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The first example of C-3 alkoxylation in mitomycins has been achieved. 3 alpha-iso-Propoxy-10-O-decarbamoylmitomycin D (4) and 3 alpha-iso-propoxymitomycin D (5) were derived from mitomycin D (3) under decarbamoylation conditions with iso-propoxide. Under similar conditions 3 alpha-iso-propoxy-10-O-decarbamoylporfiromycin (8) and 3 alpha-methoxy-10-O-decarbamoylmitomycin B (11) were also derived from porfiromycin (6) and mitomycin B (9), respectively. The mechanism of generation of these novel analogs was based on the premise that the key intermediate of hydroquinone iminium salt (14) was led through the iminium salt (13), followed by alkoxide addition and oxidation.

Glucose metabolism in Corynebacterium glutamicum was investigated using 13C nuclear magnetic resonance (13C NMR) spectroscopy. L-Glutamic acid and L-lysine producers were cultivated in medium containing [1-13C]- or [6-13C] glucose, and the 13C NMR spectrum of the culture filtrate was measured. In each fermentation, the ratio of the contributions of the Embden-Meyerhof pathway (EMP) and the hexosemonophosphate pathway (HMP) (EMP/HMP) was calculated on the basis of the 13C population at each carbon in the products. The EMP/HMP was estimated as 80/20 in glutamic acid fermentation; in contrast, it was 30-40/60-70 in lysine fermentation. These results indicate that HMP contributes much more in lysine fermentation, probably because of the greater requirement of NADPH in lysine formation from glucose.

The configuration of mitiromycin (2) was first determined by NMR experiments including the NOE technique. The absolute structure of 2 was related to mitomycin B (3) because 2 was derived from 3 under basic conditions. The ketonic form 4 was presumed to be involved in the generation of 2.

Mitomycin G (2) was derived from porfiromycin (10b) in 3 steps via the methanesulfonate (14b) in an overall yield of 39%. On the basis of the established method for the introduction of an exomethylene group in mitomycins with a 9a-methoxy group, the preparation of biologically more important 1a-demethylmitomycin G (5) from mitomycin C (1) was accomplished by the use of a protective acetyl group on the aziridine in an overall yield of 57%. 1a-Demethylmitomycin K (6) was obtained from 5 in a yield of 42%. In a preliminary evaluation of their antitumor activity, compound 5 showed superior activity against sarcoma 180 (sc-ip) to its 1a-methyl congener, i.e., mitomycin G (2).

The reaction between mitomycin A (1) and cysteamine afforded 7-N,7'-N'-bis(2-thioethyl)dimitomycin C (7), 7-N-[2-[(2-aminoethyl)dithio]ethyl]mitomycin C (8), and 7-methoxy mitosenes (10, 11). The structures of 7 and 8 were elucidated on the basis of spectroscopy and reactions between 1 and 8, and 1 and cystamine. The observation of the time course for the reaction revealed the mechanism of the formation of 7 and 8. The rapid oxidation of cysteamine by the quinone of 1 gave cystamine, which was trapped by 1 to give 8, and 8 was additionally reacted with 1 to give 7. Since 7 showed significant antitumor activities, related 7-N,7'-N'-bis(omega-thioalkyl)dimitomycins were synthesized. They also showed remarkable antitumor activities against HeLa-S3 in vitro, sarcoma 180 (sc-ip), leukemia P388 (ip-ip) in vivo. In these evaluations, compound 7 demonstrated unique potency.

L'epimere obtenu a la meme stereochimie en C-9 que la mitomycine C. L'epimerisation s'est faite via une decarbamoylation/amination/carbamoylation

Two molecular forms of salmon growth hormone (sGH), sGH I and II, have been isolated from the pituitary glands of the chum salmon (Oncorhynchus keta); a two-step extraction procedure, under alkaline (pH 10) conditions, subsequent to acid-acetone extraction was employed for extraction of the sGHs. They were then purified by isoelectric precipitation at pH 5.6, gel filtration on Sephadex G-100, and high-performance liquid chromatography on ODS. Intraperitoneal injection of sGH I and a combination of sGH I and II at doses of 0.01 μg and 0.1 μg/g body wt at different intervals resulted in a significant increase in body weight and length of juvenile rainbow trout. The GH producing cells in the pituitary of mature chum salmon were identified in the proximal pars distalis immunocytochemically with a specific antiserum; no cross-reactivity was seen in the prolactin cells in the rostral pars distalis. A molecular weight of 22,000 was estimated for both sGHs by gel electrophoresis in sodium dodecyl sulfate. Isoelectric points, by gel electrofocusing, of 5.6 and 6.0 were estimated for sGH I and II, respectively, with differences present in the amino acid composition and the N-terminal residue, suggesting that they may be genetic variants coded on two separate genes. The partial amino acid sequences of sGH I at both terminal regions have been determined.

An inosamine-idiotrophic mutant, KY11559, which produced no astromicin unless scyllo-inosamine was added to the fermentation medium, was isolated from Micromonospora olivasterospora. Biotransformation studies were performed with resting cells of this mutant and compounds assumed to be precursors of 1, 4-diaminocyclitol (fortamine). Scyllo-inosose, scyllo-inosamine and FU-10 were converted to astromicin. A number of mutants blocked in the biosynthesis of astromicin were developed from M. olivasterospora, and the intermediates accumulated by these mutants were isolated and identified. Twenty-five blocked mutants were classified into 10 groups, based on their complementation patterns by cosynthesis experiments. Further, utilizing these blocked mutants and the isolated compounds, biotransformation analyses were performed. The results showed that the amination at position 4 in fortamine occurred after formation of the pseudodisaccharide. Subsequently, the aminosugar and aminocyclitol moieties were aminated, methylated, dehydroxylated, epimerized and acylated to produce astromicin. Thus it was demonstrated that the astromicin biosynthetic pathway has a unique feature which is not found in the biosynthesis of other aminoglycoside antibiotics.

A new calmodulin antagonist, genistein, was isolated from the culture broth of Strepto-sporangium vulgare K-254. The spectral data of K-254-I indicated that the compound was identical with genistein, 4/,5,7-trihydroxyisoflavone. Genistein inhibited the Ca2+/calmodulin-depen- dent activity of cyclic nucleotide phosphodiesterase from bovine brain (IC50 = 20 μΜ) without appreciably affecting its basal activity. The inhibitory activity of genistein was antagonized by higher concentrations of calmodulin. Although phosphatidylserine did not reverse the inhibition of calmodulin, genistein inhibited the phospholipid-sensitive, Ca2 +-dependent protein kinase (protein kinase C) from bovine brain (IC50 = 35.3 μΜ). The activity of cAMP-dependent protein kinase was not affected by 700 μΜ of genistein.

The molecular and crystal structure of fortamine sulfate trihydrate, C8H18N2O4·H2SO4·3H2O, was determined by X-ray analysis. The space group is P21 with a=7.2516(9), b=11.193(1), c=9.022(1) A, β=90.55(2)°, and Z=2. The structure was solved by direct methods, and least-squares refinement using 1679 reflexions led to the final R value of 0.063. The ring conformation of fortamine sulfate is a chair form which corresponds to a chair obtained by inversion of that in the dihydrochloride. This is an extraordinary example of the cyclohexane ring inversion caused by the anion exchange in the crystal. The ring conformation of fortamines in solution and in crystal are compared and discussed.

The structures of four new protein kinase C inhibitors of microbial origin, K-252a, b, c and d were determined by spectral studies and chemical conversion.

13C NMR spectroscopy was applied to studies on histidine biosynthesis in Corynebacterium glutamicum N-730, a histidine producing mutant. When it was cultured in a medium containing [1-13C]glucose as the carbon source, the 13C isotopomer was abundantly incorporated into C-6 (28 atom%) and C-l (21%), and to a lesser extent into C-5 (7%) of the histidine accumulated. The high incorporation into C-6 was explained as being due to that most of the one-carbon unit for histidine biosynthesis is derived from serine. On the bases of this view and the 13C population value at C-6, the contributions of the Embden-Meyerhof and hexose monophosphate pathways to the histidine production were calculated to be 56 and 44%, respectively. The significance of the label incorporation into C-l and C-5 was discussed.

The pituitary of the cichlid fish tilapia secretes two prolactins (PRLs) of molecular masses 20 kDa and 24 kDa. The 20-kDa PRL has an isoelectric point in the range of those of mammalian PRLs (pI 6.7), but the 24-kDa PRL is unusually basic (pI 8.7). Partial sequence information indicates that the PRLs are homologous but distinct proteins, differing by five amino acids within the first 29 NH2-terminal residues. Homology in the known region is higher with chum salmon PRL than with known mammalian PRLs. Reversed-phase HPLC permits isolation of these two PRLs and a single tilapia growth hormone from culture medium or from the pituitary in a single step. HPLC and radio-HPLC analysis of [3H]leucine pulse-chase experiments reveal that each PRL is secreted in vitro at remarkably high rates (21 pmol per gland per hr) and that the two PRLs are released in approximately equimolar amounts, suggesting the coordinate regulation of the secretion. Both PRLs exert characteristic PRL activity in that they prevent the loss of Na+ from the plasma of hypophysectomized tilapia in fresh water.

New components of tetrocarcins (E1, E2, F and F-1) were found in the culture broth of Micromonospora chalcea KY 11091 that was known to produce tetrocarcins A, B and C. Tetrocarcin F-1 consisted of tetronolide and nitro sugar (tetronitrose). Tetrocarcins E1 and E2 consisted of F-1 and deoxy sugar (L-digitoxose). Tetrocarcin F consisted of F-1 and two deoxy sugars (their structures were not yet determined). They all showed antibacterial activities against Gram-positive bacteria and the specific activity decreased with decrease in the numbers of deoxy sugars attached to the aglycone.

13C NMR spectroscopy was applied to studying lysine biosynthesis in Corynebacterium glutamicum ATCC 21543, a lysine producing mutant. It was cultured in a medium containing [1-13C]glucose or [6–13C]glucose as the sole carbon source and the 13C NMR spectrum of the culture filtrate was measured. C labeling patterns of l-lysine produced were well explained by the putative metabolic pathways of the bacterium. Fixation of 13CO2 liberated from the labeled substrates and the operation of the tricarboxylate cycle in the fermentation were obviously observed. The dual operations of the classical diaminopimelate pathway and the diaminopimelate dehydrogenase bypath were supported. Calculation of the contribution ratios of the metabolic pathways was attempted.

The new antibiotic, XK-90, produced by Streptomyces sp. is active against Gram-positive and Gram-negative bacteria. The structure has been determined as N-acetyl-N'-(3-formyl-4-hydroxyphenyl)hydrazine (1) and is the second example of a naturally occurring antibiotic having the phenylhydrazine skeleton.

The structure of K-13, a potent inhibitor of angiotensin I converting enzyme (ACE), was determined to be a cyclic dipeptide composed of tyrosine and an unusual diamino dicarboxylic acid, isodityrosine, by spectral and chemical studies of K-13 and its derivatives.

Novel antitumor antibiotics, trioxacarcin complex, were isolated from the fermentation broth of Streptomyces bottropensis DO-45 by the use of non-ionic porous resin and silica gel chromatography. The purified components (trioxacarcins A, B and C) were characterized as new antibiotics by their physico-chemical properties and chromatographic behaviors. The mode of action of trioxacarcin A against Bacillus subtilis was found to be through the of DNA synthesis.

Fortamine, L-3-amino-1-methoxy-6-methylamino-1,3,6-trideoxy-chiro-inositol, is a component of fortimicins which are potent aminocyclitol antibiotics. The space group is P21 with a=9.454(1), b=6.6957(8), c=7.622(1) A, β=95.93(2)°, and two C8H18N2O4’s per unit cell. The structure was solved by direct methods, least-squares refinement using 932 reflexions giving the final R value of 0.033. The ring conformation of fortamine is closer to the ideal chair form than that of the fortamine moiety in fortimicin B. The strain caused by the intramolecular hydrogen bond and glycosidic bond in fortimicin B is released in fortamine.

The structure of the novel antibiotic reductiomycin has been clarified by X-ray diffraction studies. The structure was determined by the direct method and refined to an R value of 0.098. Reductiomycin has a unique chemical structure with a strong intramolecular hydrogen bond with the 0…0 distance of 2.509 A.

Mitomycin G 2 was derived from mitomycin B 5 by 3 steps in an overall yield of 43 %.

Novel antitumor antibiotics, tetrocarcin complex composed of three components (A, B and C) were isolated from the culture broth of Micromonospora chalcea KY11091 through various procedures. Tetrocarcins showed marked activity against experimental tumors such as mouse sarcoma 180 and mouse leukemia P388. Multiple injections showed better therapeutic effect against tumors. Mode of action of tetrocarcin A against Bacillus subtilis was found to be through the inhibition of RNA synthesis.

2-Aminoethyl, glycyl, (S)-4-amino-2-hydroxybutyl and (S)-4-amino-2-hydroxybutyryl groups were introduced to the 2'-amino group of 4-N-[(S)-4-amino-2-hydroxybutyl]fortimicin B (2). All of the derivatives of this type prepared showed antibacterial activity almost as strong as fortimicin A (1) and were also active against fortimicin A resistant bacteria which can inactivate fortimicin A by producing an inactivating enzyme AAC(3)-I.

The 1, 2' or 6'-amino group of fortimicin A was alkylated or acylated and the antimicrobial activities of the derivatives were compared with each other. 2'-N-Substituted fortimicins A were active against the fortimicin A resistant strain which produced AAC(3)-I. AAC(3)-I is the only enzyme which can inactivate fortimicin A. Among the derivatives prepared in the present study, 2'-N-[(S)-4-amino-2-hydroxybutyl]fortimicin A showed stronger activity than fortimicin A.

A new component, nanaomycin E, has been isolated from the culture broth of Streptomyces rosa var. notoensis, which had been found to produce nanaomycins A, B, C and D. Nanaomycin E was an epoxy derivative of nanaomycin A and was converted into nanaomycin A and 4a-epi-nanaomycin B by treatment with sodium hydrosulfite in an acidic aqueous solution. 4a-epi-Nanaomycin B was quantitatively converted into nanaomycin A under alkaline conditions.

The structure of a new antibiotic, CV-1 was determined to be 1,2-diamino-1,2-N,N'-carbonyl-1,2-dideoxy-alpha-D-glucose hydrate by spectral and chemical studies. CV-1 possessed a unique open ring hemiaminal structure. CV-1 synthesized from N-carbamoyl-D-glucosamine was identical to material isolated from fermentation.

L'epimere obtenu a la meme stereochimie en C-9 que la mitomycine C. L'epimerisation s'est faite via une decarbamoylation/amination/carbamoylation

An X-ray analysis of the α form of L-glutamic acid, C5H9NO4, has been carried out. The space group is P212121 with a=7.068(1), b=10.277(1), c=8.755(1) A and Z=4. The structure was solved by the direct method, and least-squares refinement using 855 reflexions led to the final R value of 0.034. The molecule is in a zwitter ion form, and there is a strong OH···O intermolecular hydrogen bond with the O···O distance of 2.578(2) A. The conformation of L-glutamic acid in the α form is quite different from that in the β form. The occurence frequency in various crystal structures containing glutamic acid residues shows that the conformation in the β crystal is more stable and favourable than that in the α crystal. The transition of the α form to the β form of L-glutamic acid in the mother liquor and the conditions for obtaining the α crystals are discussed based on the crystal and molecular structures of the two polymorphs.

Fortamine dihydrochloride, C8H18N2O4·2HCl, is obtained by the hydrolysis of fortimicin B with hydrochloric acid. The space group is C2 with a=16.109(2), b=10.047(1), c=8.603(1) A, β=108.69(1)°, and Z=4. The structure was solved by the heavy atom method, and least-squares refinement using 1564 reflexions led to the final R value of 0.030. The ring conformation of fortamine dihydrochloride is an inversed chair form of fortamine. The chair form is significantly compressed to relax the non-bonded interactions between axial substituents.

Pseudomonas methanica KY4634 was found to produce 5-lipoxygenase inhibitor designated KF8940, MY12-62a and MY12-62c. The inhibitors were purified by solvent extraction, silica gel column chromatography, reversed-phase low pressure liquid chromatography and crystallization. The chemical structures of KF8940, MY12-62a and MY12-62c were determined to be 2-n-heptyl-4-hydroxyquinoline-N-oxide, 2-n-heptyl-4-hydroxyquinoline and 3-n-heptyl-3-hydroxy-1,2,3,4-tetrahydroquinoline-2,4-dione, respectively, on the basis of their physico-chemical properties. Among them, KF8940 was the most potent inhibitor. The compound inhibited 5-lipoxygenase of rat basophilic leukemia cells in a dose-dependent manner and the half maximal inhibitory concentration (IC50) was 1.5 X 10(-7) M. At this concentration, KF8940 did not inhibit bovine platelet 12-lipoxygenase and cyclooxygenase, and the IC50 values for these enzyme were 3.5 X 10(-5) M and 1.7 X 10(-4) M, respectively. The results indicated that KF8940 is a potent and selective inhibitor of 5-lipoxygenase. The IC50 value of MY12-62c for 5-lipoxygenase was 1.9 X 10(-5) M and that of MY12-62a was 1.9 X 10(-5) M.

Two new antibiotics, fluopsins C and F, have been isolated from the culture broth of Pseudomonas fluorescens and characterized as bis(N-methyl-N-thioformylhydroxylaminato)copper(II) (I) and tris(N-methyl-N-thioformyl-hydroxylaminato) iron(III) (II) respectively.

Introducing the mercaptoethyl group at the 7-N position of mitomycin C 1 has led to the isolation of 7-N,7'-N'-dithiodiethylenedimitomycin C 2. The compound 2 showed excellent antitumor activity against sarcoma 180 (sc-ip) and leukemia P388 (ip-ip) in mice. As an extension of this study, we synthesized mitomycin dimers with symmetrical disulfide and mitomycin derivatives with unsymmetrical disulfide at the 7-N side chain. Among these compounds, the water soluble conjugate 3 with ethyl gamma-L-glutamyl-L-cysteinylglycinate was far more effective against sarcoma 180 and leukemia P388 than 1. During the subsequent stage of inquiry for the potent congeners of 3, the compound 4 (water solubility: greater than 500 mg/ml), designated as KW2149, with the gamma-L-glutamylcystamino group at the 7th position was finally selected for further evaluation.

The structure of KS-619-1, a potent inhibitor of Ca2+ and calmodulin-dependent cyclic nucleotide phosphodiesterase, was determined to be 8, 13-dioxo-3-(2-oxopropyl)-5, 6, 8, 13-tetrahydro-l, 7, 9, 11-tetrahydroxybenz[a]naphtacene-2-carboxylicacid by spectral studies of KS-619-1 and its methyl derivative.