Daniel J. Wherritt

Mechanistic studies of the lithium enolate of 4-fluoroacetophenone: rapid-injection NMR study of enolate formation, dynamics, and aldol reactivity.

Lithium enolates are widely used nucleophiles with a complicated and only partially understood solution chemistry. Deprotonation of 4-fluoroacetophenone in THF with lithium diisopropylamide occurs through direct reaction of the amide dimer to yield a mixed enolate-amide dimer (3), then an enolate homodimer (1-Li)(2), and finally an enolate tetramer (1-Li)(4), the equilibrium structure. Aldol reactions of both the metastable dimer and the stable tetramer of the enolate were investigated. Each reacted directly with the aldehyde to give a mixed enolate-aldolate aggregate, with the dimer only about 20 times as reactive as the tetramer at -120 °C.

A rapid injection NMR study of the reaction of organolithium reagents with esters, amides, and ketones.

Unexpectedly high rates of reaction between alkyllithium reagents and amides, compared to esters and ketones, were observed by Rapid Inject NMR and competition experiments. Spectroscopic investigations with 4-fluorophenyllithium (ArLi, mixture of monomer and dimer in THF) and a benzoate ester identified two reactive intermediates, a homodimer of the tetrahedral intermediate, stable below -100 °C, and a mixed dimer with ArLi. Direct formation of dimers suggested that the ArLi dimer may be the reactive aggregate rather than the usually more reactive monomer. In contrast, RINMR experiments with ketones demonstrated that the ArLi monomer was the reactive species.

Divergent Rhodium-Catalyzed Cyclization Reactions of Enoldiazoacetamides with Nitrosoarenes

The first cyclization reactions of enoldiazo compounds with nitrosoarenes have been developed. Under the catalysis of rhodium(II) octanoate, [3 + 2]-cyclization between enoldiazoacetamides and nitrosoarenes occurred through cleavages of the enol double bond and the amide bond, thus furnishing fully substituted 5-isoxazolone derivatives. Upon changing the catalyst to rhodium(II) caprolactamate, the reaction pathway switched to an unprecedented formal [5 + 1]-cyclization that provided multifunctionalized 1,3-oxazin-4-ones with near exclusivity under otherwise identical conditions. Mechanistic studies uncovered distinct catalytic activities and reaction intermediates, which plausibly rationalized the novel reactivity and catalyst-controlled chemodivergence. Furthermore, a mechanism-inspired enantioselective rhodium-catalyzed reaction of γ-substituted enoldiazoacetamide with nitrosobenzene produced highly enantioenriched heterocycle-linked trialkylamine.

Malonylation of glucosylated N-lauroylethanolamine: a new pathway that determines N-acylethanolamine metabolic fate in plants.

N-Acylethanolamines (NAEs) are bioactive fatty acid derivatives present in trace amounts in many eukaryotes. Although NAEs have signaling and physiological roles in animals, little is known about their metabolic fate in plants. Our previous microarray analyses showed that inhibition of Arabidopsis thaliana seedling growth by exogenous N-lauroylethanolamine (NAE 12:0) was accompanied by the differential expression of multiple genes encoding small molecule-modifying enzymes. We focused on the gene At5g39050, which encodes a phenolic glucoside malonyltransferase 1 (PMAT1), to better understand the biological significance of NAE 12:0-induced gene expression changes. PMAT1 expression was induced 3–5-fold by exogenous NAE 12:0. PMAT1 knockouts (pmat1) had reduced sensitivity to the growth-inhibitory effects of NAE 12:0 compared with wild type leading to the hypothesis that PMAT1 might be a previously uncharacterized regulator of NAE metabolism in plants. To test this hypothesis, metabolic profiling of wild-type and pmat1 seedlings treated with NAE 12:0 was conducted. Wild-type seedlings treated with NAE 12:0 accumulated glucosylated and malonylated forms of this NAE species, and structures were confirmed using nuclear magnetic resonance (NMR) spectroscopy. By contrast, only the peak corresponding to NAE 12:0-glucoside was detected in pmat1. Recombinant PMAT1 catalyzed the reaction converting NAE 12:0-glucoside to NAE 12:0-mono- or -dimalonylglucosides providing direct evidence that this enzyme is involved in NAE 12:0-glucose malonylation. Taken together, our results indicate that glucosylation of NAE 12:0 by a yet to be determined glucosyltransferase and its subsequent malonylation by PMAT1 could represent a mechanism for modulating the biological activities of NAEs in plants.

Pathway-specific metabolome analysis with 18O2-labeled Medicago truncatula via a mass spectrometry-based approach

IntroductionOxygen from carbon dioxide, water or molecular oxygen, depending on the responsible enzyme, can lead to a large variety of metabolites through chemical modification.ObjectivesPathway-specific labeling using isotopic molecular oxygen (18O2) makes it possible to determine the origin of oxygen atoms in metabolites and the presence of biosynthetic enzymes (e.g., oxygenases). In this study, we established the basis of 18O2-metabolome analysis.Methods18O2 labeled whole Medicago truncatula seedlings were prepared using 18O2-air and an economical sealed-glass bottle system. Metabolites were analyzed using high-accuracy and high-resolution mass spectrometry. Identification of the metabolite was confirmed by NMR following UHPLC–solid-phase extraction (SPE).ResultsA total of 511 peaks labeled by 18O2 from shoot and 343 peaks from root were annotated by untargeted metabolome analysis. Additionally, we identified a new flavonoid, apigenin 4′-O-[2′-O-coumaroyl-glucuronopyranosyl-(1–2)-O-glucuronopyranoside], that was labeled by 18O2. To the best of our knowledge, this is the first report of apigenin 4′-glucuronide in M. truncatula. Using MSn analysis, we estimated that 18O atoms were specifically incorporated in apigenin, the coumaroyl group, and glucuronic acid. For apigenin, an 18O atom was incorporated in the 4′-hydroxy group. Thus, non-specific incorporation of an 18O atom by recycling during one month of labeling is unlikely compared with the more specific oxygenase-catalyzing reaction.ConclusionOur finding indicated that 18O2 labeling was effective not only for the mining of unknown metabolites which were biosynthesized by oxygenase-related pathway but also for the identification of metabolites whose oxygen atoms were derived from oxygenase activity.

Metabolomics of Two Pecan Varieties Provides Insights into Scab Resistance

UHPLC-MS-based non-targeted metabolomics was used to investigate the biochemical basis of pecan scab resistance. Two contrasting pecan varieties, Kanza (scab-resistant) and Pawnee (scab-susceptible), were profiled and the metabolomics data analyzed using multivariate statistics. Significant qualitative and quantitative metabolic differences were observed between the two varieties. Both varieties were found to have some unique metabolites. Metabolites that were only present or more abundant in Kanza relative to Pawnee could potentially contribute to the scab resistance in Kanza. Some of these metabolites were putatively identified as quercetin derivatives using tandem mass spectrometry. This suggests that quercetin derivatives could be important to pecan scab resistance.

Stepwise O-Atom Transfer in Heme-Based Tryptophan Dioxygenase: Role of Substrate Ammonium in Epoxide Ring Opening

Heme-based tryptophan dioxygenases are established immunosuppressive metalloproteins with significant biomedical interest. Here, we synthesized two mechanistic probes to specifically test if the α-amino group of the substrate directly participates in a critical step of the O atom transfer during catalysis in human tryptophan 2,3-dioxygenase (TDO). Substitution of the nitrogen atom of the substrate to a carbon (probe 1) or oxygen (probe 2) slowed the catalytic step following the first O atom transfer such that transferring the second O atom becomes less likely to occur, although the dioxygenated products were observed with both probes. A monooxygenated product was also produced from probe 2 in a significant quantity. Analysis of this new product by HPLC coupled UV-vis spectroscopy, high-resolution mass spectrometry, 1H NMR, 13C NMR, HSQC, HMBC, and infrared (IR) spectroscopies concluded that this monooxygenated product is a furoindoline compound derived from an unstable epoxyindole intermediate. These results prove that small molecules can manipulate the stepwise O atom transfer reaction of TDO and provide a showcase for a tunable mechanism by synthetic compounds. The product analysis results corroborate the presence of a substrate-based epoxyindole intermediate during catalysis and provide the first substantial experimental evidence for the involvement of the substrate α-amino group in the epoxide ring-opening step during catalysis. This combined synthetic, biochemical, and biophysical study establishes the catalytic role of the α-amino group of the substrate during the O atom transfer reactions and thus represents a substantial advance to the mechanistic comprehension of the heme-based tryptophan dioxygenases.

Reassignment of the human aldehyde dehydrogenase ALDH8A1 (ALDH12) to the kynurenine pathway in tryptophan catabolism

The kynurenine pathway is the primary route for l-tryptophan degradation in mammals. Intermediates and side products of this pathway are involved in immune response and neurodegenerative diseases. This makes the study of enzymes, especially those from mammalian sources, of the kynurenine pathway worthwhile. Recent studies on a bacterial version of an enzyme of this pathway, 2-aminomuconate semialdehyde (2-AMS) dehydrogenase (AMSDH), have provided a detailed understanding of the catalytic mechanism and identified residues conserved for muconate semialdehyde recognition and activation. Findings from the bacterial enzyme have prompted the reconsideration of the function of a previously identified human aldehyde dehydrogenase, ALDH8A1 (or ALDH12), which was annotated as a retinal dehydrogenase based on its ability to preferentially oxidize 9-cis-retinal over trans-retinal. Here, we provide compelling bioinformatics and experimental evidence that human ALDH8A1 should be reassigned to the missing 2-AMS dehydrogenase of the kynurenine metabolic pathway. For the first time, the product of the semialdehyde oxidation by AMSDH is also revealed by NMR and high-resolution MS. We found that ALDH8A1 catalyzes the NAD+-dependent oxidation of 2-AMS with a catalytic efficiency equivalent to that of AMSDH from the bacterium Pseudomonas fluorescens. Substitution of active-site residues required for substrate recognition, binding, and isomerization in the bacterial enzyme resulted in human ALDH8A1 variants with 160-fold increased Km or no detectable activity. In conclusion, this molecular study establishes an additional enzymatic step in an important human pathway for tryptophan catabolism.

Cofactor Biogenesis in Cysteamine Dioxygenase: C−F Bond Cleavage with Genetically Incorporated Unnatural Tyrosine

Cysteamine dioxygenase (ADO) is a thiol dioxygenase whose study has been stagnated by the ambiguity as to whether or not it possesses an anticipated protein-derived cofactor. Reported herein is the discovery and elucidation of a Cys-Tyr cofactor in human ADO, crosslinked between Cys220 and Tyr222 through a thioether (C-S) bond. By genetically incorporating an unnatural amino acid, 3,5-difluoro-tyrosine (F2 -Tyr), specifically into Tyr222 of human ADO, an autocatalytic oxidative carbon-fluorine bond activation and fluoride release were identified by mass spectrometry and 19 F NMR spectroscopy. These results suggest that the cofactor biogenesis is executed by a powerful oxidant during an autocatalytic process. Unlike that of cysteine dioxygenase, the crosslinking results in a minimal structural change of the protein and it is not detectable by routine low-resolution techniques. Finally, a new sequence motif, C-X-Y-Y(F), is proposed for identifying the Cys-Tyr crosslink.

Chemical synthesis of 7α-hydroxypregnenolone, a neuroactive steroid that stimulates locomotor activity

Graphical abstract Figure. No Caption available. HighlightsTwo convenient approaches for the synthesis of 7&agr;‐hydroxypregnenolone are reported.Explanation of the stereoselectivity of reduction at C7‐ketone.Potential application to other steroid analogs oxygenated at C7. Abstract 7&agr;‐Hydroxypregnenolone is an endogenous neuroactive steroid that stimulates locomotor activity. A synthesis of 7&agr;‐hydroxypregnenolone from pregnenolone, which takes advantage of an orthogonal protecting group strategy, is described. In detail, the C7‐position was oxidized with CrO3 and 3,5‐dimethylpyrazole to yield a 7‐keto steroid intermediate. The resulting 7‐ketone was stereoselectively reduced to the 7&agr;‐hydroxy group with lithium tri‐sec‐butylborohydride. In contrast, reduction of the same 7‐ketone intermediate with NaBH4 resulted in primarily the 7&bgr;‐hydroxy epimer. Furthermore, in an alternative route to the target compound, the 7&agr;‐hydroxy group was successfully incorporated by direct C–H allylic benzoyloxylation of pregnenolone‐3‐acetate with CuBr and tert‐butyl peroxybenzoate followed by saponification. The disclosed syntheses to 7‐oxygenated steroids are amenable to potentially obtain other biologically active sterols and steroids.