Harnessing the Peterson Reaction for the Stereospecific Synthesis of Z-Vinyl Ethers.

Vinyl ethers are valuable synthetic intermediates which are also found as natural products, including aflatoxins, rifamycins and plasmalogens. The latter are ubiquitous phospholipids in human cells and contain a vinyl ether moiety with specifically Z configuration. Although numerous methods are available for synthesis of vinyl ethers, there is a lack of methods for obtaining Z isomers of molecules of the type RCH=CHOR' that are applicable to plasmalogens. A variant of the Peterson reaction is described that generates such molecules with very high stereoselectivity (Z/E ratio: 99 : 1). (R,R)/(S,S)-1-alkoxy-2-hydroxyalkylsilanes were synthesized from 1-trimethylsilylalkynes by a sequence of reduction with di-isobutylaluminium hydride to a (Z)-1-trimethylsilylalkene, epoxidation of the alkene to a 2-trimethylsilyl-3-substituted epoxide and regioselective, boron-trifluoride catalyzed ring-opening of the epoxide by reaction with an alcohol. Conversion of the (R,R)/(S,S)-1-alkoxy-2-hydroxyalkylsilanes to vinyl ethers (RCH=CHOR') was achieved under basic conditions as in a standard Peterson reaction. However, near exclusive formation of a Z vinyl ether was only achieved when the reaction was performed using potassium hydride in the non-polar solvent α,α,α-trifluorotoluene, more polar solvents giving increasing amounts of the E isomer. The sequence described embraces a variety of substituents and precursors, proceeds in overall high yield and is readily scalable.

Unlocking nature's antioxidants: a novel method for synthesising plasmalogens.

Plasmalogens are glycerophospholipids distinguished by their O-(Z)-vinyl ether at the sn-1 position. These lipids are implicated in several disease states requiring analytical, diagnostic and therapeutic interventions, which demand synthetic availability for a variety of structural types. By deploying the new O-protecting group 1,4-dimethoxynaphthyl-2-methyl ('DIMON') and a new stereospecific method for accessing Z-vinyl ethers, a reproducible, versatile synthetic route to plasmalogens [plasmenyl phosphocholines] has been developed. A key intermediate is (S,Z)-1-((1,4-dimethoxynaphthalen-2-yl)methoxy)-3-(hexadec-1-en-1-yloxy)propan-2-ol, which in principle, permits plasmalogen synthesis 'à la carte' at scale. The methodology compares favourably with all previous synthetic routes by virtue of the very high configurational (>99% Z) and optical purity (>99% ee), including the ability to incorporate polyunsaturated fatty acyl chains (e.g. all Z docosahexaenoic acid) reliably at the sn-2 position.

Cyclizations and fragmentations in the alkylation of 6-chloro-5-hydroxy-4-aminopyrimidines with aminoalkyl chlorides.

Substituted aminopyrimidines are an important class of compounds, in part because they frequently show biological activity. Facile synthesis of polysubstituted aminopyrimidines is highly desirable for the synthesis of screening libraries. We describe a route to 4,6-diamino-5-alkoxypyrimidines via a SNAr-alkylation-SNAr sequence from readily available 4,6-dichloro-5-methoxypyrimidine, which allows the synthesis of such compounds with regiochemical control. The extension of this approach to alkylating agents bearing amino substituents led to unexpected and, in some cases, unprecedented products resulting from intramolecular SNAr cyclization and subsequent fragmentation.

The 5,6,7,8-Tetrahydro-2-Naphthoyl-Coenzyme A Reductase Reaction in the Anaerobic Degradation of Naphthalene and Identification of Downstream Metabolites.

Anaerobic degradation of polycyclic aromatic hydrocarbons has been investigated mostly with naphthalene as a model compound. Naphthalene degradation by sulfate-reducing bacteria proceeds via carboxylation to 2-naphthoic acid, formation of a coenzyme A thioester, and subsequent reduction to 5,6,7,8-tetrahydro-2-naphthoyl-coenzyme A (THNCoA), which is further reduced to hexahydro-2-naphthoyl-CoA (HHNCoA) by tetrahydronaphthoyl-CoA reductase (THNCoA reductase), an enzyme similar to class I benzoyl-CoA reductases. When analyzing THNCoA reductase assays with crude cell extracts and NADH as electron donor via liquid chromatography-mass spectrometry (LC-MS), scanning for putative metabolites, we found that small amounts of the product of an HHNCoA hydratase were formed in the assays, but the downstream conversion by an NAD+-dependent ß-hydroxyacyl-CoA dehydrogenase was prevented by the excess of NADH in those assays. Experiments with alternative electron donors indicated that 2-oxoglutarate can serve as an indirect electron donor for the THNCoA-reducing system via a 2-oxoglutarate:ferredoxin oxidoreductase. With 2-oxoglutarate as electron donor, THNCoA was completely converted and further metabolites resulting from subsequent ß-oxidation-like reactions and hydrolytic ring cleavage were detected. These metabolites indicate a downstream pathway with water addition to HHNCoA and ring fission via a hydrolase acting on a ß'-hydroxy-ß-oxo-decahydro-2-naphthoyl-CoA intermediate. Formation of the downstream intermediate cis-2-carboxycyclohexylacetyl-CoA, which is the substrate for the previously described lower degradation pathway leading to the central metabolism, completes the anaerobic degradation pathway of naphthalene.IMPORTANCE Anaerobic degradation of polycyclic aromatic hydrocarbons is poorly investigated despite its significance in anoxic sediments. Using alternative electron donors for the 5,6,7,8-tetrahydro-2-naphthoyl-CoA reductase reaction, we observed intermediary metabolites of anaerobic naphthalene degradation via in vitro enzyme assays with cell extracts of anaerobic naphthalene degraders. The identified metabolites provide evidence that ring reduction terminates at the stage of hexahydro-2-naphthoyl-CoA and a sequence of ß-oxidation-like degradation reactions starts with a hydratase acting on this intermediate. The final product of this reaction sequence was identified as cis-2-carboxycyclohexylacetyl-CoA, a compound for which a further downstream degradation pathway has recently been published (P. Weyrauch, A. V. Zaytsev, S. Stephan, L. Kocks, et al., Environ Microbiol 19:2819-2830, 2017, https://doi.org/10.1111/1462-2920.13806). Our study reveals the first ring-cleaving reaction in the anaerobic naphthalene degradation pathway. It closes the gap between the reduction of the first ring of 2-naphthoyl-CoA by 2-napthoyl-CoA reductase and the lower degradation pathway starting from cis-2-carboxycyclohexylacetyl-CoA, where the second ring cleavage takes place.

Molecular Diversity via Tetrasubstituted Alkenes Containing a Barbiturate Motif: Synthesis and Biological Activity.

The synthesis of a molecularly diverse library of tetrasubstituted alkenes containing a barbiturate motif is described. Base-induced condensation of N1-substituted pyrimidine-2,4,6(1H,3H,5H)-triones with 5-(bis(methylthio)methylene)-2,2-dimethyl-1,3-dioxane-4,6-dione gave 3-substituted 5-(methylthio)-2H-pyrano[2,3-d]pyrimidine-2,4,7(1H,3H)-triones ('pyranopyrimidinones'), regioselectively. A sequence of reactions involving ring-opening of the pyran moiety, displacement of the methylthio group with an amine, re-formation of the pyran ring, and after its final cleavage with an amine, gave tetrasubstituted alkenes (3-amino-3-(2,4,6-trioxotetrahydropyrimidin-5(2H)-ylidene)propanamides) with a diversity of substituents. Cleavage of the pyranopyrimidinones with an aniline was facilitated in 2,2,2-trifluoroethanol under microwave irradiation. Compounds were tested against Escherichia coli, Staphylococcus aureus, the yeast Schizosaccharomyces pombe, and the pathogenic fungus Candida albicans. No compounds exhibited activity against E. coli, whilst one compound was weakly active against S. aureus. Three compounds were strongly active against S. pombe, but none was active against C. albicans.

Photochemistry of 2-butenedial and 4-oxo-2-pentenal under atmospheric boundary layer conditions.

Unsaturated 1,4-dicarbonyl compounds, such as 2-butenedial and 4-oxo-2-pentenal are produced in the atmospheric boundary layer from the oxidation of aromatic compounds and furans. These species are expected to undergo rapid photochemical processing, affecting atmospheric composition. In this study, the photochemistry of (E)-2-butenedial and both E and Z isomers of 4-oxo-2-pentenal was investigated under natural sunlight conditions at the large outdoor atmospheric simulation chamber EUPHORE. Photochemical loss rates, relative to j(NO2), are determined to be j((E)-2-butenedial)/j(NO2) = 0.14 (±0.02), j((E)-4-oxo-2-pentenal)/j(NO2) = 0.18 (±0.01), and j((Z)-4-oxo-2-pentenal)/j(NO2) = 0.20 (±0.03). The major products detected for both species are a furanone (30-42%) and, for (E)-2-butenedial, maleic anhydride (2,5-furandione) (12-14%). The mechanism appears to proceed predominantly via photoisomerization to a ketene-enol species following γ-H abstraction. The lifetimes of the ketene-enol species in the dark from 2-butenedial and 4-oxo-2-pentenal are determined to be 465 s and 235 s, respectively. The ketene-enol can undergo ring closure to yield the corresponding furanone, or further unimolecular rearrangement which can subsequently form maleic anhydride. A minor channel (10-15%) also appears to form CO directly. This is presumed to be via a molecular elimination route of an initial biradical intermediate formed in photolysis, with an unsaturated carbonyl (detected here but not quantified) as co-product. α-Dicarbonyl and radical yields are very low, which has implications for ozone production from the photo-oxidation of unsaturated 1,4-dicarbonyls in the boundary layer. Photochemical removal is determined to be the major loss process for these species in the boundary layer with lifetimes of the order of 10-15 minutes, compared to >3 hours for reaction with OH.

Identification of a novel ligand for the ATAD2 bromodomain with selectivity over BRD4 through a fragment growing approach.

ATAD2 is an ATPase that is overexpressed in a variety of cancers and associated with a poor patient prognosis. This protein has been suggested to function as a cofactor for a range of transcription factors, including the proto-oncogene MYC and the androgen receptor. ATAD2 comprises an ATPase domain, implicated in chromatin remodelling, and a bromodomain which allows it to interact with acetylated histone tails. Dissection of the functional roles of these two domains would benefit from the availability of selective, cell-permeable pharmacological probes. An in silico evaluation of the 3D structures of various bromodomains suggested that developing small molecule ligands for the bromodomain of ATAD2 is likely to be challenging, although recent reports have shown that ATAD2 bromodomain ligands can be identified. We report a structure-guided fragment-based approach to identify lead compounds for ATAD2 bromodomain inhibitor development. Our findings indicate that the ATAD2 bromodomain can accommodate fragment hits (Mr < 200) that yield productive structure-activity relationships, and structure-guided design enabled the introduction of selectivity over BRD4.

Conversion of cis-2-carboxycyclohexylacetyl-CoA in the downstream pathway of anaerobic naphthalene degradation.

The cyclohexane derivative cis-2-(carboxymethyl)cyclohexane-1-carboxylic acid [(1R,2R)-/(1S,2S)-2-(carboxymethyl)cyclohexane-1-carboxylic acid] has previously been identified as metabolite in the pathway of anaerobic degradation of naphthalene by sulfate-reducing bacteria. We tested the corresponding CoA esters of isomers and analogues of this compound for conversion in cell free extracts of the anaerobic naphthalene degraders Desulfobacterium strain N47 and Deltaproteobacterium strain NaphS2. Conversion was only observed for the cis-isomer, verifying that this is a true intermediate and not a dead-end product. Mass-spectrometric analyses confirmed that conversion is performed by an acyl-CoA dehydrogenase and a subsequent hydratase yielding an intermediate with a tertiary hydroxyl-group. We propose that a novel kind of ring-opening lyase is involved in the further catabolic pathway proceeding via pimeloyl-CoA. In contrast to degradation pathways of monocyclic aromatic compounds where ring-cleavage is achieved via hydratases, this lyase might represent a new ring-opening strategy for the degradation of polycyclic compounds. Conversion of the potential downstream metabolites pimeloyl-CoA and glutaryl-CoA was proved in cell free extracts, yielding 2,3-dehydropimeloyl-CoA, 3-hydroxypimeloyl-CoA, 3-oxopimeloyl-CoA, glutaconyl-CoA, crotonyl-CoA, 3-hydroxybutyryl-CoA and acetyl-CoA as observable intermediates. This indicates a link to central metabolism via ß-oxidation, a non-decarboxylating glutaryl-CoA dehydrogenase and a subsequent glutaconyl-CoA decarboxylase.

Cooperative hydrogen bonds form a pseudocycle stabilizing an isolated complex of isocyanic acid with urea.

The shapes of macromolecules and their complexes with small molecules are often determined by extended networks of hydrogen bonds. Here, for the first time, we provide a detailed description of a cooperative pair of hydrogen bonds to an individual molecule of urea. The structure and properties of a gas phase complex formed between urea and isocyanic acid are characterised through microwave spectroscopy and ab initio calculations at the CCSD(T)(F12*)/aug-cc-pVTZ level.

Combined PI3K and CDK2 inhibition induces cell death and enhances in vivo antitumour activity in colorectal cancer.

BACKGROUND: The phosphatidylinositol-3-kinase/mammalian target of rapamycin (PI3K/mTOR) pathway is commonly deregulated in human cancer, hence many PI3K and mTOR inhibitors have been developed and have now reached clinical trials. Similarly, CDKs have been investigated as cancer drug targets. METHODS: We have synthesised and characterised a series of 6-aminopyrimidines identified from a kinase screen that inhibit PI3K and/or mTOR and/or CDK2. Kinase inhibition, tumour cell growth, cell cycle distribution, cytotoxicity and signalling experiments were undertaken in HCT116 and HT29 colorectal cancer cell lines, and in vivo HT29 efficacy studies. RESULTS: 2,6-Diaminopyrimidines with an O(4)-cyclohexylmethyl substituent and a C-5-nitroso or cyano group (1,2,5) induced cell cycle phase alterations and were growth inhibitory (GI50<20 µM). Compound 1, but not 2 or 5, potently inhibits CDK2 (IC50=0.1 nM) as well as PI3K, and was cytotoxic at growth inhibitory concentrations. Consistent with kinase inhibition data, compound 1 reduced phospho-Rb and phospho-rS6 at GI50 concentrations. Combination of NU6102 (CDK2 inhibitor) and pictilisib (GDC-0941; pan-PI3K inhibitor) resulted in synergistic growth inhibition, and enhanced cytotoxicity in HT29 cells in vitro and HT29 tumour growth inhibition in vivo. CONCLUSIONS: These studies identified a novel series of mixed CDK2/PI3K inhibitors and demonstrate that dual targeting of CDK2 and PI3K can result in enhanced antitumour activity.

A new tool for the chemical genetic investigation of the Plasmodium falciparum Pfnek-2 NIMA-related kinase.

BACKGROUND: Examining essential biochemical pathways in Plasmodium falciparum presents serious challenges, as standard molecular techniques such as siRNA cannot be employed in this organism, and generating gene knock-outs of essential proteins requires specialized conditional approaches. In the study of protein kinases, pharmacological inhibition presents a feasible alternative option. However, as in mammalian systems, inhibitors often lack the desired selectivity. Described here is a chemical genetic approach to selectively inhibit Pfnek-2 in P. falciparum, a member of the NIMA-related kinase family that is essential for completion of the sexual development of the parasite. RESULTS: Introduction of a valine to cysteine mutation at position 24 in the glycine rich loop of Pfnek-2 does not affect kinase activity but confers sensitivity to the protein kinase inhibitor 4-(6-ethynyl-9H-purin-2-ylamino) benzene sulfonamide (NCL-00016066). Using a combination of in vitro kinase assays and mass spectrometry, (including phosphoproteomics) the study shows that this compound acts as an irreversible inhibitor to the mutant Pfnek2 likely through a covalent link with the introduced cysteine residue. In particular, this was shown by analysis of total protein mass using mass spectrometry which showed a shift in molecular weight of the mutant kinase in the presence of the inhibitor to be precisely equivalent to the molecular weight of NCL-00016066. A similar molecular weight shift was not observed in the wild type kinase. Importantly, this inhibitor has little activity towards the wild type Pfnek-2 and, therefore, has all the properties of an effective chemical genetic tool that could be employed to determine the cellular targets for Pfnek-2. CONCLUSIONS: Allelic replacement of wild-type Pfnek-2 with the mutated kinase will allow for targeted inhibition of Pfnek-2 with NCL-00016066 and hence pave the way for comparative studies aimed at understanding the biological role and transmission-blocking potential of Pfnek-2.

Elucidating the Stereochemistry of Enzymatic Benzylsuccinate Synthesis with Chirally Labeled Toluene.

Benzylsuccinate synthase is a glycyl radical enzyme that initiates anaerobic toluene metabolism by adding fumarate to the methyl group of toluene to yield (R)-benzylsuccinate. To investigate whether the reaction occurs with retention or inversion of configuration at the methyl group of toluene, we synthesized both enantiomers of chiral toluene with all three H isotopes in their methyl groups. The chiral toluenes were converted into benzylsuccinates preferentially containing (2) H and (3) H at their benzylic C atoms, owing to a kinetic isotope effect favoring hydrogen abstraction from the methyl groups. The configuration of the products was analyzed by enzymatic CoA-thioester synthesis and stereospecific oxidation using enzymes involved in benzylsuccinate degradation. Assessment of the configurations of the benzylsuccinate isomers based on loss or retention of tritium showed that inversion of configuration at the methyl group occurs when the chiral toluenes react with fumarate.

Non-enzymatic ribonucleotide reduction in the prebiotic context.

Model studies of prebiotic chemistry have revealed compelling routes for the formation of the building blocks of proteins and RNA, but not DNA. Today, deoxynucleotides required for the construction of DNA are produced by reduction of nucleotides catalysed by ribonucleotide reductases, which are radical enzymes. This study considers potential non-enzymatic routes via intermediate radicals for the ancient formation of deoxynucleotides. In this context, several mechanisms for ribonucleotide reduction, in a putative H2 S/HS(.) environment, are characterized using computational chemistry. A bio-inspired mechanistic cycle involving a keto intermediate and HSSH production is found to be potentially viable. An alternative pathway, proceeding through an enol intermediate is found to exhibit similar energetic requirements. Non-cyclical pathways, in which HSS(.) is generated in the final step instead of HS(.) , show a markedly increased thermodynamic driving force (ca. 70 kJ mol(-1) ) and thus warrant serious consideration in the context of the prebiotic ribonucleotide reduction.

Regioselective sulfamoylation at low temperature enables concise syntheses of putative small molecule inhibitors of sulfatases.

Regioselective sulfamoylation of primary hydroxyl groups enabled a 5-step synthesis (overall yield 17%) of the first reported small molecule inhibitor of sulfatase-1 and 2, ((2S,3R,4R,5S,6R)-4,5-dihydroxy-2-methoxy-6-((sulfamoyloxy)methyl)tetrahydro-2H-pyran-3-yl)sulfamic acid, which obviated the use of hydroxyl protecting groups and is a marked improvement on the reported 9-step synthesis (overall yield 9%) employing hazardous trifluoromethylsulfonyl azide. The sulfamoylation methodology was used to prepare a range of derivatives of 1, and inhibition data was generated for Sulf-2, ARSA and ARSB.

Anaerobic activation of p-cymene in denitrifying betaproteobacteria: methyl group hydroxylation versus addition to fumarate.

The betaproteobacteria "Aromatoleum aromaticum" pCyN1 and "Thauera" sp. strain pCyN2 anaerobically degrade the plant-derived aromatic hydrocarbon p-cymene (4-isopropyltoluene) under nitrate-reducing conditions. Metabolite analysis of p-cymene-adapted "A. aromaticum" pCyN1 cells demonstrated the specific formation of 4-isopropylbenzyl alcohol and 4-isopropylbenzaldehyde, whereas with "Thauera" sp. pCyN2, exclusively 4-isopropylbenzylsuccinate and tentatively identified (4-isopropylphenyl)itaconate were observed. 4-Isopropylbenzoate in contrast was detected with both strains. Proteogenomic investigation of p-cymene- versus succinate-adapted cells of the two strains revealed distinct protein profiles agreeing with the different metabolites formed from p-cymene. "A. aromaticum" pCyN1 specifically produced (i) a putative p-cymene dehydrogenase (CmdABC) expected to hydroxylate the benzylic methyl group of p-cymene, (ii) two dehydrogenases putatively oxidizing 4-isopropylbenzyl alcohol (Iod) and 4-isopropylbenzaldehyde (Iad), and (iii) the putative 4-isopropylbenzoate-coenzyme A (CoA) ligase (Ibl). The p-cymene-specific protein profile of "Thauera" sp. pCyN2, on the other hand, encompassed proteins homologous to subunits of toluene-activating benzylsuccinate synthase (termed [4-isopropylbenzyl]succinate synthase IbsABCDEF; identified subunits, IbsAE) and protein homologs of the benzylsuccinate ß-oxidation (Bbs) pathway (termed BisABCDEFGH; all identified except for BisEF). This study reveals that two related denitrifying bacteria employ fundamentally different peripheral degradation routes for one and the same substrate, p-cymene, with the two pathways apparently converging at the level of 4-isopropylbenzoyl-CoA.

Trifluoroacetic acid in 2,2,2-trifluoroethanol facilitates S(N)Ar reactions of heterocycles with arylamines.

Small-molecule drug discovery requires reliable synthetic methods for attaching amino compounds to heterocyclic scaffolds. Trifluoroacetic acid-2,2,2-trifluoroethanol (TFA-TFE) is as an effective combination for achieving SN Ar reactions between anilines and heterocycles (e.g., purines and pyrimidines) substituted with a leaving group (fluoro-, chloro-, bromo- or alkylsulfonyl). This method provides a variety of compounds containing a "kinase-privileged fragment" associated with potent inhibition of kinases. TFE is an advantageous solvent because of its low nucleophilicity, ease of removal and ability to solubilise polar substrates. Furthermore, TFE may assist the breakdown of the Meisenheimer-Jackson intermediate by solvating the leaving group. TFA is a necessary and effective acidic catalyst, which activates the heterocycle by N-protonation without deactivating the aniline by conversion into an anilinium species. The TFA-TFE methodology is compatible with a variety of functional groups and complements organometallic alternatives, which are often disadvantageous because of the expense of reagents, the frequent need to explore diverse sets of reaction conditions, and problems with product purification. In contrast, product isolation from TFA-TFE reactions is straightforward: evaporation of the reaction mixture, basification and chromatography affords analytically pure material. A total of 45 examples are described with seven discrete heterocyclic scaffolds and 2-, 3- and 4-substituted anilines giving product yields that are normally in the range 50-90 %. Reactions can be performed with either conventional heating or microwave irradiation, with the latter often giving improved yields.

Model system for irreversible inhibition of Nek2: thiol addition to ethynylpurines and related substituted heterocycles.

Recent studies have shown that irreversible inhibition of Nek2 kinase [(Never in mitosis gene a)-related kinase 2], overexpression of which is observed in several cancers, can be achieved using Michael acceptors containing an ethynyl group, which target the enzyme's cysteine 22 residue lying near the catalytic site. The model studies described herein demonstrate an analogous capture of the ethynyl moiety in a series of ethynyl-heterocycles (e.g. 6-ethynyl-N-phenyl-9H-purin-2-amine) by N-acetylcysteine methyl ester in the presence of 1,4-diazabicyclo[2.2.2]octane in either dimethyl sulfoxide or N,N-dimethylformamide. Kinetic studies showed a 50-fold range in reactivity with 7-ethynyl-N-phenyl-3H-[1,2,3]triazolo[4,5-d]pyrimidin-5-amine being the most reactive compound, whereas 4-ethynyl-N-phenyl-7H-pyrrolo[2,3-d]pyrimidin-2-amine was the least reactive. Studies of the isomeric compounds, 2-(3-((6-ethynyl-7-methyl-7H-purin-2-yl)amino)phenyl)acetamide and 2-(3-((6-ethynyl-9-methyl-9H-purin-2-yl)amino)phenyl)acetamide, revealed the N(7)-methyl isomer to be 5-fold more reactive than the 9-methyl isomer, which is ascribed to a buttressing effect in the N(7)-methyl compound. Comparison of the crystal structures of these isomers showed that the ethynyl group is significantly displaced away from the methyl group exclusively in the N(7)-methyl isomer with an sp(2) bond angle of 124°, whereas the corresponding angle in the N(9)-methyl isomer was the expected 120°. The results of this study indicate heterocyclic scaffolds that are likely to be more promising for inhibition of Nek2 and other kinases containing a reactive cysteine.

Molecular hijacking of siroheme for the synthesis of heme and d1 heme.

Modified tetrapyrroles such as chlorophyll, heme, siroheme, vitamin B(12), coenzyme F(430), and heme d(1) underpin a wide range of essential biological functions in all domains of life, and it is therefore surprising that the syntheses of many of these life pigments remain poorly understood. It is known that the construction of the central molecular framework of modified tetrapyrroles is mediated via a common, core pathway. Herein a further branch of the modified tetrapyrrole biosynthesis pathway is described in denitrifying and sulfate-reducing bacteria as well as the Archaea. This process entails the hijacking of siroheme, the prosthetic group of sulfite and nitrite reductase, and its processing into heme and d(1) heme. The initial step in these transformations involves the decarboxylation of siroheme to give didecarboxysiroheme. For d(1) heme synthesis this intermediate has to undergo the replacement of two propionate side chains with oxygen functionalities and the introduction of a double bond into a further peripheral side chain. For heme synthesis didecarboxysiroheme is converted into Fe-coproporphyrin by oxidative loss of two acetic acid side chains. Fe-coproporphyrin is then transformed into heme by the oxidative decarboxylation of two propionate side chains. The mechanisms of these reactions are discussed and the evolutionary significance of another role for siroheme is examined.

The action of coenzyme B12-dependent diol dehydratase on 3,3,3-trifluoro-1,2-propanediol results in elimination of all the fluorides with formation of acetaldehyde.

3,3,3-Trifluoro-1,2-propanediol undergoes complete defluorination in two distinct steps: first, the conversion into 3,3,3-trifluoropropionaldehyde catalyzed by adenosylcobalamin (coenzyme B12)-dependent diol dehydratase; second, non-enzymatic elimination of all three fluorides from this aldehyde to afford malonic semialdehyde (3-oxopropanoic acid), which is decarboxylated to acetaldehyde. Diol dehydratase accepts 3,3,3-trifluoro-1,2-propanediol as a relatively poor substrate, albeit without significant mechanism-based inactivation of the enzyme during catalysis. Optical and electron paramagnetic resonance (EPR) spectra revealed the steady-state formation of cob(II)alamin and a substrate-derived intermediate organic radical (3,3,3-trifluoro-1,2-dihydroxyprop-1-yl). The coenzyme undergoes Co-C bond homolysis initiating a sequence of reaction by the generally accepted pathway via intermediate radicals. However, the greater steric size of trifluoromethyl and especially its negative impact on the stability of an adjacent radical centre compared to a methyl group has implications for the mechanism of the diol dehydratase reaction. Nevertheless, 3,3,3-trifluoropropionaldehyde is formed by the normal diol dehydratase pathway, but then undergoes non-enzymatic conversion into acetaldehyde, probably via 3,3-difluoropropenal and malonic semialdehyde.

ATP-dependent/-independent enzymatic ring reductions involved in the anaerobic catabolism of naphthalene.

Polycyclic aromatic hydrocarbons are among the most hazardous environmental pollutants. However, in contrast to aerobic degradation, the respective degradation pathways in anaerobes are greatly unknown which has so far prohibited many environmental investigations. In this work, we studied the enzymatic dearomatization reactions involved in the degradation of the PAH model compounds naphthalene and 2-methylnaphthalene in the sulfate-reducing enrichment culture N47. Cell extracts of N47 grown on naphthalene catalysed the sodium dithionite-dependent four-electron reduction of the key intermediate 2-naphthoyl-coenzyme A (NCoA) to 5,6,7,8-tetrahydro-2-naphthoyl-CoA (THNCoA). The NCoA reductase activity was independent of ATP and was, surprisingly, not sensitive to oxygen. In cell extracts in the presence of various electron donors the product THNCoA was further reduced by a two-electron reaction to most likely a conjugated hexahydro-2-naphthoyl-CoA isomer (HHNCoA). The reaction assigned to THNCoA reductase strictly depended on ATP and was oxygen-sensitive with a half-life time between 30 s and 1 min when exposed to air. The rate was highest with NADH as electron donor. The results indicate that two novel and completely different dearomatizing ring reductases are involved in anaerobic naphthalene degradation. While the THNCoA reducing activity shows some properties of ATP-dependent class I benzoyl-CoA reductases, NCoA reduction appears to be catalysed by a previously unknown class of dearomatizing aryl-carboxyl-CoA reductases.