12626-73-2Relevant articles and documents
Biosynthesis of indigo in Escherichia coli expressing self-sufficient CYP102A from Streptomyces cattleya
Kim, Hee-Jung,Jang, Seyoung,Kim, Joonwon,Yang, Yung-Hun,Kim, Yun-Gon,Kim, Byung-Gee,Choi, Kwon-Young
, p. 29 - 35 (2017)
Cytochrome P450 monooxygenases (CYP) are a superfamily of heme-thiolate proteins which catalyze the incorporation of oxygen atoms into substrates. Here, a self-sufficient CYP102A from Streptomyces cattleya (CYP102A_scat) was cloned, produced recombinantly in Escherichia coli strain BL21 (DE3), and the characteristic features were investigated. However, unlike other self-sufficient CYP102A enzymes that have been reported, CYP102A_scat was found to be able to catalyze intracellular hydroxylation of indole molecules with 3-C specific regioselectivity. Consequently, E.?coli strains producing CYP102A_scat could synthesize approximately 1.0?g/L of indigo in LB media. Optimization of indigo synthesis was investigated through additional feeding of indole precursors such as glucose, L-tryptophan, and indole. Indigo production reached up to 3.8?±?0.1?g/L by adding 20?μM of extracellular indole and 0.2?mM of L-tryptophan to the LB media. To our knowledge, this is a record and the highest yield achieved so far.
Determination of acetylcholinesterase and butyrylcholinesterase activity without dilution of biological samples
Pohanka, Miroslav
, p. 1044 - 1049 (2015)
Two cholinesterases: acetylcholinesterase (AChE) and butyrylcholinesterase (BChE), are known. The enzymes are important in the body and alteration of their activity has significant use in the diagnosis of poisoning, liver function, etc. Currently available methods for the determination of cholinesterases have some major drawbacks including various interferences and the inability to be used for decreasing the enzyme activity in the presence of reversible inhibitors due to sample dilution; hence, a method for dilution free assay of cholinesterases is desired. Here, microplates were modified with indoxylacetate (100 μL of 10 mmol L-1 solution) and used for cholinesterases assay after drying at 37 °C. The fact that indoxylacetate remains stable in dry state and serves simultaneously as a chromogen and substrate provide good prerequisites for the method. The limit of detection for BChE was 0.71 U while that for AChE was 2.8 U per a 100 μL sample (solution of enzyme or plasma sample). The limit of detection is low enough to allow standard examination of cholinesterasemia. The two cholinesterases can be distinguished from each other using selective inhibitors such as donepezil and iso-OMPA. The new method was also successfully validated for the standard Ellman's assay using plasma samples with BChE activity adjusted by carbofuran. The new method based on indoxylacetate seems promising for routine tests.
A cyclodextrin-based molecular reactor to template the formation of indigoid dyes
Harper, Jason B.,Easton, Christopher J.,Lincoln, Stephen F.
, p. 5815 - 5818 (2003)
N,N′-Bis(6A-deoxy-β-cyclodextrin-6 A-yl)urea behaves as a molecular reactor to bias competing reactions of indoxyl anion and isatin-5-sulfonate in water, to give indigo and indirubin-5′-sulfonate. It appears that the cyclodextrin dimer increases the relative reactivity of the isatin-5-sulfonate, by selectively complexing the reactive form. The molecular host also aligns the isatinsulfonate with indoxyl anion to favour production of indirubin-5′-sulfonate, with the result that the ratio of indigo and indirubin-5′-sulfonate produced is altered by a factor of at least 3500, without a substantial loss of yield.
Effects of Organic Solvents on Indigo Formation by Pseudomonas sp. strain ST-200 Grown with High Levels of Indole
Doukyu, Noriyuki,Arai, Tomonori,Aono, Rikizo
, p. 1075 - 1080 (1998)
The indole tolerance level of Pseudomonas sp. strain ST-200 was 0.25 mg/ml. The level was raised to 4 mg/ml when diphenylmethane was added to the medium to 20% by volume. ST-200 grown in this two-phase culture system containing indole (1 mg/ml) and diphenylmethane (0.2 ml/ml) produced a water-soluble yellow pigment, isatic acid, and two water-insoluble and diphenylmethane-soluble pigments, blue indigo and purple indirubin. The amounts of the water-insoluble pigments corresponded to 0.5% (indigo) and 0.2% (indirubin) of the indole added to the medium. Of the conditions tried, indigo and indirubin were formed only when ST-200 was grown in the two-phase system overlaid with organic solvents with appropriate polarity.
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Bailey,Potter
, p. 215 (1922)
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Identification of broad specificity P450CAM variants by primary screening against indole as substrate
Celik, Ayhan,Speight, Robert E.,Turner, Nicholas J.
, p. 3652 - 3654 (2005)
High-throughput screening of cytochrome P450CAM libraries, for their ability to oxidise indole to indigo and indirubin, has resulted in the identification of variants with activity towards the structurally unrelated substrate diphenylmethane. The Royal Society of Chemistry 2005.
Enzymatically triggered chromogenic cross-linking agents under physiological conditions
Fujita, Hikaru,Dou, Jinghuai,Matsumoto, Nobuyuki,Wu, Zhiyuan,Lindsey, Jonathan S.
, p. 719 - 743 (2020)
The ability to cross-link molecules upon enzymatic action under physiological conditions holds considerable promise for use in diverse life sciences applications. Here, an enzymatically triggered "click reaction" has been developed by exploiting the longstanding indigo-forming reaction from indoxyl β-glucoside. The covalent cross-linking proceeds in aqueous solution, requires the presence only of an oxidant (e.g., O2), and is readily detectable owing to the blue color of the resulting indigoid dye. To achieve facile indigoid formation in the presence of a bioconjugatable tether, diverse indoxyl β-glucosides were synthesized and studied in enzyme assays with four glucosidases including from tritosomes (derived from hepatic lysosomes) and rat liver homogenates. Altogether 36 new compounds (including 15 target indoxyl-glucosides for enzymatic studies) were prepared and fully characterized in pursuit of four essential requirements: enzyme triggering, facile subsequent indigoid dye formation, bioconjugatability, and synthetic accessibility. The 4,6-dibromo motif in a 5-alkoxy-substituted indoxyl-glucoside was a key design feature for fast and high-yielding indigoid dye formation. Two attractive molecular designs include (1) an indoxyl-glucoside linked to a bicyclo[6.1.0]nonyl (BCN) group for Cu-free click chemistry, and (2) a bis(indoxyl-glucoside). In both cases the linker between the reactive moieties is composed of two short PEG groups and a central triazine derivatized with a sulfobetaine moiety for water solubilization. Glucosidase treatment of the bis(indoxyl-glucoside) in aqueous solution gave oligomers that were characterized by absorption, dynamic light-scattering, and 1H NMR spectroscopy; optical microscopy; mass spectrometry; and HPLC. Key attractions of in situ indigoid dye formation, beyond enzymatic triggering under physiological conditions without exogenous catalysts or reagents, are the chromogenic readout and compatibility with attachment to diverse molecules.
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Roy,Swan
, p. 80 (1968)
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THE STRUCTURE AND PROPERTIES OF SOME INDOLIC CONSTITUENTS IN COUROUPITA GUIANENSIS Aubl.
Bergman, Jan,Lindstroem, Jan-Olof,Tilstam, Ulf
, p. 2879 - 2882 (1985)
Extraction of the dried fruits of the cannon ball tree, Couroupita guianensis Aubl., yielded 6,12-dihydro-6,12-dioxoindoloquinazoline (tryptanthrin), 1a as well as indigo (7) indirubin (8a) and isatin.Compound 1a could readily be synthesized by condensation of isatin with isatoic anhydride (6) in pyridine.
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Perkin,Bloxam
, p. 283 (1907)
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Christopherson et al.
, p. 2779 (1978)
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Development of a practical one-pot synthesis of indigo from indole
Yamamoto, Yoshihiro,Inoue, Yoshihisa,Takaki, Usaji,Suzuki, Hiroharu
, p. 82 - 89 (2011)
A novel and highly practical one-pot synthesis of indigo from indole via 3-position selective oxidation and dimerization of the indole framework was developed. Using 0.1 mol% of molybdenum complex and 2.2 equivalents of cumene hydroperoxide in tert-butyl alcohol, the reaction was complete in 7 h and pure indigo was obtained in 81% yield as a deep-blue solid just by filtration. The described one-step method renders the pure indigo readily available on a large scale using only inexpensive raw materials.
Indigo Formation and Rapid NADPH Consumption Provide Robust Prediction of Raspberry Ketone Synthesis by Engineered Cytochrome P450 BM3
Rousseau, Olivier,Ebert, Maximilian C. C. J. C.,Quaglia, Daniela,Fendri, Ali,Parisien, Adem H.,Besna, Jonathan N.,Iyathurai, Saathanan,Pelletier, Joelle N.
, p. 837 - 845 (2020)
Natural raspberry ketone has a high value in the flavor, fragrance and pharmaceutical industries. Its extraction is costly, justifying the search for biosynthetic routes. We hypothesized that cytochrome P450 BM3 (P450 BM3) could be engineered to catalyze the hydroxylation of 4-phenyl-2-butanone, a naturally sourceable precursor, to raspberry ketone. The synthesis of indigo by variants of P450 BM3 has previously served as a predictor of promiscuous oxidation reactions. To this end, we screened 53 active-site variants of P450 BM3 using orthogonal high-throughput workflows to identify the most streamlined route to all indigo-forming variants. Among the three known and 13 new indigo-forming variants, eight hydroxylated 4-phenyl-2-butanone to raspberry ketone. Previously unreported variant A82Q displayed the highest initial rates and coupling efficiencies in synthesis of indigo and of raspberry ketone. It produced the highest total concentration of raspberry ketone despite producing less total indigo than previously reported variants. Its productivity, although modest, clearly demonstrates the potential for development of a biocatalytic route to raspberry ketone. In addition to validating indigo as a robust predictor of this promiscuous activity, we demonstrate that monitoring rapid NADPH consumption serves as an alternative predictor of a promiscuous reactivity in P450 BM3.
Exploring an anomaly: The synthesis of 7,7′-diazaindirubin through a 7-azaindoxyl intermediate
DeYoung, James R.,Lipsius, Richard J.,Patterson, Andrew C.,Shriver, James A.,Wang, Katelyn R.
, p. 36849 - 36852 (2020)
Two independent methods generating 7-azaindoxyl as an intermediate verify that 7,7′-diazaindirubin is formed exclusively over 7,7′-diazaindigo. This contrasts with long-standing knowledge related to the reactivity of indoxyl, which proceeds via a radical-initiated homodimerization process, leading to indigo. A series of experiments confirms 7-azaindoxyl as an intermediate with results suggesting a condensation pathway followed by oxidation.
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Askam,Deeks
, p. 1243 (1968)
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Transformation of isatin with P4S10 to pentathiepino[6,7-b]indole in one step
Bergman, Jan,Stlhandske, Claes
, p. 5279 - 5282 (1994)
Pentathiepino[6,7-b]indole (6) can be isolated from reactions of P4S10 with isatin in pyridine. The structure of 6 has been determined with X-ray crystallography.
CROSS-LINKING COMPOUNDS AND METHODS OF USE THEREOF
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Page/Page column 50-51, (2021/01/29)
Compounds comprising a cross-linking moiety and a protecting group are described herein along with their methods of use. The cross-linking moiety may comprise an indoxyl and the protecting group may comprise a sugar (e.g., a glucuronide or glucoside), phosphoester, or sulfoester group. The cross-linking moiety and protecting group may be attached to each other via an oxygen atom, sulfur atom, or linker. In some embodiments, the linker attaching the cross-linking moiety and protecting group is a self-immolative linker. A compound of the present invention may cross-link under physiological conditions and/or in vivo.
Structural and Biochemical Studies Enlighten the Unspecific Peroxygenase from Hypoxylon sp. EC38 as an Efficient Oxidative Biocatalyst
Ebner, Katharina,Glieder, Anton,Kroutil, Wolfgang,Mattevi, Andrea,Rinnofner, Claudia,Rotilio, Laura,Swoboda, Alexander
, p. 11511 - 11525 (2021/09/22)
Unspecific peroxygenases (UPOs) are glycosylated fungal enzymes that can selectively oxidize C-H bonds. UPOs employ hydrogen peroxide as the oxygen donor and reductant. With such an easy-to-handle cosubstrate and without the need for a reducing agent, UPOs are emerging as convenient oxidative biocatalysts. Here, an unspecific peroxygenase from Hypoxylon sp. EC38 (HspUPO) was identified in an activity-based screen of six putative peroxygenase enzymes that were heterologously expressed in Pichia pastoris. The enzyme was found to tolerate selected organic solvents such as acetonitrile and acetone. HspUPO is a versatile catalyst performing various reactions, such as the oxidation of prim- and sec-alcohols, epoxidations, and hydroxylations. Semipreparative biotransformations were demonstrated for the nonenantioselective oxidation of racemic 1-phenylethanol rac-1b (TON = 13 000), giving the product with 88% isolated yield, and the oxidation of indole 6a to give indigo 6b (TON = 2800) with 98% isolated yield. HspUPO features a compact and rigid three-dimensional conformation that wraps around the heme and defines a funnel-shaped tunnel that leads to the heme iron from the protein surface. The tunnel extends along a distance of about 12 ? with a fairly constant diameter in its innermost segment. Its surface comprises both hydrophobic and hydrophilic groups for dealing with substrates of variable polarities. The structural investigation of several protein-ligand complexes revealed that the active site of HspUPO is accessible to molecules of varying bulkiness with minimal or no conformational changes, explaining the relatively broad substrate scope of the enzyme. With its convenient expression system, robust operational properties, relatively small size, well-defined structural features, and diverse reaction scope, HspUPO is an exploitable candidate for peroxygenase-based biocatalysis.
