Bacterial terpene biosynthesis: challenges and opportunities for pathway engineering

• Eric J. N. Helfrich,
• Geng-Min Lin,
• Christopher A. Voigt and
• Jon Clardy

Beilstein J. Org. Chem. 2019, 15, 2889–2906, doi:10.3762/bjoc.15.283

• potential with molecular structure is severely restricted. The canonical terpene biosynthetic pathway uses a single enzyme to form a cyclized hydrocarbon backbone followed by modifications with a suite of tailoring enzymes that can generate dozens of different products from a single backbone. This

• a linear polyene with branching methyl groups that form the core hydrocarbon structure in a single enzyme-catalyzed step [9]. The enzyme, which is called terpene cyclase, holds the linear methyl-branched polyene in a defined conformation that initiates a series of carbocation-driven cyclizations and

• placed on terpene pathways from bacteria, as their biosynthetic pathways usually have the genes encoding the terpene cyclase and modifying enzyme in close proximity, which simplifies both analysis and pathway engineering. The review will begin with a brief description of terpene families with a special

Emission and biosynthesis of volatile terpenoids from the plasmodial slime mold Physarum polycephalum

• Xinlu Chen,
• Tobias G. Köllner,
• Wangdan Xiong,
• Guo Wei and
• Feng Chen

Beilstein J. Org. Chem. 2019, 15, 2872–2880, doi:10.3762/bjoc.15.281

• primers (Table S2, Supporting Information File 1), cloned into pEXP5 CT/TOPO vector (https://www.thermofisher.com) and fully sequenced. Terpene synthase enzyme assays pEXP5 CT/TOPO vector containing individual PpolyTPS genes was transformed into E. coli strain BL21 Codon Plus (DE3). Heterologous

• expression of individual PpolyTPS genes in E. coli and recombinant protein preparation and terpene synthase enzyme assays were performed as previously described [42]. Each PoplyTPS recombinant protein was tested with both geranyl diphosphate and farnesyl diphosphate as substrates and terpene products were

• Supporting Information File 612: Additional figures and tables. Acknowledgements Wangdan Xiong was supported by a scholarship from the China Scholarship Council. Kevin Chen from Farragut High School, Knoxville, is acknowledged for his assistance with chemical profiling and TPS enzyme assays for this project.

Palladium-catalyzed synthesis and nucleotide pyrophosphatase inhibition of benzo[4,5]furo[3,2-b]indoles

• Hoang Huy Do,
• Saif Ullah,
• Alexander Villinger,
• Joanna Lecka,
• Jean Sévigny,
• Peter Ehlers,
• Jamshed Iqbal and
• Peter Langer

Beilstein J. Org. Chem. 2019, 15, 2830–2839, doi:10.3762/bjoc.15.276

• reported diindolofurans 6a–e (Figure 3) [31]. All compounds show significant inhibition of enzyme h-NPP-3 (Table 3) and most of them of the enzyme h-NPP-1. Compound 5a, containing a phenyl substituent, and compound 5e, containing a p-methoxyphenyl group, showed a selective inhibitory response towards

• nucleotide pyrophosphatase enzyme h-NPP-3. In case of 5e, an inhibitory value IC50 ± SEM = 0.26 ± 0.01 µM was observed which, thus, might be considered as potential inhibitor of h-NPP-3. Compound 5c with an inhibitory value for h-NPP-1 of IC50 ± SEM = 1.29 ± 0.07 µM was more active against NPP1 than against

• towards h-NPP-3 (IC50 ± SEM = 0.13 ± 0.06 and 0.28 ± 0.04, respectively). In fact, they had no interaction with enzyme h-NPP-1. Compound 6a, containing a tolyl group, was active against both h-NPP-1 and h-NPP-3, but was more selective to h-NPP-1 with an inhibitory value of IC50 ± SEM = 0.11 ± 0.06 µM. In

Chemical tuning of photoswitchable azobenzenes: a photopharmacological case study using nicotinic transmission

• Lorenzo Sansalone,
• Jun Zhao,
• Matthew T. Richers and
• Graham C. R. Ellis-Davies

Beilstein J. Org. Chem. 2019, 15, 2812–2821, doi:10.3762/bjoc.15.274

• into the corresponding cis configuration [1]. The reverse reaction can be initiated with a different wavelength. In a biological context, this photochromism was exploited first by Erlanger and colleagues for enzyme inhibitors in 1968 [2], and ion channels in 1971 [3]. Their ingenious approach was

A review of asymmetric synthetic organic electrochemistry and electrocatalysis: concepts, applications, recent developments and future directions

• Munmun Ghosh,
• Valmik S. Shinde and
• Magnus Rueping

Beilstein J. Org. Chem. 2019, 15, 2710–2746, doi:10.3762/bjoc.15.264

• 2000, reported for the first time, a crucial modification of a glassy carbon electrode via chemical immobilization of ᴅ-amino acid oxidase (D-AOx) as enzyme and 1-aminopropyl-1'-methyl-4,4'-dipyridinium iodide (ADPy) as electron mediator and thus prepared electrode D-AOx/ADPy/GC. Electrochemical

• transformations using various catalysts as chiral sources has been studied in greater detail than the other strategies mentioned in this review. As per our classification, we further categorize this class into metal catalysts, enzyme catalysts and organocatalysts. Metal catalysts: In 1992, Amundsen and co-workers

• potentiostatic electrolysis of a mixture of ketones 129 with tertiary amines 128 in an undivided cell under standard conditions furnished C1-alkylated tetrahydroisoquinolines 131 in high yields with excellent enantioselectivity (Scheme 42). Enzyme catalysts: In 1981, Tischer’s group reported the electroenzymatic

Nanangenines: drimane sesquiterpenoids as the dominant metabolite cohort of a novel Australian fungus, Aspergillus nanangensis

• Heather J. Lacey,
• Cameron L. M. Gilchrist,
• Andrew Crombie,
• John A. Kalaitzis,
• Daniel Vuong,
• Peter J. Rutledge,
• Peter Turner,
• John I. Pitt,
• Ernest Lacey,
• Yit-Heng Chooi and
• Andrew M. Piggott

Beilstein J. Org. Chem. 2019, 15, 2631–2643, doi:10.3762/bjoc.15.256

• terpene synthase family, showing similarity to haloacid dehalogenase (HAD)-like hydrolases [21]. Thus, we suspected that a related enzyme may be involved in the biosynthesis of the nanangenines, and used the amino acid sequence of AstC to probe the A. nanangensis genome. We also hypothesised that the acyl

• analyses above, a biosynthetic pathway to the nanangenines was proposed (Figure 3). Unlike the ast cluster, where there are multiple HAD-like enzymes encoded (one terpene synthase and two phosphatases), the putative nanangenine cluster only encodes one such enzyme, FE257_006542. However, given that

• . The C-6 and C-8 lipid chain is likely produced by the HR-PKS encoded by gene FE257_006541, while the acylation could be attributed to the enzyme encoded by FE257_006545, which contains the conserved domain of the alpha/beta-hydrolase fold superfamily (which includes thioesterases and acyltransferases

Probing of local polarity in poly(methyl methacrylate) with the charge transfer transition in Nile red

• Aydan Yadigarli,
• Qimeng Song,
• Sergey I. Druzhinin and
• Holger Schönherr

Beilstein J. Org. Chem. 2019, 15, 2552–2562, doi:10.3762/bjoc.15.248

• polymer films, in which the values of the glass transition temperature (Tg) and segmental mobilities were found to be altered. Likewise, this holds for transport properties, including polymer nanocapsule membrane permeability [1], enzyme-triggered bacterial sensors [2][3][4][5] and intelligent self

• [1] and segment mobilities inferred [13], respectively. For other purposes the oxazine tracer dye Nile red (NR, Figure 1) served as a local probe to enable the study of degradation of enzyme labile polymersomes [14]. The same dye has been reported as probe for local permittivity in polymers, in

Current understanding and biotechnological application of the bacterial diterpene synthase CotB2

• Ronja Driller,
• Daniel Garbe,
• Norbert Mehlmer,
• Monika Fuchs,
• Keren Raz,
• Dan Thomas Major,
• Thomas Brück and
• Bernhard Loll

Beilstein J. Org. Chem. 2019, 15, 2355–2368, doi:10.3762/bjoc.15.228

• date, CotB2 represents the best studied bacterial diterpene synthase. Its reaction mechanism has been addressed by isoptope labeling, targeted mutagenesis and theoretical computations in the gas phase, as well as full enzyme molecular dynamic simulations. By X-ray crystallography different snapshots of

• , antibiotic, neuroprotective and even insecticidal activities, which makes these compounds high-value commercial targets for the chemical and pharmaceutical industry [9][10]. Structural diversity of diterpenes is created by the terpene synthase (TPS) enzyme family, which use acyclic isoprenoid precursors to

• engineering to optimize product ratios or to alter the native product portfolio of the enzyme [9][26][27]. Furthermore, production by engineered microorganisms considerably reduces the cost compared to total chemical synthesis or extraction from natural sources, since the target compounds are produced from

Isolation and biosynthesis of an unsaturated fatty acid with unusual methylation pattern from a coral-associated bacterium Microbulbifer sp.

• Amit Raj Sharma,
• Enjuro Harunari,
• Tao Zhou,
• Agus Trianto and
• Yasuhiro Igarashi

Beilstein J. Org. Chem. 2019, 15, 2327–2332, doi:10.3762/bjoc.15.225

• methyltransferase, followed by 1,2-hydride shift and deprotonation, and a subsequent reduction of the exo-methylene intermediate gives rise to a methyl group (Scheme 2) [18]. The presence of the exo-methylene intermediate was experimentally proved but the enzyme responsible for the double bond reduction has not

Harnessing enzyme plasticity for the synthesis of oxygenated sesquiterpenoids

• Melodi Demiray,
• David J. Miller and
• Rudolf K. Allemann

Beilstein J. Org. Chem. 2019, 15, 2184–2190, doi:10.3762/bjoc.15.215

• engineering; terpenes; Introduction Amorphadiene synthase (ADS) from Artemisia annua is a key enzyme involved in the biosynthesis of the antimalarial sesquiterpene drug artemisinin (1) [1][2][3][4]. ADS catalyses the Mg2+-dependent conversion of farnesyl diphosphate (FDP, 2) to amorpha-4,11-diene (3) with

• as anchimeric assistance is hampered by unknowns such as the conformation of binding to the enzyme and what effect the extra bulk of the substituents has upon the results observed, but nevertheless such empirical results will accumulate to inform future investigations. This reversal of the

Azologization and repurposing of a hetero-stilbene-based kinase inhibitor: towards the design of photoswitchable sirtuin inhibitors

• Christoph W. Grathwol,
• Nathalie Wössner,
• Sören Swyter,
• Adam C. Smith,
• Enrico Tapavicza,
• Robert K. Hofstetter,
• Anja Bodtke,
• Manfred Jung and
• Andreas Link

Beilstein J. Org. Chem. 2019, 15, 2170–2183, doi:10.3762/bjoc.15.214

• in their catalytic core domains, many isotype selective inhibitors have been developed in recent years [39][40][41][42][43][44][45]. In the case of Sirt2 it was shown that appropriate ligand binding can induce conformational changes of the enzyme, revealing a so-called selectivity pocket, which

• methylation was intended to be examined. For UV–vis spectroscopy 50 µM solutions in 5% DMSO (v/v) in enzyme assay buffer were used, as this reflects the enzyme assay conditions. However, for LC-HRMS and NMR analysis, a higher concentration of 10 mM in methanol was necessary to receive reliable chromatograms

• enzyme assay. The long irradiation periods that were necessary to obtain significant amounts of the (Z)-isomers did not permit switching of the inhibitors in the enzyme assay mixture, as the fluorescent substrate and the enzyme would be harmed by long-term UV radiation. We envisioned to replace the

Characterization of two new degradation products of atorvastatin calcium formed upon treatment with strong acids

• Jürgen Krauß,
• Monika Klimt,
• Markus Luber,
• Peter Mayer and
• Franz Bracher

Beilstein J. Org. Chem. 2019, 15, 2085–2091, doi:10.3762/bjoc.15.206

• , Germany 10.3762/bjoc.15.206 Abstract Atorvastatin calcium (Lipitor®, Sortis®) is a well-established cholesterol synthesis enzyme (CSE) inhibitor commonly used in the therapy of hypercholesterolemia. This drug is known to be sensitive to acid treatment, but only little data has been published on the

Genome mining in Trichoderma viride J1-030: discovery and identification of novel sesquiterpene synthase and its products

• Xiang Sun,
• You-Sheng Cai,
• Yujie Yuan,
• Guangkai Bian,
• Ziling Ye,
• Zixin Deng and
• Tiangang Liu

Beilstein J. Org. Chem. 2019, 15, 2052–2058, doi:10.3762/bjoc.15.202

• -overexpressing S. cerevisiae as a platform (Figure 1). By the heterologous expression of predicted terpene synthases from the genome of T. viride, an unknown sesquiterpene synthase was identified and characterised. Furthermore, a new compound produced by this enzyme and its esterified product were detected and

• synthase well-identified with products characterised in T. viride. In vitro analysis of Tvi09626 function To confirm the function of the candidate enzyme, the DNA sequence of Tvi09626 was amplified by touchdown PCR from the T. viride genome. The gene fragment was cloned into a pET28a (+) vector to

• Mg2+ or added EDTA (2.5 mM), compound 1 cannot be detected (Figure 5). This assay demonstrated that Tvi09626 was a Mg2+-dependent sesquiterpene synthase. In a kinetics analysis, the turnover rate (kcat) of the enzyme with FPP was (15 ± 0.3) × 10−2, which is similar to those of omp6 and omp7. Its

Isolation and characterisation of irinans, androstane-type withanolides from Physalis peruviana L.

• Annika Stein,
• Dave Compera,
• Bianka Karge,
• Mark Brönstrup and
• Jakob Franke

Beilstein J. Org. Chem. 2019, 15, 2003–2012, doi:10.3762/bjoc.15.196

• characteristic of Physalis species. The biosynthesis of androstanes in mammals requires three enzymatic steps starting from cholesterol (9, Figure 3B) [27]. Cholesterol (9) is converted to pregnenolone (10) by the cytochrome P450 cholesterol side-chain cleavage enzyme (P450scc), which cleaves the C20–C22 bond

• without the lactone side chain. We therefore propose that the side-chain cleavage enzyme in withanolide biosynthesis acts at a late stage, using common pathway end products such as 4ß-hydroxywithanolide E (1) as its substrates. Two mechanisms are conceivable for this transformation (Figure 3C): A non

• -oxidative Grob fragmentation could make use of a push–pull mechanism between C-17 and C-22, building on acid–base catalysis. Alternatively, an enzyme could cleave the C17–C20 diol oxidatively. Several P450 enzymes have been reported to be capable of cleaving diols, presumably via a ferric peroxo

Archangelolide: A sesquiterpene lactone with immunobiological potential from Laserpitium archangelica

• Silvie Rimpelová,
• Michal Jurášek,
• Lucie Peterková,
• Jiří Bejček,
• Vojtěch Spiwok,
• Miloš Majdl,
• Michal Jirásko,
• Miloš Buděšínský,
• Juraj Harmatha,
• Eva Kmoníčková,
• Pavel Drašar and
• Tomáš Ruml

Beilstein J. Org. Chem. 2019, 15, 1933–1944, doi:10.3762/bjoc.15.189

• enzyme were removed. The system was simulated in a periodic box of the sized of 15.65 × 15.65 × 18.00 nm3. The system was minimized and equilibrated by series of restrained simulations (3.02 ns in total). Biological assays Cell lines and their cultivation In this study, the following human cancer cell

Inherent atomic mobility changes in carbocation intermediates during the sesterterpene cyclization cascade

• Hajime Sato,
• Takaaki Mitsuhashi,
• Mami Yamazaki,
• Ikuro Abe and
• Masanobu Uchiyama

Beilstein J. Org. Chem. 2019, 15, 1890–1897, doi:10.3762/bjoc.15.184

• substrate to the enzyme’s binding site could be identified by calculating the inherent structural mobility of the carbocation intermediates. This does involve the assumption that we can neglect the influence of changes in the interior structure of the enzyme as the reaction proceeds; however, based on the

• biosynthesis, and we discuss the implications for the mechanism of fixation (preorganization) of the substrate GFPP inside the binding pocket of the enzyme. Results and Discussion For the analysis of inherent structural mobility, we firstly carried out IRC calculations using GRRM11 with Gaussian 09, obtaining

• tightly fixed by the enzyme. As we reported previously, the initial conformation of GFPP, in particular the orientation of six methyl groups (C20–C25), is critical. Therefore, we focused on these methyl groups. While the C20, C21 and C23 methyl groups are quite static in phase I, the other three methyl

N-(1-Phenylethyl)aziridine-2-carboxylate esters in the synthesis of biologically relevant compounds

• Iwona E. Głowacka,
• Aleksandra Trocha,
• Andrzej E. Wróblewski and
• Dorota G. Piotrowska

Beilstein J. Org. Chem. 2019, 15, 1722–1757, doi:10.3762/bjoc.15.168

• agonist of S1P receptors while DS-SG-45 was found inactive. Dihydrosphingosines, e.g., safingol and sphinganine itself or as components of dihydroceramides are of interest as enzyme inhibitors [74][75]. Their common vicinal aminohydroxy fragment was efficiently synthesized from the aziridine ketone (2S,1

Molecular basis for the plasticity of aromatic prenyltransferases in hapalindole biosynthesis

• Takayoshi Awakawa and
• Ikuro Abe

Beilstein J. Org. Chem. 2019, 15, 1545–1551, doi:10.3762/bjoc.15.157

• HU and HA were located at the same position, but their orientations were completely different. These data indicated that the hydrophobic interaction between the enzyme and the terpenoid moiety is important to support the prenyl acceptor, and the orientation can be altered dependently on their steric

• other enzymes. In fact, the position of the α-phosphate shifts between the Mg2+-free and -bound structures in AmbP1 and between the HU and HA structures in AmbP3, which alters the locations of the substrates in the enzyme. In the AmbP3 structures, Y225 plays an important role to form a cation shield in

• they are a unique property of AmbP1. Remarkably, the two Mg2+- binding amino acids are located at the start or end of a β-sheet (Figure 6), which causes the corresponding β-sheet to move through the metal binding. Mg-1 is likely to maintain the overall structure of the enzyme, and Mg-2 defines the

A novel three-component reaction between isocyanides, alcohols or thiols and elemental sulfur: a mild, catalyst-free approach towards O-thiocarbamates and dithiocarbamates

• András György Németh,
• György Miklós Keserű and
• Péter Ábrányi-Balogh

Beilstein J. Org. Chem. 2019, 15, 1523–1533, doi:10.3762/bjoc.15.155

• ; multicomponent reaction; one-pot; thiocarbamate; Introduction O-Thiocarbamates belong to a class of important biologically active molecules, used mainly as fungicides [1][2][3] in agricultural and pharmaceutical fields. In particular, recently antitumor [4], anesthetic [5] and enzyme inhibitory effects were

• as enzyme inhibitors [26] or antitumor agents [27]. These species are also used as valuable synthetic intermediates [28] and chemosensors for mercury and silver [29][30]. The general methods for the synthesis of O-thiocarbamates and dithiocarbamates traditionally rely on substitution reactions of the

Synthesis and biological evaluation of truncated derivatives of abyssomicin C as antibacterial agents

• Leticia Monjas,
• Peter Fodran,
• Johanna Kollback,
• Carlo Cassani,
• Thomas Olsson,
• Maja Genheden,
• D. G. Joakim Larsson and
• Carl-Johan Wallentin

Beilstein J. Org. Chem. 2019, 15, 1468–1474, doi:10.3762/bjoc.15.147

• covalent inhibitor of 4-amino-4-deoxychorismate (ADC) synthase, which is the enzyme that catalyzes the conversion of chorismate and glutamine into ADC and glutamate, the first step in the biosynthesis of p-aminobenzoic acid (PABA) in bacteria [6]. Specifically, AbC binds via a Michael addition between a

Fluorine-containing substituents: metabolism of the α,α-difluoroethyl thioether motif

• Andrea Rodil,
• Alexandra M. Z. Slawin,
• Nawaf Al-Maharik,
• Ren Tomita and
• David O’Hagan

Beilstein J. Org. Chem. 2019, 15, 1441–1447, doi:10.3762/bjoc.15.144

• significantly more rapid than the second oxidation to the sulfone. The first oxidation gave enantiomerically enriched sulfoxides (Ar–S(O)CF2CH3) in the 54–60% ee range. This could arise by the action of more than one P450 enzyme. There was no evidence of defluorination, or hydroxylation at the terminal -CH3

Complexation of a guanidinium-modified calixarene with diverse dyes and investigation of the corresponding photophysical response

• Yu-Ying Wang,
• Yong Kong,
• Zhe Zheng,
• Wen-Chao Geng,
• Zi-Yi Zhao,
• Hongwei Sun and
• Dong-Sheng Guo

Beilstein J. Org. Chem. 2019, 15, 1394–1406, doi:10.3762/bjoc.15.139

• observable signal. Subsequent to IDA, Nau and co-workers conceptualized a novel approach towards enzyme assays, termed supramolecular tandem assay (STA) (Scheme 1b) [5]. STA is envisaged as a time-resolved version of IDA and the key idea is that the competitor is not added, but rather created during the

• course of an enzymatic reaction. Thus, the progress of the reaction can be signaled by a luminescence increase or decrease with time, which enables a highly sensitive real-time luminescence monitoring of the enzymatic activity [6]. STA has been applied to screening enzyme inhibitors or activators

Anomeric sugar boronic acid analogues as potential agents for boron neutron capture therapy

• Daniela Imperio,
• Erika Del Grosso,
• Silvia Fallarini,
• Grazia Lombardi and
• Luigi Panza

Beilstein J. Org. Chem. 2019, 15, 1355–1359, doi:10.3762/bjoc.15.135

• . Galactose and fructose also allow tumor growth in the absence of glucose. Boronic acid derivatives have gained interest in the last years in different fields such as the development of enzyme inhibitors, drug delivery polymers, saccharide sensors and as boron carriers for BNCT, e.g., amino acid derivatives

Host–guest interactions between p-sulfonatocalix[4]arene and p-sulfonatothiacalix[4]arene and group IA, IIA and f-block metal cations: a DFT/SMD study

• Valya K. Nikolova,
• Cristina V. Kirkova,
• Silvia E. Angelova and
• Todor M. Dudev

Beilstein J. Org. Chem. 2019, 15, 1321–1330, doi:10.3762/bjoc.15.131

• , biopharmaceutical, biological, biomimetic (enzyme mimics, transport across membranes, ion channels, etc.) and biomedical (in cancer chemotherapy) applications, reviewed by Atwood et al. [15], Perret et al. [16], Da Silva et al. [17], Nimse et al. [18], Guo et al. [19], Agrawal et al. [20], Yousaf et al. [21]. The

Phylogenomic analyses and distribution of terpene synthases among Streptomyces

• Lara Martín-Sánchez,
• Kumar Saurabh Singh,
• Mariana Avalos,
• Gilles P. van Wezel,
• Jeroen S. Dickschat and
• Paolina Garbeva

Beilstein J. Org. Chem. 2019, 15, 1181–1193, doi:10.3762/bjoc.15.115

• ), and the C-terminal domain for its further conversion into geosmin with cleavage of 12 into acetone and the octalin 13 through a retro-Prins fragmentation (Scheme 1) [22][23][24]. The proposed neutral intermediate isolepidozene (11) has so far only been reported from the S233A enzyme variant of geosmin

• culture headspace extracts by GC–MS [31]. Compound 4 was also isolated from in vitro incubations of FPP with the recombinant enzyme and its optical rotation was shown to be opposite to the material from Eucalyptus [32], but the absolute configuration remains unknown. Production of this sesquiterpene by S

• the recombinant enzyme from Streptomyces malaysiensis [43]. The diterpene 7 is a precursor to the lysophospholipase inhibitor cyclooctatin (20) formed by the action of two genetically clustered cytochrome P450 monooxygenases CotB3 and CotB4 (Scheme 4) [40][44], while no derivatives from 8 are