Preparative synthesis of dTDP-L-rhamnose through combined enzymatic pathways

dTDP-L-rhamnose, an important precursor of O-antigen, was prepared on a large scale from dTMP by executing an one-pot reaction in which six enzymes are involved. Two enzymes, dTDP-4-keto-6-deoxy-D-glucose 3,5-epimerase and dTDP-4-keto-rhamnose reductase, responsible for the conversion of dTDP-4-keto-6-deoxy-D-glucose to dTDP-L-rhamnose, were isolated from their putative sequences in the genome of Mesorhizobium loti, functionally expressed in Escherichia coli, and their enzymatic activities were identified. The two enzymes were combined with an enzymatic process for dTDP-4-keto-6-deoxy-D-glucose involving TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-glucose 4,6-dehydratase, which allowed us to achieve a preparative scale synthesis of dTDP-L-rhamnose using dTMP and glucose-1-phosphate as starting materials. About 82% yield of dTDP-L-rhamnose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 10 mM NADH, 60 mM acetyl phosphate, and 80 mM glucose-1-phosphate. From the reaction with 20 ml volume, approximately 180 mg of dTDP-L-rhamnose was obtained in an overall yield of 60% after two-step purification, that is, anion exchange chromatography and gel filtration for desalting. The purified product was identified by HPLC, ESI-MS, and NMR, showing about 95% purity.     

Induction and detoxification of maize 1,4-benzoxazin-3-ones by insect herbivores

In monocotyledonous plants, 1,4‐benzoxazin‐3‐ones, also referred to as benzoxazinoids or hydroxamic acids, are one of the most important chemical barriers against herbivores. However, knowledge about their behavior after attack, mode of action and potential detoxification by specialized insects remains limited. We chose an innovative analytical approach to understand the role of maize 1,4‐benzoxazin‐3‐ones in plant–insect interactions. By combining unbiased metabolomics screening and simultaneous measurements of living and digested plant tissue, we created a quantitative dynamic map of 1,4‐benzoxazin‐3‐ones at the plant–insect interface. Hypotheses derived from this map were tested by specifically developed in vitro assays using purified 1,4‐benzoxazin‐3‐ones and active extracts from mutant plants lacking 1,4‐benzoxazin‐3‐ones. Our data show that maize plants possess a two‐step defensive system that effectively fends off both the generalist Spodoptera littoralis and the specialist Spodoptera frugiperda. In the first step, upon insect attack, large quantities of 2‐β‐d ‐glucopyranosyloxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA‐Glc) are formed. In the second step, after tissue disruption by the herbivores, highly unstable 2‐hydroxy‐4,7‐dimethoxy‐1,4‐benzoxazin‐3‐one (HDMBOA) is released by plant‐derived β‐glucosidases. HDMBOA acts as a strong deterrent to both S. littoralis and S. frugiperda. Although constitutively produced 1,4‐benzoxazin‐3‐ones such as 2,4‐dihydroxy‐7‐methoxy‐1,4‐benzoxazin‐3‐one (DIMBOA) are detoxified via glycosylation by the insects, no conjugation of HDMBOA in the insect gut was found, which may explain why even the specialist S. frugiperda has not evolved immunity against this plant defense. Taken together, our results show the benefit of using a plant–insect interface approach to elucidate plant defensive processes and unravel a potent resistance mechanism in maize.     

Removal of cleavage slow points from affinity tags used in the IMAC purification of recombinant proteins

The complete enzymatic removal of affinity tags from tagged recombinant proteins is often required but can be challenging when slow points for cleavage exist. This study documents a general approach to remove N‐terminal tags from recombinant proteins specifically designed to be efficiently captured by IMAC resins. In particular, site‐directed mutagenesis procedures have been used to modify the amino acid sequence of metal binding tags useful in IMAC purifications of recombinant proteins with the objective to increase cleavage efficiency with the exopeptidase, dipeptidyl aminopeptidase 1. These tags were specifically developed for application with borderline metal ions, such as Ni²⁺ or Cu²⁺ ions, chelated to the immobilized ligands, 1,4,7‐triazacyclononane (tacn) and its analogs. Due to the ability to control cleavage site structure and accessibility via site directed mutagenesis methods, these procedures offer considerable scope to obtain recombinant proteins with authentic native N‐termini, thus avoiding any impact on structural stability, humoral and cellular immune responses, or other biological functions. Collectively, these IMAC‐based methods provide a practical alternative to other procedures for the purification of recombinant proteins with tag removal. Overall, this approach is essentially operating as an integrated down‐stream purification capability.     

Enantioselective synthesis of (S)-2-amino-4-phenylbutanoic acid by the hydantoinase method

Biosynthesis of (S)-(+)-2-amino-4-phenylbutanoic acid (1) was performed by nonenantioselective hydantoinase and L-N-carbamoylase using racemic 5-[2-phenylethyl]-imidazolidine-2,4-dione (rac-2) as a substrate. The compounds involved in this biocatalysis process could be simultaneously resolved by high-performance liquid chromatography using Chirobiotic T column with a mobile phase of EtOH/H(2)O = 10/90 at pH 4.2-4.5. To our knowledge, this is the first report of the successful production of 1 by the combination of recombinant hydantoinase and L-N-carbamoylase.     

Female sex pheromone of a nettle caterpillar,Monema flavescens,in China

Monema flavescens Walker (Lepidoptera: Limacodidae) is a multivoltine, generalist moth whose larvae cause serious damage to many types of trees. Pheromone lures prepared according to a study of a Japanese population were found to be ineffective at attracting M. flavescens nettle caterpillars in China, and some studies have shown intraspecific geographical differences in the composition of sex pheromones. We therefore reexamined the sex pheromone composition of M. flavescens in a Chinese population. In this study, the electroantennographically (EAG) active compounds in an extract from Chinese virgin females of M. flavescens were identified as (E)‐8‐decen‐1‐ol (E8‐10:OH), (Z)‐7,9‐decadien‐1‐ol (Z7,9‐10:OH), (Z)‐9,11‐dodecadien‐1‐ol (Z9,11‐12:OH), and (Z)‐9,11‐dodecadienal (Z9,11‐12:Ald) via coupled gas chromatographic‐electroantennographic detection (GC‐EAD) and coupled GC‐mass spectrometry (MS). Pheromone dimorphism might occur in this species, as this mixture of compounds in Chinese females was different from that of E8‐10:OH and E7,9‐10:OH extracted from Japanese females in previous research. In wind tunnel and field tests, the males were significantly attracted to a blend of the pheromone components E8‐10:OH, Z7,9‐10:OH, and Z9,11‐12:OH in a 100:5:4 ratio. The addition of Z9,11‐12:Ald did not change the male response. The optimized three‐component lure blend may provide a useful tool for monitoring and controlling Chinese populations of M. flavescens.     

Chiral separation of pheniramine-like 3-phenyl-3-heteroarylpropylamines by CE and HPLC methods

Analytical CE and HPLC methods were developed for the chiral separation of halogen-substituted 3-phenyl-3-(2-pyridyl)propylamines 1-4 (1: 3-(4-fluorophenyl) approximately, 2: 3-(3,4-difluorophenyl) approximately, 3: 3-(4-chlorophenyl) approximately, 4: 3-(3,4-dichlorophenyl) approximately ), 3-(4-fluorophenyl)-3-(2-thiazolyl)propylamine (5), and 3-(4-fluorophenyl)-3-(1-benzylimidazol-2-yl)propylamine (6), which are building blocks for the preparation of very potent arpromidine-type histamine H(2) receptor agonists. All amines were enantioseparated by CE with resolutions of at least 1.8 using alpha-, beta-, or gamma-cyclodextrin (CD) as chiral selectors. With heparin as buffer additive for CE the optical antipodes of 1-4 and 6 were separated with resolutions > or = 1.8. On RP-18 columns the separation of the (+)-(S)-acetylmandelic acid amides of racemic 2 (R = 0.9, alpha = 1.07) and the thioureas prepared by addition of 6 to 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate (R = 2.0, alpha = 1.20) was successful, whereas the diastereomeric ureas prepared from 3 and (+)-(S)-1-(1-naphthyl)ethyl isocyanate could not be resolved. Separation of the diastereomeric isoindoles prepared from 1-5, o-phthaldialdehyde and 2,3,4,6-tetra-O-acetyl-1-thio-beta-D-glucopyranoside was achieved on a RP-18 phase (R > or = 0.4, a > or = 1.02). Direct separation of the enantiomers of 3 and 4 was achieved on a Cyclobond I column (R > or = 0.9, alpha > or = 1.07). alpha- and beta-CD were also useful as mobile phase additives for HPLC (3 and 4: RP-18 column, beta-CD, R > or = 0.4, alpha > or = 1.03; 3: RP-18 column, alpha-CD: R = 0.5, alpha = 1.04).     

Structural and spectroscopic analysis of hydrogensquarates of glycine-containing tripeptides

The hydrogensquarates of glycine-containing tripeptides glycylglycylglycine (H-Gly-Gly-Gly-OH), glycylglycylmethionine (H-Gly-Gly-Met-OH), and methionylglycylglycine (H-Met-Gly-Gly-OH) have been characterized structurally. Quantum chemical ab initio calculations, solid-state linear-dichroic infrared (IR-LD) spectroscopy, 1H and 13C NMR data, ESI-MS, HPLC-MS/MS, TGV, and DSC methods were employed. The structures consist in a positively charged peptide moiety and a negative hydrogensquarate anion (HSq), stabilized by strong intermolecular hydrogen bonds.     

One-pot enzymatic production of dTDP-4-keto-6-deoxy-D-glucose from dTMP and glucose-1-phosphate

An enzymatic production method for dTDP-4-keto-6-deoxy-D-glucose, a key intermediate of various deoxysugars in antibiotics, was developed starting from dTMP, acetyl phosphate, and glucose-1-phosphate. Four enzymes, i.e., TMP kinase, acetate kinase, dTDP-glucose synthase, and dTDP-D-glucose 4,6-dehydratase' were overexpressed using T7 promoter system in the E. coli BL21 strain, and the dTDP-4-keto-6-deoxy-D-glucose was synthesized by using the enzyme extracts in one-pot batch system. When 20 mM dTMP of initial concentration was used, Mg2+ ion, acetyl phosphate, and glucose-1-phosphate concentrations were optimized. About 95% conversion yield of dTDP-4-keto-6-deoxy-D-glucose was obtained based on initial dTMP concentration at 20 mM dTMP, 1 mM ATP, 60 mM acetyl phosphate, 80 mM glucose-1-phosphate, and 20 mM MgCl(2). The rate-limiting step in this multiple enzyme reaction system was the dTDP-glucose synthase reaction. Using the reaction scheme, about 1 gram of purified dTDP-4-keto-6-deoxy-D-glucose was obtained in an overall yield of 81% after two-step purification, i.e., anion exchange chromatography and gel filtration.     

Preparative isolation and purification of four flavonoids from the petals of nelumbo nucifera by high‐speed counter‐current chromatography

Introduction – Flavonoids, the primary constituents of the petals of Nelumbo nucifera, are known to have antioxidant properties and antibacterial bioactivities. However, efficient methods for the preparative isolation and purification of flavonoids from this plant are not currently available. Objective – To develop an efficient method for the preparative isolation and purification of flavonoids from the petals of N. nucifera by high‐speed counter‐current chromatography (HSCCC). Methodology – Following an initial clean‐up step on a polyamide column, HSCCC was utilised to separate and purify flavonoids. Purities and identities of the isolated compounds were established by HPLC‐PAD, ESI‐MS, ¹H‐NMR and ¹³C‐NMR. Results – The separation was performed using a two‐phase solvent system composed of ethyl acetate–methanol–water–acetic acid (4 : 1 : 5 : 0.1, by volume), in which the upper phase was used as the stationary phase and the lower phase was used as the mobile phase at a flow‐rate of 1.0 mL/min in the head‐to‐tail elution mode. Ultimately, 5.0 mg syringetin‐3‐O‐β‐d‐glucoside, 6.5 mg quercetin‐3‐O‐β‐d‐glucoside, 12.8 mg isorhamnetin‐3‐O‐β‐d‐glucoside and 32.5 mg kaempferol‐3‐O‐β‐d‐glucoside were obtained from 125 mg crude sample. Conclusion – The combination of HSCCC with a polyamide column is an efficient method for the preparative separation and purification of flavonoids from the petals of N. nucifera. Copyright © 2009 John Wiley & Sons, Ltd.     

Evaluation of melanin‐related metabolites as markers of solar ultraviolet‐B radiation

Ultraviolet‐B (UVB) radiation due sunlight can result in sunburns and/or suntans. Sunburn occurs only several hours after solar UVB radiation, while a suntan requires several days to several weeks to develop. In the present study, we measured serum and urine levels of melanin‐related metabolites, 5‐S‐cysteinyldopa (5‐S‐CD) and 6‐hydroxy‐5‐methoxyindole‐2‐carboxylic acid (6H5MI2C), in nine subjects exposed to normal sunlight over the course of 12 months. We collected samples in the middle of each month and examined the variation of the markers, the correlation between them, and their correlation with solar UVB radiation. Those markers exhibited a seasonal variation with lower values in the winter and higher values in the summer. Levels of 5‐S‐CD and 6H5MI2C in the serum showed 48% and 54% increases in the summer compared with those in the winter, respectively. Comparison of 5‐S‐CD in the serum and urine showed the highest correlation (r² = 0.344), followed by the pair of 5‐S‐CD and 6H5MI2C in the serum. Levels of 5‐S‐CD in the serum showed the highest correlation (r² = 0.729) with the mean solar UVB radiation during the first 10 d of the month, while 6H5MI2C in the serum was highly correlated (r² = 0.483) with solar UVB radiation during the previous month. Levels of 5‐S‐CD and 6H5MI2C in the serum appear to reflect the degrees of skin injury and pigmentation in the skin, respectively.     

Chemical constituents of Papulaspora immersa, an endophyte from Smallanthus sonchifolius (Asteraceae), and their cytotoxic activity

Papulaspora immersa H. H. Hotson was isolated from roots and leaves of Smallanthus sonchifolius (Poepp. and Endl. ) H. Rob. (Asteraceae), traditionally known as Yacon. The fungus was cultured in rice, and, from the AcOEt fraction, 14 compounds were isolated. Among them, (22E,24R)‐8,14‐epoxyergosta‐4,22‐diene‐3,6‐dione ( 4 ), 2,3‐epoxy‐1,2,3,4‐tetrahydronaphthalene‐c‐1,c‐4,8‐triol ( 10 ), and the chromone papulasporin ( 13 ) were new secondary metabolites. The spectral data of the known natural products were compared with the literature data, and their structures were established as the (24R)‐stigmast‐4‐en‐3‐one ( 1 ), 24‐methylenecycloartan‐3β‐ol ( 2 ), (22E,24R)‐ergosta‐4,6,8(14),22‐tetraen‐3‐one ( 3 ), (?)‐(3R,4R)‐4‐hydroxymellein ( 5 ), (?)‐(3R)‐5‐hydroxymellein ( 6 ), 6,8‐dihydroxy‐3‐methylisocoumarin ( 7 ), (?)‐(4S)‐4,8‐dihydroxy‐α‐tetralone ( 8 ), naphthalene‐1,8‐diol ( 9 ), 6,7,8‐trihydroxy‐3‐methylisocoumarin ( 11 ), 7‐hydroxy‐2,5‐dimethylchromone ( 12 ), and tyrosol ( 14 ). Compound 4 showed the highest cytotoxic activity against the human tumor cell lines MDA‐MB435 (melanoma), HCT‐8 (colon), SF295 (glioblastoma), and HL‐60 (promyelocytic leukemia), with IC₅₀ values of 3.3, 14.7, 5.0 and 1.6 μM , respectively. Strong synergistic effects were also observed with compound 5 and some of the isolated steroidal compounds.     

Enthalpy change on mixing a couple of S- and R-enantiomers of some chiral compounds at 298.15 K

Enthalpy change on the mixing of R- and S-enantiomers of chiral liquid compounds such as dimethyl malate (1), methyl 3-hydroxylbutanoate (2), 2-butanol (3), ethyl 4-chloro-3-hydroxylbutanoate (4), 1,3,3-trimethylbicycle-[2.2.1]heptan-2-one (5), 3,7-dimethyl-6-octenal (6), and 8-bromo-2,6-dimethyl-2-octene (7) is measured over the entire range of mole fractions at 298.15 K, albeit very small values. The mixing of chiral liquids of R-1 + S-1, R-2 + S-2, R-3 + S-3, R-6 + S-6, and R-7 + S-7 produces enthalpic destabilization over the entire range of mole fractions, while that of R-4 + S-4 and R-5 + S-5 shows enthalpic stabilization over entire compositions. Enthalpy change on mixing at an equimolar concentration and the intermolecular interaction obtained by the molecular mechanics calculations show a linear correlation, except for a few compounds measured.     

Tetramethylcyclopropyl analogue of the leading antiepileptic drug, valproic acid: evaluation of the teratogenic effects of its amide derivatives in NMRI mice

BACKGROUND: Although valproic acid (VPA) is used extensively for treating various kinds of epilepsy, it causes hepatotoxicity and teratogenicity. In an attempt to develop a more potent and safer second generation to VPA drug, the amide derivatives of the tetramethylcyclopropyl VPA analogue, 2,2,3,3‐tetramethylcyclopropanecarboxamide (TMCD), N‐methyl‐TMCD (MTMCD), 4‐(2,2,3,3‐tetramethylcyclopropanecarboxamide)‐benzenesulfonamide (TMCD‐benzenesulfonamide), and 5‐(TMCD)‐1,3,4‐thiadiazole‐2‐sulfonamide (TMCD‐thiadiazolesulfonamide) were synthesized and shown to have more potent anticonvulsant activity than VPA. Teratogenic effects of these CNS‐active compounds were evaluated in Naval Medical Research Institute (NMRI) mice susceptible to VPA‐induced teratogenicity by comparing them to those of VPA. METHODS: Pregnant NMRI mice were given a single sc injection of either VPA or TMC‐amide derivatives on gestation day 8.5, and then the live fetuses were examined to detect any external malformations on gestation day 18. After double‐staining for bone and cartilage, their skeletons were examined. RESULTS: In contrast to VPA, which induced NTDs in a high number of fetuses at 2.4–4.8 mmol/kg, TMCD, TMCD‐benzenesulfonamide, and TMCD‐thiadiazolesulfonamide at 4.8 mmol/kg and MTMCD at 3.6 mmol/kg did not induce a significant number of NTDs. TMCD‐thiadiazolesulfonamide exhibited a potential to induce limb defects in fetuses. Skeletal examination also revealed that fetuses exposed to all four of the tetramethylcyclopropanecarboxamide derivatives developed vertebral and rib abnormalities less frequently than those exposed to VPA. Our results established that TMCD, MTMCD, and TMCD‐benzenesulfonamide are distinctly less teratogenic than VPA in NMRI mice. CONCLUSIONS: The CNS‐active amides containing a tetramethylcyclopropanecarbonyl moiety demonstrated better anticonvulsant potency compared to VPA and a lack of teratogenicity, which makes these compounds good second‐generation VPA antiepileptic drug candidates. Birth Defects Research (Part A), 2008. © 2008 Wiley‐Liss, Inc.     

A new approach for the asymmetric total synthesis of umbelactone

The asymmetric total syntheses of (R)-(+)- and (S)-(-)-umbelactone were achieved by using the Sharpless asymmetric epoxidation reaction to generate the stereogenic center and a ring-closing metathesis (RCM) for the formation of the lactone structure. Starting from 3-methyl-2-buten-1-ol, the asymmetric total synthesis was achieved in an efficient 6-step protocol with an overall yield of 16%.     

Changes in the Proliferation and Differentiation of Neonatal Mouse Pink‐Eyed Dilution Melanocytes in the Presence of Excess Tyrosine

Changes in the proliferation and differentiation of epidermal melanocytes derived from newborn mice wild‐type at the pink‐eyed dilution (p) locus (P/P) and from congenic mice mutant at that locus (p/p) were investigated in serum‐free primary culture, with or without the addition of L‐Tyr. Incubation with added L‐Tyr inhibited the proliferation of P/P melanocytes in a concentration‐dependent manner and inhibition was gradually augmented as the donor mice aged. In contrast, L‐Tyr stimulated the proliferation of p/p melanoblasts–melanocytes derived from 0.5‐day‐old mice, but inhibited their proliferation when derived from 3.5‐ or 7.5‐day‐old mice. L‐Tyr stimulated the differentiation of P/P melanocytes. However, almost all cells were undifferentiated melanoblasts in control cultures derived from 0.5‐, 3.5‐ and 7.5‐day‐old p/p mice, but L‐Tyr induced their differentiation as the age of the donor mice advanced. The content of the eumelanin marker, pyrrole‐2,3,5‐tricarboxylic acid as well as the pheomelanin marker, 4‐amino‐3‐hydroxyphenylalanine in p/p melanocytes was greatly reduced compared with P/P melanocytes. However, the contents of eumelanin and its precursor, 5,6‐dihydroxyindole‐2‐carboxylic acid, as well as the contents of pheomelanin and its precursor, 5‐S‐cysteinyldopa in culture media from p/p melanocytes were similar to those of P/P melanocytes at all ages tested. L‐Tyr increased the content of eumelanin and pheomelanin two‐ to threefold in cultured cells and media derived from 0.5‐, 3.5‐ and 7.5‐day‐old mice. These results suggest that the proliferation of p/p melanoblasts–melanocytes is stimulated by L‐Tyr, and that the differentiation of melanocytes is induced by L‐Tyr as the age of the donor mice advanced, although eumelanin and pheomelanin fail to accumulate in p/p melanocytes and are released from them at all ages of skin development.     

Solvent dependence of the rotational diffusion of TOAC-spin-labeled alamethicin

Three derivatives of the hydrophobic, channel-forming peptaibiotic alamethicin (F50/5) have been synthesized, the original Aib residue at position 1, 8, or 16 being replaced with the spin-labeled amino acid TOAC (=2,2,6,6-tetramethylpiperidin-1-oxyl-4-amino-4-carboxylic acid). Electron-paramagnetic-resonance (EPR) spectroscopy was used to characterize the rotational diffusion of these compounds in five isotropic solvents of differing viscosity and polarity, including MeOH, EtOH, PrOH, i-PrOH, and hexanol (HxOH). In MeOH, the labeled alamethicins were found to rotate anisotropically as a monomer (axial ratio a/b=3). In aliphatic alcohols of increasing viscosity (and hydrophobicity), the rotational correlation times progressively increased. Even in HxOH, the (fivefold) increase in correlation time was no greater than the increase in viscosity. We conclude that TOAC-labeled alamethicins remain monomeric in these solvents of relatively high polarity.     

Microwave-assisted synthesis of 4'-azaflavones and their N-alkyl derivatives with biological activities

4'-Azaflavone (=2-(pyridin-4-yl)-4H-1-benzopyran-4-one; 4) and 3-[(pyridin-4-yl)methyl]-4'-azaflavone (5) were synthesized by a simple environmentally friendly microwave-assisted one-pot method through the cyclization of 3-hydroxy-1-(2-hydroxyphenyl)-3-(pyridin-4-yl)propan-1-one (1), (E)-2'-hydroxy-4-azachalcone (2; chalcone=1,3-diphenylprop-2-en-1-one), and 2'-hydroxy-2-[(hydroxy)(pyridin-4-yl)methyl]-4'-azachalcone (3) under solventless conditions using silica-supported NaHSO(4), followed by treatment with base. In addition, N-alkyl-substituted 4'-azaflavonium bromides 6 and 7 were prepared from compounds 4 and 5, respectively. The antimicrobial and antioxidant activities of compounds 1-7 were tested. The N-alkyl-substituted 4'-azaflavonium bromides 6 and 7 showed high antimicrobial activity against the Gram-positive bacteria and the fungus tested, with MIC values close to those of reference antimicrobials ampicilline and fluconazole. The alkylated compounds 6 and 7 also showed a good antioxidant character in the two antioxidant methods, DPPH (=1,1-diphenyl-2-picrylhydrazyl) radical-scavenging and ferric reducing/antioxidant power (FRAP) tests.