The enzymatic synthesis of ATP analogues.

The enzymatic synthesis of selected adenosine 5′-triphosphate analogues from their respective 5′-monosphosphates has been achieved using phosphoenolpyruvate synthetase. Adenosine 5′-monophosphate analogues altered at positions 1,6,7,8 or 9 of the purine ring, or at the ribose 2′- or 3′-positions are substrates with 30% conversion to the nucleoside 5′-triphosphate.     

Intermolecular indole–imidazolium complexes. II. Interaction of a block copolymer (L-tryptophan)n–(γ-ethyl-DL-glutamate)m with imidazolium hydrochloride and poly(L-histidine hydrochloride)

The charge-transfer complexes of a poly(L -tryptophan) sequence with imidazolium hydrochloride and poly(L -histidine hydrochloride) have been investigated in 2,2,2-trifluoroethanol by ultraviolet (uv), circular dichroism (CD), and fluorescence techniques. Both complexes exhibit absorption maxima centered at around 275 nm, whereas hypochromism with respect to the combined spectra of the constituents can be observed below 250 nm. All complexes show optical activity in the near uv and in the peptide absorption region, which is discussed in terms of the conformational properties of the donor. A marked decrease of the fluorescence intensity of the L -tryptophan sequence is observed upon addition of imidazolium hydrochloride and poly(L -histidine hydrochloride). From the fluorescence data the formation constant of the charge-transfer complex between the L -tryptophan sequence and imidazolium ions is also evaluated.     

Conformational studies on poly-L-tryptophan: circular dichroism and x-ray diffraction studies

Circular dichroism (CD) measurements were carried out on various copolymers of L -tryptophan and γ-ethyl L -glutamate in ethylene glycol monomethyl ether as the solvent. On increasing the L -tryptophan content of the copolymers a gradual change in the CD spectra was observed. The typical spectrum of the right-handedα-helix becomes more and more evident as the L -tryptophan content decreases. On the basis of these results we assumed that no conformational transition occurs on proceeding from pure poly (γ-ethyl L -glutamate) to pure poly-L -tryptophan in ethylene glycol monomethyl ether: therefore the conformation of poly-L -tryptophan should be that of a right-handed α-helix. Moreover we observed that the change in the CD spectra of the copolymers is gradual but not linear on increasing the tryptophan content. The deviations from linearity were attributed to interactions among side-chain chromophores whose contributions to the optical activity are not simply additive. An x-ray analysis carried out on oriented films of poly-L -tryptophan casted from solutions of the polymer in dimethylformamide shows conclusively that the solid-state conformation of the polymer is that, of an α-helix.     

Isolation and studies of the mutagenic activity in the Ames test of flavonoids naturally occurring in medical herbs

Quercetin, rhamnetin, isorhamnetin, apigenin and luteolin were isolated from medicinal herbs: Erigeron canadensis L., Anthyllis vulneraria L. and Pyrola chloranta L. The mutagenicity of these naturally occurring flavonoids was tested by the Ames method with S. typhimurium strains TA1535, TA1538, TA97, TA98, TA100 and TA102 in the presence and absence of metabolic activation. Of the above flavonoids only quercetin and rhamnetin revealed mutagenic activity in the Ames test. Quercetin induced point mutations in strains TA97, TA98, TA100 and TA102 of S. typhimurium. The presence of S9 rat liver microsome fraction markedly enhanced the mutagenic activity of quercetin in these strains. Rhamnetin appeared to be a much weaker mutagen in the Ames test. The compound induced mutations in strains TA97, TA98 and TA100 of S. typhimurium but only in the presence of metabolic activation.Comparison of the structure of the studied flavonoids with their mutagenic activity indicates that the mutagenicity of flavonoids is dependent on the presence of hydroxyl groups in the 3′ and 4′ positions of the B ring, and that the presence of a free hydroxy or methoxy group in the 7 position of the A ring also probably contributes to the appearance of mutagenic activity of flavonoids in the Ames test. It also appeared that the presence of methoxy groups, particularly in the B ring of the flavonoid molecule, markedly decreases the mutagenic activity of the compound.     

Ribonucleoside Diphosphate Adducts with Elliptinium Acetate,an Antitumor Agent

Abstract

The oxidized form of the antitumor agent elliptinium acetate is able to arylate adenosine and uridine 5′-diphosphate by attack of the 2′-0 position of the ribose and cyclisation, leading to spiro derivatives. Ring opening occurs under reducing conditions and leads to the formation of adducts at 2′ or 3′ positions. Spiro and uncyclised adducts showed low cytotoxicity against L1210 cells in vitro.     

Leaching of the allelopathic substance, -tryptophan from the foliage of mesquite (Prosopis juliflora (Sw.) DC.) plants by water spraying

The allelopathic potential of -tryptophan in the leachate from the foliage of mesquite (Prosopis juliflora (Sw.) DC.) plants was investigated. Distilled water (500 ml) was sprayed on mesquite plants from above for 50 min and water passing through the foliage was collected. The content of -tryptophan in the sample (30 ml eq.) was analyzed using a physicochemical method and the concentration was shown to be 17.9 µM. The sample (30 ml eq.) caused 69.8% inhibition of the root growth of barnyard grass (Echinochloa crus-galli L.). The concentration of authentic -tryptophan required for 69.8% inhibition of the root growth of barnyard grass was estimated at 20.0 µM from the dose-response curve. Moreover, to establish the origin of -tryptophan in the leachate, the amount of -tryptophan in both the leaves and the leachates was determined. The amount of -tryptophan containing in the foliage significantly decreased by soaking with time, whereas in the leachates the amount increased. After 60 min, its content in the leachates was nearly equivalent to that of the leaves. These results suggest that -tryptophan leached from the foliage may play an important role in the allelopathy imposed by mesquite.     

Organometallic Intermediates in the Synthesis of Nucleoside Analogs

Abstract

The common nucleosides, modified or derivatized in some way at the heterocyclic ring carbons, include examples of structures which a r e useful as biological probes and chemotherapeutic agents. Like previous authors, we will use the term “nucleoside analog” for structures related to one of the common naturally occurring nucleosides. Nucleosicle analogs can be derivatives which differ by such minor modification as replacement of hydrogen by a single atom or derivatives which are grossly modified at both the carbohydrate and the base. Examples of the former include 5-fluoro-2′-deoxyuridine, an inhibitor of thymidylate synthetase as its 5′-phosphate, and 5′-iodo-2′-deoxyuridine, a clinically useful antiviral agent. Larger groups have frequently been linked to nucleoside as probes for enzymatic processes. Side chains in “nonrestricted positions” may be used to carry spectroscopic or chemically reactive probes, or provide the means to attach a molecule to an affinity column. Ultimately with positions of bulk tolerance defined, it may be possible to design “active site directed irreversible enzyme inhibitors” as defined by B.R. Baker. Nucleoside structures in which a side chain is attached at a pyrimidine or purine carbon will undoubtedly, in some instances be the most appropriate structure. Yet, these have typically been more difficult to synthesize than analogs with side chains attached to heteroatoms.     

Separation of pure polychlorinated biphenyl isomers into two types of inducers on the basis of induction of cytochrome P-450 or P-448.

A number of isomerically pure polychlorinated biphenyls (PCBs) were tested as inducers of hepatic drug-metabolizing enzymes in the rat. The chlorinated biphenyl isomers can be categorized into two distinct groups of inducers, while commercial PCB mixtures have characteristics of both groups. Biphenyls chlorinated symmetrically in both the meta and para positions (3,4,3′,4′- and 3,4,5,3′,4′,5′-) increase the formation of cytochrome P-448, the ratio of the 455 to 430 peaks of the ethyl isocyanide difference spectrum, and aryl hydrocarbon hydroxylase and glucuronyl transferase activities, but decrease aminopyrine N-demethylase activity. These isomers are also the most toxic, as measured by weight loss. Biphenyl isomers chlorinated in both the para and ortho positions induce the formation of cytochrome P-450 rather than P-448, regardless of the chlorination of the meta position. These isomers, which include 2,4,2′,4′-tetra- and 2,4,5,2′,4′,5′-, 2,3,4,2′,3′,4′- and 2,4,6,2′,4′,6′-hexachlorobiphenyls, increase cytochrome P-450 and N-demethylase activity, but produce only a slight increase in aryl hydrocarbon hydroxylase activity, and do not alter the peak of the CO-difference spectrum or the ratio of the 455/430 peaks of the ethyl isocyanide difference spectrum. Isomers which are chlorinated in only one ring, or are chlorinated in both rings but not in the para positions, have very little activity as inducers of liver enzymes. Of the dichlorobiphenyls tested, 3,3′- and 4,4′-dichlorobiphenyls have very slight activity at extremely high doses.     

Synthesis and biological evaluation of partially fluorinated antiprogestins and mesoprogestins

A series of antiprogestins have been synthesized by partially fluorinating the steroid molecule in positions relevant for receptor binding. By introducing fluorine at the exo-methylene of the 17 spirofuran ring, we obtained partial agonists (mesoprogestins) with significant applications for antiproliferative and antiovulatory treatment strategies in gynecological therapy such as uterine fibroids, endometriosis and heavy menstrual bleeding. Compared to the standard drug RU486, our synthesized compounds exhibited considerable dissociation between antiprogestational and antiglucocorticoid PR receptors. Furthermore, our studies have shown that pure antiprogestins can be generated by partially fluorinating the 17 propenyl and propynl group or by substituting the 4′ acetyl phenyl group in the 11 position using trifluromethyl group.     

Conformational Analysis of the Deoxyribofuranose Ring in DNA by means of Sums of Proton-proton Coupling Constants: A Graphical Method

Abstract

A graphical method is presented for the conformational analysis of the sugar ring in DNA fragments by means of proton-pro ton couplings. The coupling data required for this analysis consist of sums of couplings, which are referred to as Σ1′ (= J1′2′ + J1′2″), Σ2′ (= J1′2′ + J2′3′+J2′2″), Σ2″ (= J1′2″ + J 2″3′ + J2′2″) and Σ3′ (= J2′3′ + J2″3′ + J3′4′). These sums of couplings correspond to the distance between the outer peaks of the H1′, H2′, H2″ and H3′ {³¹P} resonances, respectively, (except for Σ2′ and Σ2″ in the case of a small chemical shift difference between the H2′ and H2″ resonances) and can often be obtained from ¹H-NMR spectra via first-order measurement, obviating the necessity of a computer-assisted simulation of the fine structure of these resonances. Two different types of graphs for the interpretation of the coupling data are discussed: the first type of graph serves to probe as to whether or not the sugar ring occurs as a single conformer, and if so to analyze the coupling data in terms of the geometry of this sugar ring. In cases where the sugar ring does not occur as a single conformer, but as a blend of N-and S-type sugar puckers, the second type of graph is used to analyze the coupling data in terms of the geometry and population of the most abundant form.

It is shown that the latter type of analysis can be carried out on the basis of experimental values for merely Σ1′, Σ2′ and Σ2″, without any assumptions or restrictions concerning a relation between the geometry of the N- and S-type conformer. In addition, the question is discussed as to how insight can be gained into the conformational purity of the sugar ring from the observed fine structure of the H1′ resonance. Finally, a comparison is made between experimental coupling data reported for single-stranded and duplex DNA fragments and covalent RNA-DNA hybrids on the one hand and the predicted couplings and sums of couplings presented in this paper on the other hand.     

A flavin-dependent tryptophan 6-halogenase and its use in modification of pyrrolnitrin biosynthesis

Regioselective halogenation of electron rich substrates is catalysed by flavin-dependent halogenases. Thienodolin produced by Streptomyces albogriseolus contains a chlorine atom in the 6-position of the indole ring system and is believed to be derived from tryptophan. Using the gene of the tryptophan 7-halogenase (PrnA) from the pyrrolnitrin biosynthetic gene cluster the gene for a tryptophan 6-halogenase was cloned, sequenced and heterologously overexpressed in Pseudomonas strains. In vitro activity of the purified enzyme could only be shown in a two-component enzyme system consisting of the halogenase, a flavin reductase, NADH, FAD and halide ions. The enzyme catalyses the regioselective chlorination and bromination of l- and d-tryptophan. In its native form the enzyme is probably a homodimer with a relative molecular mass of the subunits of 63 600 (including the His-tag). Transformation of the pyrrolnitrin producer Pseudomonas chlororaphis ACN with a plasmid containing the tryptophan 6-halogenase gene lead to the formation of the new aminopyrrolnitrin derivative 3-(2′-amino-4′-chlorophenyl) pyrrole.     

TRYPTOPHAN TRANSPORT ACROSS THE SYNAPTOSOMAL MEMBRANE1

Crude mitochondrial fractions prepared from rat brains took up l -tryptophan. The component of the crude mitochondrial fraction responsible for this uptake is the synaptosome. After uptake of tryptophan occurred, rupture of synaptosomes released 97 per cent of the tryptophan unchanged. Rupture of synaptosomes abolished uptake. Penetration of the limiting membrane of synaptosomes by l -tryptophan both as influx and efflux was studied. Uptake of l -tryptophan was rapid, temperature dependent, partially inhibited by cyanide, 2-deoxy-d -glucose and ouabain, but apparently unaffected by low external sodium ion concentrations. d -tryptophan was a poor inhibiteur of l -tryptophan uptake. Concentration gradients Internal: external of up to 4:1 were achieved. Kinetic studies on l -tryptophan uptake and its competitive inhibition by l -phenylalanine indicated a saturable carrier-mediated transport system, present in the rat at birth. l -Tryptophan efflux from preloaded synaptosomes was markedly stimulated by certain arrino acids and its influx stimulated by preloading with l -tryptophan. This countertransport is further evidence for carrier-mediated or facilitated diffusion. On the basis of countertransport data there seem to be at least two systems for transporting amino acids across synaptosomal membrane. The relevance of these studies to the role of l -tryptophan as the initial precursor of brain 5-hydroxytryptamine is examined.     

Genetic engineering of Escherichia coli to enhance production of l-tryptophan

Reducing the accumulation of acetate in Escherichia coli cultures can decrease carbon efflux as by-products and reduce acetate toxicity, and therefore enable high cell density cultivation. The concentration of intracellular amino acids can be decreased by genetic modifications of the corresponding amino acid transport systems. This can increase the levels of amino acids in the fermentation broth by decreasing the feedback inhibition on the corresponding biosynthetic pathways. Here, the effects of genetic manipulation of phosphate acetyltransferase (pta), high affinity tryptophan transporter (mtr) and aromatic amino acid exporter (yddG) on l-tryptophan production were investigated. The pta mutants accumulated less acetate and showed higher capacity for producing l-tryptophan as compared with the parental strain. The strains lacking mtr, or overexpressed yddG, or with the both mtr knockout and yddG overexpression, accumulated lower concentrations of intracellular tryptophan but higher production of extracellular l-tryptophan. In the l-tryptohan fed-batch fermentation of an E. coli derived from TRTH0709/pMEL03 having deletion of pta-mtr and overexpression of yddG in a 30-L fermentor, the maximum concentration of l-tryptophan (48.68 g/L) was obtained, which represented a 15.96 % increase as compared with the parental strain. Acetate accumulated to a concentration of 0.95 g/L. The intracellular concentration of l-tryptophan was low, and the glucose conversion rate reached a high level of 21.87 %, which was increased by 15.53 % as compared with the parent strain.     

Adducts from mercury(I) and mercury(II) compounds with Bispyrazolylalkanes.X-Ray crystal structure of Bis(3,5-dimethylpyrazol-l-yl)methane(dicyano)-mercury(II)

The 1:1 adducts between thebis(3,5-dimethylpyrazol-1-yl)methane (L′-L′) or 2,2′-bis(pyrazol- 1-yl)propane (L″L″) ligand and HgX₂ (with X = Cl, CN or CO₂CF₃) have been obtained as well as [(L′L′)₂]Hg(ClO₄)₂ and the mercury(I) derivative (ligand)₂Hg₂(ClO₄)₂. The adducts have been characterized from analytical and spectral data (IR, proton and ¹³C NMR). Four-coordinated mercury is present in (L′L′)Hg(CN)₂, in which the metal-(NN)₂C ring adopts an asymmetric boat form. The molecular parameters are significantly different for the two independent molecules, the CHgC angles and the two Hg-N distances being 163.1(9)°and 2.55(1) plus 2.70(1) Å in the one case, and 148.2(8)° and 2.40(1) plus 2.51(1) Å, in the other; correspondingly the N-Hg-N angle, the ‘bite’ of the ligand, ranges from 79.0(5)° to 71.7(4)°, a value outside the range previously reported.     

Cyclopeptide alkaloids from Melochia corchorifolia

Adouetine-y′ and a new cyclopeptide alkaloid, melofoline, have been isolated from melochia corchorifolia. The latter was characterized mainly from its mass spectrum and hydrolysis products. Melofoline has N,N-dimethyl-β-hydroxyleucine as the terminal amino acid and 2-aminobutyric acid as the ring amino acid, neither of which has been found in the positions before.     

Hyper-production of l-trytophan via fermentation with crystallization

A stable and fast l-tryptophan producer, AGX1757, was isolated from Escherichia coli W3110 trpAE1 trpR tnaA, which carried pSC101-trpI15·14. Cells of AGX1757 did not lose the composite plasmid during fermentation. Whenever a fed-batch culture of AGX1757 attained an l-tryptophan concentration of about 30 g/l, indole began to appear in the broth. The emergence of indole was caused by inhibition of tryptophan synthase due to accumulated l-tryptophan. Hence, the production rate of l-tryptophan sharply decreased. A higher solubility of l-tryptophan in the supernatant of culture broth (about 32 g/l) than that in the initial medium (about 22 g/l) was attributed to some unknown interaction between l-tryptophan and certain macromolecular material(s) coming from the bacterial cells. An addition of non-ionic detergents into the supernatant was effective for decreasing the solubility of l-tryptophan, hence causing crystallization of l-tryptophan. Pluronic L-61 was supplied from outside to an extent of 0.5% in terms of wt% concentration at around 45 h of fermentation when the l-tryptophan accumulated reached about 25 g/l. This addition actually caused crystallization of l-tryptophan and, as a result, the inhibitory effect of tryptophan synthase by l-tryptophan accumulated in the broth could be alleviated. Thus far, further fermentation became possible. l-Tryptophan of more than 50 g/l was finally produced by feeding solutions of both glucose and anthranilic acid. Correspondence to: H. Tsunekawa     

Gene modification of <Emphasis Type="Italic">Escherichia coli</Emphasis> and incorporation of process control to decrease acetate accumulation and increase ʟ-tryptophan production

Acetate is a primary inhibitory metabolite in cultures of Escherichia coli, and the production of both biomass and desired products are increased by reducing the accumulation of acetate. In this study, the accumulation of acetate during ?-tryptophan production was decreased by genetic modification of ?-tryptophan-producing strain (BCTRP) and optimization of the fermentation process. The mutant (BCTRPG), which has a deletion of the integral membrane permease IICB^Glc (ptsG), produces a higher concentration of ?-tryptophan than mutants with deletions of either phosphate acetyltransferase (pta) or pta–ptsG, due to the low accumulation of acetate and other byproducts, as well as high biomass production. The appropriate dissolved oxygen (DO) level, glucose feeding mode, and pH control strategy were applied to ?-tryptophan production using the BCTRPG mutant. The BCTRPG strain with optimized conditions resulted in a reduction in acetate accumulation (71.08% reduction to 0.72 g/L) and an increase in ?-tryptophan production (35.81% increase to 17.14 g/L) compared with the BCTRP strain in the original culture condition. Meanwhile, an analysis of the metabolic flux distribution indicated that the acetate synthesis flux decreased from 19.2% (original conditions) to 8.4% (optimized conditions), and the flux of tryptophan formation with the optimized conditions was 18.5%, which was 1.89 times higher than under the original conditions. This study provided the theoretical foundation and technical support for high-level industrialization production of ?-tryptophan.     

Tuning of the redox potential of the primary electron donor in reaction centres of purple bacteria: effects of amino acid polarity and position

Mutation of residues His L168 and Phe M197 in the reaction centre from Rhodobacter sphaeroides has an unusually strong effect on the mid-point redox potential (E(m)) of the pair of bacteriochlorophylls that form the primary donor of electrons, tuning E(m) over a range of nearly 250 mV. This effect is correlated to the accompanying change in the permanent dipole of the L168 or M197 residue, suggesting it is mediated by changes in charge-dipole interactions. Comparisons with mutations made at a variety of other positions show that this correlation is particular to this residue pair, perhaps reflecting their proximity to the ring I regions of the dimer bacteriochlorophylls that form the overlap region between these molecules.