Crosslinked Aggregates of Fusion Enzymes in Microaqueous Organic Media

Baeyer-Villiger monooxygenases (BVMOs) are attractive for selectively oxidizing various ketones using oxygen into valuable esters and lactones. However, the application of BVMOs is restrained by cofactor dependency and enzyme instability combined with water-related downsides such as low substrate loading, low oxygen capacity, and water-induced side reactions. Herein, we described a redox-neutral linear cascade with in-situ cofactor regeneration catalyzed by fused alcohol dehydrogenase and cyclohexanone monooxygenase in aqueous and microaqueous organic media. The cascade conditions have been optimized regarding substrate concentrations as well as the amounts of enzymes and cofactors with the Design of Experiments (DoE). The carrier-free immobilization technique, crosslinked enzyme aggregates (CLEAs), was applied to fusion enzymes. The resultant fusion CLEAs were proven to function in microaqueous organic systems, in which the enzyme ratios, water contents (0.5–5 vol. %), and stability have been systematically studied. The fusion CLEAs showed promising operational (up to 5 cycles) and storage stability.     

Discovery and Engineering of a Novel Baeyer-Villiger Monooxygenase with High Normal Regioselectivity

Baeyer-Villiger monooxygenases (BVMOs) are remarkable biocatalysts for the Baeyer-Villiger oxidation of ketones to generate esters or lactones. The regioselectivity of BVMOs is essential for determining the ratio of the two regioisomeric products (“normal” and “abnormal”) when catalyzing asymmetric ketone substrates. Starting from a known normal-preferring BVMO sequence from Pseudomonas putida KT2440 (PpBVMO), a novel BVMO from Gordonia sihwensis (GsBVMO) with higher normal regioselectivity (up to 97/3) was identified. Furthermore, protein engineering increased the specificity constant (k_cat/K_M) 8.9-fold to 484 s⁻¹ mM⁻¹ for 10-ketostearic acid derived from oleic acid. Consequently, by using the variant GsBVMO_C308L as an efficient biocatalyst, 10-ketostearic acid was efficiently transformed into 9-(nonanoyloxy)nonanoic acid, with a space-time yield of 60.5 g L⁻¹ d⁻¹. This study showed that the mutant with higher regioselectivity and catalytic efficiency could be applied to prepare medium-chain ω-hydroxy fatty acids through biotransformation of long-chain aliphatic keto acids derived from renewable plant oils.     

Discovery and Characterization of a Baeyer-Villiger Monooxygenase Using Sequence Similarity Network Analysis

Baeyer-Villiger monooxygenases (BVMOs) are important flavin-dependent enzymes which perform oxygen insertion reactions leading to valuable products. As reported in many studies, BVMOs are usually unstable during application, preventing a wider usage in biocatalysis. Here, we discovered a novel NADPH-dependent BVMO which originates from Halopolyspora algeriensis using sequence similarity networks (SSNs). The enzyme is stable at temperatures between 10 °C to 30 °C up to five days after the purification, and yields the normal ester product. In this study, the substrate scope was investigated for a broad range of aliphatic ketones and the enzyme was biochemically characterized to identify optimum reaction conditions. The best substrate (86 % conversion) was 2-dodecanone using purified enzyme. This novel BVMO could potentially be applied as part of an enzymatic cascade or in bioprocesses which utilize aliphatic alkanes as feedstock.     

Exploring the Substrate Specificity and Enantioselectivity of a Baeyer–Villiger Monooxygenase from Dietzia sp. D5: Oxidation of Sulfides and Aldehydes

Baeyer–Villiger monooxygenases (BVMOs) are valuable enzymes for specific oxyfunctionalization chemistry. They catalyze the oxidation of ketones to esters, but are also capable of oxidizing other chemical functions, namely aldehydes and heteroatoms such as sulfur, nitrogen, selenium and boron. The oxidation specificity and enantioselectivity of a newly characterized BVMO (BVMO4) from a strain of Dietzia towards sulfide- and aldehyde substrates have been studied. BVMO4 could react with sulfides containing an aromatic group. The presence of a substituent on the aromatic group was tolerated when they were in the meta- and para position and the oxidations yielded predominantly the (R)-sulfoxides. Similarly, BVMO4 displayed a higher activity for aldehydes containing a phenyl group, but long aliphatic aldehydes, namely octanal and decanal, were also accepted as substrate by this enzyme. The major oxidation products of the aldehyde substrates were the respective carboxylic acids in contrast to formate ester that was obtained in most of the previous reports. The Baeyer–Villiger oxidation of the substrate 2-phenylpropionaldehyde was studied in further detail and the corresponding acid product was obtained with good regio- and enantioselectivity. This is a unique feature for BVMO4 and is of great interest for further exploration of an alternative biocatalytic process.     

Flavoenzyme‐Catalyzed Oxygenations and Oxidations of Phenolic Compounds

Flavin‐dependent monooxygenases and oxidases play an important role in the mineralization of phenolic compounds. Because of their exquisite regioselectivity and stereoselectivity, these enzymes are of interest for the biocatalytic production of fine chemicals and food ingredients. In our group, we have characterized several flavoenzymes that act on phenolic compounds, including 4‐hydroxybenzoate 3‐hydroxylase, 3‐hydroxyphenylacetate 6‐hydroxylase, 4‐hydroxybenzoate 1‐hydroxylase (decarboxylating), hydroquinone hydroxylase, 2‐hydroxybiphenyl 3‐monooxygenase, phenol hydroxylase, 4‐hydroxyacetophenone monooxygenase and vanillyl‐alcohol oxidase. The catalytic properties of these enzymes are reviewed here, together with insights obtained from site‐directed and random mutagenesis.     

Improving the Enantioselectivity of CHMOBrevi1 for Asymmetric Synthesis of Podophyllotoxin Precursor

(R)-β-piperonyl-γ-butyrolactones are key building blocks for the synthesis of podophyllotoxin, which have demonstrated remarkable potential in cancer treatment. Baeyer-Villiger monooxygenases (BVMOs)-mediated asymmetric oxidation is a green approach to produce chiral lactones. While several BVMOs were able to oxidize the corresponding cyclobutanone, most BVMOs gave the (S) enantiomer while Cyclohexanone monooxygenase (CHMO) from Brevibacterium sp. HCU1 gave (R) enantiomer, but with a low enantioselectivity (75 % ee). In this study, we use a strategy called “focused rational iterative site-specific mutagenesis” (FRISM) at residues ranging from 6 Å from substrate. The mutations by using a restricted set of rationally chosen amino acids allow the formation of a small mutant library. By generating and screening less than 60 variants, we achieved a high ee of 96.8 %. Coupled with the cofactor regeneration system, 9.3 mM substrate was converted completely in a 100-mL scale reaction. Therefore, our work reveals a promising synthetic method for (R)-β-piperonyl-γ-butyrolactone with the highest enantioselectivity, and provides a new opportunity for the chem-enzymatic synthesis of podophyllotoxin.     

An Alcohol Dehydrogenase from the Short-Chain Dehydrogenase/Reductase Family of Enzymes for the Lactonization of Hexane-1,6-diol

Biocatalytic production of lactones, and in particular ϵ-caprolactone (CL), have gained increasing interest as a greener route to polymer building blocks, especially through the use of Baeyer–Villiger monooxygenases (BVMOs). Despite several advances in the field, BVMOs, however, still suffer several practical limitations. Alcohol dehydrogenase (ADH)-mediated lactonization of diols in turn has received far less attention and very few enzymes have been identified for the conversion of diols to lactones, with horse-liver ADH (HLADH) remaining the catalyst of choice. Screening of a diverse panel of ADHs, AaSDR-1, a member of the short-chain dehydrogenase/reductase family, was found to produce ϵ-caprolactone from hexane-1,6-diol. Moreover, cofactor regeneration by an NADH oxidase eliminated the requirement of co-substrates, yielding water as the sole by-product. Despite lower turnover frequencies as compared to HLADH, higher selectivity was found for the production of CL, with HLADH forming significant amounts of 6-hydroxyhexanoic acid and adipic acid through aldehyde dehydrogenation/oxidation of the gem-diol intermediates. Also, CL yield were shown to be dependent on buffer choice, as structural elucidation of a Tris adduct confirmed the buffer amine to react with aliphatic aldehydes forming a Schiff-base intermediate which through further ADH oxidation, forms a tricyclic acetal product.     

Correlating structure with function in bacterial multicomponent monooxygenases and related diiron proteins

Bacterial multicomponent monooxygenases (BMMs) catalyze the O2-dependent hydroxylation of hydrocarbons at a carboxylate-bridged diiron center similar to those that occur in a variety of dimetallic oxygen-utilizing enzymes. BMMs have found numerous biodegradation and biocatalytic applications. Recent investigations have begun to reveal how BMMs perform their C-H bond activation chemistry and why these enzymes may be mechanistically different from other related diiron proteins. The structures of the BMM component proteins and of complexes between them provide insights into the tuning of the dinuclear iron center and the enzyme mechanism. Selected findings are compared and contrasted with the properties of other carboxylate-bridged diiron proteins, revealing common structural and functional themes.     

Light-driven biocatalytic oxidation and reduction reactions: scope and limitations

The quest for practical regeneration concepts for nicotinamide-dependent oxidoreductases continues. Recently we proposed the use of visible light to promote the direct reductive regeneration of a flavin-dependent monooxygenase. With this enzyme (PAMO-P3) light-driven enantioselective Baeyer-Villiger oxidations were performed. In spite of the significant reduction in the complexity achieved, catalytic performance of the novel approach did not meet the requirements for an efficient biocatalytic oxygenation system. Driven by this ultimate goal, we further investigated the limiting factors of our particular system. We discovered that oxidative uncoupling of the flavin-regeneration reaction from enzymatic O2-activation accounts for the futile consumption of approximately 95% of the reducing equivalents provided by the sacrificial electron donor, EDTA. Furthermore, it was found that the apparent turnover frequency (TOF) for PAMO-P3 in the present setup is approximately two orders of magnitude lower than in conventional setups that use NADPH as reductant. This finding was traced to sluggish electron transfer kinetics that arose from an impeded interaction between PAMO-P3-bound FAD and the reducing catalyst. The limiting factors and potential approaches for their circumvention are discussed. Furthermore, we broadened the light-driven regeneration approach to the class of flavin-dependent reductases. By using the Old Yellow Enzyme homologue YqjM as a model system, a significantly higher catalytic turnover for the enzyme catalyst was achieved, which we assign to a higher accessibility of the prosthetic group as well as to the absence of oxidative uncoupling.     

一种有发展前景的生物氧化催化剂黄素蛋白单加氧酶

在过去的数十年中,大量的黄素依赖型单加氧酶被分离并研究。研究表明,黄素蛋白单加氧酶可以催化许多种类的氧化反应,如区域选择性羟基化反应和对映选择性磺化反应。这些氧化反应用常规的化学氧化剂难以或根本不能氧化。对已获得的基因组序列的分析表明,存在更多的黄素蛋白单加氧酶,并有待于在生物催化方面的作用进行进一步的研究。此外,经选择的一些黄素蛋白单加氧酶有突出的生物催化潜能。     

聚酰胺-胺树状分子锡配合物催化性能研究

通过固相合成的方法将聚酰胺-胺树状大分子PAMAM担载于大孔硅胶上,并对其外围分别用对羟基苯甲醛、2,4-二羟基苯甲醛和邻羟基苯甲醛进行修饰,再与SnCl2.2H2O反应形成三类共计9种不同代数树状高分子锡配合物。将该类配合物用作质量分数30%双氧水氧化酮的Baeyer-Villiger(B-V)反应的催化剂,考察其催化活性,结果表明,在该类配合物作用下,2-金刚烷酮、环戊酮、环己酮、4-甲基环己酮、4-叔丁基环己酮、3-甲基-2-戊酮和4-甲基-2-戊酮都可以发生B-V氧化反应而转化为相应的酯和内酯,底物的转化率(75%～99%)和产物选择性(95%～100%)都较高。比较了不同载体(氯球、纤维素、壳聚糖、硅胶)、不同配体的金属锡配合物对B-V催氧化反应的催化效果,研究发现,载体、配体和金属担载量对配合物的催化活性均有不同程度的影响。其中,硅胶为最好的载体,而邻羟基苯甲醛为最好的配体。     

Creating Oxidase–Peroxidase Fusion Enzymes as a Toolbox for Cascade Reactions

A set of bifunctional oxidase–peroxidases has been prepared by fusing four distinct oxidases to a peroxidase. Although such fusion enzymes have not been observed in nature, they could be expressed and purified in good yields. Characterization revealed that the artificial enzymes retained the capability to bind the two required cofactors and were catalytically active as oxidase and peroxidase. Peroxidase fusions of alditol oxidase and chitooligosaccharide oxidase could be used for the selective detection of xylitol and cellobiose with a detection limit in the low-micromolar range. The peroxidase fusions of eugenol oxidase and 5-hydroxymethylfurfural oxidase could be used for dioxygen-driven, one-pot, two-step cascade reactions to convert vanillyl alcohol into divanillin and eugenol into lignin oligomers. The designed oxidase–peroxidase fusions represent attractive biocatalysts that allow efficient biocatalytic cascade oxidations that only require molecular oxygen as an oxidant.     

Exploration and mining of the bacterial terpenome

Tens of thousands of terpenoids are present in both terrestrial and marine plants, as well as fungi. In the last 5-10 years, however, it has become evident that terpenes are also produced by numerous bacteria, especially soil-dwelling Gram-positive organisms such as Streptomyces and other Actinomycetes. Although some microbial terpenes, such as geosmin, the degraded sesquiterpene responsible for the smell of moist soil, the characteristic odor of the earth itself, have been known for over 100 years, few terpenoids have been identified by classical structure- or activity-guided screening of bacterial culture extracts. In fact, the majority of cyclic terpenes from bacterial species have only recently been uncovered by the newly developed techniques of "genome mining". In this new paradigm for biochemical discovery, bacterial genome sequences are first analyzed with powerful bioinformatic tools, such as the BLASTP program or Profile Hidden Markov models, to screen for and identify conserved protein sequences harboring a characteristic set of universally conserved functional domains typical of all terpene synthases. Of particular importance is the presence of variants of two universally conserved domains, the aspartate-rich DDXX(D/E) motif and the NSE/DTE triad, (N/D)DXX(S/T)XX(K/R)(D/E). Both domains have been implicated in the binding of the essential divalent cation, typically Mg(2+), that is required for cyclization of the universal acyclic terpene precursors, such as farnesyl and geranyl diphosphate. The low level of overall sequence similarity among terpene synthases, however, has so far precluded any simple correlation of protein sequence with the structure of the cyclized terpene product. The actual biochemical function of a cryptic bacterial (or indeed any) terpene synthase must therefore be determined by direct experiment. Two common approaches are (i) incubation of the expressed recombinant protein with acyclic allylic diphosphate substrates and identification of the resultant terpene hydrocarbon or alcohol and (ii) in vivo expression in engineered bacterial hosts that can support the production of terpene metabolites. One of the most attractive features of the coordinated application of genome mining and biochemical characterization is that the discovery of natural products is directly coupled to the simultaneous discovery and exploitation of the responsible biosynthetic genes and enzymes. Bacterial genome mining has proved highly rewarding scientifically, already uncovering more than a dozen newly identified cyclic terpenes (many of them unique to bacteria), as well as several novel cyclization mechanisms. Moreover, bioinformatic analysis has identified more than 120 presumptive genes for bacterial terpene synthases that are now ripe for exploration. In this Account, we review a particularly rich vein we have mined in the genomes of two model Actinomycetes, Streptomyces coelicolor and Streptomyces avermitilis, from which the entire set of terpenoid biosynthetic genes and pathways have now been elucidated. In addition, studies of terpenoid biosynthetic gene clusters have revealed a wealth of previously unknown oxidative enzymes, including cytochromes P450, non-heme iron-dependent dioxygenases, and flavin monooxygenases. We have shown that these enzymes catalyze a variety of unusual biochemical reactions, including two-step ketonization of methylene groups, desaturation-epoxidation of secondary methyl groups, and pathway-specific Baeyer-Villiger oxidations of cyclic ketones.     

Structural and Catalytic Differences between Two FADH(2)-Dependent Monooxygenases: 2,4,5-TCP 4-Monooxygenase (TftD) from Burkholderia cepacia AC1100 and 2,4,6-TCP 4-Monooxygenase (TcpA) from Cupriavidus necator JMP134

2,4,5-TCP 4-monooxygenase (TftD) and 2,4,6-TCP 4-monooxygenase (TcpA) have been discovered in the biodegradation of 2,4,5-trichlorophenol (2,4,5-TCP) and 2,4,6-trichlorophenol (2,4,6-TCP). TcpA and TftD belong to the reduced flavin adenine dinucleotide (FADH(2))-dependent monooxygenases and both use 2,4,6-TCP as a substrate; however, the two enzymes produce different end products. TftD catalyzes a typical monooxygenase reaction, while TcpA catalyzes a typical monooxygenase reaction followed by a hydrolytic dechlorination. We have previously reported the 3D structure of TftD and confirmed the catalytic residue, His289. Here we have determined the crystal structure of TcpA and investigated the apparent differences in specificity and catalysis between these two closely related monooxygenases through structural comparison. Our computational docking results suggest that Ala293 in TcpA (Ile292 in TftD) is possibly responsible for the differences in substrate specificity between the two monooxygenases. We have also identified that Arg101 in TcpA could provide inductive effects/charge stabilization during hydrolytic dechlorination. The collective information provides a fundamental understanding of the catalytic reaction mechanism and the parameters for substrate specificity. The information may provide guidance for designing bioremediation strategies for polychlorophenols, a major group of environmental pollutants.     

Enhancement of Candida parapsilosis catalyzing deracemization of (R,S)‐1‐phenyl‐1, 2‐ethanediol: agitation speed control during cell cultivation

BACKGROUND: Microbial cells have been used widely in biosynthesis of chiral alcohols. However, research concerning the effect of oxygen supply in cultivation on biocatalytic activity of whole cells in organic synthesis is limited. This study improved the reaction efficiency of Candida parapsilosis catalyzing (R,S)‐1‐phenyl‐1,2‐ethanediol (PED) deracemization by controlling agitation during cell cultivation. RESULTS: The increase of dissolved oxygen concentration by adjusting agitation speed from 200 to 300 rpm at aeration rate 1.5 vvm significantly improved the cell growth of C. parapsilosis and the activities of two key enzymes involved in deracemization. (S)‐PED with higher optical purity of 98.23%e.e. and yield of 82.94% was formed. Compared with the initial fermentation conditions at aeration rate 0.75 vvm and agitation speed 200 rpm, enhanced oxygen supply conditions afforded better cells for highly efficient conversion under higher substrate concentration. CONCLUSION: Oxygen supply had a significant effect on cell growth and catalytic activity of C. parapsilosis catalyzing asymmetric oxidoreduction. By conveniently controlling agitation in cultivation, cell activity and key enzymes production for a complex reaction of concurrent tandem oxidation and reduction processes can easily be conducted, which could help to cultivate cells catalyzing synthesis of interested chiral compounds. Copyright © 2008 Society of Chemical Industry     

Biocatalytic C-C Bond Formation for One Carbon Resource Utilization

The carbon-carbon bond formation has always been one of the most important reactions in C1 resource utilization. Compared to traditional organic synthesis methods, biocatalytic C-C bond formation offers a green and potent alternative for C1 transformation. In recent years, with the development of synthetic biology, more and more carboxylases and C-C ligases have been mined and designed for the C1 transformation in vitro and C1 assimilation in vivo. This article presents an overview of C-C bond formation in biocatalytic C1 resource utilization is first provided. Sets of newly mined and designed carboxylases and ligases capable of catalyzing C-C bond formation for the transformation of CO₂, formaldehyde, CO, and formate are then reviewed, and their catalytic mechanisms are discussed. Finally, the current advances and the future perspectives for the development of catalysts for C1 resource utilization are provided.     

Process limitations in a whole-cell catalysed oxidation: Sensitivity analysis

Biocatalytic oxidation processes have to date presented major problems for scale-up, in part due to the complexity of the number of process variables. In this paper we have analysed the key limitations in such processes using the Baeyer-Villiger monooxygenase catalysed synthesis of optically pure lactones as an illustrative example. Limitations in product concentration, catalyst longevity and reaction rate were quantified and their effect on previously defined process metrics identified. Of particular interest is the way these metrics change with catalyst concentration. Using this assessment, the sensitivity of the metrics to potential changes to process and catalyst were analysed. We believe such an analysis is of general use to guide development efforts for a given biocatalytic reaction.     

Switch in Cofactor Specificity of a Baeyer–Villiger Monooxygenase

Baeyer–Villiger monooxygenases (BVMOs) catalyze the oxidation of ketones to esters or lactones by using molecular oxygen and a cofactor. Type I BVMOs display a strong preference for NADPH. However, for industrial purposes NADH is the preferred cofactor, as it is ten times cheaper and more stable. Thus, we created a variant of the cyclohexanone monooxygenase from Acinetobacter sp. NCIMB 9871 (CHMO_Acineto); this used NADH 4200‐fold better than NADPH. By combining structure analysis, sequence alignment, and literature data, 21 residues in proximity of the cofactor were identified and targeted for mutagenesis. Two combinatorial variants bearing three or four mutations showed higher conversions of cyclohexanone with NADH (79 %) compared to NADPH (58 %) as well as specificity. The structural reasons for this switch in cofactor specificity of a type I BVMO are especially a hydrogen‐bond network coordinating the two hydroxy groups of NADH through direct interactions and bridging water molecules.     

Biocatalytic Metal-Organic Frameworks: Promising Materials for Biosensing

The highly efficient and specific catalysis of enzymes allows them to recognize a myriad of substrates, which enables biosensing. However, the fragility of natural enzymes severely restricts their practical applications. Metal-organic frameworks (MOFs) with porous networks and attractive functions have been intelligently employed as supports to encase enzymes and protect them against harsh environments. More importantly, customizable construction and composition affords the intrinsic enzyme-like activity of some MOFs (known as nanozymes), which provides an alternative route for the construction of robust enzyme mimics. This review will introduce the concept of these biocatalytic MOFs, with special emphasis on how biocatalytic processes that operate in these materials can reverse the plight of native enzyme-based biosensing. In addition, the present challenges and future outlooks in this research field are briefly discussed.     

Laser flash photolysis generation of high-valent transition metal-oxo species: insights from kinetic studies in real time

High-valenttransition metal-oxo species are active oxidizing species in many metal-catalyzed oxidation reactions in both Nature and the laboratory. In homogeneous catalytic oxidations, a transition metal catalyst is oxidized to a metal-oxo species by a sacrificial oxidant, and the activated transition metal-oxo intermediate oxidizes substrates. Mechanistic studies of these oxidizing species can provide insights for understanding commercially important catalytic oxidations and the oxidants in cytochrome P450 enzymes. In many cases, however, the transition metal oxidants are so reactive that they do not accumulate to detectable levels in mixing experiments, which have millisecond mixing times, and successful generation and direct spectroscopic characterization of these highly reactive transients remain a considerable challenge. Our strategy for understanding homogeneous catalysis intermediates employs photochemical generation of the transients with spectroscopic detection on time scales as short as nanoseconds and direct kinetic studies of their reactions with substrates by laser flash photolysis (LFP) methods. This Account describes studies of high-valent manganese- and iron-oxo intermediates. Irradiation of porphyrin-manganese(III) nitrates and chlorates or corrole-manganese(IV) chlorates resulted in homolytic cleavage of the O-X bonds in the ligands, whereas irradiation of porphyrin-manganese(III) perchlorates resulted in heterolytic cleavage of O-Cl bonds to give porphyrin-manganese(V)-oxo cations. Similar reactions of corrole- and porphyrin-iron(IV) complexes gave highly reactive transients that were tentatively identified as macrocyclic ligand-iron(V)-oxo species. Kinetic studies demonstrated high reactivity of the manganese(V)-oxo species, and even higher reactivities of the putative iron(V)-oxo transients. For example, second-order rate constants for oxidations of cis-cyclooctene at room temperature were 6 x 10(3) M(-1) s(-1) for a corrole-iron(V)-oxo species and 1.6 x 10(6) M(-1) s(-1) for the putative tetramesitylporphyrin-iron(V)-oxo perchlorate species. The latter rate constant is 25,000 times larger than that for oxidation of cis-cyclooctene by iron(IV)-oxo perchlorate tetramesitylporphyrin radical cation, which is the thermodynamically favored electronic isomer of the putative iron(V)-oxo species. The LFP-determined rate constants can be used to implicate the transient oxidants in catalytic reactions under turnover conditions where high-valent species are not observable. Similarly, the observed reactivities of the putative porphyrin-iron(V)-oxo species might explain the unusually high reactivity of oxidants produced in the cytochrome P450 enzymes, heme-thiolate enzymes that are capable of oxidizing unactivated carbon-hydrogen bonds in substrates so rapidly that iron-oxo intermediates have not been detected under physiological conditions.