Steric and electronic properties of the cofactor's amino group control the lifetime of the central carbanion/enamine intermediate in transketolase

Transketolase (TK), a thiamin diphosphate (ThDP) dependent enzyme, catalyzes the reversible transfer of a two-carbon unit from keto- to aldo-substrates. Dihydroxyethylthiamin diphosphate (DHEThDP), formed as a result of cleavage of the donor substrate, serves as an intermediate of the TK reaction. TK from the yeast Saccharomyces cerevisiae is unique among thiamin enzymes displaying enzymatic activity after reconstitution with a methylated analogue of the native cofactor, 4′-methylamino-ThDP. The reconstitution of the apoenzyme with both ThDP and the methylated analogue can be analyzed by near UV circular dichroism. It was demonstrated that in the native holoenzyme and in the complex of TK with 4′-methylamino-ThDP the formation of the dihydroxyethyl-based carbanion/enamine took place with comparable rate constants, whereas the protonation of the reactive species was much faster in the complex with the analogue. The enzymatic activity of the enzyme reconstituted with 4′-methylamino-ThDP was 10fold higher in the ferricyanide assay. We suggest that a methylation of the 4′-amino group of ThDP impairs the resonance stabilization of the carbanion/enamine intermediate both sterically and electronically, thus allowing either a faster protonation or oxidation reaction by ferricyanide. The formation of the optically active DHE-4′-methylamino-ThDP was monitored by near UV circular dichroism spectra and corroborated by ¹H NMR analysis. The protonated form of the intermediate DHE-4′-methylamino-ThDP was released from the active sites of TK and accumulated in the medium on preparative scale.     

Synthesis and biological activity of 17α-alkynylamide derivatives of estradiol

Seven estradiol (E₂) derivatives with an alkynylamide side chain at the 17α position were synthesized starting from ethynylestradiol (EE₂). The main chemical step was the coupling reaction of the acetylide ion of EE₂ with carbon dioxide, glutaric anhydride or bromoalkyl ortho ester. The synthesis of these compounds is fast (3–6 steps according to the compound) and is easily achieved with good yield. Five compounds with different side chain lenghts were evaluated for uterotrophic and antiuterotrophic activity in the CD-1 mouse. None of the tested compounds shows estrogenic activity in this sensitive in vitro system. At low doses (1 and 3 μg), a 14–57% inhibition of E₂-induced uterine growth was observed while no additional inhibition was observed at the 10, 20 and 30 μg doses. In human breast carcinoma cells in culture, all compounds show estrogenic activity at high concentrations while only compound 39 (N-buty,N-methyl-8-[3′,17′β-dihydroxy estra-1′,3′,5′(10′)-trien-17′α-yl]-7-octynamide) possesses antiproliferative or antiestrogenic effects. No significant correlation could be demonstrated between alkynylamide side chain length and estrogenic or antiestrogenic activity. Among the compounds tested, the derivative of EE₂ possessing a five-methylene (CH₂) side chain (compound 39) possesses the best antiestrogenic activity (44 ± 7% in the CD-1 mouse uterus assay at the 3μg dose and 57 ± 4% at 0.1 nM in human ZR-75-1 cancer cells in culture).     

Biocatalytic application of nitrilases from Fusarium solani O1 and Aspergillus niger K10

The nitrilases from Fusarium solani O1 and Aspergillus niger K10 showed a broad substrate specificity for carbocyclic and nonaromatic heterocyclic amino nitriles, the preferred substrates being five-membered γ-amino nitrile (±)-1a, six-membered γ-amino nitriles (±)-3a, (±)-5a and (±)-6a, pyrrolidine-3-carbonitriles (±)-9a and (±)-10a as well as piperidine-4-carbonitriles 14a and 15a. Both enzymes showed a strong diastereopreference for cis- vs. trans-γ-amino nitriles. The electronic and steric effects of N-protecting groups affected the reactivity of the nitriles. Amides as by-products of the nitrilase-catalyzed reaction were produced from heterocyclic amino nitriles (±)-9a, (±)-10a, 14a and 15a by the A. niger enzyme but only from nitrile (±)-9a by the F. solani enzyme.     

The reaction of the mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] with guanosine and cysteine

The mutagen 1,1′-hexamethylene-bis[(5-p-chlorophenyl)-biguanide] reacts at 37°C with guanosine and guanine to yield xanthosine or xanthine and oxidizes cysteine to cystine. After treatment of a guanosine-labelled DNA sample from Escherichia coli with the mutagen xanthine could be detected as a reaction product. At a slow rate the mutagen is hydrolysed spontaneously yielding urea, 1,6-hexanediol and 4-chloroaniline. The reaction mechanisms both of the hydrolysis and of the reaction with cysteine and guanosine are discussed.     

Sulphoconjugation of steroids in porcine liver: Partial purification of the cytosolic sulphotransferases for pregnenolone and 5α-androst-16-en-3β-ol

Steroid sulphotransferase activities for 5α-androst-16-en-3β-ol and pregnenolone in porcine liver cytosol have been assayed using 3′-phosphoadenosine-5′-phospho[³⁵S]sulphate as sulphate donor. 5α-Androst-16-en-3β-ol sulphotransferase activity was obtained from porcine liver cytosol by gel filtration chromatography; activity was linear with time up to about 5 min., the optimum pH was near 8.0 and optimum temperature 37°C. Pregnenolone sulphotransferase activity was partially purified from porcine liver cytosol using DEAE-cellulose chromatography with an ionic gradient of KCl. This enzyme activity was linear with time up to 10 min and had optimum pH and temperature of 8.0 and 37°C, respectively.     

Structural versatility of peptides containing C-dialkylated glycines. An X-ray diffraction study of six 1-aminocyclopropane-1-carboxylic acid rich peptides

The molecular and crystal structures of six fully blocked, Ac₃c-rich peptides to the tetramer level were determined by X-ray diffraction. The peptides are Fmoc-(Ac₃c)₂-OMe·CH₃OH, Ac-(Ac₃c)₂-OMe, t-Boc-Ac₃c-l-Phe-OMe, pBrBz-(Ac₃c)₃-OMe·H₂O, Z-Gly-Ac₃c-Gly-OTmb·(CH₃₂CO, andt-Boc-(Ac₃c)₄-OMe·2H₂O. Type-I (I′) β-bends and distorted 3₁₀-helices were found to be typical of the tri- and tetrapeptides, respectively. In the dipeptides, too short to form β-bend conformations, other less common structural features may be observed. The average geometry of the cyclopropyl moiety of the Ac₃c residue is asymmetric and the N-C^α-C′ bond angle is significantly expanded from the regular tetrahedral value. A comparison with the structural preferences of other extensively investigated C^α,α-dialkylated α-amino acids is made and the implications for the use of the Ac₃c residue in conformational design are examined.     

Acid-catalyzed hydrolysis of α-ketoglutaramic acid : A proposed mechanism involving decarboxylation and amide hydrolysis of the γ-lactam, 2-pyrrolidone-5-hydroxy-5-carboxylic acid

The rates of the acid-catalyzed decarboxylation and amide hydrolysis of α-ketoglutaramic acid, the keto analog of glutamine, were investigated and the products of the reactions were characterized. In strong acid at 100°C, amide hydrolysis and decarboxylation occur with about equal facility, yielding α-ketoglutaric acid and 5-hydroxy-2-pyrrolidone, respectively. 5-Hydroxy-2-pyrrolidone undergoes further amide hydrolysis so that the products of complete acid hydrolysis of α-ketoglutaramic acid are ammonia (100%), carbon dioxide (50%), and approximately equal yields (50%) of α-ketoglutaric acid and succinic semialdehyde (β-formylpropionic acid). At increasing pH values, the relative rate of decarboxylation to amide hydrolysis of α-ketoglutaramic acid increases, such that, at pH values of 2 or greater, decarboxylation occurs almost exclusively. The decarboxylation product 5-hydroxy-2-pyrrolidone, was characterized chromatographically and by its infrared and pmr spectra; the compound may be regarded as the cyclized form of succinamic semialdehyde. A mechanism for the competing amide hydrolysis and decarboxylation reactions is proposed, and the potential biological significance of the decarboxylation pathway is discussed.     

Possible reactions of 1,2-naphthaquinone in the eye

1. Reactions of 1,2-naphthaquinone with amino acids, glutathione and proteins of the lens have been studied in connexion with investigations of naphthalene-induced cataract. 2. Cysteine reacts probably through its amino group with 1,2-naphthaquinone to form either purple or brown compounds with characteristic absorption spectra. 3. Glutathione reacts with 1,2-naphthaquinone through its thiol group. 4. Spectroscopic evidence suggests that 1,2-naphthaquinone reacts with the amino group of amino acids. This reaction may take place in the aqueous humour. 5. The proteins of lens react with 1,2-naphthaquinone to form brown compounds. 6. There is loss of protein thiol in this reaction and the products are less easily digestible by pancreatin than normal lens proteins. 7. The compound of α-crystallin and 1,2-naphthaquinone is soluble at neutrality, but the compounds of β-crystallins and of γ-crystallins are largely insoluble. 8. The brown reaction products of glutathione or cysteine with 1,2-naphthaquinone catalyse the oxidation of ascorbic acid in the same way as 1,2-naphthaquinone itself. 9. These results are discussed in relation to naphthalene-induced cataract.     

Oxygen-dependent increase of antioxidants in soybean embryonic axes

Oxygen, although essential for the survival of aerobic organisms leads to generation of toxic species. The effect of oxygen on enzymatic and non-enzymatic antioxidants was determined to evaluate response to oxidative stress in soybean axes. Soybean seeds were incubated over nutrient solution-saturated filter paper. Different oxygen concentrations in the incubation atmosphere were maintained by gassing either N₂ (40% O₂), air (20% O₂), a commercial mix 40% O₂ + 60% N₂ (40% O₂) or O₂ (100% O₂) in closed plastic chambers. Oxidative stress was assessed by the oxidation of 2′,7′-dichlorofluorescein diacetate. The activities of antioxidant enzymes were determined spectrophotometrically. α-Tocopherol and ubiquinol-10 contents were measured by HPLC. The weight of axes was 13 ± 1 and 27 ± 3 mg/axis in the absence and presence of 20% oxygen, respectively. 2′,7′-Dichlorofluorescein diacetate oxidation was increased from 14 ± 2 to 66 ± 5 AU/min/mg FW by supplementation of 20% oxygen. Total glutathione content was 22 ± 6 and 33 ± 6 nmol/axis in axes grown in absence of oxygen and air, respectively. Ubiquinol-10 content was not affected by oxygen. α-Tocopherol content decreased from 384 ± 94 to 14 ± 3 pmol/axis in the absence or presence of 100% oxygen, respectively. The activities of antioxidant enzymes increased in axes exposed to oxygen. Our data suggest that exposure of soybean axes to oxygen leads to oxidative stress but damage by oxygen intermediates was limited by increases in the activity of both, antioxidant substances (i.e. glutathione) and antioxidant enzymes.     

Optical resolution of hexamethylbiphenol by cholesterol esterase and porcine pancreas lipase

Both enantiomers of 2,2′-dihydroxy-4,4′,5,5′,6,6′-hexamethybiphenyl (2), a potentially useful chiral synthon, were obtained with >99% ee in high enantioselectivity by cholesterol esterase or porcine pancreas lipase (PPL)-mediated hydrolysis of the corresponding (±)-dipentanoate or (±)-dihexanoate, respectively. Absolute configuration of (S)-3-bromo-2,6′-dimethoxy-4,5,6,2′,3′,4′-hexamethyl-biphenyl (2h) was determined by X-ray analysis.     

Multinuclear (P, Pt) magnetic resonance and electrospray mass spectrometric studies of the reactions between platinum bis(dithiolates) and two potentially tetradentate phosphine ligands

The reactions of platinum(II) bis(dithiolates) Pt(S---S)₂ ((S---S)=S₂P(OEt)₂ (dtp), S₂COⁿPr (xan), S₂CNEt₂ (dtc)) with two potentially tetradentate phosphine ligands have been investigated by multinuclear magnetic resonance spectroscopy and electrospray mass spectrometry (ESMS). The phosphines used were P(CH₂CH₂PPh₂)₃ (P₃P′) and Ph₂PCH₂CH₂P(Ph)CH₂CH₂P(Ph)CH₂CH₂PPh₂ (P₂P′₂). ³¹P and ¹⁹⁵Pt NMR spectroscopies show that P₃P′ reacts in dichloromethane solution with Pt(dtp)₂ and Pt(xan)₂ to give five-coordinate [(η⁴-P₃P′)Pt(η¹-S---S)]⁺ and with Pt(dtc)₂ to give a temperature dependent mixture of [(η⁴-P₃P′)Pt(η¹-dtc)]⁺ and [(η³-P₃P′)Pt(η²-dtc)]⁺. All these formulations were confirmed by observation of the intact ions in the ES mass spectra directly from the solutions. [(η⁴-P₃P′)Pt(η¹-xan)]⁺ slowly reacts with the free xan ion to give the dithiocarbonate complex (η³-P₃P′)Pt(η²-S₂CO). The pendant phosphine in [(η³-P₃P′)Pt(η²-dtc)]⁺ undergoes various chemical reactions such as methylation and reaction with sulfur, and the cation behaves as a monodentate phosphine towards Pt(dtp)₂ to give [(η¹-dtp)(η²-dtp)Pt(η¹-μ²-η³-P₃P′)Pt(η²-dtc)]⁺ which was fully characterised by multi-NMR spectroscopy and confirmed by observation of the intact ion by ESMS. P₂P′₂ reacts with Pt(dtp)₂ to give [(P₂P′₂)Pt]²⁺, but with Pt(xan)₂ and Pt(dtc)₂ the products are [(η⁴-P₂P′₂)Pt(η¹-S---S)]⁺, but the xanthate complex slowly de-alkylates to give (η³-P₂P′₂)Pt(η²-S₂CO). The identities of the cationic P₂P′₂ species in solution were confirmed by direct observation of the intact ion by ESMS.     

Synthesis and evaluation of alpha,alpha'-disubstituted phenylacetate derivatives for T-type calcium channel blockers

We have synthesized and evaluated α,α′-disubstituted phenylacetate derivatives that were designed as T-type calcium channel blockers. Among them, compound 10e (IC₅₀ = 8.17 ± 0.48 nM) showed the most potent T-type calcium current blocking activity and higher potency than Mibefradil (IC₅₀ = 1.34 ± 0.49 μM). The PK profile and subtype selectivity over L-type calcium channel were satisfied for further animal assay using disease model.     

Dissimilation of methionine by fungi

Soil fungi that attacked methionine required a utilizable source of energy such as glucose for growth. This is an example of co-dissimilation. Experiments with one of the fungi, representative of the group, are reported. In the absence of glucose, pregrown mycelium, even when depleted of energy reserves, oxidatively deaminated methionine with accumulation of α-keto-γ-methyl mercapto butyric acid and α-hydroxy-γ-methyl mercapto butyric acid. When glucose was provided, all of the sulfur of methionine was released as methanethiol, part of which was oxidized to dimethyl disulfide. No sulfate, sulfide, or hydrosulfide products were detected. Evidence was obtained that deaminase and demethiolase were constitutive. Deamination preceded demethiolation and α-keto butyric acid accumulated as a product of the two reactions. Other carbon residues were α-hydroxy butyric acid and α-amino butyric acid. Inability of the fungus to metabolize α-keto butyrate was responsible for its inability to utilize methionine as a source of carbon and energy. Several other fungi isolated from soil grew on α-amino butyrate but could not grow on methionine owing to inability to demethiolate it.     

A study of the reactivity of (methyl 2-acetamidoacrylate)-tricarbonyliron(0) leading to a novel synthesis of β,β,β-trialkyl α-amino acids

(Methyl 2-acetamidoacrylate)tricarbonyliron(0) (3) reacts with 2 equivalents of methyllithium to give methyl N-acetylalaninate (4) and 2-acetamido-4-oxopentanoate (5) when the reaction is quenched with trifluoroacetic acid. Production of methyl N-acetylalaninate is dependent only on the presence of trifluoroacetic acid, and the ratio of 4 to 5 generated in these reactions is related to the quantity of trifluoroacetic acid used to quench them. Addition of two equivalents of methyllithium followed by tertiary haloalkanes gives protected β,β,β-trialkyl α-amino acids which may be hydrolysed to give tert-leucine (13) and the new α-amino acids 2-amino-3,3-dimethylpentanoic acid (14) and 2-amino-3,3-dimethylhexanoic acid (15).     

1-Methoxy-, 1-deoxy-11-hydroxy- and 11-Hydroxy-1-methoxy-Δ-tetrahydrocannabinols: new selective ligands for the CB2 receptor

Three series of new cannabinoids were prepared and their affinities for the CB₁ and CB₂ cannabinoid recptors were determined. These are the 1-methoxy-3-(1′,1′-dimethylalkyl)-, 1-deoxy-11-hydroxy-3-(1′,1′-dimethylalkyl)- and 11-hydroxy-1-methoxy-3-(1′,1′-dimethylalkyl)-Δ⁸-tetrahydrocannabinols, which contain alkyl chains from dimethylethyl to dimethylheptyl appended to C-3 of the cannabinoid. All of these compounds have greater affinity for the CB₂ receptor than for the CB₁ receptor, however only 1-methoxy-3-(1′,1′-dimethylhexyl)-Δ⁸-THC (JWH-229, 6e) has effectively no affinity for the CB₁ receptor (K_i=3134±110 nM) and high affinity for CB₂ (K_i=18±2 nM).     

Amino Acid Composition of the Host-specific Toxin of Helminthosporium carbonum

The host-specific toxin of Helminthosporium carbonum (C₃₂H₅₀N₆O₁₀) was hydrolyzed by 6 n HCl to yield a number of α-amino acids. The common amino acids, proline and alanine, occurred in a ratio of 1:2. Two other unstable α-amino acids that produced lower color values with ninhydrin were also produced. One of these was tentatively identified as 2-amino-2,3-dehydro-3-methylpentanoic acid by electrolytic reduction to isoleucine. Additional ninhydrin-reacting substances were produced in low yield and probably represented secondary hydrolysis products of the unstable amino acids. The finding of an α,β-unsaturated linkage in H. carbonum toxin explains the instability of the compound and may also account for its specific toxicity.     

The Crystal Structure of the Adenylation Enzyme VinN Reveals a Unique β-Amino Acid Recognition Mechanism

Adenylation enzymes play important roles in the biosynthesis and degradation of primary and secondary metabolites. Mechanistic insights into the recognition of α-amino acid substrates have been obtained for α-amino acid adenylation enzymes. The Asp residue is invariant and is essential for the stabilization of the α-amino group of the substrate. In contrast, the β-amino acid recognition mechanism of adenylation enzymes is still unclear despite the importance of β-amino acid activation for the biosynthesis of various natural products. Herein, we report the crystal structure of the stand-alone adenylation enzyme VinN, which specifically activates (2S,3S)-3-methylaspartate (3-MeAsp) in vicenistatin biosynthesis. VinN has an overall structure similar to that of other adenylation enzymes. The structure of the complex with 3-MeAsp revealed that a conserved Asp²³⁰ residue is used in the recognition of the β-amino group of 3-MeAsp similar to α-amino acid adenylation enzymes. A mutational analysis and structural comparison with α-amino acid adenylation enzymes showed that the substrate-binding pocket of VinN has a unique architecture to accommodate 3-MeAsp as a β-amino acid substrate. Thus, the VinN structure allows the first visualization of the interaction of an adenylation enzyme with a β-amino acid and provides new mechanistic insights into the selective recognition of β-amino acids in this family of enzymes.     

Isomerization of d-glucose with sodium aluminate: Mechanism of the reaction

A mechanism for the isomerization of d-glucose to d-fructose by sodium aluminate is proposed, involving transformation of a β-d-glucopyranose-1,3-aluminate complex into an α-d-fructofuranose-1,3,6-aluminate complex through an enolaluminate complex that inhibits the formation of a d-mannose-aluminate complex. The α-d-fructofuranose-1,3,6-aluminate further reacts to form a d-psicose-aluminate complex in substantial yield. Constant degradation of the 6-carbon sugars occurred during the reaction because of the high pH of the solution. The C₆ sugars were analyzed chromatographically but the degradation products were not identified.