized. The widespread occurrence of glutaminyl cyclase in both plants and animals has ensured that the number of naturally occurring pyroglutamyl peptides is rapidly growing. A recent report established formation of pyroglutamic acid (Z) from N-terminal glutamic acid in recombinant monoclonal antibodies [4]. An N-terminus pyroglutamic acid residue impedes Edman sequencing, necessitating the use of a specific enzyme, pyroglutamate aminopeptidase, to liberate a peptide with a free amino terminus, which is then amenable to automated sequence analysis [5]. During the course of studies aimed at characterizing the peptide components of Conus venom, we encountered several examples of pyroglutamyl peptides. We describe here the serendipitous finding of mild hydrolysis conditions, which permit opening of the pyroglutamyl ring, without significant cleavage of primary and secondary amide bonds.

Construction of macrocyclic structure using conformational properties of secondary and tertiary aromatic amides

A large macrocyclic compound with six para-phenylene rings and six amide moieties, which are alternately secondary and tertiary, was synthesized. In a stepwise synthesis, the final cyclization step was successful because a combination of three tertiary amides, which prefer a cis conformation, and two linear secondary amides would arrange both amino and carboxyl ends close to each other, while various sizes of macrocyclic compounds including the target cyclic trimer were generated in one-pot synthesis where three secondary amide bonds were formed.