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

Mechanistic investigations on multiproduct β-himachalene synthase from Cryptosporangium arvum

• Jan Rinkel and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2019, 15, 1008–1019, doi:10.3762/bjoc.15.99

• potential was also exploited in the preparation of sesquiterpene isotopomers, which provided insights into their EIMS fragmentation mechanisms. Keywords: enzyme mechanisms; isotopes; mass spectrometry; promiscuity; terpenes; Introduction The organic chemist usually prefers to work with pure compounds

• which lead to high requirements for the selectivity of reactions and often to tedious purification procedures, but encountering a pure compound in nature is quite rare. This does not result in reduced requirements for enzyme selectivity. The very opposite is mostly true, because proteins working in a

• a single enzyme that can produce a beneficial mixture, the advantage for the producing organism is even higher. Therefore, selectivity is not in every case the highest goal for evolution. An enzyme class, which is highly prone to a regulation of product selectivity for the production of either one

New terpenoids from the fermentation broth of the edible mushroom Cyclocybe aegerita

• Frank Surup,
• Florian Hennicke,
• Nadine Sella,
• Maria Stroot,
• Steffen Bernecker,
• Sebastian Pfütze,
• Marc Stadler and
• Martin Rühl

Beilstein J. Org. Chem. 2019, 15, 1000–1007, doi:10.3762/bjoc.15.98

• performed in triplicates using the CFX Connect™ RT-PCR Detection System (Bio-Rad Laboratories, Hercules, CA, USA). The following conditions were applied: enzyme activation at 94 °C for 20 s followed by 40 cycles of 94 °C for 30 s, 58 °C for 30 s and 72 °C for 10 s. Structures of the isolated metabolites

Heck- and Suzuki-coupling approaches to novel hydroquinone inhibitors of calcium ATPase

• Robert J. Kempton,
• Taylor A. Kidd-Kautz,
• Soizic Laurenceau and
• Stefan Paula

Beilstein J. Org. Chem. 2019, 15, 971–975, doi:10.3762/bjoc.15.94

• Abstract In this study, we explored Heck- and Suzuki-coupling methodology to modify the template 2,5-di-tert-butylhydroquinone (BHQ, 2), an inhibitor of the enzyme sarco/endoplasmic reticulum calcium ATPase (SERCA). We found that by utilizing Suzuki coupling, we could successfully attach a six-carbon

• solvent-exposed area on the surface of SERCA where it did not undergo major interactions with the enzyme [22]. Therefore, conducting the inhibition assay with the Boc-protected BHQ derivative facilitates a convenient direct comparison with inhibition results for TG-analogues that have been characterized

Towards the preparation of synthetic outer membrane vesicle models with micromolar affinity to wheat germ agglutinin using a dialkyl thioglycoside

• Dimitri Fayolle,
• Nathalie Berthet,
• Bastien Doumeche,
• Olivier Renaudet,
• Peter Strazewski and
• Michele Fiore

Beilstein J. Org. Chem. 2019, 15, 937–946, doi:10.3762/bjoc.15.90

• was measured by competitive enzyme-linked lectin assays. One of the synthetic compounds presenting two GlcNAc units showed the highest inhibitory effect of this study with an IC50 of 11 µM corresponding to a 3182-fold improvement compared to GlcNAc. These synthetic molecules were used to produce giant

• solution inevitably cause the disruption of the supramolecular assembly [23]. Analyses under saline stress were not performed as the ultimate aim is to use of those OMV models under physiological conditions. Biological evaluation of the synthesized compounds Competitive enzyme-linked lectin assays (ELLA

• microscopic observation. Enzyme-linked lectin assays: 96-well microtiter Nunc-Immuno plates (Maxi-Sorp) were coated with PAA-GlcNAc (100 μL per well, diluted from a stock solution of 5 μg mL−1 in 50 mM carbonate buffer pH 9.6) for 1 h at 37 °C. The wells were then washed with T-PBS (3 × 100 μL per well, PBS

Stereochemical investigations on the biosynthesis of achiral (Z)-γ-bisabolene in Cryptosporangium arvum

• Jan Rinkel and
• Jeroen S. Dickschat

Beilstein J. Org. Chem. 2019, 15, 789–794, doi:10.3762/bjoc.15.75

• recombinant enzyme, which resolved in an exclusive cyclisation of (R)-NPP, while (S)-NPP that is non-natural to the (Z)-γ-bisabolene synthase was specifically converted into (E)-β-farnesene. A hypothetical enzyme mechanistic model that explains these observations is presented. Keywords: biosynthesis

• ; carbocation chemistry; enzyme mechanisms; nerolidyl diphosphate; terpenes; Introduction Given the enormous impact of chirality within biomolecules for all forms of life, it is fascinating to see how nature is able to maintain and reproduce stereochemical information. This concept largely involves the

• the soil actinomycete Cryptosporangium arvum DSM 44712 was cloned into the E. coli expression vector pYE-express [13] (Table S1, Supporting Information File 1), because of its phylogenetic distance to characterised TSs (Figure S1, Supporting Information File 1). The amino acid sequence of the enzyme

Homo- and hetero-difunctionalized β-cyclodextrins: Short direct synthesis in gram scale and analysis of regiochemistry

• Gábor Benkovics,
• Mihály Bálint,
• Éva Fenyvesi,
• Erzsébet Varga,
• Szabolcs Béni,
• Konstantina Yannakopoulou and
• Milo Malanga

Beilstein J. Org. Chem. 2019, 15, 710–720, doi:10.3762/bjoc.15.66

• based on stereoselective interactions of CDs with guest molecules (chiral separations, asymmetric catalysis, enzyme mimics) [20]. However, if the target is only the side-selectivity of difunctionalization, pseudoenantiomeric mixtures of the regioisomers can be used [21]. For CD-based multifunctional

New sesquiterpenoids from the South China Sea soft corals Clavularia viridis and Lemnalia flava

• Qihao Wu,
• Yuan Gao,
• Meng-Meng Zhang,
• Li Sheng,
• Jia Li,
• Xu-Wen Li,
• Hong Wang and
• Yue-Wei Guo

Beilstein J. Org. Chem. 2019, 15, 695–702, doi:10.3762/bjoc.15.64

• the title animals in the South China Sea. The promising PTP1B inhibitory activity of laurane-type sesquiterpenoids [24] in a previously report from our group, inspired us to test the PTP1B inhibitory activity of compounds 1–4. Among them, compound 3 was inactive against PTP1B enzyme, whereas compounds

Back to the future: Why we need enzymology to build a synthetic metabolism of the future

• Tobias J. Erb

Beilstein J. Org. Chem. 2019, 15, 551–557, doi:10.3762/bjoc.15.49

• fashion from bottom-up. However, while the theoretical design of synthetic metabolic networks has made tremendous progress, the actual realization of such synthetic pathways is still lacking behind. This is mostly because of our limitations in enzyme discovery and engineering to provide the parts required

• novel enzymatic reactions for their successful realization. However, other level 4 pathway designs require the establishment of more than ten so-far unknown enzyme reactions, emphasizing the challenge to realize truly synthetic metabolic networks [13]. The challenge of finding (new) enzymes for

• million proteins were deposited into protein sequence databases, such as UniProtKB [29]. More than 40,000 enzymes were biochemically characterized and the corresponding data is available in specialized enzyme databases, such as BRENDA [30]. This wealth of biological information provides a good starting

A chemoenzymatic synthesis of ceramide trafficking inhibitor HPA-12

• Seema V. Kanojia,
• Sucheta Chatterjee,
• Subrata Chattopadhyay and
• Dibakar Goswami

Beilstein J. Org. Chem. 2019, 15, 490–496, doi:10.3762/bjoc.15.42

• envisaged that the enantiomers of 4 were ideal substrates for the asymmetric synthesis of different stereoisomers of 2. Several biocatalytic protocols for the preparation of (R) or (S)-4 were reported earlier. The hydrolysis of the corresponding acetate with crude enzyme preparations from pig liver and

• , near the optimum temperature of the enzyme (55 °C). Under these conditions, a 50% conversion was achieved within 48 h to obtain both (R)-5 and (S)-4 in 90% ee (Table 1, entry 5). A second acetylation (12% conversion, 20 h) of the resolved (S)-4 under the same conditions improved its ee to 99% (Table 1

• , entry 6). The products were isolated by filtering the insoluble enzyme from the reaction mixture, followed by concentration of the filtrate. The obtained residue was subjected to silica gel column chromatography to isolate unreacted alcohol and acetylated product. We have reused the recovered lipase at