Reactivity of ubiquinones and ubiquinols with free radicals

The reactivity of quinones 1-4 and of the corresponding quinols 5-8 towards carbon- and oxygen-centred radicals were studied. All quinones bearing at least one nuclear position free, readily react with alkyl and phenyl radicals to afford the alkylated quinones 12-24; however, quinones 1 and 3 reacted with 2-cyano-2-propyl radical to yield products (the mono- and di-ethers 9-11) derived from the attack on the carbonylic oxygen. The reactions carried out on quinones with the benzoyloxy radical led to no reaction products and in the case of Q₁₀, the isoprenic chain also remained unchanged. Quinols 5-8 reacted only with oxygencentred radicals (benzoyloxy and 2-cyano-2-propylperoxy radicals) to give the corresponding quinones. The isoprenic chain of Q₁₀ did not undergo attack even with peroxy radicals. Carbon-centred radicals resulted unable to abstract hydrogen from the studied quinols.     

Esr Spin Trapping Studies Into of Nature of the Oxidizing Species Formed in the Fenton Reaction: Pitfalls Associated With of Use Of 5,5-Dimethyl-1-Pyrroline-n-Oxide in the Detection of the Hydroxyl Radical

Several investigators have challenged the widely held view that the hydroxyl radical is the primary oxidant formed in the reaction between the ferrous ion and hydrogen peroxide. In recent studies, using the ESR spin trapping technique, Yamazaki and Piette found that the stoichiometry of oxidant formation in the reaction between Fe²⁺ and H₂O₂ often shows a marked deviation from the expected value of 1:1 (I. Yamazaki and L. H. Piette (1990) J. Am. Chem. Soc. 113, 7588-7593). In order to account for these observations, it was suggested that additional oxidizing species are formed, such as the ferryl ion (FeO²⁺), particularly when iron is present at high concentration and chelated to EDTA.

In this paper it is shown that secondary reactions, involving the redox cycling of iron and the oxidation of the hydroxyl radical adduct of the spin trap 5,5-dimethyl-1-pyrroline-N-oxide(DMPO) by iron, operate under the reaction conditions employed by Yamazaki and Piette. Consequently, the stoichiometry of oxidant formation can be rationalized without the need to envisage the formation of oxidizing species other than the hydroxyl radical. It is also demonstrated that the iron(III) complex of DETAPAC can react directly with DMPO to form the DMPO hydroxyl radical adduct (DMPO/OH) in the absence of hydrogen peroxide. Therefore, to avoid the formation of (DMPO/OH) as an artefact, it is suggested that DETAPAC should not be used as a reagent to inactivate containating adventitious iron in experiments using DMPO.     

Spin Trapping by Use of N-Tert-Butylhydroxylamine. Involvement of Fenton Reactions

Spin trapping of short-lived R. radicals is done by use of N-tert-butylhydroxylamine (1) and H²O². The hydroxylamine is oxidized to the radical t-BuN(O)H (2) which is converted into the spin trap 2-methyl-2-nitrosopropane (3). Simultaneously, hydroxyl radicals. OH are formed from H²O². The latter radical species abstracts hydrogen atoms from suitable molecules HR to give R. radicals, which are trapped with the formation of aminooxyl radicals, i. e., t-BuN(O)R (4) detectable by EPR spectroscopy. The reaction is enhanced by the presence of iron ions. The cleavage of H²O² into. OH radicals is considered to involve both a radical-driven (t-BuN(O)H 2) and an iron-driven Fenton reaction.     

Hydroxyl Radical-Induced Reactions in Polyadenylic Acid as Studied by Pulse Radiolysis: Part I. Transformation Reactions of Two Isomeric OH-Adducts

The absorption spectra of polyadenylic acid (polyA) radicals in N₂0 saturated aqueous solution have been measured as a function of time (up to 15 s) following an 0.4μS electron pulse. The spectra and their changes were analysed by comparison with those from monomeric adenine derivatives (nucleosides and nucleotides) which had been studied by Steenken.¹

The reaction of OH^· radicals with the adenine moiety in poly A results in the formation of two hvdroxvl adducts at the positions C-4 [polyA40H^·] and C-8 [polyA80H^·]. Each OH-adduct undergoes a unimol-ecular transformation reaction before any bimolecular or other unimolecular decay occurs. These reactions are characterized by different rate constants and _pH dependencies. The polyA40H^· adduct undergoes a dehydration reaction to yield a neutral N⁶ centered radical (rate constant K_deh= 1.4 × 10⁴s^-1 at ^pH7.3). This reaction is strongly inhibited by H⁺. In comparison with the analogous reactions in adenosine phosphates, the kinetic _pK value for its inhibition is two ^pH units higher. This shift is the result of the counter ion condensation or double-strand formation. The polyA80H^· adduct undergoes an imidazole ring opening reaction to yield an enol type of formamidopyrimidine radical with the resulting base damage (k^r.o. = 3.5 × 10⁴ s ^-1 at pH7.3). This reaction in contrast is strongly catalysed by H⁺and OH^-, similar as for adenosine but different compared to the nucleotides.     

人体动脉血气信号波浪式变化及连续动脉逐搏取血血气分析方法的建立*

目的: 建立动脉逐搏取血血气分析法,在人体实验中验证呼吸调控核心信号——PaO₂,PaCO₂和[H⁺]a是受呼吸影响的周期性、波浪式变化信号,而不是传统理念上误认的稳定水平信号。方法: 选择心功能正常、Allen试验阴性需要监测动脉血流动力学变化的患者6例。在左侧桡动脉穿刺,连接肝素化塑化管（3 mm×1 000 mm）,注满血液并计数血液注满所需心跳次数。用止血钳将塑化管钳闭成与心跳次数相对应的分段后,迅速置于冰水中,立即进行血气分析。选取每位患者的2个典型波浪式周期,用于分析2对最高-最低和最低-最高共4个测定值,取平均值。对相邻最高和最低值作统计学配对t检验。结果: 血液注满塑化管需要16±2次心跳,均覆盖超过2个呼吸周期。每个呼吸周期是5±0.6次心跳。PaO₂、PaCO₂、[H⁺]a和SaO₂都呈现出明显的波浪式变化（相邻高点与低点比较,P<0.05）,PaO₂、PaCO₂、[H⁺]a和SaO₂的波浪幅度分别是（11.28±1.13）mmHg,（1.77±0.89）mmHg,（1.14±0.35）nmol/L和（0.52±0.44）%;波浪幅度分别是其平均值的（7.7±1.1）%,（5.1±2.5）%,（3.1±1.0）%和（0.5±0.4）%。结论: 动脉延长管连续取血,按心跳次数分隔血样,血气分析法简单易行,为验证动脉血气受呼吸影响的周期性波浪式信号提供了可靠证据。本方法为原创,技术操作层面仍需提高熟练程度,增加志愿者和试验样本的数量进一步探索此类信号的临床检测可靠性及其与临床疾病的关系。     

Generation of reactive oxygen species in the enzymatic reduction of PbCrO4 and related DNA damage

Free radical reactions are believed to play an important role in the mechanism of Cr(VI)-induced carcinogenesis. Most studies concerning the role of free radical reactions have been limited to soluble Cr(VI). Various studies have shown that solubility is an important factor contributing to the carcinogenic potential of Cr(VI) compounds. Here, we report that reduction of insoluble PbCrO4 by glutathione reductase in the presence of NADPH as a cofactor generated hydroxyl radicals (.OH) and caused DNA damage. The .OH radicals were detected by electron spin resonance (ESR) using 5,5-dimethyl-N-oxide as a spin trap. Addition of catalase, a specific H2O2 scavenger, inhibited the .OH radical generation, indicating the involvement of H2O2 in the mechanism of Cr(VI)-induced .OH generation. Catalase reduced .OH radicals measured by electron spin resonance and reduced DNA strand breaks, indicating .OH radicals are involved in the damage measured. The H2O2 formation was measured by change in fluorescence of scopoletin in the presence of horseradish peroxidase. Molecular oxygen was used in the system as measured by oxygen consumption assay. Chelation of PbCrO4 impaired the generation of .OH radical. The results obtained from this study show that reduction of insoluble PbCrO4 by glutathione reductase/NADPH generates .OH radicals. The mechanism of .OH generation involves reduction of molecular oxygen to H2O2, which generates .OH radicals through a Fenton-like reaction. The .OH radicals generated by PbCrO4 caused DNA strand breakage.     

A novel method of measuring hydroxyl radical-scavenging activity of antioxidants using γ-irradiation

A new method using ESR spin trapping was proposed for measuring the scavenging activity of antioxidants for the hydroxyl (OH) radical. (-)-Epigallocatechin gallate (EGCg) and 5,5-dimethyl-1-pyrrolline N-oxide (DMPO) were used as the antioxidant and spin trapping agent, respectively. The conventional method using a Fenton reaction had problems associated with the estimation of activity, because the antioxidant disturbs the system for generating OH radical by coordinating on Fe²⁺ and by consuming H₂O₂, besides scavenging the spin adduct (DMPO-OH). Intense γ-irradiation was therefore used to generate OH radicals, and the intensity decrease in DMPO-OH after irradiation was followed to obtain the rate constant for the scavenging of DMPO-OH by EGCg. The intensities were extrapolated to zero time to estimate the quantity of DMPO-OH formed during γ-irradiation. By using these values, the reaction rate constant between OH radical and EGCg was calculated as a ratio to that of DMPO. It was shown that this method is useful for comparing the OH radical-scavenging activity of various antioxidants.     

Free Radicals in the Atmosphere

Like the oxidation in a flame, the oxidation in the atmosphere is mediated by free radicals. Unlike a flame, however, atmospheric oxidation needs an external source of energy: the sun light. In fact the most important radical acting in the lower atmosphere, the hydroxyl radical, OH, is produced following the UV-photolysis of ozone, O,which yields an excited oxygen atom, O'D:

OH reacts with most atmospheric trace gases, in many cases as the first and rate determining step in the reaction chain leading to oxidation. In this way a host of various other radicals (e.g. peroxy radicals), most of them very short lived, are generated. Usually these oxidation reactions form chains which regenerate OH, thus maintaining OH at a relatively high concentration level on the order of 10⁶cm∼³ during the day. The reactions which control the OH concentration will be discussed in detail. During the night radical formation is greatly diminished. It proceeds, for example, through the reaction of defines with O, and. in dry air, through reaction of defines and aldehydes with the nitrate radical, NO,.     

ADP-iron as a Fenton reactant: radical reactions detected by spin trapping, hydrogen abstraction, and aromatic hydroxylation

A mixture of ADP, ferrous ions, and hydrogen peroxide (H2O2) generates hydroxyl radicals (OH) that attack the spin trap DMPO (5,5-dimethyl-pyrollidine-N-oxide) to yield the hydroxyl free radical spin-adduct, degrade deoxyribose and benzoate with the release of thiobarbituric acid-reactive material, and hydroxylate benzoate to give fluorescent products. Inhibition studies, with scavengers of the OH radical, suggest that the behavior of iron-ADP in the reaction is complicated by the formation of ternary complexes with certain scavengers and detector molecules. In addition, iron-ADP reacting with H2O2 appears to release a substantial number of OH radicals free into solution. During the generation of OH radicals the ADP molecule was, as expected, damaged by the iron bound to it. Damage to the iron ligand in this way is not normally monitored in reaction systems that use specific detector molecules for OH radical damage. Under certain reaction conditions the ligand may be the major recipient of OH radical damage thereby leading to the incorrect assumption that the iron ligand is a poor Fenton reactant.     

Synthesis and Evaluation of DMPO-Type Spin Traps

The spin traps substituted with some groups at the 4-position of dimethyl-1-pyrroline N-oxide(DMPO) were compared with DMPO itself regarding their abilities as spin traps and their physical properties. 4,5,5-Trimethyl-l-pyroHine N-oxide (4MDMPO) and 5,5-dimethyl- 4-phenyl-l-pyrolline N-oxide (4PDMPO) were synthesized by the Bonnett method, and 5,5-dimethyl-4-hydroxymethyl-l-pyrolline N-oxide (4HMDMPO) was made by a unique method from 2(5H)-furanone. The melting points of 4MDMPO, 4PDMPO and 4HMDMPO were higher than that of DMPO. The magnitude of hydrophilicity was in the order of 4HMDMPO, DMPO, 4MDMPO, and 4PDMPO based on the partition coefficient experiments in a 1-octanol-water system. Several radicals, O₂, HO-, -CH₃, -CH₂OH, -CH(CH₃)OH, (CH₃)₃ CO and H radicals, were trapped with these DMPO derivatives for comparison with the trapping by DMPO itself. Spin adducts of O J with the three DMPO derivatives showed ESR spectra similar to that of DMPO. In spite of the formation of diastereomers arising from spin trapping, the line-width enlargement was very small. The intensities and the decay rates of the spectra of 4MDMPO-O₂^-, 4PDMPO-O₂^- 4HMDMPO-O₂^- and DMPO-O₂^- were almost equal. In the trapping of the OH radical by 4MDMPO, 4PDMPO and 4HMDMPO, the eight-line ESR spectra observed were different from the well-known four-line spectrum of DMPO-OH.     

An Efficient Chemoenzymatic Synthesis of S-(-)-Fenpropimorph

First enantioselective synthesis of S-(-)-1-[3-(4-tert-butylphenyl)-2-methyl]propyl-cis-3,5-dimethylmorpholine (6), biologically active enantiomer of the systematic fungicide fenpropimorph, is reported. It comprises reacting 4-tert-butylbenzylbromide with methyldiethylmalonate, decarbethoxylation of 2 into racemic 3-(4-tert-butylphenyl)-2-methylpropionic acid ethylester (3) in DMSO in the presence of alkali, then Pseudomonas sp. lipase catalyzed kinetic resolution of racemic 3 into S-(+)-acid (4), base-catalyzed racemization and recycling of the R-(-)-ester 3, acylation of cis-3,5-dimethylmorpholine, and final reduction of the intermediary amide 5 to provide enantiomerically pure S-(-)-6.     

Copper-Ligand Interactions and Physiological Free Radical Processes. pH-Dependent Influence of Cu2+ Ions on Fe2+-Driven OH- Generation

Prior to comparative studies on the reactivity of various copper complexes with respect to OH radicals, the influence of free Cu²⁺ ions on the superoxide-independent generation of OH radicals through Fenton assays and water gamma radiolysis has been tested in the present work.

Cu²⁺ ions have been shown to behave in a distinct manner towards each of these two production systems. As was logically expected from the noninvolvement of copper in OH^- radical production through gamma radioiysis, no influence of Cu²⁺ ions has been observed on the amount of radicals detected in that case. In contrast, Cu²⁺ ions do influence OH^- radical generation through iron-driven Fenton reactions, but differently depending on copper concentration.

When present in high concentrations, Cu²⁺ ions significantly contribute to OH^- radical production, which confirms previous observations on the reactivity of these in the presence of hydrogen peroxide. At lower levels corresponding to copper/iron ratios below unity on the contrary, Cu²⁺ ions behave as inhibitors of the OH^- production in a pH-dependent manner over the 1-6 range investigated: the lower the pH, the greater the inhibition.

The possible origin of this previously unreported inhibitory effect is discussed.     

A Hydrogen-Donating Monohydroxamate Scavenges Ferryl Myoglobin Radicals

The addition of 25μM hydrogen peroxide to 20μM metmyoglobin produces ferryl (Fe^IV = O) myoglobin. Optical spectroscopy shows that the ferryl species reaches a maximum concentration (60-70% of total haem) after 10 minutes and decays slowly (hours). Low temperature EPR spectroscopy of the high spin metmyoglobin (g = 6) signal is consistent with these findings. At this low peroxide concentration there is no evidence for iron release from the haem. At least two free radicals are detectable by EPR immediately after H₂O₂ addition, but decay completely after ten minutes. However, a longer-lived radical is observed at lower concentrations that is still present after 90 minutes. The monohydroxamate N-methylbutyro-hydroxamic acid (NMBH) increases the rate of decay of the fenyl species. In the presence of NMBH, none of the protein-bound free radicals are detectable; instead nitroxide radicals produced by oxidation of the hydroxamate group are observed. Similar results are observed with the trihydroxamate, desferoxamine. “Ferryl myoglobin” is still able to initiate lipid peroxidation even after the short-lived protein free radicals are no longer detectable (E.S.R. Newman, C.A. Rice-Evans and M.J. Davies (1991) Biochemical and Biophysical Research Communications 179, 1414-1419). It is suggested that the longer-lived protein radicals described here may be partly responsible for this effect. The mechanism of inhibition of initiation of lipid peroxidation by hydroxamate drugs, such as NMBH, may therefore be due to reduction of the protein-derived radicals, rather than reduction of ferryl haem.     

Copper-Ligand Interactions and Physiological free Radical Processes. Part 2. Influence of Cu2+ Ions on Cu+-Driven Oh Generation and Comparison with Their Effects on Fe2+-Driven Oh Production

In our search to establish a reference ·OH production system with respect to which the reactivity of copper(II) complexes could then be tested, the influence of free Cu²⁺ ions on the Cu⁺/H₂O₂ reaction has been investigated.

This influence depends on the C_Cu²⁺/C_Cu⁺ ratio. At low Cu²⁺ concentrations, ·OH damage to various detector molecules decreases with increasing Cu²⁺ concentrations until C_Cu²⁺/C_Cu⁺ reaches unity. Above this value, ·OH damage increases sharply until C_Cu²⁺/C_Cu⁺ becomes equal to 5 with salicylate and 2 with deoxyribose, ratios for which the protective effect of Cu²⁺ cancels. Finally, at higher concentrations, Cu²⁺ ions logically add their own ·OH production to that normally expected from Cu⁺ ions. The possible origin of this unprecedented alternate effect has been discussed. The possible influence of Cu⁺ ions on the generation of ·OH radicals by water gamma radiolysis has also been tested and, as already established for Cu²⁺ in a previous work, shown to be nonexistent. This definitely confirms that either form of ionised copper cannot scavenge ·OH radicals in the absence of a Iigand.     

Stereochemistry of the Enzymatic Reduction of 2-(4-Methoxybenzyl)-1-Cyclohexanone by Solanum Aviculare Cells in vitro

During the investigation of chemical properties of the dicyclic system of insect juvenile hormone analogues, biotransformation of 2-(4-methoxybenzyl)-1-cyclohexanone (1) by plant cell cultures was studied. Among other components, the cis-(1S, 2S)- and cis-(1R, 2R)-2-(4-methoxybenzyl)-1-cyclohexanol enantiomers 2a and 2b were found in the reaction mixture. Higher stereoselectivity of the biotransformation was observed for trans-(1S, 2R)-enantiomer 3a formation, which occurred in at least 60% of calculated enantiomeric excess.     

Protein damage and degradation by oxygen radicals. III. Modification of secondary and tertiary structure

Proteins which have been exposed to the hydroxyl radical (.OH) or to the combination of .OH plus the superoxide anion radical and oxygen (.OH + O2- + O2) exhibit altered primary structure and increased proteolytic susceptibility. The present work reveals that alterations to primary structure result in gross distortions of secondary and tertiary structure. Denaturation/increased hydrophobicity of bovine serum albumin (BSA) by .OH, or by .OH + O2- + O2 was maximal at a radical/BSA molar ratio of 24 (all .OH or 50% .OH + 50% O2-). BSA exposed to .OH also underwent progressive covalent cross-linking to form dimers, trimers, and tetramers, partially due to the formation of intermolecular bityrosine. In contrast, .OH + O2- + O2 caused spontaneous BSA fragmentation. Fragmentation of BSA produced new carbonyl groups with no apparent increase in free amino groups. Fragmentation may involve reaction of (.OH-induced) alpha-carbon radicals with O2 to form peroxyl radicals which decompose to fragment the polypeptide chain at the alpha-carbon, rather than at peptide bonds. BSA fragments induced by .OH + O2- + O2 exhibited molecular weights of 7,000-60,000 following electrophoresis under denaturing conditions, but could be visualized as hydrophobic aggregates in nondenaturing gels (confirmed with [3H]BSA following treatment with urea or acid). Combinations of various chemical radical scavengers (mannitol, urate, t-butyl alcohol, isopropyl alcohol) and gases (N2O, O2, N2) revealed that .OH is the primary species responsible for alteration of BSA secondary and tertiary structure. Oxygen, and O2- serve only to modify the outcome of .OH reaction. Furthermore, direct studies of O2- + O2 (in the absence of .OH) revealed no measurable changes in BSA structure. The process of denaturation/increased hydrophobicity was found to precede either covalent cross-linking (by .OH) or fragmentation (by .OH + O2- + O2). Denaturation was half-maximal at a radical/BSA molar ratio of 9.6, whereas half-maximal aggregation or fragmentation occurred at a ratio of 19.4. Denaturation/hydrophobicity may hold important clues for the mechanism(s) by which oxygen radicals can increase proteolytic susceptibility.     

Temperature-dependence of enantioselectivity and desymmetrization in the acetylation of 2-mono- and 2,2-di-substituted 1,3-propanediols by a novel lipase isolated from the yeast Cryptococcus spp. S-2

The effect of temperature on enantioselectivity and desymmetrization in the acetylation of 2-phenyl-1,3-propanediol (1a), 2-benzyl-1,3-propanediol (1b), 2-methyl-2-phenyl-1,3-propanediol (1c) and 2-benzyl-2-methyl-1,3-propanediol (1d) by a novel lipase (CSL) isolated from the yeast Cryptococcus spp. S-2 was studied. Desymmetrization of 1a, 1c and 1d by CSL-catalyzed acetylation was observed in the temperature range of -20°C to 40°C, while diacetylation of 1b occurred considerably even at 0°C. The kinetic parameters of the selectivity indicated that the acetylation of 1a is an entropy controlled process whereas the reaction of 1c and 1d is mainly controlled by the enthalpy term. In the monoacetylation of the diol 1d, the preferred configuration in the enantiomeric induction by CSL was opposite to that of immobilized porcine pancreatic lipase (PPL).     

Formation of superoxide anion during ferrous ion-induced decomposition of linoleic acid hydroperoxide under aerobic conditions

We studied the mechanism of formation of oxygen radicals during ferrous ion-induced decomposition of linoleic acid hydroperoxide using the spin trapping and chemiluminescence methods. The formation of the superoxide anion (O2*-) was verified in the present study. The hydroxyl radical is also generated through Fenton type decomposition of hydrogen peroxide produced on disproportionation of O2*-. A carbon-centered radical was detected using 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline N-oxide (DEPMPO) as a spin trap. Alkoxyl radical formation is essential for the conversion of linoleic acid hydroperoxide into the peroxyl radical by ferrous ion. It is likely that the alkoxyl radical [R1CH(O*)R2] is converted into the hydroxylcarbon radical [R1C*(OH)R2] in water, and that this carbon radical reacts with oxygen to give the alpha-hydroxyperoxyl radical [R1R2C(OH)OO*], which decomposes into the carbocation [R1C+(OH)R2] and O2*-.     

Vanadyl-induced Fenton-like reaction in RNA. An ESR and spin trapping study.

Vanadyl (VO2+) complexed to RNA reacts with hydrogen peroxide in a Fenton-like manner producing hydroxyl radicals (.OH). The hydroxyl radicals can be spin trapped with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) forming the DMPO-OH spin adduct. In addition, in the presence of ethanol the formation of the hydroxyethyl radical adduct of DMPO (DMPO-ETOH) confirms the production of hydroxyl radicals by the RNA/VO2+ complex. When the reaction between the RNA/VO2+ complex and H2O2 is carried out in the presence of the spin trap 2-methyl-2-nitrosopropane (MNP), radicals produced in the reaction of .OH with RNA are trapped. Base hydrolysis of the MNP-RNA adducts (pH 12) followed by a reduction in the pH to pH 7 after hydrolysis is complete, yields an MNP adduct with a well-resolved ESR spectrum identical to the ESR spectrum obtained from analogous experiments with poly U. The ESR spectrum consists of a triplet of sextets (aN = 1.48 mT, a beta N = 0.25 mT and a beta H = 0.14 mT), indicating that the unpaired nitroxide electron interacts with the nuclei of a beta-nitrogen and beta-hydrogen. The results suggest that the .OH generated in the RNA/VO2+ reaction with H2O2 add to the C(5) carbon of uracil forming a C(6) carbon centered radical. This radical is subsequently spin trapped by MNP.     

Biotransformation of podophyllotoxin by Hordeum vulgare cell suspension cultures

Hordeum vulgare cell suspension cultures were used to modify podophyllotoxin (1) One major product (1a) and one minor product (1b) were detected in both the culture medium and cells. To optimize the yield of compound 1a, we showed that: (1) the optimal concentration of added podophyllotoxin (1) was 33 mg L^-1; higher concentrations caused cell toxicity; (2) the stage of the cell cycle (lag/log/stationary) at which podophyllotoxin was added only marginally affected the yield of compound 1a; the optimal addition time was after lag phase, in which the yield of compound 1a reached ca. 76%, and (3) biotransformation of podophyllotoxin (1) was relatively slow; podophyllotoxin fed at 4 days after subculture resulted in yields of compound 1a of ca. 56, 64 and 76% after an additional 3, 6 and 10 days of incubation, respectively. Product 1a was purified and identified as isopicropodophyllone (1a) based on MS and NMR data.