High frequency discharge experiment

Investigations of the synthesis of organic compounds from methane and ammonia were carried out by using high frequency discharges. The radical of CN was observed in the early phase of the discharge. Therefore it is considered that the CN radical contributes to the successive reaction steps An important intermediate, a tetramer of hydrogen cyanide was observed to be formed at room temperature. The amounts of this compound progressively increased with time. It was speculated that the purines which were identified in the refluxed sample were synthesized through the above tetramer. Compounds which are thought to be heterocyclic (λ_max:250–265 nm) were initially formed in the sample trapped at liquid nitrogen temperature. After hydrolysis with hydrochloric acid, Gly, Asp, Ala,etc. were observed in the sample. Overall yield of organic compounds was estimated at 5–6% based on input carbon of methane.     

The enzymatic synthesis of antiviral agents.

The majority of potential antiviral agents which are currently undergoing clinical trials are inhibitors of the replication of nucleic acids. The most common class of these inhibitors are nucleoside analogues and the elucidation of synthetic routes to these compounds has been of interest for many years as many are anticancer agents. One synthetic development has been the application of bio-transformations to nucleoside syntheses. This topic has been reviewed recently (Shirae et al., 1991) but this review is not widely available. In the present review, the application of biotechnology to the synthesis of antiviral agents including those which are not nucleoside analogues will be discussed. Enzymatic syntheses of nucleosides can be simpler and quicker than syntheses carried out by chemical methods. The most useful enzymes are those found in catabolic pathways. Nucleoside phosphorylases and N-deoxyribosyltransferases have both been widely used for nucleoside synthesis catalysing the transfer of sugar residues from a donor nucleoside to a heterocyclic base. Enzymatic methods have also been applied to the resolution of racemic mixtures and adenosine deaminase is a convenient catalyst for the hydrolysis of amino groups on purines and purine analogues. Regioselective deprotection of nucleoside esters has been achieved with lipases and these enzymes have also been applied to the synthesis of esters of sugar-like alkaloids. The latter have potential as inhibitors of the replication of HIV. Esterases have also been used in combined chemical and enzymatic syntheses of organophosphorus antiviral agents.     

The current and future applications of microorganism in the bioremediation of cyanide contamination

Inorganic cyanide and nitrile compounds are distributed widely in the environment, chiefly as a result of anthropogenic activity but also through cyanide synthesis by a range of organisms including higher plants, fungi and bacteria. The major source of cyanide in soil and water is through the discharge of effluents containing a variety of inorganic cyanide and nitriles. Here the fate of cyanide compounds in soil and water is reviewed, identifying those factors that affect their persistence and which determine whether they are amenable to biological degradation. The exploitation of cyanides by a variety of taxa, as a mechanism to avoid predation or to inhibit competitors has led to the evolution in many organisms of enzymes that catalyse degradation of a range of cyanide compounds. Microorganisms expressing pathways involved in cyanide degradation are briefly reviewed and the current applications of bacteria and fungi in the biodegradation of cyanide contamination in the field are discussed. Finally, recent advances that offer an insight into the potential of microbial systems for the bioremediation of cyanide compounds under a range of environmental conditions are identified, and the future potential of these technologies for the treatment of cyanide pollution is discussed.     

On the "activation" of cytokinins.

A number of cytokinin analogs containing modifications in the heterocyclic moiety were prepared. These compounds were tested for activity as cytokinins and anticytokinins in the tabacco bioassay and the results were used to determine whether any position(s) of the heterocyclic nucleus of cytokinins may require derivatization as part of an over-all "activation" process. 3-substituted 4-alkylaminopyrazolo [3,4-d]pyrimidines and 4-alkylaminopyrrolo[2,3-d]pyrimidines, for example, have (substituted) carbon rather than nitrogen atoms at positions 3 and 5, respectively (analogous to position 7 in purines) and would be predicted to be metabolically stable at these positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable positions. The finding that these compounds had cytokinin activity suggested that modification at the metabolically stable position, and by extension at position 7 in cytokinin analogues which are purines, is not a prerequisite for the expression of cytokinin activity. Similar consideration of other heterocyclic analogs which have cytokinin activity suggests that the active form of a cytokinin can be the exogenous compound itself. Certain structural analogs of cytokinins were found to inhibit the growth of tobacco callus promoted by 6-(3-methyl-2-butenylamino)purine. These compounds were studied as potential cytokinin antagonists, i.e. having activity analogous to the 7-alkylamino-3-methylpyrazolo[4,3-d]pyrimidines (Hecht, S. M., 2068-2610; Skoog, F., Schmitz, R.Y., Hecht, S.M., and Bock, R. M. (1973) Phytochemistry 12, 25-37). The activity of these compounds is discussed and criteria are proposed to distinguish between those species which are specific anticytokinins and those which otherwise inhibit growth.     

Was ferrocyanide a prebiotic reagent?

Hydrogen cyanide is the starting material for a diverse array of prebiotic syntheses, including those of amino acids and purines. Hydrogen cyanide also reacts with ferrous ions to give ferrocyanide, and so it is possible that ferrocyanide was common in the early ocean. This can only be true if the hydrogen cyanide concentration was high enough and the rate of reaction of cyanide with ferrous ions was fast enough. We show experimentally that the rate of formation of ferrocyanide is rapid even at low concentrations of hydrogen cyanide in the pH range 6–8, and therefore an equilibrium calculation is valid. The equilibrium concentrations of ferrocyanide are calculated as a function of hydrogen cyanide concentration, pH and temperature. The steady state concentration of hydrogen cyanide depends on the rate of synthesis by electric discharges and ultraviolet light and the rate of hydrolysis, which depends on pH and temperature. Our conclusions show that ferrocyanide was a major species in the prebiotic ocean only at the highest production rates of hydrogen cyanide in a strongly reducing atmosphere and at temperatures of 0°C or less, although small amounts would have been present at lower hydrogen cyanide production rates. The prebiotic application of ferrocyanide as a source of hydrated electrons, as a photochemical replication process, and in semi-permeable membranes is discussed.     

Two-column gas chromatography of trimethylsilyl derivatives of biochemically significant compounds

Gas chromatographic (GC) retention data as methylene unit (MU) values are presented for the trimethylsilyl (TMS) derivatives of 250 biochemically significant compounds on two different GC stationary phases (OV-1 and OV-17). A ΔMU value is calculated for the difference in MU values on the moderately polar OV-17 and the nonpolar OV-1 columns. Some relationships between ΔMU values and structure are discussed; in general highly polar compounds have large ΔMU values and those compounds forming derivatives containing several TMS groups have low ΔMU values. Compounds in the following categories were studied: amino acids; organic acids; aromatic acids, amines, and glycine conjugates; and purines, pyrimidines, and other nitrogen heterocyclic compounds.     

Enzymatic detoxification of cyanide: clues from Pseudomonas aeruginosa Rhodanese

Cyanide is a dreaded chemical because of its toxic properties. Although cyanide acts as a general metabolic inhibitor, it is synthesized, excreted and metabolized by hundreds of organisms, including bacteria, algae, fungi, plants, and insects, as a mean to avoid predation or competition. Several cyanide compounds are also produced by industrial activities, resulting in serious environmental pollution. Bioremediation has been exploited as a possible alternative to chemical detoxification of cyanide compounds, and various microbial systems allowing cyanide degradation have been described. Enzymatic pathways involving hydrolytic, oxidative, reductive, and substitution/transfer reactions are implicated in detoxification of cyanide by bacteria and fungi. Amongst enzymes involved in transfer reactions, rhodanese catalyzes sulfane sulfur transfer from thiosulfate to cyanide, leading to the formation of the less toxic thiocyanate. Mitochondrial rhodanese has been associated with protection of aerobic respiration from cyanide poisoning. Here, the biochemical and physiological properties of microbial sulfurtransferases are reviewed in the light of the importance of rhodanese in cyanide detoxification by the cyanogenic bacterium Pseudomonas aeruginosa. Critical issues limiting the application of a rhodanese-based cellular system to cyanide bioremediation are also discussed.     

Design and synthesis of N-benzylpiperidine-purine derivatives as new dual inhibitors of acetyl- and butyrylcholinesterase

The synthesis and biological evaluation of N-benzyl-(piperidin or pyrrolidin)-purines are described. Compounds derived from N-benzylpiperidine and N-substituted purines showed moderate acetylcholinesterase inhibition. Preliminary structure–activity relationships and a superimposition of the best compound with the active conformation of donepezil have revealed structural features that have been used in the design of more potent N-benzylpiperidine inhibitors bearing an 8-substituted caffeine fragment and a methoxymethyl linker. These new compounds are interesting dual inhibitors of acetylcholinesterase and butyrylcholinesterase and have been chosen for further optimisation.     

Possible role of hydrogen cyanide in chemical evolution investigation of the proposed direct synthesis of peptides from hydrogen cyanide

The compounds formed upon oligomerization of cyanide in aqueous solution have been separated into acidic, basic, amphoteric and neutral fractions. Urea is the major constituent of the neutral fraction and oxalic acid is present in the acidic fraction. The oligomerization mixtures isolated in the acid, basic and amphoteric fractions consist of low molecular weight substances which yield amino acids on acid hydrolysis. Citrulline has been identified as a major amino acid released on acid hydrolysis of the product mixture. No amino acids are released from the oligomerization mixture by pronase or carboxypeptidase A. This catalyzed hydrolysis demonstrates that this system does not contain compounds with peptide links. The oligomerization products are susceptible to oxidizing agents but are affected little by reducing agents.     

Induction of erythroid differentiation in vitro by purines and purine analogues

The effectiveness of purines and purine analogues as inducers of erythroid differentiation in cultured murine erythroleukemia cells has been investigated. These cell lines have previously been shown to differentiate in vitro in response to dimethyl sulfoxide (DMSO) and a number of other polar solvents. Two purine analogues, 6-thioguanine and 6-mercaptopurine, as well as the naturally occurring purine, hypoxanthine, are shown to be extremely potent inducers. 6-Thioguanine is effective at a concentration of 0.06 mM, 750 fold lower than the DMSO concentration required for equivalent induction. 6-Mercaptopurine and hypoxanthine are effective inducers at a concentration of approximately 2 mM. Accumulation of globin mRNA was monitored during induction with purine inducers and shown to be similar in amount to globin mRNA levels reached in DMSO-induced cultures. Induction of differentiation by all three compounds follows a similar time course to induction with DMSO. All three compounds are potent inducers of HGPRT (hypoxanthine-guanine phosphoribosyltransferase)-negative cell lines; hence incorporation of purines into DNA is not required for induction of differentiation. Comparison of these compounds with other purines and purine analogues suggests a high degree of specificity in their interaction with a cellular target.     

The Cold Origin of Life: B. Implications Based on Pyrimidines and Purines Produced From Frozen Ammonium Cyanide Solutions

A wide variety of pyrimidines and purineswere identified as products of a dilute frozen ammoniumcyanide solution that had been held at –78°C for 27 years.This demonstrates that both pyrimidines and purines couldhave been produced on the primitive earth in a short time byeutectic concentration of HCN, even though the concentrationof HCN in the primitive ocean may have been low. We suggestthat eutectic freezing is the most plausible demonstratedmechanism by which HCN polymerizations could have producedbiologically important prebiotic compounds.     

Selection of microbial biocatalysts for the reduction of cyclic and heterocyclic ketones

The reduction of carbonyl compounds plays an important role in the synthesis of complex chiral molecules. In particular, enantiopure substituted cyclic and heterocyclic compounds are useful intermediates for the synthesis of several antiviral, antitumor, and antibiotic agents, and recently, they have also been used as organocatalysts for C-C addition. Alcohol dehydrogenases (ADH) are enzymes involved in the transformation of prochiral ketones to chiral hydroxyl compounds. While significant scientific effort has been paid to the use of aliphatic and exocyclic ketones as ADH substrates, reports on (hetero)cyclic carbonyl compounds as substrates of these enzymes are scarce. In the present study, 109 bacteria and 36 fungi were screened, resulting in 10 organisms belonging to both kingdoms capable of transforming cyclic and heterocyclic ketones into the corresponding alcohols. Among them, Erwinia chrysanthemi could quantitatively reduce cyclododecanone and Geotrichum candidum could stereoselectively reduce N-Boc-3-piperidone and N-Boc-3-pyrrolidinone to their corresponding (S)-alcohols; however, the anti-Prelog isomer was obtained when acetophenone was the substrate.     

Five-membered heteroaromatic ring fused-pyrimidine derivatives: Design,synthesis, and hedgehog signaling pathway inhibition study

A series of novel five-membered heteroaromatic ring fused-pyrimidine derivatives including purines, pyrrolo[2,3-d]pyrimidines, pyrrolo[3,2-d]pyrimidines, thieno[2,3-d]pyrimidines, thieno[3,2-d]pyrimidines and furo[3,2-d]pyrimidines have been identified to be potent inhibitors of hedgehog signaling pathway. The synthesis and SAR of these compounds are described. Among this new series of hedgehog signaling pathway inhibitors, most compounds exhibited significant inhibitory activity compared to vismodegib, indicating that the five-membered heteroaromatic ring fused-pyrimidines stand out as encouraging scaffolds among the currently reported structural skeletons for hedgehog signaling pathway inhibitors, deserving more exploration and further investigation.     

Formation of heterocyclic amines using model systems

Initially, modeling was used to identify the mutagenic heterocyclic amines and their precursors. Major precursors have been shown to be single amino acids or amino acids together with creatine or creatinine. There is also evidence that Maillard reactions are involved since heating sugar and amino acids together with creatine or creatinine has been shown to produce several of the mutagenic heterocyclic amines, especially the aminoimidazoazaarenes (AIA compounds), e.g., IQ, MeIQ, MeIQx, DiMeIQx and PhIP. Due to a low yield in the model systems, the mechanisms behind the formation of the mutagenic heterocyclic amines are still unclear and need further substantiation. The fact that some AIA compounds are also produced in the absence of sugar casts some doubts on an obligatory participation of the Maillard reaction; alternative routes might exist. Further work using isotopically labeled precursors needs to be done and so far such work has only been performed for PhiP. The formation of mutagenic heterocyclic amines is dependent on time, temperature, pH, concentration of the precursors, type of amino acid, and the presence of certain divalent ions. Water may have an impact both as a temperature regulator and as a solvent medium for the reactants.     

Biological degradation of cyanide compounds

Cyanide compounds are produced as waste products of a number of industrial processes and several routes for their removal from the environment are under investigation, including the use of biodegradation. The most recent developments in this area have come from studies of the hydrolytic and oxidative pathways for biodegradation and the conditions that affect their activity. The biodegradation of cyanide under anaerobic conditions has also recently demonstrated the feasibility for concomitant biogas generation, a possible economic benefit of the process. Significant advances have been reported in the use of plants for the phytoremediation of cyanide compounds and evidence for the biodegradation of thiocyanate and metal-cyanide complexes has become available. Despite these advances, however, physical and economic factors still limit the application of cyanide biodegradation, as do competing technologies.     

Novel inhibitors of advanced glycation endproducts

A number of natural or synthetic compounds as AGE inhibitors have been proposed, discovered or currently being advanced by others and us. We have identified two new classes of aromatic compounds; aryl- (and heterocyclic) ureido and aryl (and heterocyclic) carboxamido phenoxyisobutyric acids, and benzoic acid derivatives and related compounds, as potential inhibitors of glycation and AGE formation. Some of these novel compounds also showed "AGE-breaking" activities in vitro. Current evidence is that chelation of transition metals and/or trapping or indirect inhibition of formation of reactive carbonyl compounds are involved in the mechanisms of action of these novel AGE inhibitors and breakers. Here, we review the inhibitors of glycation and AGE-breakers published to date and present the results of our in vitro and in vivo investigations on a number of these novel AGE inhibitors. These AGE-inhibitors and AGE-breakers may find therapeutic use in the treatment of diseases that AGE formation and accumulation may be responsible for their pathogenesis such as diabetes, Alzheimer's, rheumatoid arthritis, and atherosclerosis.     

Chromatographic and related techniques for the determination of aromatic heterocyclic amines in foods

Some 20 years ago, Japanese scientists discovered a new group of highly toxic compounds, classified as heterocyclic aromatic amines, from broiled and grilled meat and fish products. Numerous studies have shown that most HAs are mutagenic and carcinogenic, and the safety of HA-containing foods has become a concern for the public. To date, more than 20 different mutagenic and/or carcinogenic heterocyclic amines have been identified in foods. This paper reviews the analysis of foods for HAs with 145 references. We survey some of the numerous methods available for the chromatographic analysis of heterocyclic amines and highlight the recent advances. We discuss chromatographic and related techniques, including capillary electrophoresis, and their coupling to mass spectrometry for the determination of these contaminants in foods. In addition, the review summarises data on the content of HAs in various cooked foods.     

Derivatives of imidazotriazine and pyrrolotriazine C-nucleosides as potential new anti-HCV agents

Previous investigations identified 2′-C-Me-branched ribo-C-nucleoside adenosine analogues, 1, which contains a pyrrolo[2,1-f][1,2,4]triazin-4-amine heterocyclic base, and 2, which contains an imidazo[2,1-f][1,2,4]triazin-4-amine heterocyclic base as two compounds with promising anti-HCV in vitro activity. This Letter describes the synthesis and evaluation of a series of novel analogues of these compounds substituted at the 2-, 7-, and 8-positions of the heterocyclic bases. A number of active new HCV inhibitors were identified but most compounds also demonstrated unacceptable cytotoxicity. However, the 7-fluoro analogue of 1 displayed good potency with a promising cytotherapeutic margin.     

An Investigation of Prebiotic Purine Synthesis from the Hydrolysis of HCN Polymers

The polymerization of concentrated NH₄CN solutions has been studied at various temperatures and ammonia concentrations. The products of the oligomerization of ammonium cyanide include adenine and guanine, as well as trace amounts of 2,6-diaminopurine. Our results indicate that the adenine yield is not strongly dependent on temperature. Guanine is produced in lower yield. The original studies by Oró and Kimball (1961) showed that the 6 N HCl hydrolysis of the NH₄CN polymerization supernatant greatly increased the adenine yield. However, this hydrolysis also decomposes adenine and other purines. Therefore, we have measured the yields from an NH₄CN polymerization as a function of hydrolysis time, and found that shorter hydrolytic periods give higher yields of adenine.We have also investigated the hydrolysis of the supernatant at pH 8, which is a more reasonable model of primitive oceanic conditions, and found that the adenine yield is comparable to that obtained with acid hydrolysis (approximately 0.1%). The yield of adenine does not decline at longer hydrolysis times because of the greater stability of adenine at pH 8. The insoluble black polymer formed from NH₄CN has been analyzed by both acid and neutral hydrolysis. In both cases adenine yields of approximately 0.05% were obtained. This suggests that the polymer may have been as important a prebiotic source of purines as the usually analyzed supernatant.     

Brain Purines in a Genetic Mouse Model of Lesch-Nyhan Disease

Abstract: Mice carrying a mutation in the gene encoding the purine salvage enzyme hypoxanthine-guanine phosphoribosyltransferase (HPRT) have recently been produced to provide an animal model for Lesch-Nyhan disease. The current-studies were conducted to characterize the consequences of the mutation on the expression of HPRT and to characterize potential changes in brain purine content in these mutants. Our results indicate that the mutant animals have no detectable HPRT-immunoreactive material on western blots and no detectable HPRT enzyme activity in brain tissue homogenates, confirming that they are completely HPRT deficient (HPRT^-). Despite the absence of HPRT-mediated purine salvage, the animals have apparently normal brain purine content. However, de novo purine synthesis, as measured by [¹⁴C]formate incorporation into brain purines, is accelerated four- to fivefold in the mutant animals. This increase in the synthesis of purines may protect the HPRT^- mice from potential depletion of brain purines despite complete impairment of HPRT-mediated purine salvage.