Asymmetric synthesis of enantiomerically and diastereoisomerically enriched 4-[F or Br]-substituted glutamic acids

A novel simple synthetic protocol for the preparation of both (2S,4R)- and (2S,4S)-FGlu, applying Michael addition of methyl α-fluoroacrylate to a Ni^II complex of glycine Schiff base with BPB, was elaborated. In addition, same reaction of mentioned complex with ethyl α-bromoacrylate leads to the Ni^II complex of the Schiff base of BPB with (2S,4R)-4-bromo-glutamic acid monoester, that can be transformed into the corresponding complexes of 1-aminocyclopropane-1,2-dicarboxylic acid. The decomposition of the diastereoisomerically pure complexes leads to corresponding enantiomerically enriched (ee > 98%) amino acids.     

Dynamics of post-denervational modifications of spinal reflex activity in albino rats

In experiments on rats, we studied the characteristics of reflex discharges in the ventral root (VR) L ₅; the discharges were evoked by stimulation of segmental (peripheral nerve or dorsal root, DR) and suprasegmental vestibular (stimulation of the round window of the labyrinth) inputs. Potentials were recorded within different time intervals (from 1 to 150 days) after transection of the sciatic nerve (SN); measures preventing regeneration of its fibers were used. Modifications of the segmental responses related to post-denervational changes included four phases: (i) latent period, (ii) post-denervational spinal hyperreflexia (PDSH), (iii) partial suppression of monosynaptic discharges (MDs) in the VR, and (iv) complete disappearance of VR MDs resulting from late post-denervational changes. The latency of post-denervational modifications was about 18–48 h after the moment of transection of the SN. Within the PDSH phase, modifications were the greatest 3 to 5 days after transection; these changes could be more adequately estimated in the case of stimulation of the DR on the side of transection and not under conditions of stimulation of the central segment of the transected SN per se. Within this phase, the amplitudes of VR MDs and responses to vestibular stimulation were augmented two to three and four to five times, as compared with the respective indices in intact animals. From the 7th to 10th day after the nerve transection, the amplitude of VR MDs progressively dropped, and on about the 20th day these discharges practically disappeared, while polysynaptic components of segmental responses were preserved. Vestibular responses within this period were, as earlier, considerably facilitated. On the 60th and 150th days (within the phase of late post-denervational modifications) there were no VR MDs after stimulation of segmental inputs, and polysynaptic responses were exclusively observed. The amplitude of discharges evoked by vestibular stimulation became lower than in the PDSH state but remained significantly higher than the control values of this parameter. Probable mechanisms of post-denervational modifications of the evoked spinal activity within different time intervals after transection of the SN are discussed. Neirofiziologiya/Neurophysiology, Vol. 39, No. 1, pp. 37–46, January–February, 2007.     

Using the same organocatalyst for asymmetric synthesis of both enantiomers of glutamic acid-derived Ni(II) complexes via 1,4-additions of achiral glycine and dehydroalanine Schiff base Ni(II) complexes

(S)- and (R)-BIMBOL were efficient PT catalysts of asymmetric Michael addition of prochiral Ni-PBP-Gly (1) to acrylic esters and malonic esters to Ni-PBP-Δ-Ala (2) correspondingly. The salient feature of the catalysis is opposite configurations of Glu prepared via the two paths with BIMBOL of the same configuration and a perspective novel catalytic procedure for the synthesis of Gla derivatives.     

Evaluation of the state of the gene pool of natural populations dwelling in the fragmented landscape of Moscow and Moscow region (with special reference to brown frogs)

A system of urban ecological genetic monitoring of vertebrates has been developed for the first time in Russia and in the world. As a model, two species of brown frogs, Rana arvalis Nilss. and R. temporaria L. (16 populations, seven isozyme loci) were used. An evaluation of the gene pool state in urban frog populations has shown that the diversity in the Moscow frog populations was lower than that in natural populations (up to 80 and 50% in respectively R. arvalis and R. temporaria). Mean heterozygosities per locus were higher in large natural populations than in small urban isolates: in R. arvalis, these values were 0.16 and 0.06; in R. temporaria, 0.34 and 0.18, respectively. The number of polymorphic loci was also higher in natural populations than in the urban ones: 4 versus 2 in R. arvalis and 5 versus 4 in R. temporaria. Using superoxiddismutase as an example, fixation of different alleles of the same locus in different small isolates was shown. The gene pool condition of all but one urban populations of brown frog was evaluated as unsatisfactory, and that of the R. arvalis populations, as critical. These changes of the gene pool are explained mainly by gene drift accompanied by inbreeding, which was caused by human-induced fragmentation of the range and a decrease in population size of the species. The results of this study was employed in the development of the Moscow governmental program on restoration of the gene pools of vanishing animal species on specially protected natural urban territories. The series of works on long-term monitoring and assessment of the state of natural populations of model species in anthropogenic landscapes of Moscow and Moscow region has laid a foundation for a new branch of science, gene urbanology.     

Gene pool state and degree of infestation by bark beetle (<Emphasis Type="Italic">Ips tipographus</Emphasis> L.) of Norway spruce (<Emphasis Type="Italic">Picea abies</Emphasis> L. Karst.) natural populations and planted stands in Moscow region

A comparative analysis of the gene pool state in natural populations and planted stands of Norway spruce and the degree of their infestation by the bark beetle in the Moscow region was conducted taking into account the dynamic state of communities (4 populations, 148 samples, 24 isoenzyme loci). The degree of infestation by the bark beetle of conditionally native communities is 0%; for planted stands, it is 90–100%; and for a short-term community, it is 15–20%. The comparison of “healthy” populations and those infested with bark beetle by average values of observed heterozygosity (H _O) detected no significant differences. However, the test on allelic frequency heterogeneity demonstrated the difference of planted stands from conditionally native populations both by three loci (Fe-2, Idh-1, Mdh-3) and by the totality of 18 polymorphic isoenzyme loci; the short-term population differs from conditionally native population only by two loci. The value of the inbreeding coefficient by the Idh-1 locus is significantly higher in both populations infested with the bark beetle than in “healthy” populations. The results of conducted studies demonstrate the necessity of continuation of the study on the gene pool state in Norway spruce populations owing to the degree of their infestation by the bark beetle along with the study on the dynamic state of the communities; this can provide a key to solving the problem of the forest preservation from pests.     

Development of microsatellite genetic markers in Siberian stone pine (<Emphasis Type="Italic">Pinus sibirica</Emphasis> Du Tour) based on the <Emphasis Type="Italic">de novo</Emphasis> whole genome sequencing

Siberian stone pine, Pinus sibirica Du Tour is one of the most economically and environmentally important forest-forming species of conifers in Russia. To study these forests a large number of highly polymorphic molecular genetic markers, such as microsatellite loci, are required. Prior to the new high-throughput next generation sequencing (NGS) methods, discovery of microsatellite loci and development of micro-satellite markers were very time consuming and laborious. The recently developed draft assembly of the Siberian stone pine genome, sequenced using the NGS methods, allowed us to identify a large number of microsatellite loci in the Siberian stone pine genome and to develop species-specific PCR primers for amplification and genotyping of 70 microsatellite loci. The primers were designed using contigs containing short simple sequence tandem repeats from the Siberian stone pine whole genome draft assembly. Based on the testing of primers for 70 microsatellite loci with tri-, tetra- or pentanucleotide repeats, 18 most promising, reliable and polymorphic loci were selected that can be used further as molecular genetic markers in population genetic studies of Siberian stone pine.     

Microsatellite analysis of clonality and individual heterozygosity in natural populations of aspen <Emphasis Type="Italic">Populus tremula</Emphasis> L.: Identification of highly heterozygous clone

Aspen Populus tremula L. (Salicaceae) is the fast-growing tree species of environmental and economic value. Aspen is capable of reproduction by both seeds and vegetative means, forming root sprouts. In an adult stand, identification of ramets of one clone among the trees of seed origin based on their morphology is difficult. A panel of 14 microsatellite loci developed for individual identification of aspen was applied for the clonal structure analysis in four natural aspen stands of the European part of Russia: Moscow and Voronezh oblasts, the Mari-El Republic, and the Republic of Tatarstan. In 52 trees from the Moscow sample, 41 multilocus genotypes were identified; in the Voronezh sample, among 30 individuals, 25 different genotypes were detected; and in the sample from Mari-El, 32 trees were represented by 13 genotypes. In the stand from Sabinsky Forestry, Tatarstan, all of the examined 29 trees were represented by a single genotype. The ancestral tree carrier of this genotype which was the most heterozygous (0.929) among all studied aspen individuals (sample mean, 0.598) obviously has spread over a large territory during several cutting and reproduction cycles, currently occupying the area of 2.2 ha. For aspen, usually suffering from Aspen trunk rot, such high viability is evidence of resistance to the main pathogens. The revealed superclone deserves further study with karyological methods and flow cytometry to determine ploidy level and analysis of the growth rate and the quality of wood for possible use in plantation forest production.     

Genetic diversity, population structure, and differentiation of Siberian larch, Gmelin larch, and Cajander larch on SSR-marker data

The genetic diversity of SSR markers was studied in six populations of Siberian larch, two popu-lations of Gmelin larch, and four populations of Cajander larch. Seven pairs of nuclear microsatellite loci were used for this analysis. 103 allelic variants were detected in 365 individuals of three species of larch. According to the AMOVA results, the variability proportion that characterizes the differences between three Larix species was twice as higher (15%) than the proportion that accounts for among-population differences within the species (6%). The differentiation of the populations of three species of larch based on SSR markers exceeded 12% (F _ST = 0.121). A significant correlation of the genetic distances with the geographic distances between populations was found (r = 0.835, P = 0.01).     

Distribution of the genetic diversity of the Siberian stone pine,Pinus sibirica Du Tour,along the latitudinal and longitudinal profiles

The Siberian stone pine (Pinus sibirica Du Tour) is one of the main forest-forming coniferous species of the boreal ecosystems of Western Siberia. We used the isozyme method to analyze 11 ecotypes representing the latitudinal and longitudinal profiles within the species range, including samples from the geographic boundaries of the distribution. The genetic structure of the ecotypes is described on the basis of the variability for 26 isozyme loci encoding for 16 enzyme systems. The greatest genetic diversity was observed in the taiga ecotypes in the central part of the studied area, while the ecotypes along the species range boundaries were shown to be genetically depauperized. Approximately 8.1 % of the observed genetic diversity is attributed to differences between the studied ecotypes. We detected high levels of genetic diversity for the Fest-2, Pgm-1, Sod-4, and a few other loci, as well as a correlation between allele frequencies and geographical locations of the populations. The results of multivariate analysis of allelic frequencies as well as cluster analysis allowed us to discriminate three major groups of ecotypes: north-eastern, central and south-western. In view of our results, we compare two hypotheses: one which attributes the spatial distribution of genetic variations to the selectivity for some of the polymorphic allozyme loci, and the other based in the history of the formation of the range of the Siberian stone pine.     

No carrier added synthesis of O-(2'-[18F]fluoroethyl)-L-tyrosine via a novel type of chiral enantiomerically pure precursor, NiII complex of a (S)-tyrosine Schiff base

O-(2′-[¹⁸F]fluoroethyl)-l-tyrosine ([¹⁸F]FET) has gained much attention as a promising amino acid radiotracer for tumor imaging with positron emission tomography (PET) due to favorable imaging characteristics and relatively long half-life of ¹⁸F (110 min) allowing remote-site application. Here we present a novel type of chiral enantiomerically pure labeling precursor for [¹⁸F]FET, based on NiII complex of a Schiff’s base of (S)-[N-2-(N′-benzylprolyl)amino]benzophenone (BPB) with alkylated (S)-tyrosine, Ni-(S)-BPB-(S)-Tyr-OCH2CH2X (X = OTs (3a), OMs (3b) and OTf (3c)). A series of compounds 3a–c was synthesized in three steps from commercially available reagents. Non-radioactive FET as a reference was prepared from 3a in a form of (S)-isomer and (R,S) racemic mixture. Radiosynthesis comprised two steps: (1) n.c.a. nucleophilic fluorination of 3a–c (4.5–5.0 mg) in the presence of either Kryptofix 2.2.2.or tetrabutylammonium carbonate (TBAC) in MeCN at 80 °C for 5 min, followed by (2) removal of protective groups by treating with 0.5 M HCl (120 °C, 5 min). The major advantages of this procedure are retention of enantiomeric purity during the ¹⁸F-introduction step and easy simultaneous deprotection of amino and carboxy moieties in 3a–c. Radiochemically pure [¹⁸F]FET was isolated by semi-preparative HPLC (C18 μ-Bondapak, Waters) eluent aq 0.01 M CH₃COONH₄, pH 4/C₂H₅OH 90/10 (v/v). Overall synthesis time operated by Anatech RB 86 laboratory robot was 55 min. In a series of compounds 3a–c, tosyl derivative 3a provided highest radiochemical yield (40–45%, corrected for radioactive decay). Enantiomeric purity was 94–95% and 96–97%, correspondingly, for Kryptofix and TBAC assisted fluorinations. The suggested procedure involved minimal number of synthesis steps and suits perfectly for automation in the modern synthesis modules for PET radiopharmaceuticals. Preliminary biodistribution study in experimental model of turpentine-induced aseptic abscess and Glioma35 rat’s tumor (homografts) in Wistar rats has demonstrated the enhanced uptake of radiotracer in the tumor area with minimal accumulation in the inflamed tissues.