Catalytic asymmetric synthesis of α-methyl-p-boronophenylalanine

p-Boronophenylalanine (l-BPA) is applied in clinical settings as a boron carrier for boron neutron capture therapy (BNCT) to cure malignant melanomas. Structural modification or derivatization of l-BPA, however, to improve its uptake efficiency into tumor cells has scarcely been investigated. We successfully synthesized (S)-2-amino-3-(4-boronophenyl)-2-methylpropanoic acid in enantioenriched form as a novel candidate molecule for BNCT. Key steps to enhance the efficiency of this synthesis were enantioselective alkylation of N-protected alanine tert-butyl ester with a Maruoka catalyst and Miyaura borylation reaction to install the boron functionality.     

The asymmetric adsorption of α-aminopropionitrile on D-quartz

-Aminopropionitrile was adsorbed on the powdered D-quartz, recovered as N-trifluoroacetylalanyl-(+)-secondarybutyl ester, and analyzed by means of gas chromatography. The asymmetric result supporting the preferential adsorption of L-antipode was obtained and its significance in chemical evolution has been discussed.     

Reverse-docking study of the TADDOL-catalyzed asymmetric hetero-Diels–Alder reaction

The reverse-docking of a TADDOL catalyst to rigid transition-state (TS) representations of an asymmetric hetero-Diels–Alder reaction is described. The resulting docking poses represent a simplified geometric model of the TS for the catalyzed reaction. The conformational space of the catalyst in proximity to the catalyst-free TS models is sampled stochastically and the energetically favored poses are subjected to a clustering procedure to highlight structural attributes compatible with organocatalysis. Each pose is scored and ranked based on its molecular-mechanics docking energy. The reverse-docking procedure reveals a clear energetic trend in favor of the experimentally preferred product enantiomers. Analysis of the best poses suggests a geometric model that is consistent with principles of molecular recognition, catalysis, and experimental data.      

Detection and quantification of α-keto-δ-(N,N-dimethylguanidino)valeric acid: A metabolite of asymmetric dimethylarginine

Nitric oxide is an ubiquitary cell signaling substance. Its enzymatic production rate by nitric oxide synthase is regulated by the concentrations of the substrate l-arginine and the competitive inhibitor asymmetric dimethylarginine (ADMA). A newly recognized elimination pathway for ADMA is the transamination to α-keto-δ-(N^G,N^G-dimethylguanidino)valeric acid (DMGV) by the enzyme alanine-glyoxylate aminotransferase 2 (AGXT2). This pathway has been proven to be relevant for nitric oxide regulation, but up to now no method exists for the determination of DMGV in biological fluids. We have developed a liquid chromatography–tandem mass spectrometry (LC–MS/MS) method for the quantification of DMGV. D₆-DMGV was used as internal standard. Samples were purified online by column switching, and separation was achieved on a porous graphitic carbon column. The calibration was linear over ranges of 10 to 200 nmol/L for plasma and 0.1 to 20 μmol/L for urine. The intra- and interday accuracies and precisions in plasma and urine were better than 10%. In plasma samples, DMGV was present in concentrations between 19.1 and 77.5 nmol/L. In urine samples, concentrations between 0.0114 and 1.03 μmol/mmol creatinine were found. This method can be used as a tool for the scientific investigation of the ADMA conversion to DMGV via the enzyme AGXT2.     

Introgression of the Haynaldia villosa genome into γ-ray-induced asymmetric somatic hybrids of wheat

To study the effect of -ray treatment on donor and derived somatic hybrids, we carried out -ray donor treatment experiments with a wide range of -ray dosages and asymmetric somatic hybridization between protoplasts of wheat (Triticum aestivum L. Jinan 177) and protoplasts of Haynaldia villosa Schur. treated with different dosages of -rays (40, 60 and 80 Gy, respectively). We first screened the putative hybrids by isozyme analysis, followed by characterization of nuclear and organellar genome composition of the hybrids. Genomic in situ hybridization on mitotic metaphases demonstrated that the donor chromosome elimination in the hybrids increased with increased -ray dosage. Intergenomic chromosome recombination/translocations were observed in the hybrids from different dosages of -rays. PCR amplification of 5S rDNA spacer sequences showed that only some of the regenerated hybrid clones inherited donor 5S rDNA sequences, suggesting that the donor DNA was also eliminated randomly. Restriction fragment length polymorphism analysis using mitochondrion (mt) and chloroplast (cp) gene-specific probes showed that the hybrid calli contained mt genomes of both parents and the cp genome of only one of the parents. Recombinations between parental mt as well as cp genes were found in the hybrid clones. Furthermore, development of the hybrid clones was dependent on the -ray dosage used for the donor treatment. Regenerated plants were only obtained from fusion combinations of low (40 Gy) and intermediate (60 Gy) dose irradiation. The possible role and significance of -rays on the introgression of small segments of donor chromosomes to the receptor is discussed.     

Polyethylene glycol-modified lipase catalyzes asymmetric alcoholysis of δ-decalactone in n-decanol

Summary Lipase from Pseudomonas cepacia was modified with 2,4-bis[O-methoxypoly(ethylene glycol)]-6-chloro-s-triazine(activated PEG₂) to form PEG-lipase. The PEG-lipase, soluble and active in organic solvents, catalyzes asymmetric alcoholysis of racemic -decalactone in alcohols to form (R)-5-hydroxydecanoic acid alkyl esters. The yield was 69% with 83% enantiomeric excess after 3 hr-reaction in n-decanol at 50°C. The advantage of this reaction is that the alcoholysis proceeds efficiently in straight hydrophobic substrates without any organic solvents.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

Non–antigen-contacting region of an asymmetric bispecific antibody to factors IXa/X significantly affects factor VIII-mimetic activity

While antibody engineering improves the properties of therapeutic antibodies, optimization of regions that do not contact antigens has been mainly focused on modifying the effector functions and pharmacokinetics of antibodies. We recently reported an asymmetric anti-FIXa/FX bispecific IgG₄ antibody, ACE910, which mimics the cofactor function of FVIII by placing the two factors into spatial proximity for the treatment of hemophilia A. During the optimization process, we found that the activity was significantly affected by IgG subclass and by modifications to the inter-chain disulfide bonds, upper hinge region, elbow hinge region, and Fc glycan, even though these regions were unlikely to come into direct contact with the antigens. Of these non–antigen-contacting regions, the tertiary structure determined by the inter-chain disulfide bonds was found to strongly affect the FVIII-mimetic activity. Interestingly, IgG₄-like disulfide bonds between Cys131 in the heavy chain and Cys114 in the light chain, and disulfide bonds between the two heavy chains at the hinge region were indispensable for the high FVIII-mimetic activity. Moreover, proline mutations in the upper hinge region and removal of the Fc glycan enhanced the FVIII-mimetic activity, suggesting that flexibility of the upper hinge region and the Fc portion structure are important for the FVIII-mimetic activity. This study suggests that these non–antigen-contacting regions can be engineered to improve the biological activity of IgG antibodies with functions similar to ACE910, such as placing two antigens into spatial proximity, retargeting effector cells to target cells, or co-ligating two identical or different antigens on the same cell.     

InCl3-catalyzed asymmetric aza-Friedel-Crafts reaction of indoles with imines generated from O-pivaloylated β-d-galactosylamine

The highly diastereoselective InCl₃-catalyzed aza-Friedel-Crafts reaction of substituted indoles with aldimines generated from Kunz’s amine was studied. The reaction afforded the desired product in good to high yields with up to >19:1 diastereoselective ratios. The O-pivaloylated β-d-galactosyl moiety could not be cleaved under the traditional acidic conditions. It was removed successfully after unmasking of the O-pivaloyl groups using MeOH/NaOMe and treatment with HOAc/H₂O subsequently, to yield the 3-indolyl aryl methanamine derivatives in high optical purity.     

The asymmetric function of Dph1–Dph2 heterodimer in diphthamide biosynthesis

JBIC Journal of Biological Inorganic Chemistry - Diphthamide, the target of diphtheria toxin, is a post-translationally modified histidine residue found in archaeal and eukaryotic translation...     

Asymmetric synthesis of β-amino alcohol by reductive cross-coupling of planar chiral ferrocenecarboxaldehyde with N-tosyl ferrocenylideneamine, and its application to asymmetric reaction

Samarium iodine-mediated cross-coupling of N-tosyl ferrocenylideneamine with planar chiral ferrocenecarboxaldehyde gave diastereoselectively anti-β-amino alcohol derivative in good yield. The obtained anti-β-amino alcohol with ferrocene ring at 1,2-positions was utilized as chiral auxiliary for asymmetric alkylation and acylation reactions.     

Metabolically driven equilibrium shift of asymmetric amination of ketones by ω-transaminase using alanine as an amino donor

Removal of a side product to overcome unfavorable equilibrium is a prerequisite for the asymmetric amination of ketones using ω-transaminase (ω-TA). Alanine has been preferred as an amino donor because its deamination product (i.e. pyruvate) is easily removable by several enzymatic methods. Here, we demonstrated that the removal of pyruvate by an innate metabolic pathway could afford equilibrium shift of the ω-TA reactions.     

Application of comparative proteome analysis to reveal influence of cultivation conditions on asymmetric bioreduction of β-keto ester by Saccharomyces cerevisiae

Industrial bakers' yeast strain Saccharomyces cerevisiae LH1 was selected for asymmetric reduction of ethyl benzoylacetate to (S)-ethyl 3-hydroxy-3-phenylpropionate. Higher reductive efficiency and higher cofactor availability were obtained with the alternation of cultivation condition (mainly growth medium). Compared to the bioreduction by yeast cells grown in malt extract (ME) medium, the concentration of substrate was increased 25-fold (up to 15.6 g/l) in the yeast peptone dextrose (YPD)-grown cells mediated bioreduction with 97.5% of enantioselective excess of (S)-product. The proteomic responses of S. cerevisiae LH1 cells to growth in aerobic batch cultures fed with either YPD or ME medium were examined and compared. Among the relative quantities of 550 protein spots in each gel, changes were shown in the expression level of 102 intracellular proteins when comparing YPD gel to ME gel. Most of the identified proteins were involved in energy metabolism and several cellular molecular biosynthetic pathway and catabolism. For YPD-grown yeast cells, not only enzymes involved in nicotinamide adenine dinucleotide phosphate regeneration, especially 6-phosphogluconate dehydrogenase, but also alcohol dehydrogenase 1 and D: -arabinose 1-dehydrogenase which had been demonstrated activity toward ethyl benzoylacetate to (S)-hydroxy ester were significantly upregulated. These changes provided us insight in the way the yeast cells adapted to a change in cultivation medium and regulated its catalytic efficiency in the bioreduction.     

Does asymmetric gene flow among matrilines maintain the evolutionary potential of the European eel?

Using evolutionary theory to predict the dynamics of populations is one of the aims of evolutionary conservation. In endangered species, with geographic range extending over continuous areas, the predictive capacity of evolutionary‐based conservation measures greatly depends on the accurate identification of reproductive units. The endangered European eel (Anguilla anguilla) is a highly migratory fish species with declining population due to a steep recruitment collapse in the beginning of the 1980s. Despite punctual observations of genetic structure, the population is viewed as a single panmictic reproductive unit. To understand the possible origin of the detected structure in this species, we used a combination of mitochondrial and nuclear loci to indirectly evaluate the possible existence of cryptic demes. For that, 403 glass eels from three successive cohorts arriving at a single location were screened for phenotypic and genetic diversity, while controlling for possible geographic variation. Over the 3 years of sampling, we consistently identified three major matrilines which we hypothesized to represent demes. Interestingly, not only we found that population genetic models support the existence of those matriline‐driven demes over a completely panmictic mode of reproduction, but also we found evidence for asymmetric gene flow amongst those demes. We uphold the suggestion that the detection of demes related to those matrilines reflect a fragmented spawning ground, a conceptually plausible consequence of the low abundance that the European eel has been experiencing for three decades. Furthermore, we suggest that this cryptic organization may contribute to the maintenance of the adaptive potential of the species.     

Pyrrolidine based chiral organocatalyst for efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes

An efficient asymmetric Michael addition of cyclic ketones to β-nitrostyrenes using secondary diamine as an organocatalyst derived from l-proline and (R)-α-methylbenzyl amine has been described. This pyrrolidine based catalyst 1 was found to be very effective to synthesize various γ-nitrocarbonyl compounds in good yield (up to 81%) with excellent stereoselectivity (up to >99:1 dr and >99% ee).     

NMR structure determination of the tetramerization domain of the Mnt repressor: An asymmetric α-helical assembly in slow exchange

The structure and dynamics of the chymotryptic tetramerization domain of the Mnt repressor of Salmonella bacteriophage P22 have been studied by NMR spectroscopy. Two sets of resonances (A and B) were found, representing the asymmetry within the homotetramer. Triple-resonance techniques were used to obtain unambiguous assignments of the A and B resonances. Intra-monomeric NOEs, which were distinguished from the inter-monomeric NOEs by exploiting ¹³C/¹⁵N-filtered NOE experiments, demonstrated a continuous -helix of approximately seven turns for both the A and B monomers. The asymmetry facilitated the interpretation of inter-subunit NOEs, whereas the antiparallel alignment of the subunits allowed further discrimination of inter-monomeric NOEs. The three-dimensional structure revealed an unusual asymmetric packing of a dimer of two antiparallel right-handed intertwined coiled -helices. The A and B forms exchange on a timescale of seconds by a mechanism that probably involves a relative sliding of the two coiled coils. The amide proton solvent exchange rates demonstrate a stable tetrameric structure. The essential role of Tyr 78 in oligomerization of Mnt, found by previous mutagenesis studies, can be explained by the many hydrophobic and hydrogen bonding interactions that this residue participates in with adjacent monomers.     

Conformational study by CD of chirally tethered naphthalene moieties: toward an understanding of the asymmetric intramolecular coupling reaction?

The carbonate (R,R)-1 and the diester (R,R)-2, precursors of optically active 1,1'-binaphthyl derivatives through asymmetric intramolecular coupling, were prepared and fully characterized. The absorption and CD spectra, together with the cholesteric induction measurement of (R,R)-1, indicate that it assumes a conformation in which the naphthalene rings are M-skewed. Since the intramolecular coupling of (R,R)-1 affords a P-twisted (S) binaphthyl derivative, a change of twist from M to P must occur during the reaction. Moreover, this conformation allows the coupling to occur along only one pathway, thus explaining the high stereoselectivity. The CD analysis of (R,R)-2 indicates that it assumes a rigid conformation as well, with the naphthalene rings fixed relative to each other. However, in this compound the naphthalene moieties can give rise to a coupling reaction following two different pathways, leading to oppositely configured binaphthyls with almost the same probability so that low stereoselectivity results. Copyright 2000 Wiley-Liss, Inc.