Structural,Functional, and Spectroscopic Characterization of the Substrate Scope of the Novel Nitrating Cytochrome P450 TxtE

A novel cytochrome P450 enzyme, TxtE, was recently shown to catalyze the direct aromatic nitration of L ‐tryptophan. This unique chemistry inspired us to ask whether TxtE could serve as a platform for engineering new nitration biocatalysts to replace current harsh synthetic methods. As a first step toward this goal, and to better understand the wild‐type enzyme, we obtained high‐resolution structures of TxtE in its substrate‐free and substrate‐bound forms. We also screened a library of substrate analogues for spectroscopic indicators of binding and for production of nitrated products. From these results, we found that the wild‐type enzyme accepts moderate decoration of the indole ring, but the amino acid moiety is crucial for binding and correct positioning of the substrate and therefore less amenable to modification. A nitrogen atom is essential for catalysis, and a carbonyl must be present to recruit the αB′₁ helix of the protein to seal the binding pocket.     

Engineered RebH Halogenase Variants Demonstrating a Specificity Switch from Tryptophan towards Novel Indole Compounds

Activating industrially important aromatic hydrocarbons by installing halogen atoms is extremely important in organic synthesis and often improves the pharmacological properties of drug molecules. To this end, tryptophan halogenase enzymes are potentially valuable tools for regioselective halogenation of arenes, including various industrially important indole derivatives and similar scaffolds. Although endogenous enzymes show reasonable substrate scope towards indole compounds, their efficacy can often be improved by engineering. Using a structure-guided semi-rational mutagenesis approach, we have developed two RebH variants with expanded biocatalytic repertoires that can efficiently halogenate several novel indole substrates and produce important pharmaceutical intermediates. Interestingly, the engineered enzymes are completely inactive towards their natural substrate tryptophan in spite of their high tolerance to various functional groups in the indole ring. Computational modelling and molecular dynamics simulations provide mechanistic insights into the role of gatekeeper residues in the substrate binding site and the dramatic switch in substrate specificity when these are mutated.     

Facile in Vitro Biocatalytic Production of Diverse Tryptamines

Tryptamines are a medicinally important class of small molecules that serve as precursors to more complex, clinically used indole alkaloid natural products. Typically, tryptamine analogues are prepared from indoles through multistep synthetic routes. In the natural world, the desirable tryptamine synthon is produced in a single step by l -tryptophan decarboxylases (TDCs). However, no TDCs are known to combine high activity and substrate promiscuity, which might enable a practical biocatalytic route to tryptamine analogues. We have now identified the TDC from Ruminococcus gnavus as the first highly active and promiscuous member of this enzyme family. RgnTDC performs up to 96 000 turnovers and readily accommodates tryptophan analogues with substituents at the 4, 5, 6, and 7 positions, as well as alternative heterocycles, thus enabling the facile biocatalytic synthesis of >20 tryptamine analogues. We demonstrate the utility of this enzyme in a two-step biocatalytic sequence with an engineered tryptophan synthase to afford an efficient, cost-effective route to tryptamines from commercially available indole starting materials.     

Unique Versatility of Ionic Liquids as Clean Decarboxylation Catalyst Cum Solvent: A Metal‐ and Quinoline‐Free Paradigm towards Synthesis of Indoles,Styrenes, Stilbenes and Arene Derivatives under Microwave Irradiation in Aqueous Conditions

Ionic liquids have been found to provide a new platform for metal‐ and quinoline‐free decarboxylation of various N‐heteroaryl and aryl carboxylic acids under microwave irradiation in aqueous condition. The method was found to possess a wide substrate scope towards the synthesis of various pharmacologically and industrially important aromatic compounds including indoles, styrenes, stilbenes, and nitro‐ or hydroxyarene derivatives. The decarboxylation of indole and α‐phenylcinnamic acids proceeded well without addition of any catalyst in neat 1‐hexyl‐3‐methylimidazolium bromide ([hmim]Br) and 1‐methylimidazolium p‐toluenesulfonic acid ([Hmim]PTSA), respectively, while addition of a mild base like aqueous sodium hydrogen carbonate (NaHCO₃) to [hmim]Br further improved the decarboxylation of hydroxylated cinnamic and aromatic acid substrates. The developed methodology not only precludes the usage of toxic metal/quinoline and harsh organic bases but also offers several inherent benefits like recyclability of reagent system, reduction in waste and hazards, short reaction time besides ease of product recovery.     

Tryptophan Synthase: Biocatalyst Extraordinaire

Tryptophan synthase (TrpS) has emerged as a paragon of noncanonical amino acid (ncAA) synthesis and is an ideal biocatalyst for synthetic and biological applications. TrpS catalyzes an irreversible, C−C bond-forming reaction between indole and serine to make l -tryptophan; native TrpS complexes possess fairly broad specificity for indole analogues, but are difficult to engineer to extend substrate scope or to confer other useful properties due to allosteric constraints and their heterodimeric structure. Directed evolution freed the catalytically relevant TrpS β-subunit (TrpB) from allosteric regulation by its TrpA partner and has enabled dramatic expansion of the enzyme's substrate scope. This review examines the long and storied career of TrpS from the perspective of its application in ncAA synthesis and biocatalytic cascades.     

Structure and Activity of the Thermophilic Tryptophan-6 Halogenase BorH

Flavin-dependent halogenases carry out regioselective aryl halide synthesis in aqueous solution at ambient temperature and neutral pH using benign halide salts, making them attractive catalysts for green chemistry. BorH and BorF, two proteins encoded by the biosynthetic gene cluster for the chlorinated bisindole alkaloid borregomycin A, are the halogenase and flavin reductase subunits of a tryptophan-6-halogenase. Quantitative conversion of l -tryptophan (Trp) to 6-chlorotryptophan could be achieved using 1.2 mol % BorH and 2 mol % BorF. The optimal reaction temperature for Trp chlorination is 45 °C, and the melting temperatures of BorH and BorF are 48 and 50 °C respectively, which are higher than the thermal parameters for most other halogenases previously studied. Steady-state kinetic analysis of Trp chlorination by BorH determined parameters of k_cat=4.42 min⁻¹, and K_M of 9.78 μm at 45 °C. BorH exhibits a broad substrate scope, chlorinating and brominating a variety of aromatic substrates with and without indole groups. Chlorination of Trp at a 100 mg scale with 52 % crude yield, using 0.2 mol % BorH indicates that industrial scale biotransformations using BorH/BorF are feasible. The X-ray crystal structure of BorH with bound Trp provides additional evidence for the model that regioselectivity is determined by substrate positioning in the active site, showing C6 of Trp juxtaposed with the catalytic Lys79 in the same binding pose previously observed in the structure of Thal.     

Selective nitration: the catalyzed nitration of o-xylene in the presence of Hg(II) salts

Certain metal cations, in particular Hg(II), can direct the course of aromatic nitration. A process is described for producing 4-nitro-o-xylene selectively with respect to 3-nitro-o-xylene in a system comprising o-xylene (1,2-dimethylbenzene), N₂O₄,acetic acid and a catalytic quantity of Hg(II). The ratio of 4-nitro-o-xylene to 3-nitro-o-xylene is 3:1 in the presence of Hg(II) as compared to 1:1 for the uncatalyzed nitration using conventional mixed acid (HNO₃/H₂SO₄). Conversion of o-xylene is upwards of 80% and no dinitroproducts are formed.     

Electrophilic Aromatic Nitration Using a Mixed Catalyst of Lithium,Molybdenum, Ytterbium on Silica Gel

A novel mixed catalyst of LiClO₄ (15% w/w), Yb(OPf)₃ (15% w/w, Pf=perfluorooctanesulfonyl), MoO₃ (15% w/w) on silica gel for electrophilic aromatic nitration reaction has been explored. The nitration reactions were accomplished by this mixed catalyst and nitric acid under solvent‐free conditions. Moreover, the mixed catalyst can be easily recovered from the aqueous layer by heating in an oven and reused for the next nitration reaction.     

Regioselective and green synthesis of nitro aromatic compounds using polymer‐supported sodium nitrite/KHSO4

Polymer supported reagents have become the subject of considerable and increasing interest as insoluble materials in the organic synthesis. Use of polymeric reagents simplifies routine nitration of aromatic compounds because it eliminates traditional purification. In this article, the use of readily available cross‐linked poly(4‐vinylpyridine) supported sodium nitrite, [P₄‐Me] NO₂, as an efficient polymeric nitrating agent in the presence of KHSO₄ is described. A good range of available aromatic compounds were also subjected to nitration in the presence of [P₄‐Me] NO₂/KHSO₄. This reagent is regioselective and chemoselective nitrating polymeric reagent for activated aromatic rings. In this procedure, the work‐up is easy, and the spent polymeric reagent is easily regenerated and reused. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Deep extractive desulfurization of oil over 12-molibdophosphoric acid encapsulated in metal-organic frameworks

A desulfurization process for model oil and real oil was investigated based on the chemical oxidation of mixed sulfur containing compounds in the presence of nitrogen compounds (indole and quinoline) using hydrogen peroxide as oxidizing agent and dodecamolibdophosphoric acid (H₃PMo₁₂O₄₀) encapsulated in a kind of metal-organic framework (HKUST-1) as PMo@HKUST-1. The effect of isopropanol, ethanol and acetonitrile as extractive solvent and 1-ring (toluene, xylene and mesitylene) and 2-ring (naphthalene) aromatic hydrocarbons in desulfurization of model oil was studied. The desulfurization of sulfur-containing compounds was accelerated in the presence of aromatic hydrocarbons. In fact, a higher desulfurization efficiency of the heterogeneous catalyst could be achieved with system containing a polar solvent in contact with an aromatic hydrocarbon. Quinoline had no effect on oxidative desulfurization (ODS) reaction, whereas indole had a slightly negative effect. Presence of aromatic compounds had slightly positive effect on ODS reaction.     

Chemiluminescence of indole and its derivatives induced by electrogenerated superoxide ion in acetonitrile solutions

Electrochemical behavior of the dioxygen (O₂)/superoxide ion (O₂ ⁻) redox couple in acetonitrile (AN) solutions containing indole compounds and no proton acceptor, and their chemiluminescence have been examined using spectroelectrochemical techniques. In AN solutions, in the presence of indole compounds having a substituent at the N-position (e.g. 1-methylindole and 1,2-dimethylindole), these indole compounds did not work as a proton donor to electrogenerated O₂ ⁻, and did not affect the redox reaction of the O₂/O₂ ⁻ couple. On the other hand, the indole compounds bearing a hydrogen at the N-position (e.g. indole, 3-methylindole (3-MI), tryptamine (TP), 3-indolemethanol and 3-indoleacetic acid) acted as a proton donor to O₂ ⁻. In addition, chemiluminescence was observed for some of the indole compounds having a hydrogen at the N-position and an electron-releasing group at the 3-position (i.e. TP, 2,3-dimethylindole, 2-(3-indole)-ethanol and 3-MI). The intensity of chemiluminescence of 3-MI was by far the strongest among those of the above four compounds. The chemiluminescent reaction of the indole compounds seemed to have close connection with electrogeneration of O₂ ⁻, which functioned as a proton acceptor to the indole compounds, and the chemical stability of a radical species (i.e. a one-electron oxidized form of conjugate base of indole compounds) due to the substituent which is present at the 3-position. The chemiluminescence was considered to occur via the formation of 1,2-dioxetane-like intermediates. The reaction mechanisms of chemiluminescence based on the electrogenerated O₂ ⁻ were briefly discussed.     

Synthese von Heterocyclen mit Hydroxymethylenketonen. XIV [1]. Zur Regioselektivität der Reaktion von Acetylacetaldehyd mit Tryptamin

Synthesis of Heterocyclic Compounds with Hydroxymethylene Ketones. XIV. Contribution to the Regioselectivity of the Reaction of Acetoacetaldehyde with Tryptamine The range of substitution products of tryptamine with acetoacetaldehyde as substituent at the basic or the indole nitrogen ( 2, 3 ) is completed by a product containing the substituent in the indole α-position ( 5 ). It is formed by ring opening of the tetrahydro-β-carboline 4 . The reaction conditions are commented and the ¹H-NMR spectra comparatively discussed. The synthesis of the azocino-indole 7 is described.     

Tetrahydroindoles as Multipurpose Screening Compounds and Novel Sirtuin Inhibitors

Indoles are privileged structures in medicinal and bioorganic chemistry that are particularly well suited to serve as platforms for diversity. Among many other therapeutic areas, the indole scaffold has been used to design aromatic compounds useful to interfere with enzymes engaged in the regulation of substrate acylation status, such as sirtuins. However, the planarity of the indole ring is not necessarily optimal for all target enzymes, especially when functionalization with aromatic side chains is required. Replacement of flat scaffolds by nonplanar molecular cores dominated by sp³ hybridization is a common strategy to avoid the disadvantages associated with poor solubility and high promiscuity, while covering less-well-explored areas of chemical space. Thus, we synthesized fragment-like tetrahydroindoles suitable for fragment-based drug discovery as well as a well-characterized small library intended as multipurpose screening compounds. For proof of principle, these compounds were screened against sirtuins 1–3, enzymes known to be addressable by indoles. We found that 2,6,6-trimethyl-4-oxo-4,5,6,7-tetrahydro-1H-indole-3-carboxamides are potent and selective SIRT2 inhibitors. Compound 16 t displayed an IC₅₀ value of 0.98 μm and could serve as exquisite starting point for hit-to-lead profiling.     

Composite Powders with the Pseudoeutectic Structure in the Al2O3 – TiN System

The processes occurring in the synthesis of composite powders in Al₂O₃ – TiN system are considered for two variants: the first variant is the reaction of carbothermic reduction of TiO₂ mixed with Al₂O₃ with simultaneous nitration, and the second one is direct nitration of metallic titanium dispersed in aluminum oxide under high-frequency heating in a “cold” crucible. The synthesis produces submicron composite powders, which are promising for application in construction ceramics.     

Synthesis of Bisindolylmethanes and Their Cytotoxicity Properties

Polymer supported dichlorophosphate (PEG-OPOCl₂) is an efficient green catalyst for the electrophilic substitution reaction of indole with aromatic aldehydes, in neat condition, to afford an excellent yield of bis(indolyl) methanes with short reaction time, at room temperature. The synthesized compounds and their anti-cancer activity are evaluated.     

One-pot Synthesis of 5-Unsubstituted 3,4-Dihydropyrimidin-2(1H)-ones from Aldehydes,Ketones, and Urea under Solvent-free Conditions

Various 5-unsubstituted 3,4-dihydropyrimidin-2(1H)-ones were synthesized efficiently by a one-pot three-component condensation of aromatic aldehydes, aromatic ketones, and urea using SnCl₂·2H₂O as a catalyst under solvent-free conditions. All products were identified by IR, NMR, MS, and elemental analysis. The novel method offers several advantages such as excellent yields (81–95%), short reaction time (5–16 min), broader substrate scope, and environmentally friendly conditions.     

An efficient method for nitration of aromatic compounds over solid acid and polymer‐supported sodium nitrite

A variety of aromatic compounds are nitrated under heterogeneous conditions using a polymer‐supported hydrochloric acid, [P₄‐(VP)]HCl, with a polymer‐supported sodium nitrite, [P₄‐VP]NO₂, or sodium nitrite in ethanol at room temperature with high yields. This methodology is useful for nitration of activated aromatic compounds. In this procedure, the work‐up is very easy, and the spent polymeric reagent can be regenerated and reused. © 2011 Wiley Periodicals, Inc. J Appl Polym Sci, 2011     

Evaluation of the accessibility of celluloses by the intrinsic viscosity ratio [η] acetone cell.nitr. / [gh] CuEn unsubst.cell.

Summary Standarization of the nitration procedure as well as of the conditions of viscosimetric measurements on cellulose nitrates and unsubstituted celluloses leads to a constant Intrinsic Viscosity Ratio [] _Acetone ^Cell.nitr. / [gh] _CuEn ^{Unsubst.cell.} = 1.95±0.05, independently from the range of DP. Since nitration under the standarized conditions occurs without degradation and delivers a reproducible constant degree of substitution of DS = 2.90±0.02, intrinsic viscosity ratios lower than 1.90 indicate a diminished accessibility of the respective cellulose. Determination of the intrinsic viscosity ratio reveals, therefore, the possibility to evaluate the accessibility of a cellulose sample.     

Investigation of the Correlations Between Nitro Group Charges and Some Properties of Nitro Organic Compounds

The so‐called nitro group charge method (NGCM) is successfully established to investigate some properties of nitro compounds including the molecular stability measured by total energy (only for isomers), the bond lengths, bond dissociation energies (BDE), and the nitrating activities, in that the method considers the molecular structure. These properties are intrinsically and especially thermodynamically consistent with each other and can be well related qualitatively and even quantitatively with nitro group charges (Q_Nitro). The correlations between Q_Nitro and the properties are: (1) for nitro isomers, the more negative the average Q_Nitro, the lower the total energy and the more stable is the isomer; (2) for any separate group of nitro compounds, the more negative Q_Nitro, the shorter the R‐nitro bond length; (3) for the bond dissociation energy, more negative Q_Nitro corresponds to a higher BDE of the R‐nitro bond; (4) by NGCM, the conditions, the reaction rates and the occurrence ratios of products of some nitration can be predicted and compared: the more negative Q_Nitro of the product, the easier and faster the nitration, and the higher the occurrence ratio of the corresponding product.     

Tryptamine-induced resistance in tryptophan decarboxylase transgenic poplar and tobacco plants against their specific herbivores

The presence of amines and their derivatives in plant tissues is known to influence insect feeding and reproduction. The enzyme tryptophan decarboxylase (TDC) catalyzes the decarboxylation of tryptophan to tryptamine, which is both a bioactive amine and a precursor of other indole derivatives. Transgenic poplar and tobacco plants ectopically expressing TDC1 accumulated elevated levels of tryptamine without affecting plant growth and development. This accumulation was consistently associated with adverse effects on feeding behavior and physiology of Malacosoma disstria Hub. (forest tent caterpillar, FTC) and Manduca sexta L. (tobacco hornworm, THW). Behavior studies with FTC and THW larvae showed that acceptability of the leaf tissue to larvae was inversely related to foliar tryptamine levels. Physiological studies with FTC and THW larvae showed that consumption of leaf tissue from the transgenic lines is deleterious to larvae growth, apparently due to a postingestive mechanism. Thus, ectopic expression of TDC1 can allow sufficient tryptamine to accumulate in poplar and tobacco leaf tissue to suppress significantly the growth of insect pests that normally feed on these plants.