Axially chiral hemiaminals from nonracemic α-amino acid derivatives (thiohydantoins): Synthesis and isomer identification

Stable, nonracemic axially chiral hemiaminals (O,N-hemiacetals) have been synthesized stereoselectively from lithium aluminum hydride (LiAlH₄) reductions of nonracemic 5-methyl- and 5-isopropyl-3-(o-aryl)-2-thioydantoins in tetrahydrofuran (THF) at room temperature in 10 min. Predominantly S -configured hemiaminals at C-4 of the heterocyclic ring were produced from the S-configured thiohydantoins at C-5 (by 80% when the C5 substituent is methyl and by 97% when it is isopropyl). The configuration at C-5 was retained during the reduction reaction. The stereochemical outcome of the axially chiral hemiaminals resulted from their conformational preferences.     

Simultaneous recognition of two chiral centers in α,β-diastereomeric amino acids by an N-carbamoyl-L-α-amino acid amidohydrolase from Alcaligenes xylosoxidans

Cells of Alcaligenes xylosoxidans containing N-carbamoyl-L--amino acid amidohydrolase strictly distinguished the configuration of not only the -carbon but also the -carbon of N-carbamoyl--methylphenylalanine, and produced threo-l--methylphenylalanine specifically from a mixture of the four stereoisomers.     

An inhibitory peptide derived from the α-subunit of the epithelial sodium channel (ENaC) shows a helical conformation

Proteolytic activation of the heteromeric epithelial sodium channel (ENaC) is thought to involve the release of inhibitory peptides from the extracellular domains of its α- and γ-subunit. Recently, we demonstrated that an α-13-mer peptide, corresponding to a putative inhibitory region within the extracellular domain of human αENaC, inhibits human αβγENaC. The aim of the present study was to investigate the structural basis of the inhibitory effect of this α-13-mer peptide. Analysis of the peptide by replica exchange molecular dynamics method, circular dichroism spectroscopy, nuclear magnetic resonance spectroscopy, and molecular dynamics simulations suggested that a helical turn at the carboxy-terminus is the preferred conformational state of the α-13-mer peptide. From this we predicted that a specific mutation (leucine 188 to alanine) should have a strong effect on the conformational preferences of the peptide. To functionally test this, we compared the effect of the wild-type α-13-mer with that of a mutant α-L188A-13-mer on ENaC currents in Xenopus laevis oocytes heterologously expressing human αβγENaC. We demonstrated that replacing the leucine 188 by alanine abolished the inhibitory effect of the α-13-mer peptide on ENaC. These findings suggest that a helical conformation in its carboxyterminal part is functionally important to mediate ENaC inhibition by the α-13-mer peptide. However, high resolution structural information on the complex of the inhibitory αENaC peptide and the channel are needed to confirm this conclusion.     

Sesquiterpene-α-amino acid quaternary ammonium hybrids from Stereum complicatum (Steraceae)

Five sesquiterpene-α-amino acid quaternary ammonium hybrids and the sesquiterpene core with no amino acid moiety linked were isolated from the n-butanol partition of the Stereum complicatum fungal culture grown on potato dextrose broth. Chemical structures were unambiguously elucidated by high resolution electrospray ionization mass spectrometry (HR-ESI-MS) in positive ion mode and NMR experiments and were identified as sterostrein Q and stereumamides A - E. The isolation and structure elucidation of these hybrid compounds is reported herein for the first time from S. complicatum. The phytotoxic, antifungal and antibacterial activities of these compounds were evaluated, but no significant activity was found. The HR-ESI-MS fragmentation pattern of the sesquiterpene-α-amino acid quaternary ammonium hybrid metabolites and its chemotaxonomic implication are discussed.     

Stereoconservative and stereoselective syntheses of rare and non-naturalα-amino acids from (S)-aspartic acid and (S)-malic acid

Summary A new strategy for stereoconservative and stereoselective syntheses of several types of amino acids starting from-functional carboxylic acids employing hexafluoroacetone as protecting and activating reagent is described. Outstanding features of this new method are the mild reaction conditions and the high yields for introduction and cleavage of the protective group allowing sensitive functional groups in the side chain to survive. Furthermore, the new concept results in saving of synthetic steps.     

Amino acid substitutions in the N-terminus, cord and α-helix domains improved the thermostability of a family 11 xylanase XynR8

The thermostability of xylanase XynR8 from uncultured Neocallimastigales rumen fungal was improved by combining random point mutagenesis with site-directed mutagenesis guided by rational design, and a thermostable variant, XynR8_VNE, was identified. This variant contained three amino acid substitutions, I38V, D137N and G151E, and showed an increased melting temperature of 8.8?°C in comparison with the wild type. At 65?°C the wild-type enzyme lost all of its activity after treatment for 30?min, but XynR8_VNE retained about 65?% activity. To elucidate the mechanism of thermal stabilization, three-dimensional structures were predicted for XynR8 and its variant. We found that the tight packing density and new salt bridge caused by the substitutions may be responsible for the improved thermostability. These three substitutions are located in the N-terminus, cord and α-helix domains, respectively. Hence, the stability of these three domains may be crucial for the thermostability of family 11 xylanases.     

RNP-T,a ribonucleoprotein from Arabidopsis thaliana,contains two RNP-80 motifs and a novel acidic repeat arranged in an α-helix conformation

We have isolated a 1148 bp long cDNA clone encoding an RNA-binding protein in Arabidopsis. Several partial cDNA clones were isolated by screening an Arabidopsis gt11 expression library for the binding of DNA. One of these clones was used as a probe to isolate a full-length clone. The 329 amino acid protein, termed RNP-T, contains in its carboxy terminus two adjacent RNP-80 motifs, a previously described 80 amino acid long conserved putative RNA-binding domain. Each RNP-80 motif includes both consensus short sequences, RNP1 and RNP2, which are separated by 33 amino acids. We have identified an acidic domain of 54 amino acids, which is located amino-terminal to the RNP-80 motifs. Seven tandem repeats of a hexamer are present within this domain. This acidic domain has a potential -helix conformation. We propose that the acidic patch might play a role in protein-protein interaction.     

The role of the delocalized α,α′-carbanion in vitamin B6 and Al(III) catalyzed transamination of α-amino and α-keto acids

The rates of the transamination reactions of α-amino acids and α-keto acids were followed by measurement of the 200 MHz proton NMR spectra of solution species as a function of time. Reaction systems measured in D₂O at 10 °C consisted of 1:1:1 molar ratios of pyridoxal:α-amino acid:Al(III) or pyridoxamine:α-keto acid:Al(III). Amino and keto acids employed are alanine, α-aminoisobutyric acid, valine, phenylglycine, pyruvic acid, and α-ketobutyric acid. A negatively charged deprotonated Schiff base coordinated to Al(III) was detected in all systems that undergo transamination (i.e., except α-aminoisobutyric acid). The intermediate resembles the aldimine Al(III) chelate with NMR resonances shifted upfield in accordance with its greater negative charge. Its equilibrium concentration is reached in the time required to reach transamination equilibrium and is maintained in solution at a ca. 10–20% of the aldimine Schiff base concentration.     

Structure-activity relationship (SAR) of the α-amino acid residue of potent tetrahydroisoquinoline (THIQ)-derived LFA-1/ICAM-1 antagonists

This letter describes the structure-activity relationship (SAR) of the ‘right-wing’ α-amino acid residue of potent tetrahydroisoquinoline (THIQ)-derived LFA-1/ICAM-1 antagonists. Novel (S)-substituted heteroaryl-bearing α-amino acids have been identified as replacements of the ‘right-wing’ (S)-2,3-diaminopropanoic acid (DAP) moiety. Improvement of potency in the Hut-78 assay in the presence of 10% human serum has also been achieved.     

Amino acid differences between the α-chains from two hemoglobins of the yellow-cheeked vole (family cricetidae)

The yellow-cheeked vole (Microtus xanthognathus) shows two electrophoretic hemoglobin components. Electrophoresis of the polypeptide chains from the separated hemoglobin components shows identical β-chains but two α-chains of different mobility, α ^f and α ^s . The composition of soluble tryptic peptides was determined for each α-chain. Amino acid differences were found in peptides αT1 and αT9; the compositions of the remainder of the homologous peptides were identical. Differences in αT1, found at α4 (α ^s -Gly-α ^f -Val) and α5 (α ^s -Thr-α ^f -Asp), were confirmed after a run to residue 20 of the fast component in an automatic sequencer. The differences in charge between αT1 peptides can account for the electrophoretic pattern of two hemoglobins. This is the first time that it has been possible to identity the residues which can account for the charge difference between the two hemoglobins observed in a Microtus species.     

Pectin biosynthesis: a solubilized α1,4-galacturonosyltransferase from tobacco catalyzes the transfer of galacturonic acid from UDP-galacturonic acid onto the non-reducing end of homogalacturonan

A solubilized α1,4-galacturonosyltransferase (GalAT) from tobacco transfers galacturonic acid (GalA) residues from UDP-GalA onto oligogalacturonide (OGA) exogenous acceptors with degrees of polymerization greater than nine (R.L. Doong and D. Mohnen 1998, Plant J 13: 363–374). The solubilized GalAT has been identified as putative polygalacturonate 4-α-galacturonosyltransferase (PGA-GalAT, EC 2.4.1.43) based on its α1,4-galacturonosyltransferase activity and similar K _m for UDP-GalA, pH optimum and V _max to those of membrane-bound PGA-GalAT (R.L. Doong et al., 1995, Plant Physiol 109: 141–152). The direction of elongation of homogalacturonan catalyzed by solubilized GalAT from microsomes of tobacco (Nicotiana tabacum L. cv. Samsun) cell suspensions has now been determined. Three different types of exogenous acceptor were used to study the direction of synthesis of homogalacturonan: unmodified OGAs, OGAs derivatized by biotinylation at the reducing end, and OGAs containing a 4,5-unsaturated GalA at the non-reducing end. The unmodified OGAs and the OGAs modified at the reducing end functioned equally well as acceptors in the galacturonosyltransferase reaction. In contrast, OGAs with the 4,5-unsaturated residue at the non-reducing end were not acceptors for homogalacturonan biosynthesis. These results show that homogalacturonan biosynthesis by solubilized GalAT occurs via the addition of GalA to the non-reducing end of the polymer chain. Received: 18 June 1998 / Accepted: 22 August 1998     

Efficient synthesis of α(2–8)-linkedN-acetyl andN-glycolylneuraminic acid disaccharides from colominic acid

Controlled acid hydrolysis of poly-(2,8)-linked homopolymers ofN-acetylneuraminic acid (colominic acid) and its homologous poly-N-glycolylneuraminic acid afforded high yields of the corresponding disaccharides useful in block synthesis of disialylated gangliosides. The poly-N-glycolylanalog was derived from de-N-acetylated colominic acid by two different reaction sequences. The first one involved reaction with acetoxyacetyl chloride followed by de-O-acetylation. The second and most interesting one requiredN-acryloylation and reductive ozonolysis.     

Mapping of the spectral density function of a Cα−Hα bond vector from NMR relaxation rates of a 13C-labelled α-carbon in motilin

Summary The peptide hormone motilin was synthesised with a ¹³C-enriched -carbon in the leucine at position 10. In aqueous solution, six different relaxation rates were measured for the ¹³C–H fragment as a function of temperature and with and without the addition of 30% (v/v) of the cosolvent d ₂-1,1,1,3,3,3-hexafluoro-2-propanol (HFP). The relaxation rates were analysed employing the spectral density mapping technique introduced by Peng and Wagner [(1992) J. Magn. Reson., 98, 308–322] and using the model-free approach by Lipari and Szabo [(1982) J. Am. Chem. Soc., 104, 4546–4570]. The fit to various models of dynamics was also considered. Different procedures to evaluate the overall rotational correlation time were compared. A single exponential time correlation function was found to give a good fit to the measured spectral densities only for motilin in 30% (v/v) HFP at low temperatures, whereas at high temperatures in this solvent, and in D₂O at all temperatures, none of the considered models gave an acceptable fit. A new empirical spectral density function was tested and found to accurately fit the experimental spectral density mapping points. The application of spectral density mapping based on NMR relaxation data for specific ¹³C–¹H vector is shown to be a highly useful method to study biomolecular dynamics. Advantages are high sensitivity, high precision and uniform sampling of the spectral density function over the frequency range.Abbreviations CD circular dichroism - NOE nuclear Overhauser enhancement - NOESY two-dimensional NOE spectroscopy - INEPT insensitive nuclei enhanced by polarisation transfer - DANTE delays alternating with nutations for tailored excitation - WALTZ-16 wideband, alternating phase, low-power technique for zero residual splitting - FID Free induction decay - ppm parts per million - TSPA 3-trimethylsilyl-(3,3,2,2-d)-propionic acid - HFP d ₂-1,1,1,3,3,3-hexafluoro-2-propanol - CPMG Carr-Purcell-Meiboom-Gill - TFD time-resolved fluorescence depolarisation - CSA chemical shift anisotropy Partly presented at the symposium Dynamics and Function of Biomolecules, Szeged, Hungary, July 31–August 2, 1993.     

Probing the mechanistic role of the long α-helix in subunit L of respiratory Complex I from Escherichia coli by site-directed mutagenesis

The C-terminus of the NuoL subunit of Complex I includes a long amphipathic α-helix positioned parallel to the membrane, which has been considered to function as a piston in the proton pumping machinery. Here, we have introduced three types of mutations into the nuoL gene to test the piston-like function. First, NuoL was truncated at its C- and N-termini, which resulted in low production of a fragile Complex I with negligible activity. Second, we mutated three partially conserved residues of the amphipathic α-helix: Asp and Lys residues and a Pro were substituted for acidic, basic or neutral residues. All these variants exhibited almost a wild-type phenotype. Third, several substitutions and insertions were made to reduce rigidity of the amphipathic α-helix, and/or to change its geometry. Most insertions/substitutions resulted in a normal growth phenotype, albeit often with reduced stability of Complex I. In contrast, insertion of six to seven amino acids at a site of the long α-helix between NuoL and M resulted in substantial loss of proton pumping efficiency. The implications of these results for the proton pumping mechanism of Complex I are discussed.     

Amino acid metabolism in several tissues of the obese Zucker rat as indicated by the tissue accumulation of α-amino[1-14C]isobutyrate

Measurements of the tissue accumulation of α-amino[1-¹⁴C]isobutyrate [1-¹⁴C]AIB) in lean (+/?) and obese (fa/fa) Zucker rats showed an augmented tissue/plasma ratio in the liver of the obese animals. In contrast, brown adipose tissue AIB accumulation was lower in the fa/fa animals. In response to a 24h starvation period AIB accumulation was significantly elevated in the liver and plasma of the lean animals and was unchanged in the liver of the fa/fa animals. The circulating concentration of alanine and branched-chain amino acids was elevated in the fa/fa animals as compared to their lean counterparts. These observations suggest that amino acid uptake is not involved in the impaired muscle development observed in the obese Zucker rat and that the ability of brown adipose tissue for amino acid utilization is decreased in the obese animals suggesting that this may partially explain the impaired thermoregulatory capacity observed in brown adipose tissue of obese Zucker rats.     

Identification of a cyclooxygenase gene from the red alga Gracilaria vermiculophylla and bioconversion of arachidonic acid to PGF(2α) in engineered Escherichia coli

Prostaglandins (PGs) are important local messenger molecules in many tissues and organs of animals including human. For applications in medicine and animal care, PGs are mostly purified from animal tissues or chemically synthesized. To generate a clean, reliable, and inexpensive source for PGs, we have now engineered expression of a suitable cyclooxygenase gene in Escherichia coli and achieved production levels of up to 2.7 mg l⁻¹ PGF_2α. The cyclooxygenase gene cloned from the red alga Gracilaria vermiculophylla appears to be fully functional without any eukaryotic modifications in E. coli. A crude extract of the recombinant E. coli cells is able to convert in vitro the substrate arachidonic acid (AA) to PGF_2α. Furthermore, these E. coli cells produced PGF_2α in a medium supplemented with AA and secreted the PGF_2α product. To our knowledge, this is the first report of the functional expression of a cyclooxygenase gene and concomitant production of PGF_2α in E. coli. The successful microbial synthesis of PGs with reliable yields promises a novel pharmaceutical tool to produce PGF_2α at significantly reduced prices and greater purity.     

Inhibition of activity of the ethylene-forming enzyme by α(p-chlorophenoxy)isobutyric acid

(p-Chlorophenoxy)isobutyric acid (PCIB) inhibited indole-3-acetic acid (IAA)-induced ethylene production in etiolated mung bean hypocotyl sections. The endogenous level of 1-aminocyclopropane-1-carboxylic acid (ACC) was not significantly affected by PCIB, indicating that PCIB exerted its effect primarily by inhibiting the activity of the ethylene-forming enzyme (EFE). This conclusion was supported by the observations that PCIB inhibited the conversion of exogenously applied ACC to ethylene. The inhibitory effect of PCIB was already evident with 0.05 mM PCIB, and it increased with time after application of the inhibitor. PCIB also significantly inhibited ethylene production in apple fruit tissues, but it only slightly reduced the level of endogenous ACC. Similar to mung bean, EFE activity in apple tissue was significantly inhibited by PCIB. The possibility that PCIB also inhibits auxin-induced ACC synthase activity is discussed.