Mineralization and co-metabolic degradation of phenoxyalkanoic acid herbicides by a pure bacterial culture isolated from an aquifer

A mecoprop [(+/-)-2-(4-chloro-2-methylphenoxy)propionic acid; MCPP]-degrading bacterium identified as Stenotrophomonas maltophilia PM was isolated from a Danish aquifer. Besides mecoprop, the bacterium was also able to degrade MCPA [(4-chloro-2-methylphenoxy)acetic acid)], MCPB [(4-chloro-2-methylphenoxy)butyric acid], 4-CPA [(4-chlorophenoxy)acetic acid], 2, 4-D [(2, 4-dichlorophenoxy)acetic acid], 2, 4-DP [(+/-)-2-(2, 4-dichlorophenoxy)propionic acid] and 2, 4-DB [(2, 4-dichlorophenoxy)butyric acid]. The bacterium was able to grow using these individual phenoxyalkanoic acids as the sole source of carbon and energy. In addition, it was able to co-metabolically degrade the phenoxyalkanoic acid 2, 4, 5-T [(2, 4, 5-trichlorophenoxy)acetic acid)] in the presence of mecoprop. At high 2, 4, 5-T concentrations (100 and 52 mg/l), however, only partial degradation of both mecoprop and 2, 4, 5-T was obtained, thus indicating the production of toxic metabolites. Bacterial yields were highest when grown on the monochlorinated phenoxyalkanoic acids as compared to the dichlorinated analogues, an exception being growth on 4CPA, which resulted in the lowest yield at all. Using [ring-U-14C]-labeled herbicides it was shown that the lower yield on 2, 4-D than on mecoprop was accompanied by greater CO2 generation, thus indicating that less energy is available from the complete oxidation of the dichlorinated phenoxyalkanoic acids than the monochlorinated analogues.     

Effects of phenoxy acid herbicides and glyphosate on nitrogenase activity (acetylene reduction) in root-associated Azospirillum, Enterobacter and Klebsiella

Abstract Nitrogenase activity (C₂H₂ reduction) in root-associated Azospirillum lipoferum, Klebsiella pneumoniae, Enterobacter agglomerans and Pseudomonas sp. isolated from roots of Finnish grasses was assayed in the presence of glyphosate, the phenoxy acid herbicides 2-methyl-4-chlorophenoxy acetic acid (MCPA), 2,4-dichlorophenoxy acetic acid (2,4-D), (±)-2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) and (±)-2-(2,4-dichlorophenoxy)propionic acid (dichlorprop), and the commercial products Roundup, Nurmikko-Hedonal, Mepro, and Dipro. In the presence of the phenoxy acid herbicides the nitrogenase activity of K. pneumoniae was significantly inhibited, but that of E. agglomerans was stimulated. With the exception of Mepro and mecoprop no phenoxy acid herbicides inhibited the nitrogenase activity of A. lipoferum and none that of Pseudomonas sp. Nurmikko-Hedonal considerably stimulated the nitrogenase activity of E. agglomerans , and Pseudomanas sp. On the other hand, the nitrogenase activity of both K. pneumoniae and E. agglomerans was considerably repressed by glyphosate and Roundup, which also inhibited the growth of the bacteria. These chemicals had no effect on the growth of A. lipoferum and Pseudomonas sp., but stimulated their nitrogenase activity.     

Biodegradation of mixtures of chlorophenoxyalkanoic acid herbicides by Alcaligenes denitrificans

An Alcaligenes denitrificans strain able to degrade (R)-2-(2-methyl-4-chlorophenoxy)propionic acid [(R)-MCPP, mecoprop] was assessed for its ability to utilise a range of chlorophenoxyalkanoic acid herbicides in single, binary, tertiary and quaternary combinations in batch culture. Degradation rates were rapid with single growth substrates; complete degradation occurred within 29 h for 2,4-dichlorophenoxyacetic acid (2,4-D), 43 h for 4-chloro-2-methylphenoxyacetic acid (MCPA) and 50 h for (R)-MCPP, respectively. After 20 h, the degradation of (RS)-2-(2,4-dichlorophenoxy)propionic acid [(RS)-2,4-DP] had ceased, with only the (R)-enantiomer being degraded. In binary combination, 2,4-D and MCPP degraded within 55 h. Degradation rates decreased when herbicides were added in tertiary and quaternary combinations. Thus, at the whole cell level, catalysis of closely related herbicides is likely to be facilitated by diverse enzymatic activity in A. denitrificans. Journal of Industrial Microbiology & Biotechnology (2000) 25, 255–259. Received 16 April 2000/ Accepted in revised form 07 August 2000     

Isolation and characterization of a 2-(2,4-dichlorophenoxy) propionic acid-degrading soil bacterium

Summary The 2-(2,4-dichlorphenoxy)propionic acid (2,4-DP)-degrading bacterial strain MH was isolated after numerous subcultivations of a mixed culture obtained by soil-column enrichment and finally identified as Flavobacterium sp. Growth of this strain was supported by 2,4-DP (maximum specific growth rate 0.2 h^–1) as well as by 2,4-dichlorophenoxyacetic acid (2,4-D), 4(2,4-dichlorophenoxy)butyric acid (2,4-DB), and 2-(4-chloro-2-methyphenoxy)propionic acid (MCPP) as sole sources of carbon and energy under aerobic conditions. 2,4-DP-Grown cells (10⁸) of strain MH degraded 2,4-dichlorophenoxyalkanoic acids, 2,4-dichlorophenol (2,4-DCP), and 4-chlorophenol at rates in the range of 30 nmol/h. Preliminary investigations indicate that cleavage of 2,4-DP results in 2,4-DCP, which is further mineralized via ortho-hydroxylation and ortho-cleavage of the resulting 3,5-dichlorocatechol. Offprint requests to: F. Streichsbier     

Syntheses and Herbicidai Activities of Phenoxypyrimidines and Phenoxytriazines

Series of phenoxypyrimidines and phenoxytriazines were prepared to be evaluated as herbicides. Among them, 2-(2,6-dichlorophenoxy)-pyrimidine (XV), 2-phenoxy-4,6-dimethyl- pyrimidine (XVII), 2-(3-methyl-4-chlorophenoxy)-4,6-bis(ethylamino)-5-triazine (LIV), 2-(2,4-dichlorophenoxy)-4,6-bis(ethylamino)-s-triazine (LVIII), and 2-(2,6-dichlorophenoxy)-4,6-bis(ethylamino)-s-triazine (LX) showed high pre-emergent herbicidai activity to radish. On the other hand, 2-chloro-4-(2,6-dichlorophenoxy)-6-methylpyrimidine (XXX) revealed high efficiency to millet. Some structure-activity relationship is discussed.     

Auxin and sugar effects on callus induction and plant regeneration frequencies from mature embryos of wheat (Triticum aestivum L.)

Summary Genetic engineering of cereals currently depends on the use of tissue culture and plant regeneration systems. In wheat (Triticum aestivum L.), immature embryos are the most widely used explant to initiate cultures, but they are inconvenient due to their temporal availability and production requirements. Mature embryos are easily stored and are readily available as mature seeds. However, plant regeneration frequencies from cultures derived from mature embryos are generally low. This research was undertaken to improve callus induction and plant regeneration from wheat mature embryos of cultivar ‘Bobwhite’. The effects of four auxins [2,4-dichlorophenoxyacetic acid (2,4-D): 3,6-dichloro-o-anisic acid (dicamba); 4-amino-3,5,6-trichloropicolinic acid (picloram): and 2-(2-methyl-4-chlorophenoxy) propionic acid (2-MCPP)], and the effect of maltose vs. sucrose under filter sterilized and autoclaved conditions were evaluated. All auxin treatments resulted in callus induction except 2 MCPP. A highly significant effect of auxin type on both callus and plantlet production was detected, though interactions were observed. The effect of sugar type was dependent on the type of auxin used. Substitution of sucrose by maltose enhanced the regenration ability of callus from embryos cultured on media containing 2,4-D and picloram, but caused an opposite effect on media containing dicamba. Picloram significantly enhanced callus growth, however, embryogenic response and plant regenerability were low. Relative to 2.4-D, dicamba (18μM) resulted in a twofold increase in the number of plants regenerated per embryo and reduced the amount of time required for plant regeneration by 3–4 wk. Mention of a trademark or proprietary product does not constitute a guarantce or warranty by the University of Wisconsin and does not imply its approval to the exclusion of other products that may be suitable.     

Characterization of (R/S)-mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid]-degrading Alcaligenes sp.CS1 and Ralstonia sp. CS2 isolated from agricultural soils

The herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] is widely applied to corn fields in order to control broad-leaved weeds. However, it is often detected in groundwater where it can be a persistent contaminant. Two mecoprop-degrading bacterial strains were isolated from agricultural soils through their capability to degrade ( R/S )-mecoprop rapidly. 16S rDNA sequencing of the isolates demonstrated that one was closely related to the genera Alcaligenes sp. (designated CS1) and the other to Ralstonia sp. (designated CS2). Additionally, these isolates demonstrated ability to grow on other related herbicides, including 2,4- D (2,4-dichlorophenoxyacetic acid), MCPA [4-chloro-2-methyl phenoxy acetic acid] and ( R/S )-2,4-DP [2-(2,4-dichlorophenoxy)propionic acid] as sole carbon sources. tfdABC gene-specific probes derived from the 2,4- D -degrading Variovorax paradoxus TV1 were used in hybridization analyses to establish whether tfd -like genes are present in mecoprop-degrading bacteria. Hybridization analysis demonstrated that both Alcaligenes sp. CS1 and Ralstonia sp. CS2 harboured tfdA , tfdB and tfdC genes on plasmids that have approximately > 60% sequence similarity to the tfdA , tfdB and tfdC genes of V. paradoxus . It is therefore likely that tfd -like genes may be involved in the degradation of mecoprop, and we are currently investigating this further.     

Induction of somatic embryogenesis using side chain and ring modified forms of phenoxy Acid growth regulators

The induction of somatic embryo development in cell cultures of alfalfa (Medicago sativa), celery (Apium graveolens), and lettuce (Lactuca sativa) was compared for 2,4-dichlorophenoxy-acetic acid (2,4-D) and various phenoxy acid growth regulators. Tests using a series of straight chain extensions to the phenoxy acid side chain indicate that phenoxybutanoic acid is active, whereas the phenoxypropanoic and phenoxypentanoic analogs are inactive for the induction of alfalfa embryogenesis. Side branching on the carbon adjacent to the phenoxy group results in optically active compounds. Racemic mixtures and the (+) enantiomers of the compounds are active for alfalfa embryo induction, whereas the (−) enantiomers are inactive and apparently do not inhibit embryogenesis in any way. Development of alfalfa embryos, as measured by plantlet formation from individual embryos, is improved by 4-(2,4-dichlorophenoxy)butanoic acid and with side branching at the carbon adjacent to the phenoxy group compared with induction with 2,4-D. Similarly, substituted phenoxy acids also enhance somatic embryo development in celery and lettuce when compared with 2,4-D. These results are discussed with reference to earlier studies on the structure activity of various synthetic auxins during cell elongation and with reference to the possible importance of auxin metabolism on subsequent somatic embryo development.     

Herbicides interacting with lanthanide(III) and calcium(II) ions: structural and biological similarities of Gd and Ca polymers

In this paper we report the synthesis and characterization of Ca(II), Gd(III) and Ce(III) complexes with chlorophenoxyalkanoic acids, which are commonly used as herbicides. The Gd(III) and Ca(II) complexes were characterized by the typical formulas [Gd(III)(L)(3)(H(2)O)(2).2dmf](n) and [Ca(L)(2)(MeOH)(2)](n) [L=[2,4-D=2,4-dichlorophenoxyacetic acid, 2,4,5-T=2,4,5-trichlorophenoxyacetic acid, MCPA=2-methyl-4-chlorophenoxy acetic acid and 2,4-DP=2-(2,4-dichlorophenoxy)propanoic acid]]. The crystal structure of the Gd(III) complex with 2,4-D shows that the compound is a one-dimensional polymer with a [Gd(III)(2)(2,4-D)(6)(H(2)O)(4)] dimeric repeat unit and the gadolinium atoms are in a nine-coordination environment, while the crystal structure of the Ca analog shows that it also has a polymeric structure with a [Ca(2)(2,4-D)(4)(CH(3)OH)(4)] dimeric repeat unit and the calcium atoms are in an eight-coordination environment. The gadolinium compound displays three different coordination modes for the carboxylato moiety, bidentate chelate, bidentate double bound and bidentate triple bound, while the calcium compound displays only one, bidentate triple bound. Coordination spheres are completed with oxygens of H(2)O or MeOH molecules, respectively. The complexes were tested against a few common bacteria by minimum inhibitory concentration (MIC) experiments and did not exhibit any antimicrobial action at concentrations up to 1600 microg/ml.     

Simultaneous degradation of the herbicides 2,4-dichlorophenoxyacetic acid and 2-(2-methyl-4-chlorophenoxy)propionic acid by mixed bacterial cultures

The simultaneous degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 2-(2-methyl-4-chlorophenoxy)propionic acid (mecoprop) was achieved by two mixed cultures in the absence of any additional carbon or energy substrates. Mecoprop was not completely degraded by either of the two cultures, nor did addition of 2,4-D affect the degradation of mecoprop. The cultures completely degraded 2,4-D, and the degradation was uninfluenced by the addition of mecoprop. Nearly complete dechlorination of the mixture of two herbicides was achieved by both cultures, on the basis of the total amount of the two herbicides degraded. During the course of the reaction, however, the expected values of chloride were not met. Cell growth continued after the degradation of the parent substrates ceased. Although the mecoprop degradation did not continue to completion, spectral and growth data indicated that the metabolites which had accumulated during the reaction were degraded upon further incubation.     

Auxin binding to corn coleoptile membranes: Kinetics and specificity

Summary Detailed examination of binding over the range 10^-7–10^-6 M suggests that membrane preparations from coleoptiles of Zea mays L., cv Kelvedon 33 contain at least two sets of high affinity binding sites for 1-naphthylacetic acid (NAA), with dissociation constants of 1.8×10^-7 M (site 1) and 14.5×10^-7 M (site 2). Similar studies with 3-indolylacetic acid (IAA) also indicate two sets of binding sites, whose concentrations are closely comparable to those deduced for NAA. A substantial proportion of the total binding activity is retained in a detergent-solubilized preparation. Using [¹⁴C]NAA the interactions of a range of analogues with each of the binding sites have been examined with the aid of double reciprocal plots. The specificity of site 2 is compatible with that expected for an auxin receptor, in that only active auxins, antiauxin transport inhibitors are able to compete with [¹⁴C]NAA for the binding sites. Site 1 on the other hand is less specific, since it appears to bind all compounds tested, including physiologically inactive analogues.Abbreviations NAA 1-naphthylacetic acid - IAA 3-indolylacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - 2,6-D 2,6-dichlorophenoxyacetic acid - 2,4,5-T 2,4,5-trichlorophenoxyacetic acid - 2-CPIB -(2-chlorophenoxy)-isobutyric acid - 2,4-B 2,4-dichlorobenzoic acid - 2,6-B 2,6-dichlorobenzoic acid - TIBA 2,3,5-triiodobenzoic acid - NPA 1-N-naphthylphthalamic acid     

Characterization of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing <Emphasis Type="Italic">Methylobacterium oryzae</Emphasis> and interactions with auxins and ACC regulation of ethylene in canola (<Emphasis Type="Italic">Brassica campestris</Emphasis>)

The possible interaction of the plant hormones auxin and ethylene and the role of 1-aminocyclopropane-1-carboxylate (ACC) deaminase containing bacteria on ethylene production in canola (Brassica campestris) in the presence of inhibitory concentrations of growth regulators were investigated. The effects of auxin (indole-3-acetic acid and 2,4-dichlorophenoxy acetic acid), auxin transport inhibitor 2-(p-chlorophenoxy)-2-methylpropionic acid, ethylene precursor 1-aminocyclopropane-1-carboxylate and ethylene synthesis inhibitor l-α-(2-aminoethoxyvinyl)glycine hydrochloride on root elongation were concentration dependent. Exogenous addition of growth regulators influences the enzyme activities of ethylene production and we have presented here evidences that support the hypothesis that inhibitory effects of auxin on root elongation are independent of ethylene. Additionally, we have proved that inoculation of ACC deaminase containing Methylobacterium oryzae sequester ACC exuded from roots and hydrolyze them lowering the concentration of ACC in root exudates. However, the inhibitory actions of exogenous additions of auxins could not be ameliorated by bacterial inoculation that reduces ethylene concentration in canola seedlings.     

A rapid method to screen degradation ability in chlorophenoxyalkanoic acid herbicide-degrading bacteria

AIMS: An agar medium containing a range of related chlorophenoxyalkanoic acid herbicides, 2,4-dichlorophenoxyacetic acid (2,4-D), 2-methyl-4-chlorophenoxyacetic acid (MCPA), racemic mecoprop, (R)-mecoprop and racemic 2,4-DP (2-(2,4-dichlorophenoxy) propionic acid) was developed to assess the catabolic activity of a range of degradative strains. METHODS AND RESULTS: The medium was previously developed containing 2,4-D as a carbon source to visualise degradation by the production of dark violet bacterial colonies. Strains isolated on mecoprop were able to degrade 2,4-D, MCPA, racemic mecoprop, (R)-mecoprop and racemic 2,4-DP, whereas the 2,4-D-enriched strains were limited to 2,4-D and MCPA as carbon sources. Sphingomonas sp. TFD44 solely degraded the dichlorinated compounds, 2,4-D, racemic 2,4-DP and 2,4-DB (2,4-dichlorophenoxybutyric acid). However, Sphingomonas sp. AW5, originally isolated on 2,4,5-T, was the only strain to degrade the phenoxybutyric compound MCPB (4-chloro-2-methylphenoxybutyric acid). CONCLUSION: This medium has proved to be a very effective and rapid method for screening herbicide degradation by bacterial strains. SIGNIFICANCE AND IMPACT OF THE STUDY: This method reduces the problem of assessing the biodegradability of this family of compounds to an achievable level.     

Analogues of auxin modifying growth and development of some monocot and dicot plants

The dependence of morphogenetic processes in the formation of vegetative and generative organs in spring oilseed rape and barley on exogenously applied physiological analogues of auxin: 2,4-D (2,4-dichlorphenoxyacetic acid), NAA (naphthalene-1-acetic acid), TA-12 (1-[2-chloroethoxycarbonyl-methyl]-4-naphthalenesulfonic acid calcium salt) and TA-14 (1-[2-dimethylaminoethoxicarbonylmethyl]naphtalene chlormethylate) were investigated. The experiments were performed with hypocotyl tissue cultures of oilseed rape and barley microspores in vitro. The auxin analogues applied revealed differences of morphogenetic competence in dedifferentiation-redifferentiation processes that occurred in oilseed rape cultures. TA-12 and TA-14 applied together with NAA and BA (6-benzylaminopurine) caused more intensive callus growth in comparison with 2,4-D. Rhizogenesis was induced when 2,4-D was substituted by TA-12. Compound TA-14, unlike TA-12, facilitated the appearance and development of cotyledons in callus tissues. Hower the compounds TA-12 and TA-14 have no positive effect in monocot plant — barly anther culture for callogenesis and regeneration in comparison to indole-3-acetic acid (IAA). TA-14 and TA-12 showed similar but not identical auxin properties and demonstrated high efficiency as modifiers of rape-dicot plant growth and morphogenesis.     

Inhibitory Effects of Turf Pesticides on Bacillus popilliae and the Prevalence of Milky Disease

Fourteen pesticides (fungicides, herbicides, and insecticides) were tested to determine whether they had deleterious effects on the bioinsecticide Bacillus popilliae, the causal agent of milky disease. All of these pesticides reduced levels of spore viability, spore germination, and/or vegetative cell growth when they were tested over a range of concentrations from 0 to 1,000 ppm of active ingredient, and the fungicides had the greatest detrimental effects. As determined by tests in water, the level of spore viability was significantly reduced by chlorothalonil, iprodione, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and 2-[(4-chloro-o-tolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid. In tests performed with iprodione, loss of viability was evident at concentrations less than the concentration calculated to result from recommended use. Tests performed in soil demonstrated that triadimefon, chlorothalonil, (2,4-dichlorophenoxy)acetic acid plus 2-(2,4-dichlorophenoxy)propionic acid, and pendimethalin at concentrations resulting from recommended rates of application reduced spore titers. Spore germination did not occur in the continued presence of 2-[(4-chloro-otolyl)oxy]propionic acid plus (2,4-dichlorophenoxy)acetic acid, isofenphos, and chlordane, whereas exposure of spores to triadimefon or pendimethalin for 2 days stimulated germination. The tests to determine effects on spore germination were inconclusive for all other pesticides. Triadimefon, chlorothalonil, iprodione, pendimethalin, and chlorpyrifos at concentrations less than the concentrations recommended for use inhibited vegetative cell growth of B. popilliae, and chlordane at a concentration that was twice the concentration expected to result from the recommended rate of application repressed cell growth. My data support the hypothesis that use of synthetic pesticides can contribute to a low incidence of milky disease in white grubs.     

Parthenocarpic fruit set in triploid watermelon induced by CPPUand 2,4-D applications

The localized application of the synthetic cytokinin CPPU ((2-chloro-4-pyridyl)-N-phenyl urea) to ovaries at flower opening was as effective as free pollination in setting parthenocarpic fruit in the triploid watermelon cultivar ‘Reina de Corazones’, and increased yield per unit land area by at least 50%, simply due to the lack of requirement for diploid pollen producing plants within the orchard. The application of the synthetic auxin 2,4-D (2,4-dichlorophenoxy acetic acid) as a full coverage spray, was also effective in setting fruit; total yield was however 10% smaller than in the CPPU-treated plots, but the cost of application was much less expensive. These applications had no adverse effect on fruit quality, and their effectiveness in commercial watermelon production was evaluated over 4 years. Localized applications of 2,4-D to ovaries were less effective in setting fruit, and increased hollow fruit.     

Plant and soil enantioselective biodegradation of racemic phenoxyalkanoic herbicides

The biodegradation of the chiral phenoxyalkanoic herbicides 2-(2,4-dichlorophenoxy)propionic aid (2,4-DP) and 2-(4-chloro-2-methylphenoxy)propionic acid (MCPP) was investigated using enantioselective HPLC and chiroptical detection. Racemic mixtures of 2,4-DP and MCPP were applied to three species of turf grass, four species of broadleaf weeds, and soil. Preferential degradation of the S-(-) enantiomer of each herbicide was observed in most species of broadleaf weeds and soil, while the degradation in all species of grass occurred without enantioselectivity. The biodegradation in all systems appeared to follow pseudo first-order kinetics with the fastest degradation occurring in broadleaf weeds, followed by the grasses. The slowest degradation was observed in soil. The results of this work illustrate the need to characterize both enantiomers of chiral agrochemicals in order to have an accurate understanding of their distribution and fate in the environment.     

Detection and identification of substituted phenols as intermediates of concurrent bacterial degradation of the phenoxy herbicides MCPP and 2,4-D

The concurrent bacterial degradation of 2-(2-methyl-4-chlorophenoxy)propionic acid and 2,4-dichlorophenoxyacetic acid was studied using a stirred tank reactor and a bacterial culture which had been originally derived by enrichment with MCPP. High pressure liquid chromatographic methodology was used to measure both herbicides and it also resolved the corresponding phenols as intermediates, i.e., 2-methyl-4-chlorophenol and 2,4-dichlorophenol. Gas chromatography-mass spectrometry was used to verify the intermediates. UV scans of spent cultures showed that the wave-length of maximum absorption shifted from 282 nm to 280 nm toward the end of incubation, but the characteristic peaks of maximum absorption of these compounds could not be used resolved because of the overlap.     

Enantiomers of 2-methyl- and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid: Preparation by resolution,determination of absolute configuration,and Curtius rearrangement

Both enantiomers of 2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 2 and 2,4-dimethyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid 3 were prepared via resolution of the corresponding racemic carboxylic acids with (R)- and (S)-1-phenylethylamine, respectively. Absolute configuration of (−)-(R)-2-methyl-3-oxo-3,4-dihydro-2H-1,4-benzoxazine-2-carboxylic acid was determined by X-ray crystallography. Curtius rearrangement of acyl azides prepared from enantiomers of these heterocyclic carboxylic acids carried out in benzyl alcohol afforded enantiomers of the corresponding benzyl carbamates, which upon hydrogenolysis gave racemic 2-amino-2-methyl-3,4-dihydro-2H-1,4-benzoxazin-3-one 4 and 2-amino-2,4-dimethyl-3,4-dihydro-2H-1,4h-benzoxazin-3-one 5. Chirality 10:791–799, 1998. © 1998 Wiley-Liss, Inc.