Synthesis of meta-substituted arene bioisosteres from [3.1.1]propellane.

Small-ring cage hydrocarbons are popular bioisosteres (molecular replacements) for commonly found para-substituted benzene rings in drug design1. The utility of these cage structures derives from their superior pharmacokinetic properties compared with their parent aromatics, including improved solubility and reduced susceptibility to metabolism2,3. A prime example is the bicyclo[1.1.1]pentane motif, which is mainly synthesized by ring-opening of the interbridgehead bond of the strained hydrocarbon [1.1.1]propellane with radicals or anions4. By contrast, scaffolds mimicking meta-substituted arenes are lacking because of the challenge of synthesizing saturated isosteres that accurately reproduce substituent vectors5. Here we show that bicyclo[3.1.1]heptanes (BCHeps), which are hydrocarbons for which the bridgehead substituents map precisely onto the geometry of meta-substituted benzenes, can be conveniently accessed from [3.1.1]propellane. We found that [3.1.1]propellane can be synthesized on a multigram scale, and readily undergoes a range of radical-based transformations to generate medicinally relevant carbon- and heteroatom-substituted BCHeps, including pharmaceutical analogues. Comparison of the absorption, distribution, metabolism and excretion (ADME) properties of these analogues reveals enhanced metabolic stability relative to their parent arene-containing drugs, validating the potential of this meta-arene analogue as an sp3-rich motif in drug design. Collectively, our results show that BCHeps can be prepared on useful scales using a variety of methods, offering a new surrogate for meta-substituted benzene rings for implementation in drug discovery programmes.

Studies on enmetazobactam clarify mechanisms of widely used ß-lactamase inhibitors.

ß-Lactams are the most important class of antibacterials, but their use is increasingly compromised by resistance, most importantly via serine ß-lactamase (SBL)-catalyzed hydrolysis. The scope of ß-lactam antibacterial activity can be substantially extended by coadministration with a penicillin-derived SBL inhibitor (SBLi), i.e., the penam sulfones tazobactam and sulbactam, which are mechanism-based inhibitors working by acylation of the nucleophilic serine. The new SBLi enmetazobactam, an N-methylated tazobactam derivative, has recently completed clinical trials. Biophysical studies on the mechanism of SBL inhibition by enmetazobactam reveal that it inhibits representatives of all SBL classes without undergoing substantial scaffold fragmentation, a finding that contrasts with previous reports on SBL inhibition by tazobactam and sulbactam. We therefore reinvestigated the mechanisms of tazobactam and sulbactam using mass spectrometry under denaturing and nondenaturing conditions, X-ray crystallography, and NMR spectroscopy. The results imply that the reported extensive fragmentation of penam sulfone­derived acyl­enzyme complexes does not substantially contribute to SBL inhibition. In addition to observation of previously identified inhibitor-induced SBL modifications, the results reveal that prolonged reaction of penam sulfones with SBLs can induce dehydration of the nucleophilic serine to give a dehydroalanine residue that undergoes reaction to give a previously unobserved lysinoalanine cross-link. The results clarify the mechanisms of action of widely clinically used SBLi, reveal limitations on the interpretation of mass spectrometry studies concerning mechanisms of SBLi, and will inform the development of new SBLi working by reaction to form hydrolytically stable acyl­enzyme complexes.

Interactions of hydrolyzed ß-lactams with the L1 metallo-ß-lactamase: Crystallography supports stereoselective binding of cephem/carbapenem products.

L1 is a dizinc subclass B3 metallo-ß-lactamase (MBL) that hydrolyzes most ß-lactam antibiotics and is a key resistance determinant in the Gram-negative pathogen Stenotrophomonas maltophilia, an important cause of nosocomial infections in immunocompromised patients. L1 is not usefully inhibited by MBL inhibitors in clinical trials, underlying the need for further studies on L1 structure and mechanism. We describe kinetic studies and crystal structures of L1 in complex with hydrolyzed ß-lactams from the penam (mecillinam), cephem (cefoxitin/cefmetazole), and carbapenem (tebipenem, doripenem, and panipenem) classes. Despite differences in their structures, all the ß-lactam-derived products hydrogen bond to Tyr33, Ser221, and Ser225 and are stabilized by interactions with a conserved hydrophobic pocket. The carbapenem products were modeled as Δ1-imines, with (2S)-stereochemistry. Their binding mode is determined by the presence of a 1ß-methyl substituent: the Zn-bridging hydroxide either interacts with the C-6 hydroxyethyl group (1ß-hydrogen-containing carbapenems) or is displaced by the C-6 carboxylate (1ß-methyl-containing carbapenems). Unexpectedly, the mecillinam product is a rearranged N-formyl amide rather than penicilloic acid, with the N-formyl oxygen interacting with the Zn-bridging hydroxide. NMR studies imply mecillinam rearrangement can occur nonenzymatically in solution. Cephem-derived imine products are bound with (3R)-stereochemistry and retain their 3' leaving groups, likely representing stable endpoints, rather than intermediates, in MBL-catalyzed hydrolysis. Our structures show preferential complex formation by carbapenem- and cephem-derived species protonated on the equivalent (ß) faces and so identify interactions that stabilize diverse hydrolyzed antibiotics. These results may be exploited in developing antibiotics, and ß-lactamase inhibitors, that form long-lasting complexes with dizinc MBLs.

Natural and synthetic 2-oxoglutarate derivatives are substrates for oncogenic variants of human isocitrate dehydrogenase 1 and 2.

Variants of isocitrate dehydrogenase (IDH) 1 and 2 (IDH1/2) alter metabolism in cancer cells by catalyzing the NADPH-dependent reduction of 2-oxoglutarate (2OG) to (2R)-hydroxyglutarate. However, it is unclear how derivatives of 2OG can affect cancer cell metabolism. Here, we used synthetic C3- and C4-alkylated 2OG derivatives to investigate the substrate selectivities of the most common cancer-associated IDH1 variant (R132H IDH1), of two cancer-associated IDH2 variants (R172K IDH2, R140Q IDH2), and of WT IDH1/2. Absorbance-based, NMR, and electrochemical assays were employed to monitor WT IDH1/2 and IDH1/2 variant-catalyzed 2OG derivative turnover in the presence and absence of 2OG. Our results reveal that 2OG derivatives can serve as substrates of the investigated IDH1/2 variants, but not of WT IDH1/2, and have the potential to act as 2OG-competitive inhibitors. Kinetic parameters reveal that some 2OG derivatives, including the natural product 3-methyl-2OG, are equally or even more efficient IDH1/2 variant substrates than 2OG. Furthermore, NMR and mass spectrometry studies confirmed IDH1/2 variant-catalyzed production of alcohols in the cases of the 3-methyl-, 3-butyl-, and 3-benzyl-substituted 2OG derivatives; a crystal structure of 3-butyl-2OG with an IDH1 variant (R132C/S280F IDH1) reveals active site binding. The combined results highlight the potential for (i) IDH1/2 variant-catalyzed reduction of 2-oxoacids other than 2OG in cells, (ii) modulation of IDH1/2 variant activity by 2-oxoacid natural products, including some present in common foods, (iii) inhibition of IDH1/2 variants via active site binding rather than the established allosteric mode of inhibition, and (iv) possible use of IDH1/2 variants as biocatalysts.

Spectroscopic studies reveal details of substrate-induced conformational changes distant from the active site in isopenicillin N synthase.

Isopenicillin N synthase (IPNS) catalyzes formation of the ß-lactam and thiazolidine rings of isopenicillin N from its linear tripeptide l-δ-(α-aminoadipoyl)-l-cysteinyl-d-valine (ACV) substrate in an iron- and dioxygen (O2)-dependent four-electron oxidation without precedent in current synthetic chemistry. Recent X-ray free-electron laser studies including time-resolved serial femtosecond crystallography show that binding of O2 to the IPNS-Fe(II)-ACV complex induces unexpected conformational changes in α-helices on the surface of IPNS, in particular in α3 and α10. However, how substrate binding leads to conformational changes away from the active site is unknown. Here, using detailed 19F NMR and electron paramagnetic resonance experiments with labeled IPNS variants, we investigated motions in α3 and α10 induced by binding of ferrous iron, ACV, and the O2 analog nitric oxide, using the less mobile α6 for comparison. 19F NMR studies were carried out on singly and doubly labeled α3, α6, and α10 variants at different temperatures. In addition, double electron-electron resonance electron paramagnetic resonance analysis was carried out on doubly spin-labeled variants. The combined spectroscopic and crystallographic results reveal that substantial conformational changes in regions of IPNS including α3 and α10 are induced by binding of ACV and nitric oxide. Since IPNS is a member of the structural superfamily of 2-oxoglutarate-dependent oxygenases and related enzymes, related conformational changes may be of general importance in nonheme oxygenase catalysis.

Substrate promiscuity of enzymes from the sesquiterpene biosynthetic pathways from Artemisia annua and Tanacetum parthenium allows for novel combinatorial sesquiterpene production.

The therapeutic properties of complex terpenes often depend on the stereochemistry of their functional groups. However, stereospecific chemical synthesis of terpenes is challenging. To overcome this challenge, metabolic engineering can be employed using enzymes with suitable stereospecific catalytic activity. Here we used a combinatorial metabolic engineering approach to explore the stereospecific modification activity of the Artemisia annua artemisinic aldehyde ∆11(13) double bond reductase2 (AaDBR2) on products of the feverfew sesquiterpene biosynthesis pathway (GAS, GAO, COS and PTS). This allowed us to produce dihydrocostunolide and dihydroparthenolide. For dihydroparthenolide we demonstrate that the preferred order of biosynthesis of dihydroparthenolide is by reduction of the exocyclic methylene of parthenolide, rather than through C4-C5 epoxidation of dihydrocostunolide. Moreover, we demonstrate a promiscuous activity of feverfew CYP71CB1 on dihydrocostunolide and dihydroparthenolide for the production of 3ß-hydroxy-dihydrocostunolide and 3ß-hydroxy-dihydroparthenolide, respectively. Combined, these results offer new opportunities for engineering novel sesquiterpene lactones with potentially improved medicinal value.

Terpene synthase genes in eukaryotes beyond plants and fungi: Occurrence in social amoebae.

Terpenes are structurally diverse natural products involved in many ecological interactions. The pivotal enzymes for terpene biosynthesis, terpene synthases (TPSs), had been described only in plants and fungi in the eukaryotic domain. In this report, we systematically analyzed the genome sequences of a broad range of nonplant/nonfungus eukaryotes and identified putative TPS genes in six species of amoebae, five of which are multicellular social amoebae from the order of Dictyosteliida. A phylogenetic analysis revealed that amoebal TPSs are evolutionarily more closely related to fungal TPSs than to bacterial TPSs. The social amoeba Dictyostelium discoideum was selected for functional study of the identified TPSs. D. discoideum grows as a unicellular organism when food is abundant and switches from vegetative growth to multicellular development upon starvation. We found that expression of most D. discoideum TPS genes was induced during development. Upon heterologous expression, all nine TPSs from D. discoideum showed sesquiterpene synthase activities. Some also exhibited monoterpene and/or diterpene synthase activities. Direct measurement of volatile terpenes in cultures of D. discoideum revealed essentially no emission at an early stage of development. In contrast, a bouquet of terpenes, dominated by sesquiterpenes including ß-barbatene and (E,E)-α-farnesene, was detected at the middle and late stages of development, suggesting a development-specific function of volatile terpenes in D. discoideum. The patchy distribution of TPS genes in the eukaryotic domain and the evidence for TPS function in D. discoideum indicate that the TPS genes mediate lineage-specific adaptations.

Roles of 2-oxoglutarate oxygenases and isopenicillin N synthase in ß-lactam biosynthesis.

Covering: up to 2017 2-Oxoglutarate (2OG) dependent oxygenases and the homologous oxidase isopenicillin N synthase (IPNS) play crucial roles in the biosynthesis of ß-lactam ring containing natural products. IPNS catalyses formation of the bicyclic penicillin nucleus from a tripeptide. 2OG oxygenases catalyse reactions that diversify the chemistry of ß-lactams formed by both IPNS and non-oxidative enzymes. Reactions catalysed by the 2OG oxygenases of ß-lactam biosynthesis not only involve their typical hydroxylation reactions, but also desaturation, epimerisation, rearrangement, and ring-forming reactions. Some of the enzymes involved in ß-lactam biosynthesis exhibit remarkable substrate and product selectivities. We review the roles of 2OG oxygenases and IPNS in ß-lactam biosynthesis, highlighting opportunities for application of knowledge of their roles, structures, and mechanisms.

Two Diterpene Synthases for Spiroalbatene and Cembrene†A from Allokutzneria albata.

Two bacterial diterpene synthases from the actinomycete Allokutzneria albata were investigated, resulting in the identification of the structurally unprecedented compound spiroalbatene from the first and cembrene A from the second enzyme. Both enzymes were thoroughly investigated in terms of their mechanisms by isotope labeling experiments, site-directed mutagenesis, and variation of the metal cofactors and pH value. For spiroalbatene synthase, the pH- and Mn2+ -dependent formation of the side product thunbergol was observed, which is biosynthetically linked to spiroalbatene.

Mechanisms of the Diterpene Cyclases ß-Pinacene Synthase from Dictyostelium discoideum and Hydropyrene Synthase from Streptomyces clavuligerus.

Two diterpene cyclases, one from the social amoeba Dictyostelium discoideum and the other from the bacterium Streptomyces clavuligerus, with products containing a Z-configured double bond between the original C2 and C3 of geranylgeranyl diphosphate, were extensively investigated for their mechanisms through isotopic labelling experiments. The participation of geranyllinalyl diphosphate, in analogy to the role of linalyl and nerolidyl diphosphate for mono- and sesquiterpene biosynthesis, as an intermediate towards diterpenes with a Z-configured C2=C3 double bond is discussed.

Isoafricanol synthase from Streptomyces malaysiensis.

Genome sequencing of Streptomyces malaysiensis DSM 4137 revealed the presence of four terpene cyclase genes, one of which was characterised as (+)-isoafricanol synthase. Its cyclisation mechanism was extensively studied using isotopically labelled precursors. Several enzymes with high homology that likely also function as (+)-isoafricanol synthases are encoded in a number of other genome sequenced streptomycetes.

Mechanistic Investigations of Two Bacterial Diterpene Cyclases: Spiroviolene Synthase and Tsukubadiene Synthase.

The mechanisms of two diterpene cyclases from streptomycetes-one with an unknown product that was identified as the spirocyclic hydrocarbon spiroviolene and one with the known product tsukubadiene-were investigated in detail by isotope labeling experiments. Although the structures of the products were very different, the cyclization mechanisms of both enzymes proceed through the same initial cyclization reactions, before they diverge towards the individual products, which is reflected in the close phylogenetic relationship of the enzymes.

18-Hydroxydolabella-3,7-diene synthase - a diterpene synthase from Chitinophaga pinensis.

The product obtained in vitro from a diterpene synthase encoded in the genome of the bacterium Chitinophaga pinensis, an enzyme previously reported to have germacrene A synthase activity during heterologous expression in Escherichia coli, was identified by extensive NMR-spectroscopic methods as 18-hydroxydolabella-3,7-diene. The absolute configuration of this diterpene alcohol and the stereochemical course of the terpene synthase reaction were addressed by isotopic labelling experiments. Heterologous expression of the diterpene synthase in Nicotiana benthamiana resulted in the production of 18-hydroxydolabella-3,7-diene also in planta, while the results from the heterologous expression in E. coli were shown to be reproducible, revealing that the expression of one and the same terpene synthase in different heterologous hosts may yield different terpene products.

Experimental and Theoretical Studies on Corvol Ether Biosynthesis.

The biosynthesis of corvol ethers A and B, two sesquiterpenes from Kitasatospora setae, proceeds with involvement of either one 1,3- or two sequential 1,2-hydride shifts. Quantum chemical calculations revealed that the sequence of two 1,2-hydride shifts is energetically favoured. Labelling experiments were in agreement with this finding. In addition, the stereochemical course of a reprotonation step was investigated by incubation of (13)C-labelled isotopomers of farnesyl diphosphate in water and in deuterium oxide.

Position-Specific Mass Shift Analysis: A Systematic Method for Investigating the EI-MS Fragmentation Mechanism of epi-Isozizaene.

The EI-MS fragmentation mechanism of the bacterial sesquiterpene epi-isozizaene was investigated through enzymatic conversion of all 15 synthetic ((13) C1 )FPP isotopomers with the epi-isozizaene synthase from Streptomyces albus and GC-MS and GC-QTOF analysis including MS-MS. A systematic method, which we wish to call position-specific mass shift analysis, for the identification of the full set of fragmentation reactions was developed.

A method for investigating the stereochemical course of terpene cyclisations.

Three sesquiterpene cyclases from Streptomyces scabei 87.22, Streptomyces venezuelae ATCC 10712 and Streptomyces clavuligerus ATCC 27064 were characterised and their products were identified as (-)-neomeranol B, (+)-isodauc-8-en-11-ol and (+)-intermedeol, respectively. The stereochemical courses of the terpene cyclisations were investigated by use of various (13)C-labelled FPP isotopomers. A quick and easy test was developed that allows to distinguish reprotonations of olefinic double bonds in neutral intermediates from the two stereoheterotopic faces. The method makes use of incubating (13)C-FPP isotopomers labelled at the reprotonated carbon in deuterium oxide and subsequent HSQC analysis of the product. A 1,7-cyclisation towards (+)-isodauc-8-en-11-ol was followed by use of (1,7-(13)C2)FPP. Surprisingly, the (+)-isodauc-8-en-11-ol also accepted (2Z,6E)-FPP resulting in the same product profile as obtained from (2E,6E)-FPP.

Pristinol, a Sesquiterpene Alcohol with an Unusual Skeleton from Streptomyces pristinaespiralis.

A terpene cyclase from Streptomyces pristinaespiralis was characterized as the synthase for (+)-(2S,3S,9R)-pristinol. The structure of this sesquiterpene alcohol, which has a new carbon skeleton, was established by NMR spectroscopy and single-wavelength anomalous-dispersion X-ray crystallography. Extensive isotopic labelling experiments were performed to distinguish between various possible cyclization mechanisms of the terpene cyclase and to decipher the EI-MS fragmentation mechanism for pristinol.

Lessons from 1,3-Hydride Shifts in Sesquiterpene Cyclizations.

Stereospecifically labelled precursors were subjected to conversion by seven bacterial sesquiterpene cyclases to investigate the stereochemistry of their initial 1,10-cyclisation-1,3-hydride shift cascades. Enzymes with products of known absolute configuration showed a coherent stereochemical course, except for (-)-α-amorphene synthase, for which the obtained results are better explained by an initial 1,6-cyclisation. The link between the absolute configuration of the product and the stereochemical course of the 1,3-hydride shifts enabled assignment of the absolute configurations of three enzyme products, which were confirmed independently through the absolute configuration of the common byproduct germacrene D-4-ol.

Terpene Cyclases from Social Amoebae.

Genome sequences of social amoebae reveal the presence of terpene cyclases (TCs) in these organisms. Two TCs from Dictyostelium discoideum converted farnesyl diphosphate into (2S,3R,6S,9S)-(-)-protoillud-7-ene and (3S)-(+)-asterisca-2(9),6-diene. The enzyme mechanisms and EI-MS fragmentations of the products were studied by labeling experiments.

The EIMS fragmentation mechanisms of the sesquiterpenes corvol ethers A and B, epi-cubebol and isodauc-8-en-11-ol.

Farnesyl diphosphate (FPP) and all fifteen positional isomers of ((13)C1)FPP were enzymatically converted by the bacterial terpene cyclases corvol ether synthase from Kitasatospora setae, the epi-cubebol synthase from Streptosporangium roseum, and the isodauc-8-en-11-ol synthase from Streptomyces venezuelae. The enzyme products were analysed by GC-MS and GC-QTOF MS(2) and the obtained data were used to delineate the EIMS fragmentation mechanisms of the two sesquiterpene ethers corvol ethers A and B, and the sesquiterpene alcohols epi-cubebol and isodauc-8-en-11-ol.