Dieter Sicker

Role of natural benzoxazinones in the survival strategy of plants.

Benzoxazinoid acetal glucosides are a unique class of natural products abundant in Gramineae, including the major agricultural crops maize, wheat, and rye. These secondary metabolites are also found in several dicotyledonous species. Benzoxazinoids serve as important factors of host plant resistance against microbial diseases and insects and as allelochemicals and endogenous ligands. Interdisciplinary investigations by biologists, biochemists, and chemists are stimulated by the intention to make agricultural use of the benzoxazinones as natural pesticides. These natural products are not only constituents of a plant defense system but also part of an active allelochemical system used in the competition with other plants. This review covers biological and chemical aspects of benzoxazinone research over the last decade with special emphasis on recent advances in the elucidation of the biosynthetic pathway.

Elucidation of the Final Reactions of DIMBOA-Glucoside Biosynthesis in Maize: Characterization of Bx6 and Bx7

Benzoxazinoids were identified in the early 1960s as secondary metabolites of the grasses that function as natural pesticides and exhibit allelopathic properties. Benzoxazinoids are synthesized in seedlings and stored as glucosides (glcs); the main aglucone moieties are 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) and 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA). The genes of DIBOA-glc biosynthesis have previously been isolated and the enzymatic functions characterized. Here, the enzymes for conversion of DIBOA-glc to DIMBOA-glc are identified. DIBOA-glc is the substrate of the dioxygenase BENZOXAZINLESS6 (BX6) and the produced 2,4,7-trihydroxy-2H-1,4-benzoxazin-3-(4H)-one-glc is metabolized by the methyltransferase BX7 to yield DIMBOA-glc. Both enzymes exhibit moderate Km values (below 0.4 mm) and kcat values of 2.10 s−1 and 0.25 s−1, respectively. Although BX6 uses a glucosylated substrate, our localization studies indicate a cytoplasmic localization of the dioxygenase. Bx6 and Bx7 are highest expressed in seedling tissue, a feature shared with the other Bx genes. At present, Bx6 and Bx7 have no close relatives among the members of their respective gene families. Bx6 and Bx7 map to the cluster of Bx genes on the short arm of chromosome 4.

A 2-oxoglutarate-dependent dioxygenase is integrated in DIMBOA-biosynthesis.

Benzoxazinoids are secondary metabolites of grasses that function as natural pesticides. While many steps of DIMBOA biosynthesis have been elucidated, the mechanism of the introduction of OCH(3)-group at the C-7 position was unknown. Inhibitor experiments in Triticum aestivum and Zea mays suggest that a 2-oxoglutarate-dependent dioxygenase catalyses the hydroxylation reaction at C-7. Cloning and reverse genetics analysis have identified the Bx6 gene that encodes this enzyme. Bx6 is located in the Bx-gene cluster of maize.

White biotechnology for green chemistry: fermentative 2-oxocarboxylic acids as novel building blocks for subsequent chemical syntheses

Functionalized compounds, which are difficult to produce by classical chemical synthesis, are of special interest as biotechnologically available targets. They represent useful building blocks for subsequent organic syntheses, wherein they can undergo stereoselective or regioselective reactions. “White Biotechnology” (as defined by the European Chemical Industry [http://www.europabio.org/white_biotech.htm], as part of a sustainable “Green Chemistry,”) supports new applications of chemicals produced via biotechnology. Environmental aspects of this interdisciplinary combination include: Use of renewable feedstockOptimization of biotechnological processes by means of: New “high performance” microorganismsOn-line measurement of substrates and products in bioreactorsAlternative product isolation, resulting in higher yields, and lower energy demand In this overview we describe biotechnologically produced pyruvic, 2-oxopentaric and 2-oxohexaric acids as promising new building blocks for synthetic chemistry. In the first part, the microbial formation of 2-oxocarboxylic acids (2-OCAs) in general, and optimization of the fermentation steps required to form pyruvic acid, 2-oxoglutaric acid, and 2-oxo-d-gluconic acid are described, highlighting the fundamental advantages in comparison to chemical syntheses. In the second part, a set of chemical formula schemes demonstrate that 2-OCAs are applicable as building blocks in the chemical synthesis of, e.g., hydrophilic triazines, spiro-connected heterocycles, benzotriazines, and pyranoic amino acids. Finally, some perspectives are discussed.

Benzoxazinones in plants: Occurrence, synthetic access, and biological activity

Abstract Acetal glycosides of the 2-hydroxy-2H-1,4-benzoxazin-3(4H)-one skeleton are naturally occurring in Acanthaceae, Poaceae, Ranunculaceae, and Scrophulariaceae, i.e. in plants of different taxonomic positions. They act as plant own resistance factors towards pests, like microbial diseases, insects and fungi. Structurally, they are unique because only in their case a nitrogen atom is part of the aglyconic cyclohemiacetal unit. In case of a pest attack, the glycosides undergo a two step degradation, consisting in an enzymatic deglycosylation followed by a chemical ring contraction of the 1,4-benzoxazinone aglycone to form a benzoxazolinon-2(3H)-one derivative. The latter process is taking place, when plants release such aglycones into the environment by root exudation. Both series of degradation products are bioactive towards pests and can also act as allelochemicals The driving forcefor all investigations is the possibility to make agricultural use of the results in the growing of main cereals, like maize, rye, and wheat (Poaceae). A future goal consists in the gene transfer for the benzoxazinone biosynthesis into other plants of agricultural interest. A detailed overview on natural occurring benzoxazinone acetal glucosides, benzoxazinone aglycones, and benzoxazolinones is presented. Three subjects of the benzoxazinone research are especially emphasised: synthetic access to aglucones and glucosides, medical effects of structures derived from natural product leads and molecular allelopathy, i.e. detoxification strategies of plants coexisting with benzoxazinone forming species in comparison with those belonging to other plant associations. Rapidaccess to further fields of research, like biosynthesis, plant-pest interaction, and molecular mode of action is given by citation of leading references.

(2R)-2-β-D-glucopyranosyloxy-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one from Secale cereale

Abstract (2 R )-2-β- d -Glucopyranosyloxy-4-hydroxy-2H-1,4-benzoxazin-3(4H)-one was isolated from Secale cereale for the first time as a pure substance. The absolute configuration was determined as the 2 R -type by spectroscopic methods.

Syntheses with a Chiral Building Block from the Citric Acid Cycle: (2R,3S)‐Isocitric Acid by Fermentation of Sunflower Oil

The citric acid cycle constitutes a main metabolic process. Since its discovery in 1937 by H. A. Krebs, all of its intermediates have been prepared in multigramm amounts—with one exception: (2R,3S)-isocitric acid (1, dSthreo-isocitric acid). As a new member of the chiral pool it would be an interesting starting material for organic synthesis. This chiral a-hydroxy tricarboxylic acid is mainly accompanied by its constitutional isomer, citric acid (2). However, attempts to separate 1 from 2 have so far been successful only on an analytical scale. Though experiments have been carried out to achieve synthetic access to ent-isocitric acid (ent-1), again only milligram quantities were obtained. As a result of the scarce availability of 1 there is virtually no application known for it in synthesis. In databases only (2R,3S)-isocitric acid trimethyl ester (3), (2R,3S)-isocitric acid lactone-2,3-dicarboxylic acid dimethyl ester (5), (2R,3S)-isocitric acid lactone2,3-dicarboxylic acid (6), and (2R,3S)-isocitric acid lactone2,3-dicarboxylic acid anhydride (7) are listed superficially. Surprisingly, no attempts have been made to obtain 1 by fermentation, although a large number of yeasts are known to produce and excrete citric acid and (2R,3S)-isocitric acid in varying ratios when grown on long-chain n-alkanes or glucose. So far, these fermentations have been optimized for high levels of citric acid excretion. Herein we describe a combination of ecologically desirable biotechnological and chemical methods yielding enantiopure (2R,3S)-isocitric acid (1) and its derivatives in kilogram amounts, thus representing an unadulterated application of the “white biotechnology for green chemistry” concept. We discovered that the thiamine auxotrophic yeast Yarrowia lipolytica excretes organic acids in high percentage when it is grown on vegetable oils with an excess of thiamine under nitrogen-limited, aerobic conditions. Our aim was to achieve the highest possible ratio of isocitric to citric acid and concomitant high isocitric acid concentration. We succeeded in producing isocitric acid concentrations of 93 gL 1 and 1/2 ratios of 1.14:1 on the pilot-plant scale in the cultivation of wild-type Y. lipolytica EH59 on refined sun flower oil—a hitherto unrivalled achievement especially with regard to the use of renewable vegetable raw materials. After filtration of the biomass, electrodialysis was performed to convert the obtained trisodium salts into the free acids, before the removal of water was accomplished under reduced pressure. Then, it was time to search for an adequate process to separate the two isomers. Esterification of the highly viscous concentrated solution yielded the corresponding triesters of both tricarboxylic acids. From this mixture citric acid trimethyl ester 4 crystallized as a colorless solid, while (2R,3S)-isocitric acid trimethyl ester 3 did not, as it is a liquid under standard conditions. Utilizing this formerly unknown fact, separation of the isomeric esters 3 and 4 could be carried out simply by filtration of 4 from 3 (Scheme 1). In view of the intended application of 1 in

Glycoside carbamates from benzoxazolin-2(3H)-one detoxification in extracts and exudates of corn roots

Zea mays was incubated with the natural phytotoxin benzoxazolin-2(3H)-one (BOA) to investigate the detoxification process. A hitherto unknown detoxification product, 1-(2-hydroxyphenylamino)-1-deoxy-beta-gentiobioside 1,2-carbamate (3), was isolated and identified. A reinvestigation of known BOA detoxification products by NMR methods led to the finding that the structure of benzoxazolin-2(3H)-one-N-beta-glucoside (1) first reported from Avena sativa has to be revised. In fact, the correct structure is that of the isomeric 1-(2-hydroxyphenylamino)-1-deoxy-beta-glucoside 1,2-carbamate 2, which is structurally related to 3. It was now shown with a synthetic mixture of 1 and 2 that 1 underwent spontaneous isomerization to form 2 in solution. Thus, N-glucosylation of BOA in the plant led finally to the carbamate 2. In contrast to BOA-6-O-glucosylation, BOA-induced N-glucosylation appears first after 6-8 h of incubation. As soon as N-glucosylation is possible, BOA-6-O-glucoside is not further accumulated, whereas the amount of glucoside carbamate increases continuously during the next 40 h. Synthesis of gentiobioside carbamate seems to be a late event in BOA detoxification. All detoxification products are released into the environment via root exudation.

3-beta-D-glucopyranosyl-benzoxazolin-2(3H)-one - A detoxification product of benzoxazolin-2(3H)-one in oat roots

Abstract Another detoxification product of Avena sativa roots incubated with the natural phytotoxin benzoxazolin-2(3 H )-one was isolated from their methanolic extracts and identified as 3-β- d -glucopyranosyl-benzoxazolin-2(3 H )-one. The structure was proven by synthesis. The ability of oat to produce the N-glucoside is supposed to be one reason for a less pronounced sensitivity to the phytotoxin. Methanolic extracts of Avena sativa roots previously incubated with the natural phytotoxin benzoxazolin-2(3 H )-one contain, in addition to two already described detoxification products, a third one of hitherto unknown structure. The compound has now been identified by NMR and mass spectral methods as 3-β- d -glucopyranosyl-benzoxazolin-2(3 H )-one. This structure was confirmed by an independent synthesis. The ability of oat to produce the N-glucoside is supposed to be one reason for a less pronounced sensitivity to the phytotoxin.

Evaluation of Dimboa Analogs as Antifeedants and Antibiotics Towards the Aphid Sitobion avenae in Artificial Diets

A total of 25 compounds including benzoxazinones, benzoxazolinones, and N-glyoxylamide derivatives were tested as antifeedants and antibiotics towards the aphid Sitobion avenae in diet bioassays. The antifeedant and mortality indexes increased with the presence of electron-donating groups in the 7 position of the benzoxazinone moiety, the replacement of the oxygen atom by sulfur in the heterocyclic ring, the presence of a hemiacetal instead of an acetal at C-2 of the benzoxazine moiety (and hence the possibility of ring opening), and the presence of a hydroxyl group at C-4 of the benzoxazine moiety (hydroxamic acid) instead of a hydrogen atom (lactam). The results support earlier hypotheses on the chemical bases for the mode of action of these compounds.