Concerning the presence and formation of ascorbic acid in yeasts

The selective, sensitive method of analysis of ascorbic acid by high performance liquid chromatography with electrochemical detection (HPLC/EC) has been used to determine the ascorbic acid content of cell extracts from yeasts grown in glucose-free medium, 0.3 M D-glucose, and 0.112 M L-galactono-1,4-lactone. Saccharomyces cerevisiae (strain G-25 and its tetraploid) and a commercial baker's yeast contained less than 2 μg ascorbic acid g^?1 wet wt. of cells when grown for 22 h in glucose-free medium. In 0.3 M D-glucose, only the commercial baker's yeast gave a slight increase (2–50 μg g^?1 wet wt. in 22 h). In 0.112 M L-galactono-1,4-lactone, all three strains produced ascorbic acid (372–587 μg g^?1 wet wt. in 22 h). Lypomyces starkeyi, a species previously reported to contain a significant amount of ascorbic acid (Heick et al., Can. J. Biochem., 47 (1972) 752), was essentially devoid of ascorbic acid under all three conditions of incubation although it did contain an HPLC/EC reactive peak (R_T = 0.87 relative to ascorbic acid) that was readily oxidized by charcoal in the presence of oxygen. The identity of this new compound remains to be determined.     

Reverse-phase high-performance liquid chromatography of hydrophobic proteins and fragments thereof

Reverse-phase high-performance liquid chromatography (HPLC) resolution and recovery of cytochrome P-450 and bovine rhodopsin, both integral membrane proteins, and large peptides derived from P-450 LM₂ were enhanced by utilizing ternary solvents. Surprisingly, most test materials eluted later in the gradient when using mixtures of acetonitrile and propanol in the mobile phase compared to using either solvent alone. Of the supports tested, the best recovery of hydrophobic cytochrome P-450 LM₄ was experienced on the less retentive CN-bonded phase. Two alternate solvents for HPLC of polypeptides are proposed: (1) 0.02–0.1 m hexafluoroacetone/NH₃, pH 7.2 for highly acidic peptides; and (2) 6 m formic acid/0.13 m trimethylamine, pH 1.5, vs 4 m formic acid/0.09 m trimethylamine in propanol for relatively insoluble peptides. Anomalous side reactions between formic acid and peptides can cause HPLC peak broadening, increased retention, and decreased resolution. These deleterious effects are thought to be due in part to formyl esterification of serine and threonine residues and appear to be reversible by aminoethanol treatment.     

Enhancing the production of an antifungal compound from Anabaena laxa through modulation of environmental conditions and its characterization

An investigation was undertaken to optimize the physiological conditions and characterize the bioactive compound responsible for fungicidal activity in Anabaena laxa. Fungicidal activity against Pythium debaryanum in the A. laxa cultures increased, when grown under continuous light (CL) at the stationary phase (28 d), with further enhancement on doubling phosphorus levels and high pH (9.0) in the growth medium. Preparatory thin layer chromatography analyses revealed the peptide nature of the fungicidal metabolite, with highest activity in a spot with R_f 0.13. The bioactive fraction (in terms of fungicidal activity) was isolated and identified using HPLC with a retention time of 14.7. Fourier transform infrared (FT-IR) spectrum of the purified bioactive fraction indicated the cyclic peptide nature of the antifungal compound. The structural elucidation using ¹H, and ¹³C NMR analyses revealed the same number and type of carbons as present in previously reported majusculamide C from Lyngbya majuscula. GC–MS indicated a similar major mass ion fragment spectra, with peaks as previously obtained from majusculamide C. This represents a first report on biosynthesis of a fungicidal compound in A. laxa, with structural similarities to majusculamide C.     

Metabolism of Arachidonic Acid to Epoxyeicosatrienoic Acids. Hydroxyeicosatetraenoic Acids, and Prostaglandins in Cultured Rat Hippocampal Astrocytes

Abstract: We have recently shown that brain slices are capable of metabolizing arachidonic acid by the epoxy-genase pathway. The purpose of this study was to begin to determine the ability of individual brain cell types to form epoxygenase metabolites. We have examined the astrocyte epoxygenase pathway and have also confirmed metabolism by the cyclooxygenase and lipoxygenase enzyme systems. Cultured rat hippocampal astrocyte homogenate, when incubated with radiolabeled [³H]-arachidonic acid, formed products that eluted in four major groups designated as R_17–30, R_42–50, R_51–82, and R_83–90 based on their retention times in reverse-phase HPLC. These fractions were further segregated into as many as 13 peaks by normal-phase HPLC and a second reverse-phase HPLC system. The principal components in each peak were structurally characterized by gas chromatography/electron impact-mass spectrometry. Based on HPLC retention times and gas chromatography/electron impactmass spectrometry analysis, the more polar fractions (R_17–30) contained prostaglandin D₂ as the major cyclooxygenase product. Minor products included 6-keto prostaglandin F_1α, prostaglandin E₂, prostaglandin F_2α, and thromboxane B₂. Fractions R_42–50, R_51–82. and R_83–90 contained epoxygenase and lipoxygenase-like products. The major metabolite in fractions R_83–90 was 5, 6-epoxyeicosatrienoic acid (EET). Fractions R_51–82 contained 14, 15-and 8, 9-EETs, 12-and 5-hydroxyeicosatetraenoic acids, and 8, 9-and 5, 6-dihydroxyeicosatrienoic acids (DHETs). In fractions R_42–50, 14, 15-DHET was the major product. When radiolabeled [³H]14, 15-EET was incubated with astrocyte homogenate, it was rapidly metabolized to [³H]14, 15-DHET. The metabolism was inhibited by submicromolar concentration of 4-phenylchalcone oxide, a potent inhibitor of epoxide hydrolase activity. Formation of other polar metabolites such as triols or epoxyalcohols from 14, 15-DHET was not observed. In conclusion, astro-cytes readily metabolize arachidonic acid to 14, 15-EET, 5, 6-EET, and their vicinal-diols. Previous studies suggest these products may affect neuronal function and cerebral blood flow.     

Isolation, Identification and Study of Antimicrobial Property of a Bioactive Compound in an Indian Medicinal Plant Acalypha indica (Indian-Nettle)

Summary Fresh, dried and powdered samples of leaf, stem and root of Acalypha indica were subjected to fractional distillation in a soxhlet apparatus using solvents such as hexane, chloroform, acetone and methanol. The plant extracts and a synthetic antifungal compound, Clotrimazole (authentic standard) were subjected to TLC and HPLC analyses. The R_f (relative front) value of Clotrimazole was 0.371. The plant’s leaf, root and stem extracts also gave distinct spots respectively at R_f value of 0.371 ± 0.0009. In HPLC, the TLC-separated active compound and Clotrimazole resolved at 1.90 ± 0.2 min (retention time). The amounts of active compound present in root, leaf and stem extracts were 538, 415 and 171 μg/g respectively. From the results of our study, we infer that the active compound isolated from Acalypha indica is more potent in controlling Candida albicans, Aspergillus niger and Escherichia coli. The Active compound present in the plant had more than 100% activity when compared to standard Clotrimazole.     

Urinary excretion of C-20-reduction products of corticosterone

1. A 200 mg. portion of corticosterone was ingested by a healthy man and the urine collected. Part of the urine was treated with the gastric juice of Helix pomatia and extracted with ethyl acetate, and the extract fractionated with Girard T. Paper-chromatographic separation of the non-ketonic fraction in the Bush (1952) system B₅ revealed the presence of two unknown polar components. 2. The unknown compounds did not possess a reducing (blue tetrazolium) or a reducible (potassium borohydride) grouping. Both contained a terminal α-glycollic fragment as shown by the formation of formaldehyde, and of a non-volatile aldehyde on oxidation with sodium bismuthate. 3. Unknown compound (I) had paper-chromatographic mobilities identical with those of 5β-pregnane-3α,11β,20β,21-tetraol. The oxidation product of compound (I) had a retention time (gas–liquid chromatography) on an SE30 column identical with that of 3α,11β-dihydroxy-21-nor-5β-pregnan-20-al. The retention times of various derivatives agreed with those produced in an identical manner on the standard, and accordingly compound (I) is formulated as 5β-pregnane-3α,11β,20ξ,21-tetraol. 4. Unknown compound (II) had a higher R_F than compound (I), and its oxidation product had a longer retention time than that of compound (I). From the group effects observed in paper and gas–liquid chromatography, compound (II) is tentatively formulated as 5α-pregnane-3α,11β,20ξ,21-tetraol. The 5α/5β ratio found was about 2·0.     

Detection of microgram quantities of carrier ampholytes in electrofocused proteins by thin-layer chromatography

A rapid and sensitive method for the detection of carrier ampholyte contamination in electrofocused proteins is described. Samples containing proteins and carrier ampholytes were applied to cellulose thin-layer chromatographic sheets and developed in 10% trichloroacetic acid. Proteins and large-molecular-weight carrier ampholytes were precipitated at the origin while 10% trichloroacetic acid-soluble carrier ampholytes migrated as a diffuse ninhydrin (nitrogen)-positive area at an R_f greater than 0.50. We found that 1.25 μg of carrier ampholytes contained enough 10% trichloroacetic acid-soluble components to be detected by thinlayer chromatography. Using this assay, we investigated techniques designed to remove carrier ampholytes from an electrofocused protein. Removal of large-molecular-weight components from carrier ampholytes by dialysis through a 3500 M_r cutoff membrane did not facilitate separation of carrier ampholytes from streptococcal pyrogenic exotoxin type C by dialysis or gel chromatography. Also, this protein binds irreversibly to mixed-bed ion-exchange resin. The best method for separating carrier ampholytes from streptococcal pyrogenic exotoxin type C was by electrodialysis at pH 4.0. Following electrodialysis, estimated carrier ampholyte contamination in this protein was less than 1 part in 500 parts (by weight).     

The presence of 12β-deoxydecarbamoylsaxitoxin in the Japanese toxic dinoflagellate Alexandrium determined by simultaneous analysis for paralytic shellfish toxins using HILIC-LC–MS/MS

A differential screening study using high-resolution (HR)-hydrophilic interaction chromatography (HILIC)-electrospray ionization (ESI)–quadrupole time-of-flight mass spectrometry (Q-TOF MS) was conducted to identify saxitoxin (STX) analogues in the marine dinoflagellate toxic sub-clone Alexandrium tamarense Axat-2 and the non-toxic sub-clone UAT-014-009 derived from the same Japanese isolate. One unknown compound was identified only in the toxic sub-clone and was found to have the molecular formula C₉H₁₆N₆O₂. This structure differed from that of decarbamoyl STX (dcSTX; C₉H₁₆N₆O₃) by the loss of a single oxygen. A 12-deoxy-dcSTX standard (a mixture of 12α- and β-deoxy-dcSTX) was chemically prepared from dcSTX by reduction with sodium borohydride. The unknown compound in the toxic strain of A. tamarense was identified as 12β-deoxy-dcSTX by comparison of its HR-HILIC-LC–MS retention time and HR–MS/MS spectrum with those of the chemically prepared standard, and the identification was confirmed by high-sensitivity HPLC analysis with post-column fluorescent derivatization. Moreover, two Japanese isolates of A. catenella showing toxin profiles different from that of A. tamarense were also found to contain 12β-deoxy-dcSTX. Previously, 12β-deoxy-dcSTX was isolated from the freshwater cyanobacterium Lyngbya wollei, which produces a unique set of STX analogues. This study is the first evidence of the presence of 12β-deoxy-dcSTX in marine dinoflagellates.     

A toxic amino acid, 2(S)3(R)-2-amino-3-hydroxypent-4-ynoic acid from the fungus Sclerotium rolfsii

An amino acid, lethal to New Hampshire chickens (LD₅₀, 150 mg/kg) was isolated from dried sclerotia of the fungus Sclerotium rolfsii (Sacc.). Purification of the rather unstable compound was effected on a cation exchange column by means of displacement chromatography and the amino acid was crystallised from 80% methanol. A structure was assigned to the compound on the basis of available chemical and physical data, namely 2(S),3(R)-2- amino-3-hydroxypent-4-ynoic acid. Confirmation of this structure was gained by direct and indirect synthetic procedures.     

Production of allantoin, rabdosiin and rosmarinic acid in callus cultures of the seacoastal plant Mertensia maritima (Boraginaceae)

A callus culture of the extreme halophyte seacoastal plant Mertensia maritima (Boraginaceae) was established from apical shoots of the plant using a modified Murashige and Skoog medium supplemented with 6-benzyladenine (0.5?mg/L) and ??-naphthaleneacetic acid (2.0?mg/L). Three main compounds, (?)-R-allantoin, (+)-rabdosiin and rosmarinic acid, were isolated from extracts of M. maritima calli by liquid chromatography and identified by ¹H and ¹³C NMR, UV, ECD and HPLC?CMS. Quantitative HPLC analysis showed that the calli produce (+)-rabdosiin (0.14% dry wt), rosmarinic acid (0.74% dry wt) and (?)-R-allantoin (3.7% dry wt). Allantoin was detected in plant cell cultures for the first time. All of these metabolites were also present in lower quantities in different parts of the plant. The presence of rabdosiin and rosmarinic acid, in combination with the skin-conditioning agent (?)-R-allantoin, represents a potentially useful novel composition for skin protection.     

Gentiopicrin-producing endophytic fungus isolated from Gentiana macrophylla

Gentiana macrophylla is a traditional Chinese medicinal plant. Its dominant active constituents are secoiridoids, mainly gentiopicrin. The objective of this study was to determine whether endophytic fungi isolated from this plant produce the bioactive ingredient gentiopicrin. Primary screening was done by Dragendorff's reaction and the strain re-selection was done with thin-layer chromatography (TLC) and high-performance liquid chromatography (HPLC) to identify the fermentation products of the selected strains. In this study, 20 strains of endophytic fungi were isolated from G. macrophylla, and the extracts from five strains had a positive Dragendorff's reaction. Two strains (QJ16 and QJ18) had a component with the same R_f value in TLC as that of authentic gentiopicrin and one ingredient of the QJ18 extract had a retention time identical with that of authentic gentiopicrin in HPLC. Therefore, the fungus appears to produce the bioactive ingredient gentiopicrin, as does its host plant, and could be used for the production of gentiopicrin by fermentation.     

Enzymatic analysis with chondrosulfatases of constituent disaccharides of sulfated chondroitin sulfate and dermatan sulfate isomers by high-performance liquid chromatography

Various under-sulfated, monosulfated, and over-sulfated chondroitin sulfate and dermatan sulfate isomers were analyzed in terms of disaccharide units before or after desulfation with chondrosulfatases in addition to digestion with chondroitinases. The unsaturated disaccharides were separable by a high-performance liquid chromatography (HPLC) method using a resin made from a sulfonized styrene-divinylbenzene copolymer. The retention times of the parent sulfated unsaturated disaccharides and newly generated unsaturated mono- or nonsulfated disaccharides were reproducible. On desulfation of the parent sulfated unsaturated disaccharides with chondrosulfatases, almost all ΔDi-S showed the same retention times as those of standard ΔDi-S from known components. Following digestion of ΔDi-diS_B with chondro-4-sulfatase as well as ΔDi-diS_D or ΔDi-diS_G with chondro-6-sulfatase, three ΔDi-monoS with the same retention time were detected with the HPLC method. These newly generated ΔDi-monoS₂ showed that the structure is N-acetyl-d-galactosamine, uronic acid 2-sulfate.     

Structure–activity relationship study on α1 adrenergic receptor antagonists from beer

Hordatine A and aperidine have been previously isolated from beer as active ingredients, which bind to muscarinic M₃ receptor. In addition, these compounds have exhibited antagonist activity against the α_1A adrenoceptor. Although the relative structures of these two molecules have previously been determined, the absolute stereochemistry was unclear. Hence, to elucidate the absolute stereochemistry of natural hordatine A, we synthesized each enantiomer of hordatine A and aperidine from optically pure dehydrodi-p-coumaric acid. Several additional related compounds were also synthesized for structure–activity relationship studies. Chiral column HPLC analysis demonstrated that the absolute stereochemistry of natural hordatine A is (2S,3S), while based on the isomerization mechanism, the stereochemistry of aperidine is (2R,3S). The α_1A adrenoceptor binding activity of (2R,3R)-hordatine A is the most potent among the enantiomeric pairs of hordatines and aperidines. Furthermore, the related, synthetic compound, (2R,3R)-methyl benzofurancarboxylate exhibits antagonist activity against the α_1A adrenoceptor at a lower concentration than that of hordatine A.     

Occurrence and identification of jasmonic acid and its amino acid conjugates induced by osmotic stress in barley leaf tissue

The effect of osmotically active substances on the alteration of endogenous jasmonates was studied in barley (Hordeum vulgare L. cv. Salome) leaf tissue. Leaf segments were subjected to solutions of d-sorbitol, d-mannitol, polyethylene glycol 6000, sodium chloride, or water as a control. Alterations of endogenous jasmonates were monitored qualitatively and quantitatively using immunoassays. The structures of jasmonates isolated were determined on the basis of authentic substances by capillary gas chromatography-mass spectrometry. The stereochemistry of the conjugates was confirmed by high performance liquid chromatography with diastereoisomeric references. In barley leaves, jasmonic acid and its amino acid conjugates, for example, with valine, leucine, and isoleucine, are naturally occurring jasmonates. In untreated leaf segments, only low levels of these native jasmonates were found. After treatment of the leaf tissues with sorbitol, mannitol, as well as with polyethylene glycol, an increase of both jasmonic acid and its conjugates could be observed, depending on the stress conditions used. In contrast, salt stress was without any stimulating effect on the levels of endogenous jasmonates. From barley leaf segments exposed to sorbitol (1m) for 24 h, jasmonic acid was identified as the major accumulating compound. Jasmonic acid-amino acid conjugates increased likewise upon stress treatment.Abbreviations JM methyl jasmonate - JA jasmonic acid - JIP(s) jasmonate-induced protein(s) - PEG polyethylene glycol - RIA radioimmunoassay - ELISA enzyme-linked immunosorbent assay - HPLC high performance liquid chromatography - GC-MS gas chromatography-mass spectrometry - R _t retention time - IAA indole-3-acetic acid     

Synthesis of (R,S)-isoproterenol,an inhibitor of tau aggregation,as an 11C-labeled PET tracer via reductive alkylation of (R,S)-norepinephrine with [2-11C]acetone

(R,S)-Isoproterenol inhibits the formation of toxic granular tau oligomers associated with neuronal loss and development of cognitive disorders, and is an attractive drug candidate for Alzheimer’s disease. To elucidate its behavior in the brain by positron emission tomography, we synthesize (R,S)-[¹¹C]isoproterenol by reductive alkylation of (R,S)-norepinephrine with [2-¹¹C]acetone, which was in turn synthesized in situ under improved conditions afforded a decay-corrected radiochemical yield of 54%. The reductive alkylation using NaBH(OAc)₃ as reducing agent in the presence of benzoic acid in DMSO/DMF (60:40 v/v) at 100 °C for 10 min gave (R,S)-[¹¹C]isoproterenol in an 87% radio-high performance liquid chromatography (HPLC) analytical yield. HPLC separation using a strong cation exchange column, followed by pharmaceutical formulation in the presence of d/l-tartaric acid, afforded (R,S)-[¹¹C]isoproterenol with a total radioactivity of 2.0 ± 0.2 GBq, a decay-corrected radiochemical yield of 19 ± 2%, chemical and radiochemical purities of 71% and >99%, respectively, and a molar activity of 100 ± 13 GBq/μmol (n = 3). The overall synthesis time from the end of the bombardment to pharmaceutical formulation was 48 min. A preliminary preclinical PET study in a rat demonstrated the potential of the radioligand for the evaluation of the penetration of (R,S)-isoproterenol in human brain.     

Dual-column high-performance liquid chromatographic urinary organic acid profiling

Two-dimensional high-performance liquid chromatography (HPLC) is increasingly used for the separation and identification of compounds in biological matrices. Conventional two-dimensional HPLC involves either heart-cutting or column-switching techniques. These techniques work well but are very time consuming when the analysis involves many compounds and requires more than a few minutes to completely elute from the column. We have modified the column-switching technique by utilizing two columns in series, an Aminex HPX-87H organic acid column followed by a 15-cm 5-μm C₁₈ analytical column. Both columns are compatible with the isocratic pH 2.5 H₂SO₄ mobile phase employed in the organic acid profiling. The dual-column system affords better resolution of urinary organic acids than does either column separately. Reversing the column order does not dramatically affect the elution pattern (peak shape, peak height, and R_f values are approximately the same). The dualcolumn HPLC configuration works well as a rapid means of screening urinary carboxylic acids prior to subsequent definitive analyses of abnormal samples.     

The effect of ethylene, octanoic acid and a plant-derived smoke extract on the germination of light-sensitive lettuce seeds

The effects of an aqueous plant-derived smoke extract, octanoic acid and ethylene on germination of light-sensitive Grand Rapids lettuce seeds were investigated. The smoke extract brought about a concentration dependent increase in germination and a complete inhibition of germination at high concentrations. Octanoic acid could not induce germination. Ethylene at concentrations over 5 L L^–1 increased lettuce seed germination, but not to the same degree as smoke. Aqueous smoke in combination with ethylene showed a synergistic effect on germination at suboptimal smoke concentrations. At high smoke concentrations the effect of ethylene was almost completely inhibited. Octanoic acid in combination with ethylene brought about a higher level of germination than with ethylene alone, but only at the highest concentration of octanoic acid tested (1 mM). Standardized hexane and dichloromethane-partitioned smoke extracts and octanoic acid were subjected to TLC separation. The R _f -fractions in the smoke lanes showing activity in the lettuce seed bioassay did not correspond to the R _f -value of octanoic acid. As aqueous smoke can withstand autoclaving and can be separated by TLC and HPLC without loosing activity it is unlikely that the activity of aqueous smoke is linked to ethylene. It thus appears that the active compound in smoke is neither octanoic acid nor ethylene.Abbreviations TLC thin layer chromatography - HPLC High performance liquid chromatography     

Investigations on the Nature of the Auxin-Wave in the Cambial Region of Pine Stems : Validation of IAA as the Auxin Component by the Avena Coleoptile Curvature Assay and by Gas Chromatography-Mass Spectrometry-Selected Ion Monitoring

The major auxin of Scots pine (Pinus silvestris L.) which is transported basipetally into agar strips from the cambial region of the stem was quantified by the Went Avena coleoptile curvature assay before and after reversed phase C₁₈ high performance liquid chromatography (HPLC), and then identified by full spectrum gas chromatography-mass spectrometry (GC-MS) as indole-3-acetic acid (IAA). The IAA was subsequently quantified by GC-MS-selected ion monitoring (SIM) using an internal standard of [¹³C]-(C₆)-IAA. The amount of IAA collected into 22-millimeter long agar strips during 10 minutes of contact with the stem cambial region was estimated by GC-MS-SIM and the Went bioassay to be 2.3 and 2.1 nanograms per strip, respectively. The GC-MS technique thus confirmed the results obtained by the Went curvature assay. The Avena curvature assay revealed the presence of at least one other, more polar (based on HPLC retention time) auxin that diffused into the agar strips with the IAA. Its bioactivity was only 5% of the IAA fraction. Its HPLC retention time was earlier than IAA-glucoside, IAA-aspartate, or IAA-glycine, but the same as IAA-inositol. No significant amounts of inhibitors or synergists of IAA activity on the Avena assay were found in extracts corresponding to one or five strips of agar. Thus, the direct bioassay of the agar strips immediately after their removal from the cambial region of P. silvestris stem sections reflects the concentration of the native IAA. For both P. silvestris and lodgepole pine (Pinus contorta) a wavelike pattern of auxin stimulation of Avena curvature was found in agar strips exposed for only 10 minutes to the basal ends of an axial series of 6-millimeter long sections from the cambial region of the stem. This wavelike pattern was subsequently confirmed for P. contorta both by Avena curvature assay and by GC-MS-SIM of HPLC fractions at the retention time of [³H]IAA. The wavelike pattern of auxin diffusing from the cambial region of Pinus has thus been determined to consist primarily of IAA and this pattern has now been quantitated using both the Went Avena curvature assay and GC-MS-SIM with [¹³C]-C₆-IAA as an internal standard.     

Antinociceptive and antidepressant-like action of endomorphin-2 analogs with proline surrogates in position 2

In our efforts to develop new candidate drugs with antinociceptive and/or antidepressant-like activity, two novel endomorphin-2 (EM-2, Tyr-Pro-Phe-Phe-NH₂) analogs, containing proline surrogates in position 2 were synthesized using commercially available racemic trans-4-phenylpyrrolidine-3-carboxylic acid (4-Ph-β-Pro). The obtained mixture of two diastereoisomeric peptides (2a and 2b) was separated by HPLC and both enantiopure analogs were used in the in vitro and in vivo studies. To assign the absolute configuration to the 4-Ph-β-Pro residues in both peptides, the stereoselective synthesis of (3R,4S)-4-phenylpyrrolidine-3-carboxylic acid was performed and this enantiomer was introduced into position 2 of EM-2 sequence. Based on the HPLC retention times we were able to assign the absolute configuration of 4-Ph-β-Pro residues in both peptide analogs. Analog 2a incorporating (3R,4S)-4-Ph-β-Pro residue produced strong analgesia in mice after intracerebroventricular (icv) administration which was antagonized by the μ-opioid receptor (MOR) antagonist, β-funaltrexamine (β-FNA). This analog also influenced an emotion-related behavior of mice, decreasing immobility time in the forced swimming and tail suspension tests, without affecting locomotor activity. The antidepressant-like effect was reversed by the δ-selective antagonist, naltrindole (NLT) and κ-selective nor-binaltorphimine (nor-BNI). Thus, the experiments with selective opioid receptor antagonists revealed that analgesic action of analog 2a was mediated through the MOR, while the δ- and κ-receptors (DOR and KOR, respectively) were engaged in the antidepressant-like activity. Analog 2b with (3S,4R)-4-Ph-β-Pro in position 2 showed no antinociceptive or antidepressant-like activity in animal studies.     

Chemical investigation of drug-like compounds from the Australian tree,Neolitsea dealbata

Two of the four parameters in the ‘rule of five’, molecular weight and log P, which can be detected and predicted by mass spectrometry and compound retention on reversed-phase HPLC, were used as guidelines in natural product isolation. A new aporphine alkaloid, (6aR)-normecambroline (1), was isolated from the bark of Neolitsea dealbata (R. Br.) Merr. Its structure was determined on the basis of NMR, MS and CD analysis. It is the first time the absolute configuration of the roemerine-N-oxide was assigned for both roemerine-N_α-oxide (3) and roemerine-N_β-oxide (4). Physico-chemical property evaluation demonstrated all alkaloids had no Lipinski violation. Compound 1 inhibited selectively against cervical cancer cells (HeLa) with an IC₅₀ of 4.0 μM.