Effects of four benzoxazinoids on gibberellin-induced alpha-amylase activity in barley seeds

Germination of barley seeds was inhibited by 2,4-dihydroxy-7-methoxy-2H-1,4-benzoxazin-3(4H)-one (DIMBOA) and 2,4-dihydroxy-2H-1,4-benzoxazin-3(4H)-one (DIBOA) at concentrations greater than 0.03mmol/L, and 6-methoxy-benzoxazolin-2(3H)-one (MBOA) and benzoxazolin-2(3H)-one (BOA) at concentrations greater than 0.1mmol/L. These benzoxazinoids also inhibited the induction of alpha-amylase activity in the barley seeds, and inhibited gibberellin-induced alpha-amylase activity in de-embryonated barley seeds. Significant inhibition in the germination and alpha-amylase induction were observed as concentrations of DIMBOA, DIBOA, MBOA and BOA increased. These results suggest that DIMBOA, DIBOA, MBOA and BOA may inhibit the germination of barley seeds by inhibiting the gibberellin-induced process, leading to alpha-amylase production. The inhibitory activities of germination and alpha-amylase induction of DIMBOA and DIBOA were greater than those of their degraded substances MBOA and BOA, respectively, and the inhibitory activities of DIMBOA and MBOA were greater than those of their demethoxylated analogues DIBOA and BOA, respectively.     

Effects of benzisoxazole and benzisothiazole on tomato plant regeneration in vitro

The effects of two synthetic auxins, BOA and BIA, on plant regeneration in vitro have been studied on explants of tomato cotyledons. The activity of these substances on cell elongation has also been tested on pea stem segments. It has been found that BOA is particularly effective in inducing the formation of shoots but has a weak activity on cell elongation, while BIA, which is more effective in inducing cell elongation, is less active in morphogenesis. It is concluded that (1) the two activities are not related to each other, (2) the receptors involved in the two processes are probably different, (3) thechemical structure of the auxin may be an important factor in organogenetic processes.Abbreviations BIA 1,2-benzisothiazole-3-acetic acid - BOA 1,2-benzisoxazole-3-acetic acid - IAA indoleacetic acid - MS Murashige & Skoog medium     

Allelochemical stress inhibits growth, leaf water relations, PSII photochemistry, non-photochemical fluorescence quenching, and heat energy dissipation in three C3 perennial species

In this study, the effect of two allelochemicals, benzoxazolin-2(3H)-one (BOA) and cinnamic acid (CA), on different physiological and morphological characteristics of 1-month-old C(3) plant species (Dactylis glomerata, Lolium perenne, and Rumex acetosa) was analysed. BOA inhibited the shoot length of D. glomerata, L. perenne, and R. acetosa by 49%, 19%, and 19% of the control. The root length of D. glomerata, L. perenne, and R. acetosa growing in the presence of 1.5 mM BOA and CA was decreased compared with the control. Both allelochemicals (BOA, CA) inhibited leaf osmotic potential (LOP) in L. perenne and D. glomerata. In L. perenne, F(v)/F(m) decreased after treatment with BOA (1.5 mM) while CA (1.5 mM) also significantly reduced F(v)/F(m) in L. perenne. Both allelochemicals decreased ΦPSII in D. glomerata and L. perenne within 24 h of treatment, while in R. acetosa, ΦPSII levels decreased by 72 h following treatment with BOA and CA. There was a decrease in qP and NPQ on the first, fourth, fifth, and sixth days after treatment with BOA in D. glomerata, while both allelochemicals reduced the qP level in R. acetosa. There was a gradual decrease in the fraction of light absorbed by PSII allocated to PSII photochemistry (P) in R. acetosa treated with BOA and CA. The P values in D. glomerata were reduced by both allelochemicals and the portion of absorbed photon energy that was thermally dissipated (D) in D. glomerata and L. perenne was decreased by BOA and CA. Photon energy absorbed by PSII antennae and trapped by 'closed' PSII reaction centres (E) was decreased after CA exposure in D. glomerata. BOA and CA (1.5 mM concentration) decreased the leaf protein contents in all three perennial species. This study provides new understanding of the physiological and biochemical mechanisms of action of BOA and CA in one perennial dicotyledon and two perennial grasses. The acquisition of such knowledge may ultimately provide a rational and scientific basis for the design of safe and effective herbicides.     

Experimental and theoretical studies of anthelmintics: oxfendazole and its imidazo[1,2-a]pyridine-2-carbamate isomer

Mice experimentally infected with Trichinella spiralis were used to test the therapeutic effectiveness of an anthelmintic, methyl 6-(phenylsulfinyl)imidazo[1,2-a]pyridine-2-carbamate, against the immature and adult worms during the intestinal phase of infection. A single oral dose of 100 mg kg-1 of the drug on the third day after exposure to infection was totally ineffective against the adult worms as determined at necropsy on day 6. Neither higher unit dosages of the drug, division of the daily oral dose, nor increasing the length of the treatment period from 1 to 4 days enhanced drug activity in vivo. Furthermore the drug was inactive as a single oral dose against the immature worms at all of the dosages tested (12.5-400 mg kg-1). These results are in marked contrast to those obtained previously with oxfendazole (methyl 5[6]-(phenylsulfinyl)benzimidazole-2-carbamate) under comparable experimental conditions and clearly indicate that the two compounds are not anthelmintically equivalent in the T. spiralis-infected mouse system in spite of their similar structural features. A quantum mechanical study of these drugs was undertaken and a hypothesis for the inactivity of the imidazo[1,2-a]pyridine-2-carbamate isomer is proposed.     

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.     

Fdb1 and Fdb2, Fusarium verticillioides loci necessary for detoxification of preformed antimicrobials from corn

Fusarium verticillioides is a fungus of significant economic importance because of its deleterious effects on plant and animal health and on the quality of their products. Corn (Zea mays) is the primary host for F. verticillioides, and we have investigated the impact of the plant's antimicrobial compounds (DIMBOA, DIBOA, MBOA, and BOA) on fungal virulence and systemic colonization. F. verticillioides is able to metabolize these antimicrobials, and genetic analyses indicated two loci, Fdb1 and Fdb2, were involved in detoxification. Mutation at either locus caused sensitivity and no detoxification. In vitro physiological complementation assays resulted in detoxification of BOA and suggested that an unknown intermediate compound was produced. Production of the intermediate compound involved Fdbl, and a lesion in fdb2 preventing complete metabolism of BOA resulted in transformation of the intermediate into an unidentified metabolite. Based on genetic and physiological data, a branched detoxification pathway is proposed. Use of genetically characterized detoxifying and nondetoxifying strains indicated that detoxification of the corn antimicrobials was not a major virulence factor, since detoxification was not necessary for development of severe seedling blight or for infection and endophytic colonization of seedlings. Production of the antimicrobials does not appear to be a highly effective resistance mechanism against F. verticillioides.     

Biotransformation of 2-benzoxazolinone and 2-hydroxy-1,4-benzoxazin-3-one by endophytic fungi isolated from Aphelandra tetragona

The biotransformation of the phytoanticipins 2-benzoxazolinone (BOA) and 2-hydroxy-1,4-benzoxazin-3-one (HBOA) by four endophytic fungi isolated from Aphelandra tetragona was studied. Using high-performance liquid chromatography-mass spectrometry, several new products of acylation, oxidation, reduction, hydrolysis, and nitration were identified. Fusarium sambucinum detoxified BOA and HBOA to N-(2-hydroxyphenyl)malonamic acid. Plectosporium tabacinum, Gliocladium cibotii, and Chaetosphaeria sp. transformed HBOA to 2-hydroxy-N-(2-hydroxyphenyl)acetamide, N-(2-hydroxyphenyl)acetamide, N-(2-hydroxy-5-nitrophenyl)acetamide, N-(2-hydroxy-3-nitrophenyl)acetamide, 2-amino-3H-phenoxazin-3-one, 2-acetylamino-3H-phenoxazin-3-one, and 2-(N-hydroxy)acetylamino-3H-phenoxazin-3-one. BOA was not degraded by these three fungal isolates. Using 2-hydroxy-N-(2-hydroxyphenyl)[(13)C(2)]acetamide, it was shown that the metabolic pathway for HBOA and BOA degradation leads to o-aminophenol as a key intermediate.     

2-3H-Benzoxazolinone (BOA) induces loss of salt tolerance in salt-adapted plants

In order to test the stress hypothesis of allelopathy of Reigosa et al. (1999, 2002) , the combined action of a well-established allelochemical compound (2-3 H -benzoxazolinone, BOA) and a common abiotic stress (salt stress) were investigated in lettuce ( Lactuca sativa L.). In a previous study ( Baerson et al. 2005 ), we demonstrated that the primary effects of BOA are related to the expression of genes involved in detoxification and stress responses, which might serve to simultaneously alleviate biotic and abiotic stresses. Through analysis of the same physiological and biochemical parameters previously studied for BOA alone ( Sánchez-Moreiras & Reigosa 2005 ), we observed specific effects of salt stress alone, as well as for the two stresses together (BOA and salt). This paper demonstrates that plants showing tolerance to salt stress (reduced stomatal density, increased proline content, higher K⁺ concentration, etc .) become salt sensitive (markedly low Ψw values, high putrescine content, increased lipid peroxidation, etc .) when simultaneously treated with the allelochemical BOA. We also report additional information on the mechanisms of action of BOA, and general stress responses in this plant species.     

1,2-Diacylglycerols but not phorbol esters stimulate sphingomyelin hydrolysis in GH3 pituitary cells

It has recently been proposed that degradation products of sphingolipids may serve as physiologic inhibitors of protein kinase C. The present study was performed to determine the effect of 1,2-diacylglycerols and phorbol esters, known activators of protein kinase C, on sphingomyelin metabolism. 1,2-Dioctanoylglycerol (diC8) caused time- and concentration-dependent reduction in the level of sphingomyelin labeled to equilibrium with [3H]choline. diC8 (200 micrograms/ml) reduced [3H]sphingomyelin to 81 +/- 3% of control (p less than 0.005) by 15 min, and the level was 58 +/- 5% of control after 1 h; an EC50 for this event was 56 micrograms/ml. To evaluate the mechanisms of stimulated hydrolysis, the sphingoid base backbone of sphingomyelin was labeled with [14C] serine, and the effects of diC8 were quantitated. diC8 (100 micrograms/ml) reduced the level of sphingomyelin to 66 +/- 7% of control by 1 h from 375 +/- 12 pmol/10(6) cells to 245 +/- 26 pmol/10(6) cells. There was a concomitant increase in ceramide from 89 +/- 4 pmol/10(6) cells to 252 +/- 27 pmol/10(6) cells consistent with activation of the enzyme, sphingomyelinase (EC 3.1.4.12). In support of this contention, 1,2-diacylglycerols appeared to enhance the activity of an acid, but not a neutral, sphingomyelinase in homogenates of GH3 cells. The 1,2-diacylglycerol, 1-oleyl-2-acetylglycerol, produced similar effects. In contrast, the phorbol esters, 12-O-tetradecanoylphorbol 13-acetate and phorbol 12,13-dibutyrate, failed to stimulate sphingomyelin hydrolysis. Further, these effects of the 1,2-diacylglycerols occurred in cells down-modulated for protein kinase C. These studies demonstrate that 1,2-diacylglycerols stimulate sphingomyelin hydrolysis by a mechanism independent of the protein kinase C which mediates phorbol ester action. This is the first report of stimulated sphingomyelin hydrolysis by a physiologic effector molecule.     

Structure-based design of 7-carbamate analogs of geldanamycin

The 7-carbamate groups of geldanamycin and its 17-(2-dimethylaminoethyl)amino-17-demethoxy derivative (17-DMAG) bind the N-terminal domain of Hsp90 by establishing a network of hydrogen bonds which involve four buried water molecules. In this study, a structure-based approach was used to investigate the effects of displacing some of these waters by modification of the 7-carbamate. A general loss of binding to human Hsp90 was observed, except for replacement of the carbamate with a hydroxamate group which gave an analog with weak activity. Modeling of Hsp90-ligand interactions suggested that the hydroxamate was not able to displace the buried water molecules, while bulkier substituents able to do so proved inactive.     

Reaction of urethane with nucleic acids in vivo

1. [1-(14)C]Ethyl carbamate, ethyl [carboxy-(14)C]carbamate, [1-(14)C]ethanol and sodium hydrogen [(14)C]carbonate were injected intraperitoneally into C57 mice, and nucleic acids and proteins were separated from the liver and lungs with phenol as described by Kirby (1956). 2. Chromatographic analysis of the hydrolytic products of the urethane-labelled RNA showed the presence of a single radioactive compound differing in behaviour from the major pyrimidine nucleotides and purines. 3. The products from RNA labelled by [1-(14)C]ethyl carbamate or ethyl [carboxy-(14)C]carbamate appeared chromatographically identical but could not be detected in the RNA of mice given [1-(14)C]ethanol or sodium hydrogen [(14)C]-carbonate. 4. The labelled product appeared to be the ethyl ester of cytosine-5-carboxylic acid formed by the reaction of urethane with RNA in vivo. 5. A direct reaction between labelled urethane or the labelled metabolite of urethane, [1-(3)H]-ethyl N-hydroxycarbamate, and RNA was not detected.     

Effect of 1,2-benzisoxazole-3-acetic acid on plant regeneration from protoplasts of Nicotiana tabacum

Summary Benzisoxazole-3-acetic acid, a new synthetic growth regulator, was administered to protoplast cultures from Nicotiana tabacum and subsequently to the developed microcalluses, to test its activity on plant regeneration from protoplasts in different culture conditions. Such activity, compared to that of naphthalene-acetic acid, proved to be rather low in the stage of cellular division and microcallus formation but particulary high in the stage of shoot induction from microcallus, thus confirming that the activity of this compound is mainly morphogenetic.Abbreviations BAP (6-benzyl-aminopurine) - BOA (1,2-benzisoxazole-3-aceticacid) - NAA (1-naphthalene-acetic acid)     

Uptake and translocation of phytochemical 2-benzoxazolinone (BOA) in radish seeds and seedlings

The molecular aspects of phytochemical interactions between plants, especially the process of phytochemical translocation by the target plant, remain challenging for those studying allelopathy. 2-Benzoxazolinone (BOA) is a natural chemical produced by rye (Secale cereale) and is known to have phytotoxic effects on weed seeds and seedlings. The translocation of BOA into target plants has been poorly investigated. Therefore, the total absorption of [ring U 14C] BOA was estimated by oxidizing whole seedlings of Raphanus sativus cv. for 8 days and quantifying the radioactivity. Non-radiolabelled BOA in seedlings was also estimated by HPLC. BOA applied at 10(-3) M was readily taken up by germinated radish at a rate of 1556 nmol g(-1) FW. At these same concentrations, BOA reduced radish germination by 50% and caused a delay in radicle elongation. Exogenous BOA was responsible for the observed germination inhibition. At a concentration of 10(-5) M, BOA was taken up by germinated seeds (31 nmol g(-1) FW), but this quantity did not affect radish germination. Labelled BOA was not mineralized in the culture medium during seedling growth as no 14CO2 was recovered. Both 10(-3) and 10(-5) M BOA were translocated into radish organs, mainly into roots and cotyledons. These organs were then identified as potential physiological target sites. Cotyledons remained the target sink (44% of the total radioactivity). The kinetics of BOA uptake at 10(-3) and 10(-5) M in radish seedlings was identical: BOA accumulation was proportional to its initial concentration. A comparison between radioactivity and HPLC quantification for 10(-3) M BOA indicated that BOA (along with some metabolites) could effectively be recovered in radish organs using chromatography.     

Biological oxidation of hydrogen sulfide under steady and transient state conditions in an immobilized cell biofilter

The removal of hydrogen sulfide (H(2)S) was investigated in a lab scale biofilter packed with biomedia, encapsulated by sodium alginate and polyvinyl alcohol (PVA). The main H(2)S oxidation products were SO(4)(2-), SO(3)(2-), S(2-) and S(0). The immobilized cell biofilter required no separate acclimatization period and showed high removal efficiencies (RE) within the first few days of experiments. The removal efficiencies in the biofilter were consistently greater than 99% even when H(2)S loading was 6 g m(-3)h(-1). The maximum elimination capacity achieved in this study is 8 g H(2)Sm(-3)h(-1) at a loading rate of 13 g H(2)Sm(-3) h(-1). The response of the immobilized cells to fluctuations in inlet concentration and flow rate was determined by subjecting the biofilter to inlet loads of up to 10 g H(2)Sm(-3)h(-1). The biofilter responded effectively to these shock loading conditions and convalesced rapidly within 4-8h. Pressure drop values were consistently less throughout the operational period. The results from this study suggest that an immobilized cell biofilter is effective in treating H(2)S under steady and transient operating conditions.     

Synthesis and structure determination of some oxadiazole-2-thione and triazole-3-thione galactosides

The syntheses of 5-pyridyl-3(beta-D-galactopyranosyl)-1,3,4-oxadiazole-2-thiones 3a-3c and 5-pyridyl-2(beta-D-galactopyranosyl)-4-benzyl-1,2, 4-triazole-3-thiones 6a-6c are reported. The existence of N-galactosides--not S-galactosides--was proven by IR and 15N NMR spectroscopy. The structures of the final products and the intermediates were elucidated by IR, 1H, 13C and 15N NMR spectroscopy and mass spectrometry.     

Metallothionein induction in early larval stages of tilapia (Oreochromis mossambicus)

Amounts of whole-body metallothionein (MT) in tilapia (Oreochromis mossambicus) larvae increased to a peak (1,500 ng mg(-1) protein) 1 d after hatching (H1), decreased rapidly thereafter, and was maintained at a constant level (700 ng mg(-1)) 3 d after hatching (H3). Waterborne Cd(2+) could stimulate MT expression in newly hatched (H0) larvae in dose-dependent and time-dependent patterns. H0 larvae, which were treated with 35 microg L(-1) Cd(2+) for 24 h, showed a 1.7-fold increase in the MT amount (174.0+/-64.7) and a 6. 5-fold increase in accumulated Cd(2+) but no significant change in Ca(2+) content, compared with the H0 control (MT, 102.6+/-48.1). H3 larvae with the same treatment revealed about a 10-fold increase in accumulated Cd(2+), a 10% decrease in Ca(2+) content, but no change in MT (261.2+/-120.0), compared with the H3 control (MT, 330+/-74.0). H0 larvae could synthesize more MT to bind Cd(2+) for detoxification in 35 microg L(-1) Cd(2+), a dose that would not affect normal physiology or survival of H0 larvae. On the other hand, 35 microg L(-1) Cd(2+) caused H3 larvae to experience hypocalcemia, an abnormal physiological condition, in which H3 larvae could not synthesize sufficient MT, thus causing greater than 25% mortality. These results indicate for the first time that the inducibility of MT by waterborne Cd(2+) is development dependent, being correlated with inconsistent sensitivities to Cd(2+) during larval development.     

Non-enzymatic glutathione conjugation of 2-nitroso-6-methyldipyrido [1,2-a: 3',2'-d] imidazole (NO-Glu-P-1) in vitro: N-hydroxy-sulfonamide, a new binding form of arylnitroso compounds and thiols

In order to study the possible detoxification mechanisms of the carcinogenic arylamine, 2-amino-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (Glu-P-1), the in vitro non-enzymatic reaction of 2-nitroso-6-methyldipyrido[1,2-a: 3',2'-d]imidazole (NO-Glu-P-1) with reduced glutathione (GSH) was examined at pH 7.4 under both aerobic and anaerobic conditions. Two GSH-arylamine adducts were isolated and found to contain the Glu-P-1 and GSH moieties in a 1:1 molar ratio via an N-S linkage. Their structures were assigned as sulfinamide (-NH-SO-) and N-hydroxy-sulfonamide (-N(OH)-SO2-) by their behaviour under acidic and basic conditions and by UV-VIS, 1H-NMR, infrared and mass spectrometries. Also, a N-hydroxy-sulfonamide adduct was produced when NO-Glu-P-1 and cysteine were reacted at pH 7.4. The N-hydroxy-sulfonamide structure is a new binding form between arylnitroso compounds and thiols. The formation of these adducts may also take place in vivo as a detoxification of toxic arylamines since GSH is abundant in organs such as liver or kidney.     

Characterization of novel glycolipids from the giant cockroach (Blaberus colosseus)

A novel class of glycolipids, assigned the trivial name blaberosides, was isolated from whole head tissues of the giant cockroach (Blaberus colosseus). The class consists of two closely related families, blaberoside I and blaberoside II, each containing species differing by 26 atomic mass units. The structure of these gentiobiose-based glycoglycerolipids was elucidated by chromatographic behavior, nuclear magnetic resonance spectroscopy, mass spectrometry, and analysis of chemical degradation products and derivatives. Species in the blaberoside I family have been identified as 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a-D- glucopyranosyl]-3-(hexadecyloxy)-1-(3-hydroxy-11-eicosenoyl)-1,2-p ropanediol (blaberoside Ia) and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D-glucopyranosyl)-bet a- D-glucopyranosyl]-3-(6-octadeceloxy)-1-(3-hydroxy-11-eicosenoyl )-1,2- propanediol (blaberoside Ib). Two smaller homologs of the blaberoside II family were discerned to be 2-O-[6'-O-(6"-O-3-hydroxy-11- eicosenoyl-beta-D-glucopyranosyl)-beta-D-glucopyranosyl]-3-(hex ade cyloxy)- 1,2-propanediol (blaberoside IIa), and 2-O-[6'-O-(6"-O-3-hydroxy-11-eicosenoyl-beta-D- glucopyranosyl)-beta-D-glucopyranosyl]-3-(4-octadeceloxy)-1,2-prop anediol (blaberoside IIb). These compounds are unique because they are animal origin glyceroglycolipids with a highly flexible gentiobiose backbone, and a beta-linkage of the carbohydrate to the glycerol ether at the 2 position rather than the usual 1 position.     

Enantiotopic differentiation in horse-liver alcohol-dehydrogenase-catalyzed oxidoreduction studied with novel substrates having organometallic moieties

Horse-liver alcohol-dehydrogenase-catalyzed oxidation of 1,2-bis(hydroxymethyl)ferrocene (1) gave (1R)-(+)-1-formyl-2-hydroxymethylferrocene (3) (86 +/- 2% enantiomeric excess, e.e.), while the reduction of the corresponding dialdehyde 1,2-diformylferrocene (2) gave the antipode (1S)-(-)-3 (94 +/- 2% e.e.). This fact indicates that the pro-R group in both 1 and 2 was preferentially converted by the enzyme. When one of two substituents on the substrate was replaced by a methyl group or moved to the beta-site, the stereoselectivity in the reaction decreased as evidenced by the enantiomeric purity of the products (5-64% e.e.). Treatment of racemic 1-hydroxyethylferrocene (14) with horse-liver alcohol dehydrogenase (HLADH) gave optically pure (R)-(-)-14 together with acetylferrocene. The reduction of 2 with HLADH, NAD and (2H6)ethanol gave (-)-(1S,2R)-1-formyl-2-[(R)-hydroxy(2H1)methyl]ferrocene and that of 1,2-di[(2H)formyl]ferrocene with HLADH, NAD and ethanol gave (-)-(1S,2R)-1-(2H)formyl-2-[(S)-hydroxy(2H1)methyl]ferrocene. These configurations indicate that the enzymic reduction occurred on the re-face of pro-R formyl group. The re-face selectivity was also found in the enzymic reduction of (eta 6-benzaldehyde)tricarbonylchromium and its (2H)formyl analogue. Docking of 2 into the active site of HLADH was examined using computer graphics. It has been suggested that the enantioselectivity to the pro-R side in the oxidoreduction of 1 and 2 by HLADH is a natural consequence of the re-face selectivity, which is caused by a steric interaction between the ligand and the side chain of Phe-93 or the Zn complex and strengthened by an interaction between the unreactive polar alpha-substituent and the protein, probably by hydrogen bond formation.