Epimeric Products Research Articles

A novel cellobiose 2-epimerase from anaerobic halophilic Iocasia fonsfrigidae and its ability to convert lactose in fresh goat milk into epilactose.

Cellobiose 2-epimerase (CE) has received great attention due to its potential applications in the food and pharmaceutical industries. In this study, a novel CE from mesophilic anaerobic halophilic bacterium Iocasia fonsfrigidae strain SP3-1 (IfCE) was successfully expressed in Escherichia coli and characterized. Unlike other CEs, the purified IfCE shows only epimerization activity toward β-1,4-glycosidic linkages of disaccharides, including mannobiose, cellobiose and lactose, but not for monosaccharides, β-1,4-glycosidic linkages of trisaccharides and α-1,4-glycosidic linkages of disaccharides. Only one epimerization product was obtained from the action of IfCE against mannobiose, cellobiose and lactose. Under optimum conditions, 31.0% of epilactose, a rare and low-calorie prebiotic sweetener with medicinal and pharmacological properties, was obtained from 10 mg mL-1 lactose. IfCE was highly active against lactose under NaCl concentrations up to 500 mmol L-1, possibly due to the excessive basic (arginine and lysine) and acidic (aspartic and glutamic acids) amino acid residues, which are localized on the surface of the halophilic enzyme structure. These residues may protect the enzyme from Cl- and Na+ ions from the environment, respectively. Under normal conditions, IfCE was able to convert lactose present in fresh goat milk to epilactose with a conversion yield of 31% in 10 min. In addition, IfCE has been investigated as a safe enzyme for human allergen. The results suggested that IfCE is a promising candidate to increase the quality and value of milk and dairy products by converting lactose that causes digestive problems in people with lactose intolerance into epilactose. © 2024 Society of Chemical Industry.

Features of the intramolecular Hosomi–Sakurai reaction of allylic cyclopentenylsilane containing an enol ether moiety

A new intramolecular variant of the Hosomi–Sakurai reaction of 5-(2-methoxyvinyl)-4-((phenylthio)methyl)cyclopent-2-en-1-yl)trimethylsilane is presented, which is a by-process between hydrolysis to aldehyde and acetalization to dimethylacetal. The key steps are the generation of methoxy methylene carbocation from enol ether during acid catalysis and its subsequent attack on the allylsilane moiety with formation of norbornene derivative. In turn, the corresponding aldehyde undergoes Hosomi–Sakurai cyclization by interaction with BF3·Et2O in acetonitrile, as well as by reaction with TBAF in THF through various transition states, leading to epimeric alcohols of the same norbornene structure. At the same time, the starting enol ether by treatment with TBAF undergoes exclusively protodesilylation and hydrolysis, while with BF3·Et2O it gives mixtures of epimeric products of topology both bicyclo[2.2.1]heptane and bicyclo[3.2.0]heptane.

Structure-activity relationships of rationally designed AMACR 1A inhibitors

α-Methylacyl-CoA racemase (AMACR; P504S) is a promising novel drug target for prostate and other cancers. Assaying enzyme activity is difficult due to the reversibility of the ‘racemisation’ reaction and the difficulties in the separation of epimeric products; consequently few inhibitors have been described and no structure–activity relationship study has been performed. This paper describes the first structure–activity relationship study, in which a series of 23 known and potential rational AMACR inhibitors were evaluated. AMACR was potently inhibited (IC50 = 400–750 nM) by ibuprofenoyl-CoA and derivatives. Potency was positively correlated with inhibitor lipophilicity. AMACR was also inhibited by straight-chain and branched-chain acyl-CoA esters, with potency positively correlating with inhibitor lipophilicity. 2-Methyldecanoyl-CoAs were ca. 3-fold more potent inhibitors than decanoyl-CoA, demonstrating the importance of the 2-methyl group for effective inhibition. Elimination substrates and compounds with modified acyl-CoA cores were also investigated, and shown to be potent inhibitors. These results are the first to demonstrate structure–activity relationships of rational AMACR inhibitors and that potency can be predicted by acyl-CoA lipophilicity. The study also demonstrates the utility of the colorimetric assay for thorough inhibitor characterisation.

Glucopyranosylidene-Spiro-Thiazolinones: Synthetic Studies and Determination of Absolute Configuration by TDDFT-ECD Calculations.

Reactions of O-peracylated C-(1-bromo-β-d-glucopyranosyl)formamides with thioamides furnished the corresponding glucopyranosylidene-spiro-thiazolin-4-one. While O-debenzoylations under a variety of conditions resulted in decomposition, during O-deacetylations the addition of MeOH to the thiazolinone moiety was observed, and with EtOH and water similar adducts were isolated or detected. The structure and stereochemistry of the new compounds were established by means of NMR and electronic circular dichroism (ECD) data supported by time-dependent density functional theory ECD (TDDFT-ECD) calculations. TDDFT-ECD calculations could efficiently distinguish the proposed epimeric products having different absolute configuration in the spiro heterocyclic ring.

Energy Landscape of Alginate-Epimerase Interactions Assessed by Optical Tweezers and Atomic Force Microscopy.

Mannuronan C-5 epimerases are a family of enzymes that catalyze epimerization of alginates at the polymer level. This group of enzymes thus enables the tailor-making of various alginate residue sequences to attain various functional properties, e.g. viscosity, gelation and ion binding. Here, the interactions between epimerases AlgE4 and AlgE6 and alginate substrates as well as epimerization products were determined. The interactions of the various epimerase–polysaccharide pairs were determined over an extended range of force loading rates by the combined use of optical tweezers and atomic force microscopy. When studying systems that in nature are not subjected to external forces the access to observations obtained at low loading rates, as provided by optical tweezers, is a great advantage since the low loading rate region for these systems reflect the properties of the rate limiting energy barrier. The AlgE epimerases have a modular structure comprising both A and R modules, and the role of each of these modules in the epimerization process were examined through studies of the A- module of AlgE6, AlgE6A. Dynamic strength spectra obtained through combination of atomic force microscopy and the optical tweezers revealed the existence of two energy barriers in the alginate-epimerase complexes, of which one was not revealed in previous AFM based studies of these complexes. Furthermore, based on these spectra estimates of the locations of energy transition states (x β), lifetimes in the absence of external perturbation (τ 0) and free energies (ΔG #) were determined for the different epimerase–alginate complexes. This is the first determination of ΔG # for these complexes. The values determined were up to 8 kBT for the outer barrier, and smaller values for the inner barriers. The size of the free energies determined are consistent with the interpretation that the enzyme and substrate are thus not tightly locked at all times but are able to relocate. Together with the observed different affinities determined for AlgE4-polymannuronic acid (poly-M) and AlgE4-polyalternating alginate (poly-MG) macromolecular pairs these data give important contribution to the growing understanding of the mechanisms underlying the processive mode of these enzymes.

Novel cellobiose 2-epimerases for the production of epilactose from milk ultrafiltrate containing lactose

A selected number of enzymes have recently been assigned to the emerging class of cellobiose 2-epimerases (CE). All CE convert lactose to the rare sugar epilactose, which is regarded as a new prebiotic. Within this study, the gene products of 2 potential CE genes originating from the mesophilic bacteria Cellulosilyticum lentocellum and Dysgonomonas gadei were recombinantly produced in Escherichia coli and purified by chromatography. The enzymes have been identified as novel CE by sequence analysis and biochemical characterizations. The biochemical characterizations included the determination of the molecular weight, the substrate spectrum, and the kinetic parameters, as well as the pH and temperature profiles in buffer and food matrices. Both identified CE epimerize cellobiose and lactose into the C2 epimerization products glucosylmannose and epilactose, respectively. The epimerization activity for lactose was maximal at pH 8.0 or 7.5 and 40°C in defined buffer systems for the CE from C. lentocellum and the CE from D. gadei, respectively. In addition, biotransformations of the foodstuff milk ultrafiltrate containing lactose were demonstrated. The CE from D. gadei was produced in a stirred-tank reactor (12 L) and purified using an automatic system. Enzyme production and purification in this scale indicates that a future upscaling of CE production is possible. The bioconversions of lactose in milk ultrafiltrate were carried out either in a batch process or in a continuously operated enzyme membrane reactor (EMR) process. Both processes ran at an industrially relevant low temperature of 8°C to reduce undesirable microbial growth. The enzyme was reasonably active at the low process temperature because the CE originated from a mesophilic organism. An epilactose yield of 29.9% was achieved in the batch process within 28 h of operation time. In the continuous EMR process, the epilactose yield in the product stream was lower, at 18.5%. However, the enzyme productivity was approximately 6 times higher because the continuous epilactose formation was carried out for about 6 d without further addition of biocatalyst. Within this time, 24g of epilactose in 2.8 L of permeate were produced. The batch and the EMR process showed that the milk ultrafiltrate, which is a sidestream of the milk protein production, might be upgraded to a dairy product of higher value by the enzymatic in situ production of epilactose.

Crystallographic and molecular dynamics simulation analysis of Escherichia coli dihydroneopterin aldolase.

BackgroundDihydroneopterin aldolase (DHNA) catalyzes the conversion of 7,8-dihydroneopterin to 6-hydroxymethyl-7,8-dihydropterin and also the epimerization of DHNP to 7,8-dihydromonapterin. Previously, we determined the crystal structure of Staphylococcus aureus DHNA (SaDHNA) in complex with the substrate analogue neopterin (NP). We also showed that Escherichia coli DHNA (EcDHNA) and SaDHNA have significantly different binding and catalytic properties by biochemical analysis. On the basis of these structural and functional data, we proposed a catalytic mechanism involving two proton wires.ResultsTo understand the structural basis for the biochemical differences and further investigate the catalytic mechanism of DHNA, we have determined the structure of EcDHNA complexed with NP at 1.07-Å resolution [PDB:2O90], built an atomic model of EcDHNA complexed with the substrate DHNP, and performed molecular dynamics (MD) simulation analysis of the substrate complex. EcDHNA has the same fold as SaDHNA and also forms an octamer that consists of two tetramers, but the packing of one tetramer with the other is significantly different between the two enzymes. Furthermore, the structures reveal significant differences in the vicinity of the active site, particularly in the loop that connects strands β3 and β4, mainly due to the substitution of nearby residues. The building of an atomic model of the complex of EcDHNA and the substrate DHNP and the MD simulation of the complex show that some of the hydrogen bonds between the substrate and the enzyme are persistent, whereas others are transient. The substrate binding model and MD simulation provide the molecular basis for the biochemical behaviors of the enzyme, including noncooperative substrate binding, indiscrimination of a pair of epimers as the substrates, proton wire switching during catalysis, and formation of epimerization product.ConclusionsThe EcDHNA and SaDHNA structures, each in complex with NP, reveal the basis for the biochemical differences between EcDHNA and SaDHNA. The atomic substrate binding model and MD simulation offer insights into substrate binding and catalysis by DHNA. The EcDHNA structure also affords an opportunity to develop antimicrobials specific for Gram-negative bacteria, as DHNAs from Gram-negative bacteria are highly homologous and E. coli is a representative of this class of bacteria.

Epimers of Azaspiracids: Isolation, Structural Elucidation, Relative LC-MS Response, and in Vitro Toxicity of 37-epi-Azaspiracid-1

Since azaspiracid-1 (AZA1) was identified in 1998, the number of AZA analogues has increased to over 30. The development of an LC-MS method using a neutral mobile phase led to the discovery of isomers of AZA1, AZA2, and AZA3, present at ~2-16% of the parent analogues in phytoplankton and shellfish samples. Under acidic mobile phase conditions, isomers and their parents are not separated. Stability studies showed that these isomers were spontaneous epimerization products whose formation is accelerated with the application of heat. The AZA1 isomer was isolated from contaminated shellfish and identified as 37-epi-AZA1 by nuclear magnetic resonance (NMR) spectroscopy and chemical analyses. Similar analysis indicated that the isomers of AZA2 and AZA3 corresponded to 37-epi-AZA2 and 37-epi-AZA3, respectively. The 37-epimers were found to exist in equilibrium with the parent compounds in solution. 37-epi-AZA1 was quantitated by NMR, and relative molar response studies were performed to determine the potential differences in LC-MS response of AZA1 and 37-epi-AZA1. Toxicological effects were determined using Jurkat T lymphocyte cells as an in vitro cell model. Cytotoxicity experiments employing a metabolically based dye (i.e., MTS) indicated that 37-epi-AZA1 elicited a lethal response that was both concentration- and time-dependent, with EC50 values in the subnanomolar range. On the basis of EC50 comparisons, 37-epi-AZA1 was 5.1-fold more potent than AZA1. This data suggests that the presence of these epimers in seafood products should be considered in the analysis of AZAs for regulatory purposes.

Temperature influences epimerization and composition of flavanol monomers, dimers and trimers during cocoa bean roasting

Cocoa consumption is suggested to promote many health benefits, since cocoa is a rich source of flavanols; but amounts and profiles of flavanols depend strongly on the bean type, origin and manufacturing process. Roasting is known as a crucial step in technical treatment of cocoa, which leads to flavanol losses and modifications, especially the epimerization of (−)-epicatechin to (−)-catechin. This study monitors the influence of cocoa bean roasting on the composition of flavanol monomers to trimers, with special focus on epimerization, which was quantified for procyanidin dimers, and also observed for trimers for the first time. Five dimeric and two trimeric potential epimerization products were detected and the extent of epimerization during cocoa roasting was shown to be a function of temperature. The data also showed remarkable variations in the change of flavanol content. The quantified flavanols decreased about 50% in Java beans and increased about 30% in Ivory Coast beans, despite being roasted under equal conditions.

Palladium‐Catalyzed Synthesis and Isolation of Functionalized Allylboronic Acids: Selective, Direct Allylboration of Ketones

Palladium‐Catalyzed Synthesis and Isolation of Functionalized Allylboronic Acids: Selective, Direct Allylboration of Ketones

Uncovering Biphasic Catalytic Mode of C5-epimerase in Heparan Sulfate Biosynthesis

Heparan sulfate (HS), a highly sulfated polysaccharide, is biosynthesized through a pathway involving several enzymes. C(5)-epimerase (C(5)-epi) is a key enzyme in this pathway. C(5)-epi is known for being a two-way catalytic enzyme, displaying a "reversible" catalytic mode by converting a glucuronic acid to an iduronic acid residue, and vice versa. Here, we discovered that C(5)-epi can also serve as a one-way catalyst to convert a glucuronic acid to an iduronic acid residue, displaying an "irreversible" catalytic mode. Our data indicated that the reversible or irreversible catalytic mode strictly depends on the saccharide substrate structures. The biphasic mode of C(5)-epi offers a novel mechanism to regulate the biosynthesis of HS with the desired biological functions.

Sn-Beta zeolites with borate salts catalyse the epimerization of carbohydrates via an intramolecular carbon shift

Carbohydrate epimerization is an essential technology for the widespread production of rare sugars. In contrast to other enzymes, most epimerases are only active on sugars substituted with phosphate or nucleotide groups, thus drastically restricting their use. Here we show that Sn-Beta zeolite in the presence of sodium tetraborate catalyses the selective epimerization of aldoses in aqueous media. Specifically, a 5 wt% aldose (for example, glucose, xylose or arabinose) solution with a 4:1 aldose:sodium tetraborate molar ratio reacted with catalytic amounts of Sn-Beta yields near-equilibrium epimerization product distributions. The reaction proceeds by way of a 1,2 carbon shift wherein the bond between C-2 and C-3 is cleaved and a new bond between C-1 and C-3 is formed, with C-1 moving to the C-2 position with an inverted configuration. This work provides a general method of performing carbohydrate epimerizations that surmounts the main disadvantages of current enzymatic and inorganic processes.

A convenient method for synthesis of tetraoxazole peptide macrocycles

G-quadruplex DNA plays important regulatory roles in the maintenance of telomere length and transcriptions inhibition of such oncogenes as c-myc, and thus has become an attractive target for the development of anti-cancer therapeutic agents. Poly-oxazole macrocycles are a promising new class of G-quadruplex binding ligands. Herein is described the synthesis of a tetraoxazole peptide macrocycle and an epimerization product. The synthetic unit was prepared by cyclization-oxidation of a diserine with one hydroxyl protected. Two-cycle coupling of the unit gave a linear tetraoxazole amide. The targeted molecule and the epimerization product were obtained after macrocyclization of the linear precursor and following removal of the protecting benzyl groups. With structural similarities to the most potent G-quadruplex stabilizer telomestatin, these two molecules might potentially be used to probe the biological significance of G-quadruplex' in vivo.

Synthesis and Characterization of Organic Impurities in Bortezomib Anhydride Produced by a Convergent Technology

A profile of impurities in bortezomib anhydride, produced by a recently developed convergent technology, has been characterized. HPLC-MS analysis of the drug essence revealed three impurities: an epimer of bortezomib, resulting from partial racemization of l-phenylalanine’s stereogenic center during the chemical synthesis, and two epimeric products of oxidative degradation of bortezomib, in which boron is replaced by the OH group. The impurities were obtained by chemical synthesis and characterized by physical methods.

Intramolecular interactions in dimedone and phenalen-1,3-dione adducts of 2(4)-pyridinecarboxaldehyde: Enol–enol and ring-chain tautomerism, strong hydrogen bonding, zwitterions

Abstract The 2:1 adducts of dimedone and phenalen-1,3-dione with 2- and 4-pyridine carboxaldehyde, in spite of similar chemical behavior of their diketone precursors, have quite different tautomeric structure both in solid state and in solution. 2,2′-(Pyridin-2-ylmethanediyl)-bis(5,5-dimethyl-cyclohexane-1,3-dione) 5 exists as an equilibrium mixture of its dienol tautomer 5а′ with two intramolecular H-bonds ОН⋯О С and OH⋯N and the epimeric products of its reversible cyclization, that is, 4a-hydroxy-9-(pyridin-2-yl)-2,3,4,4a,6,7,9,9a-octahydro-5-H-xanthene-1,8-diones 5b (major) and 5c (minor), the latter appears only in polar media like DMSO. 2,2′-(Pyridin-4-ylmethanediyl)bis(5,5-dimethylcyclohexane-1,3-dione) 4, like other 2:1 dimedone–aldehyde adducts, both in solution and in solid state exists as dienol with two intramolecular H-bonds ОН⋯О С. 4-[Bis(1H-phenalen-1,3(2H)-dione)methyl]pyridine 6 in nonpolar media like chloroform exists as dienol, but crystallizes from this solvent as zwitter-ion 6b with one very strong ionic hydrogen bond O H⋯O and protonated pyridine nitrogen. The same zwitterion is formed in polar media (DMSO). For 2-[bis(1H-phenalen-1,3(2H)-dione)-methyl]-pyridine 7, fast exchange between its dienol tautomer 7a and zwitter-ion 7b occurs even in CD2Cl2, whereas in DMSO the equilibrium shifts towards zwitter-ion 7b.

Usage of α-picoline borane for the reductive amination of carbohydrates

The reaction of reductive amination, widely used for carbohydrates, was reviewed in our lab, especially in the context of the analytical determination of carbohydrate enantiomers. The best conditions for the technique have been evaluated, showing that under optimal working conditions it is possible to use α-picoline borane as a reducing agent instead of sodium cyanoborohydride without affecting the selectivity or the yield. The main reason for the variation of the technique was that, in the presence of acetic acid, secondary epimeric products were produced with α-picoline borane due to an Amadori rearrangement. This new modification assures lower toxicity, and thus a more environmental-friendly reaction system. Preparative scale synthesis can also be efficiently made with this reducing reagent.

A Synthetic Route to Fully Substituted Chiral Cyclopentylamine Derivatives: Precursors of Carbanucleosides

Removal of silyl protection from D-glucose derived substrate 6 afforded 7, which upon acetonide deprotection followed by reaction with N-benzylhydroxylamine furnished two isomeric isoxazolidinocyclopentane derivatives via spontaneous cyclization of an in situ generated nitrone. The methyl xanthate derivative of the tertiary hydroxyl group of one isomer was isolated and subjected to radical deoxygenation reaction to form epimeric products, while with the other isomer it underwent spontaneous 1,2-elimination to form a mixture of the two possible endocyclic olefins. Hydrogenolytic cleavage of the isoxazolidine rings of the purified products followed by insertion of 5-amino-4-chloropyrimidine moiety and purine ring construction smoothly afforded structurally unique carbanucleoside analogues. Various spectroscopic methods on the synthesized compounds and X-ray analysis on one important intermediate were used to assign the structures and stereochemistry of the products.

Capillary electrophoresis analysis of the degradation of the aspartyl tripeptide Phe-Asp-GlyOH at pH 2.0 and 7.4 under forced conditions

The degradation of the tripeptide l-Phe-α- l-Asp-GlyOH was studied at 80 °C and pH 2.0 and 7.4 by capillary electrophoresis. Separation of most known as well as unknown degradation products was achieved in a 50 mM sodium phosphate buffer, pH 3.0. The diastereomers l-Phe-α- l-Asp-GlyOH/ l-Phe-α- d-Asp-GlyOH could only be separated upon addition of 16 mg/ml carboxymethyl-β-cyclodextrin and 5% acetonitrile to the background electrolyte. Compound identification was performed by capillary electrophoresis-electrospray ionization-mass spectrometry. In addition to Asp isomerization and epimerization products as well as hydrolysis products four diketopiperazine derivatives were identified. Moreover, two degradation products were observed containing the amino acids Asp, Gly and Phe but the unequivocal assignment could not be accomplished based on the mass spectra. Following validation with regard to linearity, range, limit of detection, limit of quantitation and precision the assay was applied to the analysis of the incubation solutions. While peptide backbone hydrolysis dominated at pH 2.0, isomerization and enantiomerization yielding β-Asp and d-Asp peptides as well as cyclization to diketopiperazine derivatives were observed at pH 7.4. The diketopiperazines were the dominant reaction products amounting to about 85% of the compounds detected after the maximal incubation time of 240 h. A kinetic model was used to fit the concentration versus time data.

D‐Fructose‐6‐phosphate Aldolase in Organic Synthesis: Cascade Chemical‐Enzymatic Preparation of Sugar‐Related Polyhydroxylated Compounds

Novel aldol addition reactions of dihydroxyacetone (DHA) and hydroxyacetone (HA) to a variety of aldehydes catalyzed by D-fructose-6-phosphate aldolase (FSA) are presented. In a chemical-enzymatic cascade reaction approach, 1-deoxynojirimycin and 1-deoxymannojirimycin were synthesized starting from (R)- and (S)-3-(N-Cbz-amino)-2-hydroxypropanal, respectively. Furthermore, 1,4-dideoxy-1,4-imino-D-arabinitol and 1,4,5-trideoxy-1,4-imino-D-arabinitol were prepared from N-Cbz-glycinal. 1-Deoxy-D-xylulose was also synthesized by using HA as the donor and either 2-benzyloxyethanal or 2-hydroxyethanal as acceptors. In both cases the enzymatic aldol addition reaction was fully stereoselective, but with 2-hydroxyethanal 17 % of the epimeric product at C2, 1-deoxy-D-erythro-2-pentulose, was observed due to enolization/epimerization during the isolation steps. It was also observed that D-(-)-threose is a good acceptor substrate for FSA, opening new synthetic possibilities for the preparation of important novel complex carbohydrate-related compounds from aldoses. To illustrate this, 1-deoxy-D-ido-hept-2-ulose was obtained stereoselectively by the addition of HA to D-(-)-threose, catalyzed by FSA. It was found that the reaction performance depended strongly on the donor substrate, HA being the one that gave the best conversions to the aldol adduct. The examples presented in this work show the valuable synthetic potential of FSA for the construction of chiral complex polyhydroxylated sugar-type structures.

Intermediate Release by ADP-l-glycero-d-manno-heptose 6-Epimerase

ADP-l-glycero-d-manno-heptose 6-epimerase (HldD or AGME, formerly RfaD) catalyzes the interconversion of ADP-beta-d-glycero-d-manno-heptose (ADP-d,d-Hep) and ADP-beta-l-glycero-d-manno-heptose (ADP-l,d-Hep). The latter compound provides the heptose moiety that is used in lipopolysaccharide biosynthesis by Gram-negative bacteria. Several lines of evidence suggest that the enzyme uses a direct oxidation/reduction mechanism involving a tightly bound NADP+ cofactor. An initial oxidation at C-6'' gives a 6''-keto intermediate, and a subsequent reduction on the opposite face of the carbonyl group generates the epimeric product. The reorientation required for the nonstereoselective reduction could take place within a single active site, or it could involve the release of the intermediate and rebinding in an altered conformation. To distinguish between these possibilities, two isotopically labeled substrates (ADP-d,d-Hep) were prepared that contained 18O and 2H isotopes at C-7'' and C-6'', respectively. A crossover experiment was used to determine whether unlabeled or doubly labeled products were formed upon epimerization of a mixture of the two singly labeled compounds. After an initial epimeric equilibrium was reached, no crossover could be detected, indicating that intermediate release is not intrinsic to the overall mechanism. After extended incubation, however, scrambling of the labels could be detected, indicating that a low background rate of intermediate release does occur. To directly detect the release of the intermediate, the labeled compounds were independently epimerized in the presence of a ketone-trapping reagent, phenylhydrazine. The corresponding phenylhydrazones were identified by mass spectrometry, and the absence of any 2H isotope in the adduct obtained from the deuterated starting compound confirmed that the oxidation had occurred at C-6''.