Enzymatic synthesis of L-tryptophan from D,L-2-amino-Δ2-thiazoline-4-carboxylic acid and indole by Pseudomonas sp. TS1138 L-2-amino-Δ2-thiazoline-4-carboxylic acid hydrolase, S-carbamyl-L-cysteine amidohydrolase and Escherichia coli L-tryptophanase

L-Tryptophan (L-Trp) is an essential amino acid. It is widely used in medical, health and food products, so a low-cost supply is needed. There are 4 methods for L-Trp production: chemical synthesis, extraction, enzymatic synthesis, and fermentation. In this study, we produced a recombinant bacterial strain pET-tnaA of Escherichia coli which has the L-tryptophanase gene. Using the pET-tnaA E. coli and the strain TS1138 of Pseudomonas sp., a one-pot enzymatic synthesis of L-Trp was developed. Pseudomonas sp. TS1138 was added to a solution of D,L-2-amino-Δ2-thiazoline-4-carboxylic acid (DL-ATC) to convert it to L-cysteine (L-Cys). After concentration, E. coli BL21 (DE 3) cells including plasmid pET-tnaA, indole, and pyridoxal 5’-phosphate were added. At the optimum conditions, the conversion rates of DL-ATC and L-Cys were 95.4% and 92.1%, respectively. After purifying using macroporous resin S8 and NKA-II, 10.32 g of L-Trp of 98.3% purity was obtained. This study established methods for one-pot enzymatic synthesis and separation of L-Trp. This method of producing L-Trp is more environmentally sound than methods using chemical synthesis, and it lays the foundations for industrial production of L-Trp from dl-ATC and indole.     

Isolation and genetic improvement of Pseudomonas sp. strain HUT-78, capable of enzymatic production of L-cysteine from DL-2-amino-Δ2-thiazoline-4-carboxylic acid

Microorganisms able to bioconvert DL-2-amino-Δ(2)-thiazoline-4-carboxylic acid (DL-ATC) into L-cysteine were originally isolated from 10 soil samples with DL-ATC as the sole nitrogen source. Ninety-seven L-cysteine-producing bacterial strains were screened out and obtained in pure culture. Among them, a strain, designated as HUT-78, was selected as the best producer, with a molar bioconversion rate of 60%. Based on the 16S rRNA gene sequence analysis, this isolate was placed within the genus Pseudomonas. A novel mutant of this strain with a significantly reduced activity of L-cysteine desulfhydrase, a L-cysteine-decomposing enzyme, was derived by UV-mutagenesis. This mutant, designated as mHUT-78, exhibited a 42% increase in L-cysteine producing activity. Moreover, the bioconversion reactions in both the parent and the mutant strain were significantly accelerated by co-overexpression of the two key enzymes, AtcB and AtcC, involved in the bioconversion reaction.     

The self-disproportionation of enantiomers (SDE) via column chromatography of β-amino-α,α-difluorophosphonic acid derivatives

Amino Acids - This work presents the first study of the self-disproportionation of enantiomers via chromatography (SDEvC) of β-aminophosphonic acid esters, several of which have been...     

Synthesis of the disaccharides methyl 4-O-(2′/3′-O-sulfo-β-d-glucopyranosyluronic acid)-2-amino-2-deoxy-α-d-glucopyranoside disodium salts,related to heparin biosynthesis

The synthesis of the disaccharides methyl 4-O-(2/3-O-sulfo--d-glucopyranosyluronic acid)-2-amino-2-deoxy--d-glucopyranoside3 and4 as, disodium salts is described. Allyl 4,6-O-benzylidene--d-glucopyranoside6 was converted to trichloroacetimidate20 Glycosylation of20 with5 promoted by BF₃·OEt₂ gave disaccharide21. Deacetylation of21 followed by monoacetylation of the resultant diol22 afforded the two monoacetylated disaccharides23 and24. Sulfation and deprotection of each disaccharide gave the desired sulfated compounds3 and4.     

Reaction of 2-amino-2-deoxyheptoses with cyclic β-dicarbonyl compounds

The reaction between 2-amino-2-deoxyaldoses and β-dicarbonyl compounds yields polyhydroxyalkylpyrroles. Thus, 6,6-dimethyl-2-(D-galacto-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4a), 6,6-dimethyl-2-(D-gluco-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4b), and 6,6-dimethyl-2-(D-manno-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (4c) have been obtained from 5,5-dimethylcyclohexane-1,3-dione (2) and 2-amino-2-deoxyheptoses having D-glycero-L-gluco (1a), D-glycero-D-ido (1b), and D-glycero-D-talo (1c) configurations, respectively. 2-Amino-2-deoxy-D-glycero-L-manno-heptose (1d), the epimer of 1a, also reacts with 2, to yield 4a. In a similar way, 1a, 1b, and 1c react with cyclohexane-1,3-dione (3), to give 2-(D-galacto-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5a), 2-D-gluco-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5b), and 2-(D-manno-pentitol-1-yl)-4,5,6,7-tetrahydroindol-4-one (5c), respectively.     

Ab Initio studies of receptor interactions with AMPA ((S)-2-amino-3-(3-hydroxy-5-methyl-4-isoxazolyl) propionic acid ) and Kainic acid (2S-(2α,3β,4β))-2-carboxy-4-(1-methylethenyl)-3-pyrrolidineacetic acid

The optimum geometries and binding energies of the complexes formed by AMPA and Kainic acid, as well as their anions with tyrosine, proline and some tripeptides are investigated with quantum chemical calculations (HF/6-31G**). It was found that receptors featuring the Tyr-Ala-Pro sequence exhibit stronger binding energies to the substrates than the Tyr-Ser-Pro and Tyr-Ser-Ser. As expected, the anions are more bound than the neutral species. This work can lead to investigations on the effect of AMPA receptors mutations on the brain functions, possibly related to criminal tendencies.     

Molecular orbital studies on nucleoside antibiotics,VII. Conformation of 2′-amino- 2′-deoxyguanosine

Conformational properties of the nucleoside antibiotic 2-amino-2-deoxyguanosine have been investigated by the PCILO method along with those of its parent nucleoside, guanosine. This antibiotic, formed by replacement of the 2-hydroxyl group by an amino group in guanosine, shows anti-tumor activity and also inhibits RNA and protein syntheses. Both C(2)-endo and C(3)-endo sugar conformations have been considered in the computations. The results indicate striking similarity between the conformations of the antibiotic and the parent nucleoside, particularly in simulated aqueous environment. The biological implication of this result in terms of the antibiotic activity is discussed.     

Inhibition of δ-aminolevulinic acid synthetase and δ-aminolevulinic acid dehydrase by L-2-amino-4-methoxy-trans-3-butenoic acid in the rat

The rate limiting enzyme of heme biosynthesis, δ-aminolevulinic acid synthetase (ALA synthetase), and the second enzyme in the heme biosynthetic pathway, δ-aminolevulinic acid dehydrase (ALA dehydrase), were inhibited by the olefinic amino acid L-2-amino-4-methoxy - $\text{trans}$-3-butenoic acid (AMTB). Administration of AMTB (20 mg/kg; i.p.) to rats inhibited ALA synthetase and ALA dehydrase in control animals and in animals with markedly elevated activity of ALA synthetase which resulted from the administration of 3,5-dicarbethoxy-1,4-dimethyl-collidine (DDC, 200 mg/kg, i.p.) or allylisopropylacetamide (200 mg/kg, s.c.). AMTB also blocked the synthesis of rat hepatic porphyrins and inhibited the increase in the urinary excretion of δ-aminolevulinic acid and porphobilinogen following DDC (150 mg/kg, p.o.) administration. Preincubation of AMTB with liver mitochondria or a soluble fraction of liver decreased the activity of mitochondrial ALA synthetase and soluble ALA dehydrase, respectively.     

Quantitative analysis and comparative decarboxylation of aminomalonic acid, β-carboxyaspartic acid,and γ-carboxyglutamic acid

Methods for quantitative analysis of the carboxylated amino acids, aminomalonic acid, β-carboxyaspartic acid, and γ-carboxyglutamic acid, are presented. These substances are acid labile and thus can be measured only after alkaline hydrolysis of proteins and peptides. Half-times for decarboxylation in 1 m HCl at 100°C are: aminomalonic acid (1.2 min); β-carboxyaspartic acid (1.7 min); and γ-carboxyglutamic acid (8.6 min). This property is useful for unequivocal identification in complex hydrolysates.     

Parallel RNA-strand recognition by 2′-amino-β-l-LNA

A short synthetic route to the first β-l-ribo configured locked nucleic acid (LNA), that is, 2′-amino-β-l-LNA thymine phosphoramidite 6, has been developed from bicyclic nucleoside 1. Incorporation of 2′-amino-β-l-LNA thymine monomers into α-DNA strands results in probes forming stable duplexes with complementary RNA in parallel orientation.     

Asialo-α1-acid glycoprotein resialylated with 9-amino-5-N-acetyl-d-neuraminic acid is resistant towards bacterial,viral and mammalian sialidases

We demonstrate that 9-amino-NeuAc transferred to asialo-₁-acid glycoprotein resists cleavage by bacterial, viral and mammalian sialidases. This is the first synthetic sialic acid analogue, which can be activated and transferred to glycoprotein, but is not a sialidase (EC 3.2.1.18) substrate.Abbreviations HPLC high performance liquid chromatography - BSA bovine serum albumin - NeuAc N-acetyl-d-neuraminic acid, 5-acetamido-3,5-dideoxy-d-glycero-d-galacto-non-2-ulosonic acid - 9-Amino-NeuAc 9-amino-5-N-acetyl-d-neuraminic acid, 5-acetamido-9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid - CMP-NeuAc cytidine-5-monophospho-N-acetyl-d-neuraminic acid - CMP-9-amino-NeuAc cytidine-5-monophospho-9-amino-5-N-acetyl-d-neuraminic acid - 9-azido-NeuAc 5-acetamido-9-azido-3,5,9-trideoxy-d-glycero-d-galacto-non-2-ulosonic acid. Enzymes EC 3.2.1.18 sialidase, acylneuraminylhydrolase - EC 2.4.99.1 Galß1-4GlcNAc a(2-6)-sialytransferase     

N-Aminoacyl and N-hydroxyacyl derivatives of diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside: Synthesis,antimicrobial and hemolytic activities

Diosgenyl 2-amino-2-deoxy-β-d-glucopyranoside is a semisynthetic saponin with antimicrobial and antitumor activities. To search for more effective analogues, N-aminoacyl and N-hydroxyacyl derivatives of this saponin were synthesized conventionally and with microwave assistance, and tested against the human pathogenic fungi and Gram-positive and Gram-negative bacteria. None of the tested compounds exhibit activity against Gram-negative bacteria. Almost all of the synthesized N-aminoacyl saponins exhibit antifungal activity and act effectively against Gram-positive bacteria, some better than the parent compound. The best acting saponins are the same size and possess sarcosine or l- or d-alanine attached to the parent glucosaminoside. Shorter and longer aminoacyl residues are less advantageous. d-Alanine derivative is the most effective against Gram positive bacteria. Structure-activity relationship (SAR) analysis indicates that the free α-amino group in aminoacyl residue is necessary for antimicrobial activities of the tested saponins. (N-Acetyl)aminoacyl and N-hydroxyacyl analogs are inactive. Measurements of the hemolytic activities demonstrate that the best acting saponins are not toxic towards human red blood cells.     

Rational design,synthesis, and structure–activity relationships of 5-amino-1H-pyrazole-4-carboxylic acid derivatives as protein tyrosine phosphatase 1B inhibitors

A series of novel amino-carboxylic based pyrazole as protein tyrosine phosphatase 1B (PTP1B) inhibitors were designed on the basis of structure-based pharmacophore model and molecular docking. Compounds containing different hydrophobic tail (1,2-diphenyl ethanone, oxdiadizole and dibenzyl amines) were synthesized and evaluated in PTP1B enzymatic assay. Structure–activity relationship based optimization resulted in identification of several potent, metabolically stable and cell permeable PTP1B inhibitors.     

Role of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor regulation in stress-induced pain chronification

Persistent postsurgical pain is a serious issue in public health, which has received increased interest in recent years. Previous studies have reported that psychological factors promote the development of chronic postsurgical pain. However, it is unclear how chronification of postsurgical pain occurs. The α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor(AMPA) phosphorylation in the central nervous system plays a critical role in synaptic plasticity and contributes to central sensitization and chronic pain development. Here, we discuss the role of AMPA receptor regulation in stress-induced pain chronification after surgery.     

Fluorescent probes in model membranes. II. monolayer studies of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole, d-3-aminodesoxyequilenin and n-octadecylnaphthyl-2-amino-6-sulfonic acid with host-lipid tetradecanoic acid

Film studies at the air-water interface have been carried out for pure films of 2,2′-(vinylenedi-p-phenylene)bisbenzoxazole (VPBO), d-3-aminodesoxy-equlenin (EQ) and N-octadecylnapthyl-2-amino-6-sulfonic acid (ONS), and for mixed films with tetradecanoic acid for the first two fluorescent probes. Pure film isotherms indicate highly rigid non-monomolecular films for both VPBO and EQ, revealing the presence of strong intermolecular forces. In mixed films with tetradecanoic acid VPBO rapidly segregates with resultant film loss over a wide concentration range. EQ, however, can be stabilized by the host-lipid at low concentrations. This, coupled with an ability to only slightly affect the host-lipid liquid-condensed/liquid-expanded phase change, suggests that EQ can be regarded as “non-perturbing” and should be retained in condensed lipid phases.ONS, because of its unusual polar headgroup, resembled hexadecanoic acid more than octadecanoic acid. While difficulties in spreading ONS precluded the study of mixed films, the indications are that it would be a satisfactory expanded lipid state probe if mixing can be brought about.     

The “ON”-bipolar agonist,L-2-amino-4-phosphonobutyrate,blocks light-evoked cone contraction in xenopus eye cups

Rhythmic photoreceptor metabolism in relationship to light-dark cycles is now thought be regulated through a retinal feed-back mechanism with dopamine serving as a principal signal initiating light-evoked events. In order to test the hypothesis that depolarizing ON-bipolar neurons participate in the retinal signalling pathway, we determined the effects of L-2-amino-4-phosphonobutyrate (L-APB) on light-evoked cone contraction in eye cups fromXenopus laevis. L-APB blocked the response stereospecifically when applied over a broad concentration range. The high specificity of L-APB in retina suggests that sign-inverting bipolar neurons which depolarize in light are in the signalling pathway. One possibility is that this pathway conveys signals that regulate dopamine release.Special issue dedicated to Dr. Frederick E. Samson.     

Sequence-specific DNA binding by long hairpin pyrrole–imidazole polyamides containing an 8-amino-3,6-dioxaoctanoic acid unit

With the aim of improving aqueous solubility, we designed and synthesized five N-methylpyrrole (Py)–N-methylimidazole (Im) polyamides capable of recognizing 9-bp sequences. Their DNA-binding affinities and sequence specificities were evaluated by SPR and Bind-n-Seq analyses. The design of polyamide 1 was based on a conventional model, with three consecutive Py or Im rings separated by a β-alanine to match the curvature and twist of long DNA helices. Polyamides 2 and 3 contained an 8-amino-3,6-dioxaoctanoic acid (AO) unit, which has previously only been used as a linker within linear Py–Im polyamides or between Py–Im hairpin motifs for tandem hairpin. It is demonstrated herein that AO also functions as a linker element that can extend to 2-bp in hairpin motifs. Notably, although the AO-containing unit can fail to bind the expected sequence, polyamide 4, which has two AO units facing each other in a hairpin form, successfully showed the expected motif and a K_D value of 16 nM was recorded. Polyamide 5, containing a β-alanine–β-alanine unit instead of the AO of polyamide 2, was synthesized for comparison. The aqueous solubilities and nuclear localization of three of the polyamides were also examined. The results suggest the possibility of applying the AO unit in the core of Py–Im polyamide compounds.     

Evaluation of urinary 1-hydroxypyrene,S-phenylmercapturic acid,trans,trans-muconic acid, 3-methyladenine, 3-ethyladenine, 8-hydroxy-2′-deoxyguanosine and thioethers as biomarkers of exposure to cigarette smoke

The objective was to evaluate the utility of urinary 1-hydroxypyrene (1-OHP), S-phenylmercapturic acid (S-PMA), trans,trans-muconic acid (t,t-MA), 3-methyladenine (3-MeAd), 3-ethyladenine (3-EtAd), 8-hydroxy-2′-deoxyguanosine (8-OHdG) and thioethers as biomarkers for assessing the exposure in adult smokers who switched from smoking conventional cigarettes to candidate potential reduced exposure products (PREP) or who stopped smoking. Two electrically heated smoking systems (EHCSS) were used as prototype cigarettes that have significant reductions in a number of mainstream smoke constituents as measured by smoking machines relative to those from conventional cigarettes. Urine samples were collected from a randomized, controlled, forced-switching study in which 110 adult smokers of a conventional cigarette brand (CC1) were randomly assigned to five study groups. The groups included the CC1 smoking group, a lower-tar conventional cigarette (CC2) smoking group, EHCSS1 group, EHCSS2 group and a no smoking group that were monitored for 8 days. Biomarkers were measured at baseline and day 8. The daily excretion levels of these biomarkers were compared among the groups before and after switching, and the relationships between the daily excretion levels of these biomarkers and cigarette smoking-related exposure were investigated using Pearson product-moment correlation and multiple regression analyses. It was concluded that under controlled study conditions: (1) 1-OHP, S-PMA and t,t-MA are useful biomarkers that could differentiate exposure between smoking conventional and EHCSS cigarettes or between smoking conventional cigarettes and no smoking; between S-PMA and t,t-MA, the former appeared to be more sensitive; (2) 3-MeAd could only differentiate between smoking conventional cigarettes and no smoking; the results for 3-EtAd were not conclusive because contradictory results were observed; (3) 8-OHdG had a questionable association with smoking and therefore the utility of this biomarker for smoking-related exposure could not be established; and (4) urinary excretion of thioethers as a biomarker lacked sensitivity to demonstrate a clear dose–response relationship in conventional cigarette smokers, although it could differentiate the excretion levels between those subjects who smoked a conventional cigarette and those who stopped smoking.