Determination of the enantiomeric purity of (−−) 2-(N-propyl-n-2-thienylethylamino)-5-hydroxytetralin (n-0923) by chiral stationary phase HPLC

The determination of the enantiomeric impurity, i.e., the percentage of (+) N?0437 (= N?0924) in several batches of (??) N-0437 (= N-0923) by chiral HPLC is described. Enantiomeric impurities were calculated based on the peak areas of the two baseline separated enantiomers in the chromatogram. The enantiomeric impurities found in different batches ranged from 0.02% to 0.11%. Calibration curves of the two isomers of N-0437 (Fig. 1,) were made twice to study the reproducibility and linearity of the method. The absorbance ratio, N-0923/N-0924, was found to be 1.02 with a relative standard deviation (RSD) of 9% over the whole concentration range used for the calibration curves.     

Comparison between cellulose and amylose tris(3,5-dimethylphenylcarbamate) chiral stationary phases for enantiomeric separation of 17 amidotetralins

Direct enantiomeric separations of 17 chiral amidotetralins by means of high performance liquid chromatography were performed on stationary phases composed of tris(3,5-dimethylphenylcarbamate) derivatives of cellulose and amylose, coated on silica gel. The enantiomers of 15 out of 17 amidotetralins were resolved with a resolution of more than 1.5 by at least one of the chiral stationary phases. The stationary phases showed complementary results with regard to the separation of the amidotetralins, that is, pairs that did not separate on the cellulose-type column were well separated on the amylose-type column, and vice versa. There was no significant correlation between the chromatographic properties of the chiral stationary phases. © 1993 Wiley-Liss, Inc.     

Blood drugs and other analytical challenges (Methodological Surveys in Biochemistry,Vol. 7) : Edited by E. Reid,Ellis Horwood (Wiley), Chichester, 1978, X + 355 pp., price £ 19.50, ISBN 0-85312-124-9

A high-performance liquid chromatographic procedure has been developed for the separation and fluorometric detection of guanidino compounds in physiologic fluids. All guanidino compounds were separated on a 17 × 0.46 cm cation-exchange column using a stepwise pH gradient. The chromatographic system was designed to enable the use of the specific reagent 9,10-phenanthrenequinone as a means of monitoring the guanidino compounds of physiologic fluids. This new analytical method is so sensitive that it enables the analysis at the picomole level. Our automatic guanidino-compound analyzer was successfully applied to the quantitative determination of all guanidino compounds in physiologic fluids from normal controls and uremic patients.     

Age and biostratigraphic significance of the Punung Rainforest Fauna, East Java, Indonesia, and implications for Pongo and Homo

The Punung Fauna is a key component in the biostratigraphic sequence of Java. It represents the most significant faunal turnover on the island in the last 1.5 million years, when Stegodon and other archaic mammal species characteristic of earlier Faunal stages were replaced by a fully modern fauna that included rainforest-dependent species such as Pongo pygmaeus (orangutan). Here, we report the first numerical ages for the Punung Fauna obtained by luminescence and uranium-series dating of the fossil-bearing deposits and associated flowstones. The Punung Fauna contained in the dated breccia is of early Last Interglacial age (between 128+/-15 and 118+/-3 ka). This result has implications for the age of the preceding Ngandong Fauna, including Homo erectus remains found in the Ngandong Terrace, and for the timing of Homo sapiens arrival in Southeast Asia, in view of claims for a modern human tooth associated with the Punung breccia.     

Dragon's Paradise Lost: Palaeobiogeography,Evolution and Extinction of the Largest-Ever Terrestrial Lizards (Varanidae)

Background

The largest living lizard species, Varanus komodoensis Ouwens 1912, is vulnerable to extinction, being restricted to a few isolated islands in eastern Indonesia, between Java and Australia, where it is the dominant terrestrial carnivore. Understanding how large-bodied varanids responded to past environmental change underpins long-term management of V. komodoensis populations.

Methodology/Principal Findings

We reconstruct the palaeobiogeography of Neogene giant varanids and identify a new (unnamed) species from the island of Timor. Our data reject the long-held perception that V. komodoensis became a giant because of insular evolution or as a specialist hunter of pygmy Stegodon. Phyletic giantism, coupled with a westward dispersal from mainland Australia, provides the most parsimonious explanation for the palaeodistribution of V. komodoensis and the newly identified species of giant varanid from Timor. Pliocene giant varanid fossils from Australia are morphologically referable to V. komodoensis suggesting an ultimate origin for V. komodoensis on mainland Australia (>3.8 million years ago). Varanus komodoensis body size has remained stable over the last 900,000 years (ka) on Flores, a time marked by major faunal turnovers, extinction of the island''s megafauna, the arrival of early hominids by 880 ka, co-existence with Homo floresiensis, and the arrival of modern humans by 10 ka. Within the last 2000 years their populations have contracted severely.

Conclusions/Significance

Giant varanids were once a ubiquitous part of Subcontinental Eurasian and Australasian faunas during the Neogene. Extinction played a pivotal role in the reduction of their ranges and diversity throughout the late Quaternary, leaving only V. komodoensis as an isolated long-term survivor. The events over the last two millennia now threaten its future survival.     

Singlet oxygenation in microemulsion catalysed by vanadium chloroperoxidase

Non-ionic microemulsions compatible with the enzyme vanadium chloroperoxidase were designed to perform singlet oxygenation of apolar substrates. The media were based on mono- and polydisperse ethoxylated fatty alcohols (C_iE_j), octane and aqueous buffer. “Fish” diagrams were determined to identify the Winsor-boundaries and to formulate a monophasic Winsor IV microemulsion with a minimal surfactant concentration, ensuring less singlet oxygen (¹O₂) loss than in an aqueous system, thus creating a high oxygenation efficiency. The enzyme was shown to be fully stable in the microemulsion for at least 10 h, converting H₂O₂ into a constant flow of ¹O₂ in the aqueous microdomains. Part of the ¹O₂ diffuses into the organic compartments prior to fast physical deactivation of ¹O₂ by water molecules. In the apolar domains ¹O₂ quantitatively converts the model substrate 9,10-dimethylanthracene into its corresponding endoperoxide. Near-IR chemiluminescence measurements confirm that the ¹O₂ signal in the microemulsion is higher than in simple aqueous buffer. In a well-stirred (water/octane) biphasic system endoperoxide formation is also observed but the conversion rate is much lower, most likely due to stronger physical quenching of ¹O₂.     

Impact of substituents on the enantioseparation of racemic 2-amidotetralins on polysaccharide stationary phases. I. chiralcel OD

The direct enantiomeric separation of 32 racemic 2-amidotetralins on the commercially available tris-(3,5-dimethylphenylcarbamate) derivative of cellulose, coated on silica gel (Chiralcel OD), is presented. To date, the selection of a column for the chiral separation of a racemic mixture is done empirically. Studying the impact of small changes in the chemical structure of a series of amidotetralins on the separation behavior may help to give an insight in the chiral recognition mechanism. The amidotetralins differed structurally in three of their substituents, which were never directly located on the chiral carbon atom. The enantiomers of 24 out of 32 amidotetralins could be resolved with a resolution >1.5. Hydrogen bonding and π-π interactions are supposed to be the major analyte-chiral stationary phase (CSP) interactions. However, the spatial arrangement of the enantiomers may play an important role too. Increasing the bulkiness of the acyl substituent led to an increase in the resolution (R_S), whereas a more bulky substituent on the aromatic ring resulted in a very low resolution. The introduction of a chlorine atom into the acyl substituent additionally increased the resolving power. Chirality 8:574–578, 1996. © 1997 Wiley-Liss, Inc.     

X-ray crystal structures of active site mutants of the vanadium-containing chloroperoxidase from the fungus Curvularia inaequalis

The X-ray structures of the chloroperoxidase from Curvularia inaequalis, heterologously expressed in Saccharomyces cerevisiae, have been determined both in its apo and in its holo forms at 1.66 and 2.11?Å resolution, respectively. The crystal structures reveal that the overall structure of this enzyme remains nearly unaltered, particularly at the metal binding site. At the active site of the apo-chloroperoxidase structure a clearly defined sulfate ion was found, partially stabilised through electrostatic interactions and hydrogen bonds with positively charged residues involved in the interactions with the vanadate in the native protein. The vanadate binding pocket seems to form a very rigid frame stabilising oxyanion binding. The rigidity of this active site matrix is the result of a large number of hydrogen bonding interactions involving side chains and the main chain of residues lining the active site. The structures of single site mutants to alanine of the catalytic residue His404 and the vanadium protein ligand His496 have also been analysed. Additionally we determined the structural effects of mutations to alanine of residue Arg360, directly involved in the compensation of the negative charge of the vanadate group, and of residue Asp292 involved in forming a salt bridge with Arg490 which also interacts with the vanadate. The enzymatic chlorinating activity is drastically reduced to approximately 1% in mutants D292A, H404A and H496A. The structures of the mutants confirm the view of the active site of this chloroperoxidase as a rigid matrix providing an oxyanion binding site. No large changes are observed at the active site for any of the analysed mutants. The empty space left by replacement of large side chains by alanines is usually occupied by a new solvent molecule which partially replaces the hydrogen bonding interactions to the vanadate. The new solvent molecules additionally replace part of the interactions the mutated side chains were making to other residues lining the active site frame. When this is not possible, another side chain in the proximity of the mutated residue moves in order to satisfy the hydrogen bonding potential of the residues located at the active site frame.     

Peroxidase and phosphatase activity of active-site mutants of vanadium chloroperoxidase from the fungus Curvularia inaequalis. Implications for the catalytic mechanisms

Mutation studies were performed on active-site residues of vanadium chloroperoxidase from the fungus Curvularia inaequalis, an enzyme which exhibits both haloperoxidase and phosphatase activity and is related to glucose-6-phosphatase. The effects of mutation to alanine on haloperoxidase activity were studied for the proposed catalytic residue His-404 and for residue Asp-292, which is located close to the vanadate cofactor. The mutants were strongly impaired in their ability to oxidize chloride but still oxidized bromide, although they inactivate during turnover. The effects on the optical absorption spectrum of vanadium chloroperoxidase indicate that mutant H404A has a reduced affinity for the cofactor, whereas this affinity is unchanged in mutant D292A. The effect on the phosphatase activity of the apoenzyme was investigated for six mutants of putative catalytic residues. Effects of mutation of His-496, Arg-490, Arg-360, Lys-353, and His-404 to alanine are in line with their proposed roles in nucleophilic attack, transition-state stabilization, and leaving-group protonation. Asp-292 is excluded as the group that protonates the leaving group. A model based on the mutagenesis studies is presented and may serve as a template for glucose-6-phosphatase and other related phosphatases. Hydrolysis of a phospho-histidine intermediate is the rate-determining step in the phosphatase activity of apochloroperoxidase, as shown by burst kinetics.     

Unique Dental Morphology of Homo floresiensis and Its Evolutionary Implications

Homo floresiensis is an extinct, diminutive hominin species discovered in the Late Pleistocene deposits of Liang Bua cave, Flores, eastern Indonesia. The nature and evolutionary origins of H. floresiensis’ unique physical characters have been intensively debated. Based on extensive comparisons using linear metric analyses, crown contour analyses, and other trait-by-trait morphological comparisons, we report here that the dental remains from multiple individuals indicate that H. floresiensis had primitive canine-premolar and advanced molar morphologies, a combination of dental traits unknown in any other hominin species. The primitive aspects are comparable to H. erectus from the Early Pleistocene, whereas some of the molar morphologies are more progressive even compared to those of modern humans. This evidence contradicts the earlier claim of an entirely modern human-like dental morphology of H. floresiensis, while at the same time does not support the hypothesis that H. floresiensis originated from a much older H. habilis or Australopithecus-like small-brained hominin species currently unknown in the Asian fossil record. These results are however consistent with the alternative hypothesis that H. floresiensis derived from an earlier Asian Homo erectus population and experienced substantial body and brain size dwarfism in an isolated insular setting. The dentition of H. floresiensis is not a simple, scaled-down version of earlier hominins.