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 feedstock Optimization of biotechnological processes by means of: New "high performance" microorganisms On-line measurement of substrates and products in bioreactors Alternative 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.

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.

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.

A formylating agent by dehydration of the natural product DIMBOA

The natural aglucone 2,4-dihydroxy-7-methoxy-2H-1, 4-benzoxazin-3(4H)-one (DIMBOA, 1) of maize underwent spontaneous dehydration and rearrangement to form 3-formyl-6-methoxybenzoxazolin-2(3H)-one (FMBOA, 2) on reaction with N-ethoxycarbonyl-trichloroacetaldimine. Compound 2 was proven to be a reactive formyl donor toward N-, O-, and S-nucleophiles, which may be important in case 2 is formed under biological conditions.

Detoxification of Benzoxazolinone Allelochemicals from Wheat by Gaeumannomyces graminis var. tritici, G. graminis var. graminis, G. graminis var. avenae, and Fusarium culmorum.

The ability of phytopathogenic fungi to overcome the chemical defense barriers of their host plants is of great importance for fungal pathogenicity. We studied the role of cyclic hydroxamic acids and their related benzoxazolinones in plant interactions with pathogenic fungi. We identified species-dependent differences in the abilities of Gaeumannomyces graminis var. tritici, Gaeumannomyces graminis var. graminis, Gaeumannomyces graminis var. avenae, and Fusarium culmorum to detoxify these allelochemicals of gramineous plants. The G. graminis var. graminis isolate degraded benzoxazolin-2(3H)-one (BOA) and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) more efficiently than did G. graminis var. tritici and G. graminis var. avenae. F. culmorum degraded BOA but not MBOA. N-(2-Hydroxyphenyl)-malonamic acid and N-(2-hydroxy-4-methoxyphenyl)-malonamic acid were the primary G. graminis var. graminis and G. graminis var. tritici metabolites of BOA and MBOA, respectively, as well as of the related cyclic hydroxamic acids. 2-Amino-3H-phenoxazin-3-one was identified as an additional G. graminis var. tritici metabolite of BOA. No metabolite accumulation was detected for G. graminis var. avenae and F. culmorum by high-pressure liquid chromatography. The mycelial growth of the pathogenic fungi was inhibited more by BOA and MBOA than by their related fungal metabolites. The tolerance of Gaeumannomyces spp. for benzoxazolinone compounds is correlated with their detoxification ability. The ability of Gaeumannomyces isolates to cause root rot symptoms in wheat (cultivars Rektor and Astron) parallels their potential to degrade wheat allelochemicals to nontoxic compounds.

Synthesis of 4-acetylbenzoxazolin-2(3H)-one reported from zea mays

A three-step alternative synthesis of 4-acetylbenzoxazolin-2(3H)-one (4) is reported. Starting from inexpensive 3-hydroxyacetophenone (1) 3-hydroxy-2-nitroacetophenone (2) is prepared by nitration followed by catalytic hydrogenation to yield 2-amino-3-hydroxyacetophenone (3) in which a C=O unit is inserted by means of bis(trichloromethyl)carbonate (triphosgene) in the presence of triethylamine to afford 4 in 35% overall yield.

Chemoimmunotherapy of metastatic murine renal cell carcinoma using flavone acetic acid and interleukin 2.

Metastatic renal cell carcinoma (RCC) remains largely incurable. We used a murine RCC (Renca) in BALB/c mice to investigate the treatment possibilities with chemoimmunotherapy using in vivo boosters of natural killer (NK) activity. Diffuse pulmonary metastases were induced by intravenous (i.v.) inoculation with 100,000 Renca cells. All untreated control animals died within one month from pulmonary metastases. Chemoimmunotherapy using the NK immunostimulator flavonic-8-acetic acid (FAA) at 200 mg./kg. i.v. was given on the third day post tumor inoculation, followed by four consecutive days of twice daily intraperitoneal (i.p.) administration of 10,000 units human recombinant interleukin-2 (rIL-2). This chemoimmunotherapy regimen consistently cured 70% of tumor-bearing animals. Mice cured by this chemoimmunotherapy regimen did not reject subsequent reinoculation with Renca, indicating absence of specific antitumor immunity as a result of the treatment. While FAA and rIL-2 have no demonstrable in vitro cytotoxicity for Renca, they are excellent boosters of in vivo NK activity. These data suggest a potential alternative treatment method for metastatic RCC, a tumor type for which no efficient cytostatic drugs are available.

Improved lymphocyte cytotoxicity against murine renal cell carcinoma.

In this paper we describe the generation of antibody dependent cellular cytotoxicity against a murine renal cell carcinoma. Using human recombinant interleukin-2 and in vitro adherence to plastic, we generated lymphokine activated killer and adherent lymphokine activated killer cells. Adherent lymphokine activated killer cells had significant (p less than 0.05) higher unrestricted cytotoxicity than LAK cells. Using a rabbit antibody against Renca developed in our laboratory, we induced significant (p less than 0.01) antibody dependent cellular cytotoxicity using fresh spleen, lymphokine activated killer and adherent lymphokine activated killer cells. The strongest antibody dependent cellular cytotoxicity killing was mediated by adherent lymphokine activated killer cells and was restricted only to the renal cell carcinoma target. Using FACS cell surface analysis and antibody and complement depletion of selected effector cell subsets, we also demonstrate that the antibody dependent cellular cytotoxicity effector cell population consists of asialoGM1+ Lyt 2.1- natural killer cells. This first description of antibody dependent cellular cytotoxicity against renal cell carcinoma by activated natural killer cells suggests a novel method for more efficient use of cytotoxic effector cells against this type of cancer.