An Isotopic Labelling Strategy to Study Cytochrome P450 Oxidations of Terpenes

The cytochrome P450 monooxygenase CYP267B1 from Sorangium cellulosum was applied for the enzymatic oxidation of the sesquiterpene alcohols T‐muurolol and isodauc‐8‐en‐11‐ol. Various isotopically labelled geranyl and farnesyl diphosphates were used for product identification from micro‐scale reactions, for the determination of the absolute configurations of unknown compounds, to follow the stereochemical course of a cytochrome P450‐catalysed hydroxylation step, and to investigate kinetic isotope effects. Overall, this study demonstrates that isotopically labelled terpene precursors are highly useful to follow cytochrome P450 dependent oxidations of terpenes.     

Synthesis of 6,6- and 7,7-Difluoro-1-acetamidopyrrolizidines and Their Oxidation Catalyzed by the Nonheme Fe Oxygenase LolO

LolO, a 2-oxoglutarate-dependent nonheme Fe oxygenase, catalyzes both the hydroxylation of 1-exo-acetamidopyrrolizidine (AcAP), a pathway intermediate in the biosynthesis of the loline alkaloids, and the cycloetherification of the resulting alcohol. We have prepared fluorinated AcAP analogues to aid in continued mechanistic investigation of the remarkable LolO-catalyzed cycloetherification step. LolO was able to hydroxylate 6,6-difluoro-AcAP (prepared from N,O-protected 4-oxoproline) and then cycloetherify the resulting alcohol, forming a difluorinated analogue of N-acetylnorloline and providing evidence for a cycloetherification mechanism involving a C(7) radical as opposed to a C(7) carbocation. By contrast, LolO was able to hydroxylate 7,7-difluoro-AcAP (prepared from 3-oxoproline) but failed to cycloetherify it, forming (1R,2R,8S)-7,7-difluoro-2-hydroxy-AcAP as the sole product. The divergent LolO-catalyzed reactions of the difluorinated AcAP analogues provide insight into the LolO cycloetherification mechanism and indicate that the 7,7-difluorinated compound, in particular, may be a useful tool to accumulate and characterize the iron intermediate that initiates the cycloetherification reaction.     

Characterization of a Self-Sufficient Cytochrome P450 Monooxygenase from Deinococcus apachensis for Enantioselective Benzylic Hydroxylation

A self-sufficient cytochrome P450 monooxygenase from Deinococcus apachensis (P450DA) was identified and successfully overexpressed in Escherichia coli BL21(DE3). P450DA would be a member of the CYP102D subfamily and assigned as CYP102D2 according to the phylogenetic tree and sequence alignment. Purification and characterization of the recombinant P450DA indicated both NADH and NADPH could be used by P450DA as a reducing cofactor. The recombinant E. coli (P450DA) strain was functionally active, showing excellent enantioselectivity for benzylic hydroxylation of methyl 2-phenylacetate. Further substrate scope studies revealed that P450DA is able to catalyze benzylic hydroxylation of a variety of compounds, affording the corresponding chiral benzylic alcohols in 86–99 % ee and 130–1020 total turnover numbers.     

Sesquiterpene Synthase‐Catalysed Formation of a New Medium‐Sized Cyclic Terpenoid Ether from Farnesyl Diphosphate Analogues

Terpene synthases catalyse the first step in the conversion of prenyl diphosphates to terpenoids. They act as templates for their substrates to generate a reactive conformation, from which a Mg²⁺‐dependent reaction creates a carbocation–PP_i ion pair that undergoes a series of rearrangements and (de)protonations to give the final terpene product. This tight conformational control was exploited for the (R)‐germacrene A synthase– and germacradien‐4‐ol synthase–catalysed formation of a medium‐sized cyclic terpenoid ether from substrates containing nucleophilic functional groups. Farnesyl diphosphate analogues with a 10,11‐epoxide or an allylic alcohol were efficiently converted to a 11‐membered cyclic terpenoid ether that was characterised by HRMS and NMR spectroscopic analyses. Further experiments showed that other sesquiterpene synthases, including aristolochene synthase, δ‐cadinene synthase and amorphadiene synthase, yielded this novel terpenoid from the same substrate analogues. This work illustrates the potential of terpene synthases for the efficient generation of structurally and functionally novel medium‐sized terpene ethers.     

Development of Colorimetric HTS Assay of Cytochrome P450 for ortho‐Specific Hydroxylation,and Engineering of CYP102D1 with Enhanced Catalytic Activity and Regioselectivity

A current challenge in high‐throughput screening (HTS) of hydroxylation reactions by P450 is a fast and sensitive assay for regioselective hydroxylation against millions of mutants. We have developed a solid‐agar plate‐based HTS assay for screening ortho‐specific hydroxylation of daidzein by sensing formaldehyde generated from the O‐dealkylation reaction. This method adopts a colorimetric dye, pararosaniline, which has previously been used as an aldehyde‐specific probe within cells. The rationale for this method lies in the fact that the hydroxylation activity at ortho‐carbon position to C? OH correlates with a linear relationship to O‐dealkylation activity on chemically introduced methoxy group at the corresponding C? OH. As a model system, a 4′,7‐dihydroxyisoflavone (daidzein) hydroxylase (CYP102D1 F96V/M246I), which catalyzes hydroxylation at ortho positions of the daidzein A/B‐ring, was examined for O‐dealklyation activity, by using permethylated daidzein as a surrogate substrate. By using the developed indirect bishydroxylation screening assay, the correlation coefficient between O‐dealkylation and bishydroxylation activity for the template enzyme was 0.72. For further application of this assay, saturation mutants at A273/G274/T277 were examined by mutant screening with a permethylated daidzein analogue substrate (A‐ring inactivated in order to find enhanced 3′‐regioselectiviy). The whole‐cell biotransformation of daidzein by final screened mutant G1 (A273H/G274E/T277G) showed fourfold increased conversion yield, with 14.3 mg L^?1 production titer and greatly increased 3′‐regioselectiviy (3′/6=11.8). These results show that there is a remarkably high correlation (both in vitro and in vivo), thus suggesting that this assay would be ideal for a primary HTS assay for P450 reactions.     

Benzylic Fragmentation (BFR), an Aromatic Analogue of E1cB Including a Homolytic Variant,as either Nucleofugal or Homolytic Path to Aryl Stabilized Carbanions or Ketyls,respectively

BFR is the decisive step of novel reductions at benzyl‐type carbons that may complement available methods. BFR is mechanistically important for understanding and control of curious findings in radical anion chemistry. Driving force of BFR is rearomatization of a dihydroarene species usually derived from anthracene. This intermediate 1 arises (solvent THF) either from coupling of anthracenidyl A ^°− with a benzyl radical or from nucleophilic reaction of anthracene hydride AH ⁻ with a benzylic electrophile followed by CH ₂ deprotonation (excess AH ⁻ ) of the generated 9‐benzyl‐dihydroanthracene. 1 spontaneously undergoes BFR either heterolytically or homolytically depending on the stabilities of carbanion 2 (or even dianion 2 , e.g. from chalcone) and ketyl 3 . BFR is more rapid with counterion Na⁺ than with Li⁺ . The overall reaction is a selective one electron (innersphere eletron transfer) or two electron reduction of a benzylic electrophile, sometimes under expected subsequent rearrangement. This account reports on mechanism and scope of these reductions; a few related cases are described.     

An efficient route to selective bio-oxidation catalysts: an iterative approach comprising modeling, diversification, and screening, based on CYP102A1

Perillyl alcohol is the terminal hydroxylation product of the cheap and readily available terpene, limonene. It has high potential as an anti‐tumor substance, but is of limited availability. In principle, cytochrome P450 monooxygenases, such as the self‐sufficient CYP102A1, are promising catalysts for the oxidation of limonene or other inert hydrocarbons. The wild‐type enzyme converts (4R)‐limonene to four different oxidation products; however, terminal hydroxylation at the allylic C7 is not observed. Here we describe a generic strategy to engineer this widely used enzyme to hydroxylate exclusively the exposed, but chemically less reactive, primary C7 in the presence of other reactive positions. The approach presented here turns CYP102A1 into a highly selective catalyst with a shifted product spectra by successive rounds of modeling, the design of small focused libraries, and screening. In the first round a minimal CYP102A1 mutant library was rationally designed. It contained variants with improved or strongly shifted regio‐, stereo‐ and chemoselectivity, compared to wild‐type. From this library the variant with the highest perillyl alcohol ratio was fine‐tuned by two additional rounds of molecular modeling, diversification, and screening. In total only 29 variants needed to be screened to identify the triple mutant A264V/A238V/L437F that converts (4R)‐limonene to perillyl alcohol with a selectivity of 97 %. Focusing mutagenesis on a small number of relevant positions identified by computational approaches is the key for efficient screening for enzyme selectivity.     

Synthesis of Methylene‐Bridge Polyarenes through Palladium‐Catalyzed Activation of Benzylic Carbon‐Hydrogen Bond

In the presence of palladium(II) acetate [Pd(OAc)₂] and an N‐heterocyclic carbene (NHC) ligand, fluorene derivatives can be generated in good to excellent yields from 2‐halo‐2′‐methylbiaryls through the benzylic C H bond activation (14 examples; 81–97% yields). The scope and limitations of this protocol have been examined. A wide range of functional groups, such as alkyl, alkoxy, ester, nitrile, and others, is able to tolerate the reaction conditions herein. The cyclization of an isotope‐labelled biphenyl gave the corresponding product with a primary kinetic isotope effect (k_H/k_D=4.8:1), which indicates that the rate‐determining step of this reaction is the activation of the benzylic C H bond. Moreover, indenofluorenes were also accessed in excellent results from terphenyls (3 examples; 91–92% yields). The cascade reaction of 2,6‐dichloro‐2′‐methylbiphenyl with diphenylacetylene produced 8,9‐diphenyl‐4H‐cyclopenta[def]phenanthrene in 60% yield through the activation of an aryl and a benzylic C H bond.     

Tandem enzyme‐catalysed reduction/ cis‐dihydroxylation of 2,2,2‐trifluoroace‐ tophenone: chemoenzymatic routes to new enantiopure phenol and benzylic alcohol reagents

Factors that control the competition between toluene dioxgenase‐catalysed arene cis‐dihydroxylation and dehydrogenase‐catalysed ketone reduction have been studied, using whole cells of Pseudomonas putida UV and three alkylaryl ketones. The triol metabolite, obtained from 2,2,2‐trifluoroacetophenone, has been used in the synthesis of single enantiomer chiral phenols and benzylic alcohols. Potential applications of the methylether derivatives of the chiral phenols and benzylic alcohols, as resolving agents, have been found. Copyright © 2007 Society of Chemical Industry     

Synthesis,photochemical characterization,and thermal stability of a series of substituted formamidines as ultraviolet light absorbers

The three‐step reactions of ethyl 4‐aminobenzoate, formic acid, and halohydrocarbons afforded 10 N‐substituted‐N,N′‐diaryl‐formamidine derivatives ( F1 – 10 ) as ultraviolet absorbers. These N‐substituted formamidines were characterized by ¹H NMR, ¹³C NMR, FT‐IR, and ESI‐MS spectroscopies. The UV–vis absorbance and fluorescence properties of the compounds F1 – 10 were investigated in different solvents and in the presences of different metal ions. The effects of the amount of Al³⁺, Pb²⁺, Zr⁴⁺ ions on the UV–vis absorbance and fluorescence properties of compound F1 were also investigated. Moreover, the thermal stability of the compounds F1 – 10 was evaluated as well as the intermediate N,N′‐bis(4‐ethoxycarbonylphenyl)‐formamidine. J. VINYL ADDIT. TECHNOL., 25:E108–E113, 2019. © 2019 Society of Plastics Engineers     

Enzymatic Fluorination of Biotin and Tetrazine Conjugates for Pretargeting Approaches to Positron Emission Tomography Imaging

The use of radiolabelled antibodies and antibody‐derived recombinant constructs has shown promise for both imaging and therapeutic use. In this context, the biotin–avidin/streptavidin pairing, along with the inverse‐electron‐demand Diels–Alder (iEDDA) reaction, have found application in pretargeting approaches for positron emission tomography (PET). This study reports the fluorinase‐mediated transhalogenation [5′‐chloro‐5′‐deoxyadenosine (ClDA) substrates to 5′‐fluoro‐5′‐deoxyadenosine (FDA) products] of two antibody pretargeting tools, a FDA‐PEG‐tetrazine and a [¹⁸F]FDA‐PEG‐biotin, and each is assessed either for its compatibility towards iEDDA ligation to trans‐cyclooctene or for its affinity to avidin. A protocol to avoid radiolytically promoted oxidation of biotin during the synthesis of [¹⁸F]FDA‐PEG‐biotin was developed. The study adds to the repertoire of conjugates for use in fluorinase‐catalysed radiosynthesis for PET and shows that the fluorinase will accept a wide range of ClDA substrates tethered at C‐2 of the adenine ring with a PEGylated cargo. The method is exceptional because the nucleophilic reaction with [¹⁸F]fluoride takes place in water at neutral pH and at ambient temperature.     

Mechanistic aspects of biotransformations by the monooxygenase system of Methylosinus trichosporium OB3b

Mechanistic aspects of the mode of action of the soluble monooxygenase system of Methylosinus trichosporium OB3b have been investigated. The hydroxylation of 4-deuteroanisole in the 4-position was shown to proceed via an NIH shift with a deuterium retention of 66 %, indicative of an epoxide intermediate. Isotopic studies using ethylbenzene and ethylbertzene-d₁₀ showed an isotopic effect, kH/kD = 4.5 for benzylic hydroxylation, and an inverse isotopic effect, kH/kD = 0.75 for arene hydroxylation. It is suggested that a stepwise mechanism (hydrogen abstraction and hydroxylation) could be involed.     

Oxidation and Ammoxidation of Aromatics

An overview of the state of the art in the direct oxygen or nitrogen insertion to aromatic rings and side‐chains by hydroxylation, acetoxylation, partial oxidation and ammoxidation is presented. The influence of a variety of catalysts and oxidants on the yields of hydroxylated products of aromatic species is discussed in more detail. The survey is also focussed on the usage of H₂O₂ as an effective oxidising agent for hydroxylation reactions. Acetoxylation of methyl‐substituted aromatic compounds to their corresponding esters in a single step is indeed an interesting area from an industrial point of view. Hence, the topics covering benzylic acetoxylation, although they are under a developmental stage for commercial exploitation, are also reviewed. The present contribution also covers the main directions of selective oxidation/ammoxidation of aromatic compounds to useful products, surveys recent developments and provides an updated discussion of the state of the art in the field of oxidation and ammoxidation of aromatics. Additionally, a comparative study of the vapour phase oxidation and ammoxidation of different alkyl aromatics to their corresponding aldehydes and nitriles using various heterogeneous catalysts is presented. Besides, the achievements and limitations of the catalysts/processes are emphasised. Furthermore, the present article includes a discussion of common features and differences in mechanistic steps of oxidation and ammoxidation reactions investigated by in situ FITR spectroscopy. The influence of acid‐base properties of catalyst surfaces in connection with electronic effects of the substituents on the performance of the catalysts is also described.     

Toluene Dioxygenase‐Catalysed Oxidation of Benzyl Azide to Benzonitrile: Mechanistic Insights for an Unprecedented Enzymatic Transformation

Enzymatic dioxygenation of benzyl azide by toluene dioxygenase (TDO) produces significant amounts of the cis‐cyclohexadienediol derived from benzonitrile, along with the expected azido diols. We demonstrate that TDO catalyses the oxidation of benzyl azide to benzonitrile, which is further dioxygenated to produce the observed cis‐diol. A proposed mechanism for this transformation involves initial benzylic monooxygenation followed by a nitrene‐mediated rearrangement to form an oxime, which is further dehydrated to afford the nitrile. To the best of our knowledge, this is the first report of enzymatic oxidation of an alkyl azide to a nitrile. In addition, the described oxime‐dehydration activity has not been reported for Rieske dioxygenases.     

Targeted Fluorination of a Nonsteroidal Anti‐inflammatory Drug to Prolong Metabolic Half‐Life

In drug design, one way of improving metabolic stability is to introduce fluorine at a metabolically labile site. In the early stages of drug design, identification of such sites is challenging, and a rapid method of assessing the effect of fluorination on a putative drug’s metabolic stability would be of clear benefit. One approach to this is to employ micro‐organisms that are established as models of drug metabolism in parallel with the synthesis of fluorinated drug analogues. In this study, we have used the filamentous fungus Cunninghamella elegans to identify the metabolically labile site of the nonsteroidal anti‐inflammatory drug flurbiprofen, to aid in the design of fluorinated derivatives that were subsequently synthesised. The effect of the additional fluorine substitution on cytochrome P450‐catalysed oxidation was then determined via incubation with the fungus, and demonstrated that fluorine substitution at the 4′‐position rendered the drug inactive to oxidative transformation, whereas substitution of fluorine at either 2′ or 3′ resulted in slower oxidation compared to the original drug. This approach to modulating the metabolic stability of a drug‐like compound is widely applicable and can be used to address metabolic issues of otherwise good lead compounds in drug development.     

On the Trapping of Vinylgold Intermediates

An internal aryl‐substituted ortho‐alkynylphenol and a similar aniline with stoichiometric amounts of N,N′‐bis(2,6‐diisopropylphenyl)imidazol‐2‐ylidene‐gold tosylate [(IPr)AuOTs] and triethylamine gave the aurated heterocycles as stable intermediates of the corresponding gold(I)‐catalysed hydrooxylation and hydroamination reactions. X‐ray crystal structure analyses of both products could be obtained. A similar internal alkyl‐substituted ortho‐alkynylphenol gave only the cycloisomerised product, no aurated intermediate could be detected.     

In Vitro Double Oxidation of n‐Heptane with Direct Cofactor Regeneration

A novel concept for the direct oxidation of cycloalkanes to the corresponding cyclic ketones in a one‐pot synthesis in water with molecular oxygen as sole oxidizing agent was reported recently. Based on this concept we have developed a new strategy for the double oxidation of n‐heptane to enable a biocatalytic resolution for the direct synthesis of heptanone and (R)‐heptanols in a one‐pot reaction. The bicatalytic cascade employs an NADH driven P450 BM3 monooxygenase variant (WT^NADH, 19A12^NADH or CM1^NADH) and an (S)‐enantioselective alcohol dehydrogenase (RE‐ADH). In the initial step n‐heptane is hydroxylated under consumption of NADH to produce (R/S)‐heptanol. In the second oxidation step the (S)‐heptanol enantiomers are transformed to the corresponding ketones, reducing and thereby regenerating the cofactor. Characterization of initial hydroxylation step revealed high turnover frequencies (TOF) of up to 600 min⁻¹, as well as high coupling efficiencies using NADH as cofactor (up to 44%). In the cascade reaction a nearly 2‐fold improved product formation was achieved, compared to the single hydroxylation reaction. The total product concentration reached 1.1 mM, corresponding to a total turnover number (TTN) of 2500. Implementation of an additional cofactor regeneration system (D ‐glucose/glucose dehydrogenase) enabled a further enhancement in product formation with a total product concentration of 1.8 mM and a TTN of 3500.     

TEMPO‐Copper(II) Diimine‐Catalysed Oxidation of Benzylic Alcohols in Aqueous Media

In situ generated copper(II)‐diimine complexes combined with TEMPO (2,2,6,6‐tetramethylpiperidinyl‐1‐oxyl radical) were studied in the oxidation of benzylic alcohols, the focus being on enviromentally benign reaction conditions. In this respect, reactions were studied in aqueous alkaline solutions and dioxygen was used as an end oxidant. This simple catalytic system turned out to be highly efficient and selective in the oxidation of primary and secondary benzylic alcohols to their corresponding carbonyl compounds. Under optimised reaction conditions [5 mol % of TEMPO, 3 mol % of copper(II ) diimine, pH 12.6–13.5, 80 °C, 10 bar O₂] benzyl alcohol was quantitatively and selectively oxidised to benzaldehyde. According to ESI‐MS studies, coordination of TEMPO, as well as deprotonated benzyl alcohol to the parent copper‐diimine complex in aqueous solutions is feasible. Supported by these observations a plausible reaction mechanism is proposed for the oxidation reaction.     

A flavoprotein monooxygenase that catalyses a Baeyer-Villiger reaction and thioether oxidation using NADH as the nicotinamide cofactor

A gene from the marine bacterium Stenotrophomonas maltophilia encodes a 38.6 kDa FAD‐containing flavoprotein (Uniprot B2FLR2) named S. maltophilia flavin‐containing monooxygenase (SMFMO), which catalyses the oxidation of thioethers and also the regioselective Baeyer–Villiger oxidation of the model substrate bicyclo[3.2.0]hept‐2‐en‐6‐one. The enzyme was unusual in its ability to employ either NADH or NADPH as nicotinamide cofactor. The K_M and k_cat values for NADH were 23.7±9.1 μM and 0.029 s^?1 and 27.3±5.3 μM and 0.022 s^?1 for NADPH. However, k_cat/K_M value for the ketone substrate in the presence of 100 μM cofactor was 17 times greater for NADH than for NADPH. SMFMO catalysed the quantitative conversion of 5 mM ketone in the presence of substoichiometric concentrations of NADH with the formate dehydrogenase cofactor recycling system, to give the 2‐oxa and 3‐oxa lactone products of Baeyer–Villiger reaction in a ratio of 5:1, albeit with poor enantioselectivity. The conversion with NADPH was 15 %. SMFMO also catalysed the NADH‐dependent transformation of prochiral aromatic thioethers, giving in the best case, 80 % ee for the transformation of p‐chlorophenyl methyl sulfide to its R enantiomer. The structure of SMFMO reveals that the relaxation in cofactor specificity appears to be accomplished by the substitution of an arginine residue, responsible for recognition of the 2′‐phosphate on the NADPH ribose in related NADPH‐dependent FMOs, with a glutamine residue in SMFMO. SMFMO is thus representative of a separate class of single‐component, flavoprotein monooxygenases that catalyse NADH‐dependent oxidations from which possible sequences and strategies for developing NADH‐dependent biocatalysts for asymmetric oxygenation reactions might be identified.     

Characterisation of the Major Synthetic Products of the Reactions Between Butanone and Hydrogen Peroxide

The reactions between butanone and hydrogen peroxide, both catalysed and un‐catalysed, were investigated and spectral and sensitiveness data reported. The major product of the un‐catalysed reaction, 2‐hydroxy,2‐hydroperoxybutane, displayed a Figure of Insensitiveness (F of I) of 10, Temperature of Ignition (T of I) of 132 °C, and initiated when 128 N of frictional force or an electrostatic discharge (ESD) of 4.5 J was applied. Differential scanning calorimetric analyses revealed an onset of decomposition at 128 °C, peak maximum of 140 °C, and decomposition energy of 203 J g⁻¹. The major product of the cooled (5 °C) acid catalysed reaction between butanone and hydrogen peroxide, 2,2′‐dihydroperoxy‐2,2′‐dibutyl peroxide, displayed a F of I of<10, T of I of 110 °C and initiated upon application of 5 N of friction or a 0.45 J ESD. Calorimetry showed a melt at 38.3 °C, an onset of exothermic decomposition at 127 °C and the evolution of 1292 J g⁻¹. The major product of the raised temperature (20 °C) acid catalysed synthesis, 1,4,7‐trimethyl‐1,4,7‐triethyl‐1,4,7‐cyclononatriperoxane, displayed F of I of<10 and initiated upon application of 5 N of friction or a 0.45 J ESD. Calorimetry revealed an onset to melting at 28.9 °C, an onset to thermal decomposition at 128 °C, and decomposition energy of 1438 J g⁻¹.