Intermediate Amino Research Articles

In situ (1)H NMR study of the biodegradation of xenobiotics: application to heterocyclic compounds.

In vivo or in situ nuclear magnetic resonance (NMR) offers a powerful tool to study the degradation of xenobiotics by microorganisms. Most studies reported are based on the use of heteronuclei, and experiments with xenobiotics have been limited because specifically labeled xenobiotics are not commercially available, with the exception of (19)F and (31)P. (1)H NMR is, thus, of great interest in this area. To avoid problems caused by the presence of water and intrinsic metabolite signals, some studies were performed using a deuterated medium or specific detection of protons linked to the (13)C-(15)N enriched pattern. We report here the application of in situ (1)H NMR, performed directly on culture media, to study the metabolism of heterocyclic compounds. In this review, we show that a common pathway is involved in the biodegradation of morpholine, piperidine, and thiomorpholine by Mycobacterium aurum MO1 and Mycobacterium sp. RP1. In all cases, the first step is the cleavage of the C-N bond, which results in an amino acid. Thiomorpholine is first oxidized to sulfoxide before the opening of the ring. The second step is the deamination of the intermediate amino acid, which leads to the formation of a diacid. We have shown that the cleavage of the C-N bond and the oxidation of thiomorpholine are initiated by reactions involving a cytochrome P450.

A novel route to chlorodimethylsilane

A new efficient laboratory method of preparation of chlorodimethylsilane (Cl(CH3)2SiH) has been elaborated, which is a modification of the Eaborn et al. method (34) and is based on a transsilylation reaction of substituted (amino)dimethylhydrosilanes, R2NSiMe2H (R2 = Me2, Et2, (CH2)n, etc.) with dimethyldichlorosilane (Me2SiCl2). The reaction proceeds at reflux, at 70°C, preferably with an excess of Me2SiCl2. The most important feature of this novel method is a recovery of intermediate (amino)chlorodimethylsilanes (R2NSiMe2Cl), which can be again reduced to R2NSiMe2H. The transsilylation mechanism has been proven by reaction of (diethylamino)methylphenylsilane with Me2SiCl2. The products of this latter reaction are HMePhSiCl and R2NSiMe2Cl, thus a disproportionation mechanism has been excluded. New substituted bis(amino)dimethylsilanes ((R2N)2SiMe2), (amino)dimethylchlorosilanes (R2NSiMe2Cl), and (amino)dimethylhydrosilanes (R2NSiMe2H) have been synthesized and characterized by NMR and IR.Key words: chlorodimethylsilane, (CH3)2SiHCl, synthesis, new laboratory method, transsilylation mechanism.

Common degradative pathways of morpholine, thiomorpholine, and piperidine by Mycobacterium aurum MO1: evidence from (1)H-nuclear magnetic resonance and ionspray mass spectrometry performed directly on the incubation medium.

In order to see if the biodegradative pathways for morpholine and thiomorpholine during degradation by Mycobacterium aurum MO1 could be generalized to other heterocyclic compounds, the degradation of piperidine by this strain was investigated by performing (1)H-nuclear magnetic resonance directly with the incubation medium. Ionspray mass spectrometry, performed without purification of the samples, was also used to confirm the structure of some metabolites during morpholine and thiomorpholine degradation. The results obtained with these two techniques suggested a general pathway for degradation of nitrogen heterocyclic compounds by M. aurum MO1. The first step of the degradative pathway is cleavage of the C---N bond; this leads formation of an intermediary amino acid, which is followed by deamination and oxidation of this amino acid into a diacid. Except in the case of thiodiglycolate obtained from thiomorpholine degradation, the dicarboxylates are completely mineralized by the bacterial cells. A comparison with previously published data showed that this pathway could be a general pathway for degradation by other strains of members of the genus Mycobacterium.

Multiple mechanisms control phosphorylation of PHAS-I in five (S/T)P sites that govern translational repression.

Control of the translational repressor, PHAS-I, was investigated by expressing proteins with Ser/Thr --> Ala mutations in the five (S/T)P phosphorylation sites. Results of experiments with HEK293 cells reveal at least three levels of control. At one extreme is nonregulated phosphorylation, exemplified by constitutive phosphorylation of Ser82. At an intermediate level, amino acids and insulin stimulate the phosphorylation of Thr36, Thr45, and Thr69 via mTOR-dependent processes that function independently of other sites in PHAS-I. At the third level, the extent of phosphorylation of one site modulates the phosphorylation of another. This control is represented by Ser64 phosphorylation, which depends on the phosphorylation of all three TP sites. The five sites have different influences on the electrophoretic properties of PHAS-I and on the affinity of PHAS-I for eukaryotic initiation factor 4E (eIF4E). Phosphorylation of Thr45 or Ser64 results in the most dramatic decreases in eIF4E binding in vitro. However, each of the sites influences mRNA translation, either directly by modulating the binding affinity of PHAS-I and eIF4E or indirectly by affecting the phosphorylation of other sites.

A novel route to chlorodimethylsilane

A new efficient laboratory method of preparation of chlorodimethylsilane (Cl(CH3)2SiH) has been elaborated, which is a modification of the Eaborn et al. method (34) and is based on a transsilylation reaction of substituted (amino)dimethylhydrosilanes, R2NSiMe2H (R2 = Me2, Et2, (CH2)n, etc.) with dimethyldichlorosilane (Me2SiCl2). The reaction proceeds at reflux, at 70°C, preferably with an excess of Me2SiCl2. The most important feature of this novel method is a recovery of intermediate (amino)chlorodimethylsilanes (R2NSiMe2Cl), which can be again reduced to R2NSiMe2H. The transsilylation mechanism has been proven by reaction of (diethylamino)methylphenylsilane with Me2SiCl2. The products of this latter reaction are HMePhSiCl and R2NSiMe2Cl, thus a disproportionation mechanism has been excluded. New substituted bis(amino)dimethylsilanes ((R2N)2SiMe2), (amino)dimethylchlorosilanes (R2NSiMe2Cl), and (amino)dimethylhydrosilanes (R2NSiMe2H) have been synthesized and characterized by NMR and IR.Key words: chlorodimethylsilane, (CH3)2SiHCl, synthesis, new laboratory method, transsilylation mechanism.

Aberrations of ammonia metabolism in ornithine carbamoyltransferase-deficient spf-ash mice and their prevention by treatment with urea cycle intermediate amino acids and an ornithine aminotransferase inactivator

Sparse fur with abnormal skin and hair (spf-ash) mice are deficient in ornithine carbamoyltransferase (OCT) activity, but their OCT protein is kinetically normal. We administered ammonium chloride to spf-ash mice, in order to analyze ammonia metabolism and to find a rationale for the therapy of OCT deficiency. Ammonia concentration in the liver of spf-ash mice increased to a level much higher than in the control. Ammonium chloride injection caused an increase in ornithine (Orn) 5 min after injection and an increase in the sum of Orn, citrulline (Cit) and arginine (Arg) for at least 15 min in the liver of control mice, but no increase in Orn, Cit and Arg in the liver of spf-ash mice. Treatment of spf-ash mice with Arg 5–20 min prior to the injection of ammonium chloride kept the hepatic ammonia concentration at a level comparable to that without the load. A significant reciprocal relationship between ammonia and Orn concentrations in the liver of spf-ash mice 5 min after an ammonium chloride load with or without Arg strongly suggests that ammonia disposal is dependent on the supply of Orn. In spf-ash mice loaded with tryptone as a nitrogen source, Arg supplementation showed a dramatic decrease in urinary orotic acid excretion in a dose-dependent manner. Similar effects were observed with Cit and Orn at the same dose, and a long-lasting effect with an ornithine aminotransferase inactivator, 5-(fluoromethyl)ornithine, at a much lower dose. The rate of urea formation in liver perfused with ammonium chloride was lower in spf-ash mice than in controls, but with the addition of Orn to the medium it increased to a similar level in control and spf-ash mice. These results indicate that OCT is not saturated with Orn in vivo under physiological conditions and that the administration or enrichment of the urea cycle intermediate amino acids enhances the OCT reaction so that the ammonia metabolism of OCT-deficient spf-ash mice is at least partially normalized.

Sexual development of Aspergillus nidulans in tryptophan auxotrophic strains.

The interplay between sexual development and amino acid biosynthesis in the ascomycete Aspergillus nidulans was studied. The growth and differentiation of four tryptophan auxotrophic strains that are unable to regulate their tryptophan biosynthesis, were examined. These strains are sterile on medium containing low tryptophan concentrations. Fruitbody formation was restored by supplementation with high concentrations of tryptophan and was promoted by supplementation with indole or auxin. Tryptophan supplementation resulted in auxin production of A. nidulans. Fertility of ascospores of the tryptophan auxotrophic strains could only be partially re-established. trpC transcript levels and enzyme activities remained stable in cleistothecia and conidiophore extracts as compared to those of mycelium, while levels of gene transcripts involved in glycolysis were lower in fruitbodies and conidiospores. Auxotrophic strains unable to form fruitbodies at intermediate amino acid supplementation levels turned on the cross-pathway regulatory system that is induced by amino acid starvation. We conclude that there is a connection between the genetic network of cross-pathway control and sexual development in A. nidulans.

No detectable NO synthesis from L-arginine or N(G)-hydroxy-L-arginine in fMLP-stimulated human blood neutrophils despite production of nitrite, nitrate, and citrulline from N(G)-hydroxy-L-arginine.

Nitric oxide (NO) is a well-documented effector molecule in rodent phagocytes but its synthesis in human neutrophils has been controversial. In this study, NO production in human neutrophils activated by chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (fMLP) was measured in the presence of L-arginine (L-Arg) and N(G)-hydroxy-L-arginine (OH-L-Arg), the precursor and intermediate amino acids in NO synthesis, respectively. Incubation of fMLP-activated neutrophils with OH-L-Arg resulted in a production of nitrite, nitrate, and citrulline that was greater than with unstimulated neutrophils but was not inhibited by the NOS inhibitors L-NMMA and L-NIO or the cytochrome P450 inhibitor troleandomycin and was not seen when OH-L-Arg was replaced with L-Arg. This nitrite, nitrate, and citrulline production was not associated with any detectable NO synthesis because no increases in cyclic GMP were observed in the presence of phosphodiesterase inhibitors and in the presence or absence of superoxide dismutase. Moreover, no increases in the formation of the reaction product of NO with superoxide, peroxynitrite, were observed on addition of either OH-L-Arg or L-Arg to activated neutrophils, as assessed either by dihydrorhodamine oxidation or protein nitration. This suggests that, in spite of the production of nitrite, nitrate, and citrulline, commonly used indicators of NO formation, normal human blood neutrophils, are not producing detectable amounts of either NO or peroxynitrite when stimulated with fMLP in the presence of OH-L-Arg.

X‐ray photoelectron spectroscopy analysis of Mo metal surface exposed to sulfate‐reducing bacteria

Molybdenum (Mo) is known for its roles in microbial enzymes and in enhancing the corrosion resistance of engineering alloys. In this study, Mo coupons were exposed to a sulfate-reducing bacterial (SRB) culture Desulfovibrio desulfuricans and then changes in surface chemical states were analyzed by XPS and pitting resistance by potentiodynamic polarization in deaerated 0.1 M HCl. Further investigation was conducted using XPS after potentiostatic polarization of the SRB-exposed Mo coupons at −160 mV SCE in deaerated 0.1 M HCl. The samples were analyzed with and without the presence of biofilm. Control samples were prepared in uninoculated medium. X-ray photoelectron spectroscopy analysis revealed two effects: the surface stability of Mo coupon was compromised due to MoS2 formation during exposure to SRB; and molybdate formation induced by bacterial H2 S production was inhibitory to sulfate reduction by forming Mo(V)–S complexes with intermediate sulfur-containing amino groups and proteins. The Mo(V)–S complexes were sparingly soluble and thus protective in the hydrochloric solution. Copyright © 1999 John Wiley & Sons, Ltd.

X-ray photoelectron spectroscopy analysis of Mo metal surface exposed to sulfate-reducing bacteria

Molybdenum (Mo) is known for its roles in microbial enzymes and in enhancing the corrosion resistance of engineering alloys. In this study, Mo coupons were exposed to a sulfate-reducing bacterial (SRB) culture Desulfovibrio desulfuricans and then changes in surface chemical states were analyzed by XPS and pitting resistance by potentiodynamic polarization in deaerated 0.1 M HCI. Further investigation was conducted using XPS after potentiostatic polarization of the SRB-exposed Mo coupons at - 160 mV SCE in deaerated 0.1 M HCI. The samples were analyzed with and without the presence of biofilm. Control samples were prepared in uninoculated medium. X-ray photoelectron spectroscopy analysis revealed two effects: the surface stability of Mo coupon was compromised due to MoS 2 formation during exposure to SRB; and molybdate formation induced by bacterial H 2 S production was inhibitory to sulfate reduction by forming Mo(V)-S complexes with intermediate sulfur-containing amino groups and proteins. The Mo(V)-S complexes were sparingly soluble and thus protective in the hydrochloric solution.

The “interferon sensitivity determining region” of hepatitis C virus is a stable sequence element

Background/Aims: A sequence of 40 amino acids within the nonstructural protein 5A of hepatitis C virus (HCV) has been suggested to be an interferon sensitivity determining region (ISDR). The variations in the ISDR after 12–14 years of chronic infection and the correlation between ISDR and interferon response were studied in patients who were infected by the same HCV isolate. Methods: We determined theHCV-ISDRs of 13 chronically infected patients by direct sequencing of polymerase chain reaction products. All patients were infected by isolate HCV-AD78, but differed with respect to their sensitivity to interferon. Four patients were complete responders, two patients were non-responders, and seven showed a partial response. Results: The ISDR of HCV-AD78 differed from a prototypical HCV-1b sequence in one amino acid and was therefore classified as an intermediate type. Direct sequencing of the HCV-ISDRs of the patients 12–14 years after infection, but before interferon therapy, revealed a rate of 2.2×10 −3 nucleotide substitutions per site per year, resulting in only single intermediate type amino acid exchanges. All sequences ranked with the intermediate type. Moreover, during interferon treatment no selection to a wild type ISDR was observed in five partial responders. Conclusions: Within the homogeneous patient group examined here, no correlation was found between the ISDR and the interferon response. Recent studies found only a small number of mutant type ISDRs in Europe. Additionally, our results indicate that the ISDR is a stable sequence element. This provides an explanation for the divergent data relating to the importance of the ISDR in different geographical regions.

Stereoselective and Enantioselective Synthesis of anti-1-(Trifluoromethyl) Amino Alcohols

anti-(Trifluoromethyl) β-amino alcohols 2 have been prepared in good yields and with 90% diastereoisomeric excess through a reaction of 1-(trifluoromethyl) epoxy ethers 3 with dimethylaluminum amide, followed by the in situ chelation-controlled stereoselective reduction of the intermediate amino ketone. The salen-mediated chiral epoxidation of 1-(trifluoromethyl) enol ether 4a led to the homochiral epoxy ethers 11a and 12a in a good enantiomeric excess. Reaction with dimethylaluminum amide followed by a reduction step provided the chiral amino alcohols 15a and 16a, respectively.

NOx Abatement in Exhaust from Lean-Burn Combustion Engines by Reduction of NO2 over Silver-Containing Zeolite Catalysts

Resistance to sulfur, long life, and high activity are characteristic of silver-containing zeolite catalysts with Brønsted acid centers, which can be used for the reduction of NO2 to N2 in the presence of hydrocarbons [Eq. (1)]. In the proposed reaction mechanism, N2 formation proceeds via intermediate nitro, amino, and diazonium compounds.

Aziridinium ions from phenylglycinol — A new approach to the synthesis of chiral diamines

A new two-step method [(i) N, N-dialkylation; (ii) aziridinium ion formation followed by amine attack] for the synthesis of chiral diamines from phenylglycinol is described. The two steps can be carried out sequentially without isolation of the intermediate amino alcohols to give diamines in good yields (62–76%). An improved protocol for the N, N-dialkylation of phenylglycinol is also reported.

Glacial advances and stratigraphy in Otto Fiord and adjacent areas, Ellesmere Island, Northwest Territories

Hvitland and Svartfjeld peninsulas have been glaciated at least two or three times since the Late Pliocene. The earliest recognized glaciation is constrained by amino acid ratios on in situ pelecypods from marine sediments stratigraphically overlying till in Otto Fiord. High amino acid ratios suggest that the shells may be Pliocene and related to recently discovered Late Pliocene Hvitland beds at White Point. The same samples yielded finite radiocarbon ages in the 30.2–34.8 ka BP range, which cautions against their acceptance. A second, mid Quaternary glaciation is suggested by intermediate amino acid ratios from in situ pelecypods and shell-bearing till covering coastal uplands along Nansen Sound. During the last glaciation the peninsula was covered by coalesced ice caps, with glacial tongues descending tributary valleys and extending into the fiords. Deglaciation of a tributary valley in outer Otto Fiord proceeded before 11.6 ka BP and before 9.1 ka BP in mid Otto Fiord, but major ice margins remained at the coast until 8–8.5 ka BP. Lateral meltwater channels record systematic ice retreat up tributary valleys into the interior of the peninsulas. The highest recognized marine limits are marked by prominent deltas at least 164 m asl on western Hvitland Peninsula. The marine limit declines eastward inside Otto, Jugeborg, and Hare fiords. The highest strandlines could not be dated; however, by ~8.3 ka BP relative sea level stood at 103 m asl.

A Novel Synthesis of 3,3-(Spiro)substituted Azetidines

A smooth and efficient new synthesis for 3,3-disubstituted azetidines, starting from readily available nitriles, was established: α-hydroxymethylation of the starting materials, followed by O-tosylation and LiAlH 4 -reduction of the key intermediates thus obtained, led ― via spontaneous cyclization of the intermediate amino derivatives ― to 3,3disubstituted azetidines. Scope and limitations of this new method were studied with respect to generalized applicability: the target compounds were accessible in good yields for a variety of starting materials (cyclic and acyclic di(hetero)aryl, (hetero)arylalkyl, dialkyl, as well as basic moieties). The products thus obtained may be of interest for ensuing conversions due to their unblocked nitrogen

Expression of mutant ELH prohormones in AtT-20 cells: the relationship between prohormone processing and sorting.

Posttranslational processing of many proteins is essential to the synthesis of fully functional molecules. The ELH (egg-laying hormone) prohormone is cleaved by endoproteases in a specific order at a variety of basic residue processing sites to produce mature peptides. The prohormone is first cleaved at a unique tetrabasic site liberating two intermediates (amino and carboxy) which are sorted to different classes of dense core vesicles in the bag cell neurons of Aplysia. When expressed in AtT-20 cells, the ELH prohormone is also first cleaved at the tetrabasic site. The amino-terminal intermediate is then sorted to the constitutive pathway, and a portion of the carboxy-terminal intermediate is sorted to the regulated pathway. Here, we use mutant constructs of the ELH prohormone expressed in AtT-20 cells to examine the relationship between prohormone processing and consequent sorting. Prohormone which has a dibasic site in place of the tetrabasic site is processed and sorted similarly to wild type. Furthermore, mutant prohormone which lacks the tetrabasic site is processed at an alternative site comprising three basic residues. In these mutant prohormones, mature ELH is still produced and stored in dense core vesicles while amino-terminal products are constitutively secreted. However, deletion of the tetrabasic and tribasic sites results in the rerouting of the amino-terminal intermediate products from the constitutive pathway to the regulated secretory pathway. Thus, in the ELH prohormone, the location of the proteolytic processing events within the secretory pathway and the order of cleavages regulate the sorting of peptide products.

Amination of 3,5‐Dinitropyridines with Liquid Ammonia/Potassium Permanganate

Abstract3,5‐Dinitropyridine and some of its derivatives are aminated with a liquid ammonia solution of potassium permanganate to the corresponding 2‐, 4‐ and 6‐ mono‐ and (or) di‐ and (or) triamino‐substituted compounds. The intermediate amino σ‐adducts of 3,5‐dinitropyridines are detected by 1H‐NMR spectroscopy. Quantum‐mechanical calculations for a few 3,5‐dinitropyridines suggest that the experimentally observed regioselectivity of the amination is a charge‐controlled process.

Separation of immunoglobulin G by high-performance pseudo-bioaffinity chromatography with immobilized histidine I. Preliminary report on the influence of the silica support and the coupling mode

High-performance liquid affinity chromatography with immobilized histidine as a preudo-biospecific ligand has been used for the fractionation of human immunoglobulin G (IgG). Histidine was immobilized onto silica in two different modes: directly onto silica after epoxy activation or using an intermediate amino derivatization of silica and then coupling histidine using water-soluble carbodiimide. The behaviours and capacities of the obtained affinity supports as well as the influence of pH, silica type, pore diameter and coupling mode have been studied. IgG was effectively separated from human plasma and high maximal binding capacities were obtained.

Synthesis of 1H-isoindoles by a novel rearrangement of some isoquinolin-4(1H)-ones

The isoquinolinones 8 undergo acid-induced rearrangement to yield the isomeric 1,1-dihydro-3-aroylisoindoles 9 in good yield. The reaction involves hydrolysis of the CN bond to afford a putative intermediate amino diketone 10. Cyclization within this is controlled by two factors (i) the stability of the CN group in the starting material and product and (ii) the relative reactivity of the two carbonyl groups.