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Novel streptonigrin 2-amide derivatives have been efficiently synthesized without protection by using 3,3′-(phenylphosphoryl)bis(1,3-thiazolidine-2-thione)(PPBTT) as a chemoselective condensing agent.

Sarcosine oxidase (EC 1.5.3.1) produced by Streptomyces sp. KB210-8SY was purified by ion exchange chromatography on DEAE-sepharose CL-6B, affinity chromatography on sarcosyl-AH-sepharose 4B and gel filtration on sephadex G-150 to electrophoretic homogeneity.The molecular weight of the enzyme was estimated to be 44000 by sodium dodecyl sulfate (SDS) -polyacrylamide gel electrophoresis and gel filtration on Sephadex G-150. The enzyme showed the maximum activity at pH 8 and was stable at pH 7-9. In addition to sarcosine, the enzyme oxidized N-methyl-L-leucine, N-methyl-DL-alanine and N-methyl-DL-valine to lesser extents. The apparent Km values for sarcosine, N-methyl-L-leucine, N-methyl-DL-alanine and N-methyl-DL-valine were 0.91, 0.58, 1.6 and 6.7 mM, respectively. The enzyme was inactivated by N-bromosuccinimide, hydroxylamine hydrochloride, SDS, Zn2+, Ni2+ and Hg2+ but not by ethylenediaminetetraacetate, p-chloromercuribenzoate or p-toluenesulfonyl chloride.The taxonomic characteristics of strain KB210-8SY are closely related to those of Streptomyces flavovirens and S. misakiensis.

Thirteen heterocyclic quinones (5 quinoline quinones, 7 isoquinoline quinones, 1 indole quinone) were tested for their effects on avian myeloblastosis virus reverse transcriptase, growth of murine lymphoblastoma L5178Y cells, respiration of rat liver mitochondria and oxidation of NADH by Clostridium kluyveri diaphorase in comparison with those of streptonigrin, in which the quinoline quinone moiety is considered to play a crucial role. Most of the quinoline quinones and isoquinoline quinones inhibited reverse transcriptase to the same extent as streptonigrin with the ID50 values ranging between 1 and 5 micrograms/ml, whereas the ID50 value of the indole quinone derivative, 4,7-dihydro-2,3-dimethylindole-4,7-dione, was 80 micrograms/ml. The cytotoxicities of the quinones were much lower than that of streptonigrin; the ID50 values of the quinones were higher than 0.15 micrograms/ml. In particular, the ID50 value of the ortho-quinoline quinone derivative, 8-methoxy-7-methyl-5,6-dihydroquinoline-5,6-dione, was as high as 16 micrograms/ml, while the 50% inhibition of cell growth was seen in the presence of 0.0025 micrograms/ml streptonigrin. The membrane transport of the quinones was evaluated by comparing the effects on oxygen consumption by mitochondria and oxidation of NADH by bacterial diaphorase, being proven not to be responsible for their lower cytotoxicities.

In contrast to the formation of 2,2,4,4-tetrakis(trifluoromethyl)-1,3-dithietane from hexafluoropropene, potassium fluoride, and sulfur, perfluoro-2-butene and perfluoro-pentenes react with potassium fluoride and sulfur to form perfluoro-1,3-dithioles via intermediate thioenolate anions.

The usefulness of 2-hydryl-2-( F -methyl)- F -propanoyl fluoride as a building block for the synthesis of trifluoro methylated compounds has been shown in the synthesis of the title compounds by the reaction of the acid fluoride with 1,3-dimethylthiourea and 1,3-dimethylurea followed by intramolecular cyclization.

Inhibition of avian myeloblastosis virus (AMV) reverse transcriptase by natural and synthetic quinones including antibiotics could be accounted for by an oxidation-reduction reaction. The quinones were shown to function as electron acceptors as revealed by the catalytic oxidation of NADH by Clostridium kluyveri diaphorase which was in excellent agreement with enzyme inhibition activity. The kinetics of inhibition of AMV reverse transcriptase by three synthetic quinones with different core structures, i.e., 6-methoxy-5, 8-dihydroquinoline-5, 8-dione, 5, 8-dihydroisoquinoline-5, 8-dione and 1, 4-naphthoqumone, were studied. These quinones inhibited reverse transcriptase in the same manner as streptonigrin (STN) and were shown to act at a single class of reaction site(s) on the enzyme molecule. In contrast, the quinones with bulky substituents, i.e., 7-(2-nitrophenethylamino)-5, 8-dihydroisoquinoline-5, 8-dione and 7-methoxy6-methyl-3-piperidino-5, 8-dihydroisoquinoline-5, 8-dione, were inactive as inhibitors of reverse transcriptase, whereas they retained competent catalytic activities in the oxidation of NADH by C. kluyveri diaphorase. Based on these observations, the existence of a specific site of interaction on the enzyme molecule, referred to as a quinone pocket, was proposed. The quinone pocket might play a crucial role in the early sequence of events leading to the inhibition of reverse transcriptase by quinones including STN and sakyomicin A (SKM). Access of SKM to a quinone pocket might be restricted due to its bulky structure in the vicinity of the quinone group. This is inferred from unsuccessful inhibition of reverse transcriptase by the quinones with bulky substituents, resulting in much poorer inhibition of reverse transcriptase in spite of more potent electron acceptor activity in the oxidationreduction system as compared with those of STN.

Sarcosine oxidase (EC 1.5.3.1) produced by Bacillus sp. B-0618 was purified by ion exchange chromatography on diethyl aminoethyl-cellulose and gel filtration on Sephadex G-100 and G-150. The molecular weight of the enzyme was estimated to be 42000 by gel filtration on Sephadex G-150 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme exhibited an absorption spectrum characteristic of flavoprotein. The enzyme showed the maximum activity at pH 8.5-9 and was stable at pH 7-10. The pI value was 4.7 as determined by isoelectric focusing. Although sarcosine is the preferred substrate, the enzyme also oxidized N-methyl-DL-alanine, N-methyl-L-leucine and N-methyl-DL-valine to lesser extents. The apparent Km values of sarcosine, N-methyl-DL-alanine, N-methyl-L-leucine and N-methyl-DL-valine were 12.2, 6.8, 106 and 173 mM, respectively. The enzyme was inactivated by N-bromosuccinimide, Zn2+, Fe3+ and Hg2+, but not by ethylenediaminetetraacetate, p-chloromercuribenzoate, monoiodoacetate or p-toluenesulfonylchloride.

A neutral metalloendopeptidase, produced by Streptomyces, which has anti-inflammatory activity against carrageenan-induced edema in rats was purified by affinity chromatography on N-benzyloxycarbonylglycyl-D-leucylaminohexyl-Sepharose (Z-Gly-D-Leu-AH-Sepharose) to electrophoretic homogeneity. The enzyme was adsorbed on a Z-Gly-D-Leu-AH-Sepharose column equilibrated with phosphate buffer (pH 7.0). The column was washed with phosphate buffer (pH 5.6) containing 2 M urea and eluted with acetate buffer (pH 4.1) supplemented with 2 M urea. The caseinolytic activity of the enzyme, which was lost on treatment with ethylenediaminetetraacetate, was recovered by the addition of ZnCl2 to the reaction mixture. The enzyme showed the maximum caseinolytic activity at pH between 7.0 and 8.0, and was stable in the pH range of 5.0-10.0. The molecular weight was estimated to be 59000. The enzyme preferentially hydrolyzed N-benzyloxycarbonylglycyl-L-leucine amide, N-benzyloxycarbonylglycyl-L-phenylalanine amide and N-benzyloxycarbonylglycyl-L-leucyl-L-tyrosine among the synthetic substrates tested in this work. The producing microbe was identified as Streptomyces mauvecolor based on the results of taxonomic studies.

3β-Hydroxysteroid oxidase activity was detected in the broth filtrate of strain H 1109 MY 12. Based on taxonomic studies, this strain was shown to be a new species of genus Streptoverticillium and the name Stv. cholesterolicum is proposed for this strain. The enzyme was purified by ammonium sulfate precipitation and affinity column chromatography on crystalline cholesterol, and the product showed a single band on SDS-polyacrylamide gel electrophoresis. The enzyme showed optimum activity at pH 7.0-7.5 and was stable over a rather wide pH range of 4.0 to 12.5. Cholesterol and dihydrocholesterol were oxidized rapidly. The Km value for the oxidation of cholesterol by this enzyme was about 0.4mM. The enzyme activity was greately inhibited by HgCl2, and AgNO3. FeCl3 and CuSO4 were also inhibitory though to lesser extents. Iodine and N-bromosuccinimide completely inhibited the enzyme activity at concentrations of less than 0.01 mM. Neither metal-binding agents nor p-chloromercuribenzoic acid had any inhibitory effect on the oxidation of cholesterol by this enzyme. The molecular weight of the enzyme was estimated to be 56000 by SDS-polyacrylamide gel electrophoresis. The enzyme was proved to be a flavoprotein containing flavin adenine dinucleotide as a prosthetic group.

The title compounds were readily synthesized in one pot by the reaction of methyltriethylammonium 2-( F -methyl)- F -propenolate, formed directly by the reaction of methyl 2-( F -methyl)-2-hydryl- F -propyl ether with two equivalents of triethylamine in DMF, and S-alkylisothiourea hydrogen halide, followed by the reaction with triethylamine again. Derivatizations via nucleophilic substitution of the fluorine atom at the 6-position were performed after alkylation of the title compounds leading to the preparation of various trifluoromethyl substituted pyrimidines.

Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies. Against AMV reverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH, with the ID50 values ranging 2-8 micrograms/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but not STN-CONH(CH2)3N(CH3)2. Doxorubicin-resistant murine lymphoblastoma L5178Y cells showed collateral sensitivity to both STN-COOH and STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.

The naphthoquinone moiety was proven to be essential to the biological activities of sakyomicin A using various naphthoquinone derivatives. Among the naphthoquinones tested, juglone (5-hydroxy-1, 4-naphthoquinone) which resembles the partial structure of sakyomicin A was the most active in cytotoxicity against murine lymphosarcoma L5178Y cells, electron acceptor function in the oxidation of NADH by Clostridium kluyveri diaphorase or rat liver mitochondria and inhibition against avian myeloblastosis virus reverse transcriptase. The significantly lower cytotoxicity of sakyomicin A as compared with juglone was attributable to its poor membrane transport. The inhibition of reverse transcriptase activity may result from the interaction between a sulfhydryl group in the active center of the enzyme and quinone groups of the naphthoquinones and sakyomicin A.

The rearrangement of terminal allylic hexafluoroisopropyl sulfides to the internal olefinic sulfide occurs under either photochemical or thermal conditions. In the case of the formation of E and Z isomers, the E isomer was predominant. The rearrangement is suggested as a unimolecular, associative radical mechanism, via kinetic studies, use of inhibitors, or radical initiator.

Etude comparative de l'activite inhibitrice de differentes flavones et isoflavones vis-a-vis de la reverse transcriptase du virus de la myeloblastose aviaire. Une relation entre la structure et l'activite inhibitrice est mise en evidence

Antitumor antibiotic streptonigrin (STN-COOH) is a potent inhibitor of avian myeloblastosis virus (AMV) and human immunodeficiency virus reverse transcriptases. The carboxyl group at 2'-position of STN-COOH was modified to give esters, hydrazide, amides and amino acid derivatives for biological studies. Against AMV reverse transcriptase, the hydrazide, amides and amino acid derivatives showed inhibitory activity, which compared favorably to that of STN-COOH, with the ID50 values ranging 2-8 μg/ml. In contrast, the esters lacked this activity except for those having a dimethylamino group in the substituent. Splenomegaly caused by Friend leukemia virus infection was significantly inhibited by STN-COOH and STN-COO(CH2)3N(CH3)2, but not STN-CONH(CH2)3N(CH3)2. Doxorubicin-resistant murine lymphoblastoma L5178Y cells showed collateral sensitivity to both STN-COOH and STN-COO(CH2)3N(CH3)2 not only in vitro but also in vivo.

The structures of three neoenactin congeners, designated as neoenactins B1, B2 and M2 were elucidated by 1H and 13C NMR and mass spectroscopic studies. Another minor component, neoenactin M1, isolated from the crude mixture, was identified as lipoxamycin. All three new compounds were structurally related to neoenactin A. While B1 was proved to be a positional isomer of B2, M2 was found to be a dihydro derivative of A. All the compounds were active against yeasts and fungi and potentiated the activities of polyene antifungal antibiotics such as trichomycins A and B and amphotericin B.

In contrast to the formation of 2,2,4,4-tetrakis(trifluoromethyl)-1,3-dithietane from hexafluoropropene, potassium fluoride, and sulfur, perfluoro-2-butene and perfluoro-pentenes react with potassium fluoride and sulfur to form perfluoro-1,3-dithioles via intermediate thioenolate anions.

The inhibition of human immunodeficiency virus (HIV) reverse transcriptase by certain antibiotics and related compounds was studied in comparison with that of avian myeloblastosis virus (AMV) reverse transcriptase and cellular DNA polymerases alpha and beta. In general, compounds that inhibited HIV reverse transcriptase also inhibited AMV reverse transcriptase. For example, 10 micrograms/ml of the isoquinoline quinones used in this study inhibited approximately 80% of the activity of reverse transcriptases of HIV and AMV, but did not inhibit the activity of DNA polymerases alpha and beta even at 50 micrograms/ml. AMV enzyme was more sensitive than HIV enzyme to colistin, enduracidins A and B, janiemycin, glysperin A, and thielavins A and B. The streptonigrin alkyl esters, however, inhibited HIV reverse transcriptase only. Sakyomicin A, luzopeptins, ellagic acid and suramine inhibited the activities of reverse transcriptases and cellular DNA polymerases.

Though all the streptonigrin derivatives with modifications in the carboxyl group on C2' were active as electron acceptors in the oxidation of NADH by Clostridium kluyveri diaphorase as well as streptonigrin, the glycine derivative did not show any marked effect on the KCN-insensitive oxidation of glutamate by rat liver mitochondria, suggesting a poor membrane transport of the glycine derivative. Sakyomicin A also induced the KCN-insensitive oxidation of glutamate by mitochondria, while a trace activity was observed by mitomycin C and the effect of doxorubicin was negligible. Like streptonigrin, the autoxidation of a reduced form (hydroquinone) of sakyomicin A to a quinone accompanied the generation of H2O2. However, exogenous NADH was oxidized by mitochondria in the presence of sakyomicin A but not streptonigrin.

Extracellular creatine amidinohydrolase (creatinase, EC 3.5.3.3) produced by Alcaligenes sp.nov. was purified to electrophoretic homogeneity by ion exchange chromatography on diethyl-aminoethyl-cellulose, gel filtration on Sephadex G-75 and hydrophobic chromatography on phenyl-Sepharose CL-4B. The molecular weight of the enzyme was estimated to be 51000 by gel filtration on Sephadex G-200 and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The enzyme showed the maximum activity at pH 8 and was stable at pH 5-9. The pI value was 4.7 as determined by isoelectric focusing. The enzyme catalyzed hydrolysis of creatine to sarcosine and urea, and the Km and Vmax values for creatine were 17.2 mM and 105 μmol/min/mg protein, respectively. The enzyme was markedly inactivated by p-chloromercuribenzoate (PCMB). Besides PCMB, the enzyme was inactivated by N-bromosuccinimide, Zn2+, Cu2+ or Hg2+.

The structures of neoenactins (NEs) NL1 and NL2, novel antimycotic antibiotics produced by Streptoverticillium olivoreticuli in a precursor-oriented fashion, were elucidated by 1H and 13C NMR and mass spectroscopic studies. The structures of both antibiotics are closely related to that of NE-A, the major component of NE congeners, being classified in the group of hydroxamic acid antimycotic antibiotics in which L-serine and a diketo amine form a hydroxamic acid structure. To study the role of the carbonyl groups in the biological activities of the hydroxamic acid antimycotic antibiotics, NE-A was modified by reaction with various carbonyl reagents. In terms of antimycotic activity, the derivatives are classified into two distinct groups; the first ones are fairly comparable to but not exceeding and the second ones are less active than NE-A depending on their tendency to revert to NE-A by hydrolysis. In general, the biological activities of the derivatives are inversely proportional to their stabilities to hydrolysis.