Studies on Chrysanthemic Acid

Ribose-5-phosphate ketol-isomerase, an enzyme isomerizing ribose-5-phosphate to ribulose-5-phosphate, is isolated from Candida utilis which is grown in a medium containing xylose. The enzyme is also purified by means of fractionation with ammonium sulfate, acetone, and by DEAE-cellulose column chromatography.

The enzyme has its optimum pH at 7.5 and optimum temperature at 50°C.

Michaelis-Menten constant for d-ribose-5-phosphate is 7.38 × 10^?4 M and activation energy of the enzyme reaction is 10,525 calories.

The enzyme activity is inhibited by p-CMB, EDTA and sodium pyrophosphate, and activated by the addition of magnesium ion.

Extract of Candida utilis contains polyol: NAD oxidoreductase which catalyzes the conversion of polyols to the corresponding ketoses.

By fractionation with ammonium sulfate and on DEAE-cellulose column chromatography, the purity of enzyme has been increased about 14-fold.

The relatively high activity with both xylitol and sorbitol suggests that they may be the natural substances for the enzyme.

Evidence suggests that this enzyme relates to the metabolism of d-xylose in Candida utilis.     

Studies on Chrysanthemic Acid

Pyrethrin II, cinerin II, allethrin II, pyrethrin II isomer, and allethrin II isomer were prepared by esterification of rethrolons with (+)-trans-pyrethric acid and (+)-trans-methyl-2,2-dimethyl-3-(2′-carboxy-l′-propenyl) cyclopropanecarboxylate and their relative toxicities to pyrethrin I, cinerin I and allethrin I against houseflies were measured by counting “mortality” and “knock-down percent”     

Studies on Chrysanthemic Acid

(±) -trans-2,2-Dimethyl-3- (2′-methyl-2′-propenyl) cyclopropan-l-carboxylic acid (VII) was obtained by the treatment of (±) -pyrocin (IV) with thionyl chloride and absolute ethanol saturated with dry hydrogen chloride followed by the cyclization action of sodium tert-amylate in dry benzene and alkaline hydrolysis. This was converted into (±) -trans-chrysanthemic acid (VIII) by the catalytic action of p-toluenesulfonic acid.     

Studies on Chrysanthemic Acid

Synthesis of (±)-trans-chrysanthemic acid from (±)-1′-hydroxydihydro-trans-chrysanthemic acid by the dehydration with p-toluene-sulfonic acid was attempted. However, the attempt was found to be unsuccessful giving a compound believed to be methyl methyl 2,6 dimethylhepta-3.6-diene-5-carboxylate upon dehydration.

A cleavage upon cyclopropane ring was confirmed by deriving the acid obtained by the hydrolysis of the above ester to already known 2,6-dimethyl-heptane-5-carboxylic acid.

Analogous mode of dehydration and cleavage upon the ester of (±)-2,2-dimethyl-3-trans-hydroxylbenzyl-cyclopropane-l-carboxylic acid was also observed to give 1-phenyl-4-methyl-penta-1,3-diene-3-carboxylic acid. On the other hand, (±)-trans-caronic acid being derived to (±)-1′-oxo-2′-hydroxy-dihydro-trans-chrysanthemic acid, the synthesis of (±)-trans-chrysanthemic acid from (±)-trans-caronic acid became possible using (±)-1′-oxo-2′-hydroxy-dihydro-trans-chrysanthemic acid as a relay substance.     

Studies of Chrysanthemic Acid

(±) -cis and trans-2,2-dimethyl-3- (2′-cyano-l′-propenyl) cyclopropane carboxylic acids and their optically active forms were synthesized starting from chrysanthemic acids via oximes of 2,2-dimethyl3- (2′-formyl-l′-propenyl) cyclopropane carboxylates. Their allethrolone esters were also prepared which showed insecticidal activity.     

Gas Chromatographic Separation and Determination of Optical Isomers of Chrysanthemic Acid

Separation and determination of the optical isomers of chrysanthemic acid has been carried out by gas chromatography. The diastereoisomeric esters of chrysanthemic acid with l-menthol were separated on a column of 2% QF-1 coated on Chromosorb W. d-trans, l-trans and dl-cis Chrysanthemic acids were resolved but d-cis and l-cis acids were not separatable from one another on any column tested. These isomers of chrysanthemic acid were not isomerized during esterification and gas chromatographic operation, and their ratios were determined from their peak area ratios. The l-bornyl esters of the isomers of chrysanthemic acicf were not so easily separatable as the l-menthyl esters.     

Novel Active Chrysanthemic Esters

The rates of recycling and turnover of glucose in 5-day- and 21-day-old lambs and adult sheep were measured by the method of single simultaneous injection of glucose-6-³H and glucose-6-¹⁴C. The ³H/¹⁴C ratio decreased linearly with time and was 0.58 and 0.60 in lambs of 5-day- and 21-day-old, respectively, and 0.82 in adult sheep at 120 min after injection of the labeled glucose. The pool size and turnover rate of glucose considerably decreased with age. The rate of glucose recycling was significantly higher in lambs of both ages (22.0 and 26.2%, respectively) than in adult sheep (11.1 %).     

Studies on Abscisic Acid

Methyl α-ionylideneacetates were oxidized with selenium dioxide to a mixture of methyl 3′-keto-β-ionylideneacetates and a small amount of methyl 4′-keto-α-ionylidene-acetates followed by treatment with active manganese dioxide. By a similar oxidation methyl 3′-keto-β-ionylideneacetates were prepared from methyl β-ionylidene acetates. Methyl 4′-keto-α-ionylideneacetates were obtained by oxidation of methyl α-ionylideneacetates with tert-butyl chromate. Dehydrobromination of methyl bromoionylideneacetate, obtained by bromination of methyl 2-trans-α-ionylideneacetate with N-bromosuccinimide, gave a mixture of methyl 2-trans-dehydro-β-ionylideneacetate and methyl 2-cis-dehydro-β-ionylideneacetate. The growth inhibitory activities of these sesquiterpene carboxylic acids and keto esters on rice seedlings were tested.     

Studies on Abscisic Acid

Photosensitized oxygenation of dehydro-β-ionylidene-ethanol afforded 1′-hydroxy-4′keto-α-ionylidene-ethanol, which was oxidized with active MnO₂ to give 1′-hydroxy-4′-keto-α-ionylidene-acetaldehyde. The Wittig reaction of α-ionylideneacetaldehyde with carbethoxymethylenetriphenylphosphorane or the phosphorane prepared from ethyl γ-bromosenecioate gave ethyl α-ionylidene-crotonate or ethyl α-ionylidenesenecioate. Vitamin A₂ acid ethyl ester was converted to the hydroxy-keto-ester by photosensitized oxygenation. About the above synthesized compounds were examined growth inhibitory activities on rice seedlings.     

Studies on Abscisic Acid

Oxidation of methvl 2-trans-β-ionylideneacetate with X-bromosuccinimide afforded methyl 2-cis and trans-3′-hydroxy-β-ionylideneacetates. NaBH₄ reduction of methyl 2-cis-3′-keto-β-ionylideneacetate and ethyl 4′-keto-α-ionylideneacetate gave methyl 2-cis-3′-hydroxy-β-ionylideneacetate and ethyl 4′-hydroxy-α-ionyiideneacetate respectively. Further, methyl 4′-methoxy-epoxy-α-ionylideneacetate was prepared by epoxidation of methyl 4′-methoxy-α-ionylideneacetate. And then methyl 4′-hydroxy-l′, 2′-dihydro-β-ionylideneacetate was synthesized from ethyl 4-keto-α-cyclogeranate. Growth inhibitory activities of the above compounds on rich seedlings were examined.     

Studies on Abscisic Acid

Methyl α-cyclocitrylideneacetate was successively oxidized with selenium dioxide and chromium trioxide-pyridine complex to give methyl 1′-hydroxy-α-cyclocitrylideneacetate and a mixture of methyl 3′-keto-β-cyclocitrylideneacetate and methyl 4′-keto-α-cyclocitrylideneacetate. Further, oxidation of methyl α-cyclocitrylideneacetate with tert-butyl chromate afforded methyl 4′-keto-α-cyclocitrylideneacetate and methyl 1′-hydroxy-4′-keto-α-cyclocitry-lineacetate. Similarly, methyl α-cyclogeranate was oxidized to methyl 3-keto-β-cyclogeranate and methyl 4-keto-α-cyclogeranate. Methyl l′-hydroxy-4′-keto-α-cyclocitrylideneacetate, methyl l-hydroxy-4-keto-α-cyclogeranate and their related compounds did not show growth inhibitory activities on rice seedlings.     

Studies on Sorbic Acid

Sorbate inhibited the growth of baker’s yeast at the logarithmic and stationary phases and its inhibition showed the Type II mode proposed by Tamiya et al.

Microscopic observation of the yeast cells during growth demonstrated that the sorbate inhibition was fungistatic, but not fungicidal. The respiration of the yeast was mano-metrically determined and the mechanism of the inhibition was suggested that sorbate would competitively combine with coenzyme A and acetate and would consequently inhibit the enzyme reaction relating coenzyme A. In addition, it was clarified that sorbyl-coenzyme A was also determined by the method of the enzymatic acetylation of sulfanilamide. This would suggest that the sorbyl moiety might be transferred to 4-amino radical of sulfanilamide enzymatically as well as in the case of acetyl-coenzyme A.     

Studies on Abscisic Acid

Epoxidation of methyl dehydro-β-ionylideneacetates with perbenzoic acid afforded methyl 1′, 2′-epoxy-dehydro-β-ionylideneacetates and then methyl 1′, 2′-, 3′, 4′-di-epoxy-dehydro-β-ionylideneacetates. 1′,2′-Epoxy-dehydro-β-ionone, obtained byepoxidation of dehydro-β-ionone, was treated with carbethoxymethylenetriphenlphosphorane to give ethyl 1′, 2′-epoxy-dehydro-β-ionylideneacetates. Further, sensitive photooxidation of ethyl dehydro-β-ionylidenecrotonate, followed by alkaline hydrolysis, gave 1′-hydroxy-4′-keto-α-ionylidenecrotonic acid. Growth inhibitory activities of the above compounds on rice seedlings were examined.     

Studies on Abscisic Acid

Oxidation of 2-cis-α-ionylidene-ethanol (II) with active MnO₂ afforded a mixture of 2-cis and 2-trans-α-ionylideneacetaldehydes (III and IV). Reduction of methyl epoxy-α- and -β-ionylideneacetates (Vb, Xb XXIb and XXIIb) with LiAlH₄ gave the diols (VI, XI, XXIII and XXIV). The Wittig reaction of the hydroxyketones (XIII and XVIII) with carbethoxymethylenetriphenylphosphorane, followed by alkaline hydrolysis, yielded 5-(1′-and 2′-hydroxy-2′,6′,6′-trimethyl-1′-cyclohexyl)-3-methylpentadienoic acids (XIVa, XVa, XIXa and XXa). The reaction of α-cyclocitrylideneacetaldehyde (XXVII) and dihydro-α-ionone (XXXIII) with carbethoxymethylenetriphenylphosphorane afforded ethyl 3-demethyl-α-ionyli-deneacetate (XXVIIIb) and ethyl dihydro-α-ionylideneacetates (XXXIVb and XXXVb). Physiological activities of the above synthesized compounds on rice seedlings were examined.     

13C-NMR Studies on Halomethylvinyl Chrysanthemic Acids and Esters-Spin-lattice Relaxation Time and 13C-1H Coupling Constants

New types of stable chrysanthemic acids and esters were synthesized, and their ¹³C-NMR were examined and fully analyzed. The configurations of the cyclopropanecarboxylic acid and halomethylvinyl group were reflected on the spin-lattice relaxation time of the substituted methyl carbon involved in their structure. The long-range spin-spin coupling constants (³J_CH) correlated well to the NOE and T₁ measurements, which can generally be used to distinguish the geometry of the substituted double bond.     

Studies on l-Glutamic Acid Fermentation

Micrococcus glutamicus, a glutamate-produeing bacterium, is known to have strong activity of l-glutamic acid dehydrogenase which requires NADP as co-enzyme. In this paper, the NADP-speeifie l-glutamic acid dehydrogenase was purified from M. glutamicus by means of heat treatment with sodium sulfate, precipitation with acetic acid and diethyl-amino-ethyl (DEAE) cellulose column chromatography. The activity of the purified enzyme preparation reached 200-fold as high as that of the crude extract. Some properties of the purified enzyme were investigated. As a result, it was found that the highly purified enzyme preparation acted not only on l-glutamic acid (l-GA) but also on α, ε-diaminopimelic acid (α, ε-DAP) in the presence of NADP. Some of the probable consideration for the dehydrogenation of l-GA and α, ε-DAP are noted.     

Studies on l-Glutamic Acid Fermentation

The authors have carried out a series of studies on l-glutamic acid fermentation with a strain of Brevibacterium divaricatum nov. sp. in the previous papers.

In this paper, some metabolism of l-glutamic acid and oxidative decomposition of several organic acids concerning the tricarboxylic acid cycle by the resting cells have been studied. The results suggest that l-glutamic acid is one of the final fermentative products of this bacterium, and the tricarboxylic acid cycle is working as a glutamic acid forming cycle.

The presence of glucokinase, phosphoglucoisomerase, phosphofructokinase, aldolase, DPN-linked glyceraldehyde-3-phosphate dehydrogenase, and TPN-linked glucose-6-phosphate dehydrogenase in cell-free extracts of this bacterium was also demonstrated.     

Studies on Lactic Acid Fermentation

Resting cells of l. fermentum convert glyceraldehyde to equimolar lactic acid and neither the evolution of carbon dioxide nor the uptake of oxygen was observed. Glyceraldehyde-3-phosphate and 3-phosphoglycerate were identified as intermediates which were equally labeled with inorganic P³² in reaction systems, and the presence of triokinase was suggested.     

Studies on l-Glutamic Acid Fermentation

In the previous paper, most of the enzymes of the Embden-Meyerhof-Parnas pathway and glucose-6-phosphate dehydrogenase have been demonstrated to be present in cell-free extracts of Brevibacterium divaricatum, No. 1627. In this paper, the presence of condensing enzyme, aconitase, TPN-linked isocitric dehydrogenase, succinic dehydrogenase, fumarase, DPN-linked malic dehydrogenase, TPN-linked malic enzyme, oxalacetic carboxylase, isocitritase and malate synthetase in cell-free extracts of this bacterium was also demonstrated. From these results it was concluded that a strain of Brevibacterium divaricatum which has been found to contain all of the enzymes of the tricarboxylic acid cycle, would be capable of forming the key enzymes of the glyoxylate bypass as well. It suggests that the accumulation of α-ketoglutarate involves the glyoxylate bypass besides the tricarboxylic acid cycle in this bacterium.