Asymmetric synthesis of (1R,2S,3R)-3-methylcispentacin and (1S,2S,3R)-3-methyltranspentacin by kinetic resolution of tert-butyl (+/-)-3-methylcyclopentene-1-carboxylate

Conjugate addition of lithium dibenzylamide to tert-butyl (+/-)-3-methylcyclopentene-1-carboxylate occurs with high levels of stereocontrol, with preferential addition of lithium dibenzylamide to the face of the cyclic alpha,beta-unsaturated acceptor anti- to the 3-methyl substituent. High levels of enantiorecognition are observed between tert-butyl (+/-)-3-methylcyclopentene-1-carboxylate and an excess of lithium (+/-)-N-benzyl-N-alpha-methylbenzylamide (10 eq.) (E > 140) in their mutual kinetic resolution, while the kinetic resolution of tert-butyl (+/-)-3-methylcyclopentene-1-carboxylate with lithium (S)-N-benzyl-N-alpha-methylbenzylamide proceeds to give, at 51% conversion, tert-butyl (1R, 2S, 3R,alphaS)-3-methyl-2-N-benzyl-N-alpha-methylbenzylaminocyclopentane-1-c arboxylate consistent with E > 130, and in 39% yield and 99 +/- 0.5% de after purification. Subsequent deprotection by hydrogenolysis and ester hydrolysis gives (1R, 2S, 3R)-3-methylcispentacin in > 98% de and 98 +/- 1% ee. Selective epimerisation of tert-butyl (1R, 2S, 3R, alphaS)-3-methyl-2-N- benzyl-N-alpha-methylbenzylaminocyclopentane-1-carboxylate by treatment with (KOBu)-Bu-t in (BuOH)-Bu-t gives tert-butyl (1S, 2S, 3R, alphaS)-3-methyl-2-N-benzyl-N-alpha-methylbenzylaminocyclopentane-1-carb oxylate in quantitative yield and in > 98% de, with subsequent deprotection by hydrogenolysis and ester hydrolysis giving (1S, 2S, 3R)-3-methyltranspentacin hydrochloride in > 98% de and 97 +/- 1% ee.

Constitutive oligomerization of human D-2 dopamine receptors expressed in Spodoptera frugiperda 9 (Sf9) and in HEK293 cells - Analysis using co-immunoprecipitation and time-resolved fluorescence resonance energy transfer

Human D-2Long (D-2L) and D-2Short (D-2S) dopamine receptor isoforms were modified at their N-terminus by the addition of a human immunodeficiency virus (HIV) or a FLAG epitope tag. The receptors were then expressed in Spodoptera frugiperda 9 (Sf9) cells using the baculovirus system, and their oligomerization was investigated by means of co-immunoprecipitation and time-resolved fluorescence resonance energy transfer (FRET). [H-3] Spiperone labelled D-2 receptors in membranes prepared from Sf9 cells expressing epitope-tagged D-2L or D-2S receptors, with a pK(d) value of approximate to 10. Co-immunoprecipitation using antibodies specific for the tags showed constitutive homo-oligomerization of D-2L and D-2S receptors in Sf9 cells. When the FLAG-tagged D-2S and HIV-tagged D-2L receptors were co-expressed, co-immunoprecipitation showed that the two isoforms can also form hetero-oligomers in Sf9 cells. Time-resolved FRET with europium and XL665-labelled antibodies was applied to whole Sf9 cells and to membranes from Sf9 cells expressing epitope-tagged D-2 receptors. In both cases, constitutive homo-oligomers were revealed for D-2L and D-2S isoforms. Time-resolved FRET also revealed constitutive homo-oligomers in HEK293 cells expressing FLAG-tagged D-2S receptors. The D-2 receptor ligands dopamine, R-(-) propylnorapomorphine, and raclopride did not affect oligomerization of D-2L and D-2S in Sf9 and HEK293 cells. Human D-2 dopamine receptors can therefore form constitutive oligomers in Sf9 cells and in HEK293 cells that can be detected by different approaches, and D-2 oligomerization in these cells is not regulated by ligands.

Interaction of the D-2short dopamine receptor with G proteins: analysis of receptor G protein selectivity

The human D-2short (D-2S) dopamine receptor has been expressed together with the G proteins Gi2 and Go in insect cells using the baculovirus system. Levels of receptor were determined using [H-3]spiperone binding. Levels of G protein heterotrimer were determined using quantitative Western blot and using [S-35]GTPgammaS saturation binding experiments. Levels of the receptor and G protein and the receptor/G protein ratio were similar in the two preparations. Stimulation of [S-35]GTPgammaS binding by a range of agonists occurred with higher relative efficacy and in some cases higher potency in the preparation expressing Go, indicating that interaction of the D-2S receptor is more efficient with this G protein. The effects of various G protein-selective agents on 10,11-dihydroxy-N-n-propylnorapomorphine ([H-3]NPA) binding were used to examine the receptor/G protein complex in the two preparations. Suramin inhibited [H-3]NPA binding with slightly higher potency in the Gi2 preparation, whereas GppNHp inhibited [H-3]NPA binding with greater potency (similar to6-fold) in the Go preparation. This may imply that the G protein is more readily activated in the D-2S/Go preparation. [H-3]Spiperone binding occurred with an increased B-max in the presence of suramin in the Go preparation but not in the Gi2 preparation, suggesting a higher affinity interaction between the free receptor and this G protein. It is concluded that the higher efficiency activation of Go by the D-2S receptor may be a function of higher affinity receptor/G protein interaction as well as a greater ability to activate the G protein. (C) 2003 Elsevier Science Inc. All rights reserved.

Monatin and its stereoisomers: chemoenzymatic synthesis and taste properties

The sweet natural compound monatin 1 has two stereogenic centres and the (2S,4S) absolute configuration has been attributed to the natural isomer. We obtained all four stereoisomers as pure compounds by a six-step synthetic sequence. The stereogenic centre at C-4 was introduced stereoselectively by a regio- and enantiospecific enzymatic hydrolysis of the racemic ethyl dicarboxylate 4 using a protease from Aspergillus oryzae. The absolute configuration of the intermediate products was assigned by X-ray diffraction of chiral derivatives. The stereogenic centre at C-2 was introduced non-specifically, and the resulting diastereomeric mixtures were separated by RP-HPLC. The absolute configurations of the final products were established by comparing retention times on a chiral HPLC column with those of known samples. The four stereoisomers were submitted to tasting trials and three of them, particularly the (2R,4R) isomer, were found to be intensely sweet. A sample of natural monatin analysed under the same conditions is shown to contain all the four stereoisomers. The relative stereoisomeric content in the plant, as well as the possible isomerisation of the chiral centres during extraction and manipulation of monatin samples, are important points that need to be clarified by extensive analysis of the natural extracts. ((c) Wiley-VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2005).

Asymmetric aziridine synthesis by aza-Darzens reaction of N-diphenylphosphinylimines with chiral enolates. Part 2: inversion of diastereoselectivity

The aza-Darzens ('ADZ') reactions of N-diphenylphosphinyl ('N-Dpp') imines with chiral enolates derived from N-bromoacetyl 2S-2,10-camphorsultam proceed in generally good yield to give N-diphenylphosphinyl aziridinoyl sultams. However, the stereoselectivity of the reaction is dependent upon the structure of the imine substituent: when the chiral enolate was reacted with arylimines substituted in the ortho-position, mixtures of cis- and trans-2'R,3'R-aziridines were obtained, often with a complete selectivity in favour of the trans-isomer. (c) 2006 Elsevier Ltd. All rights reserved.

Asymmetric aziridine synthesis by aza-Darzens reaction of N-diphenylphosphinylimines with chiral enolates. Part 1: formation of cis-aziridines

Theaza-Darzens ('ADZ') reactions off-diphenylphosphinyl (W-Dpp') imines with chiral enolates derived from oxazolidinones and camphorsultam have been Studied. Whilst oxazolidinone enolates reacted poorly in terms of aziridination, the use of the chiral enolate derived from both antipodes of N-bromoacetyl 2, 10-camphorsultam, 2R-(5) and 2S-(5), with N-diphehenylphosphinyl aryl and tert-butylimines proceeded in generally good yield to give, respectively, (2'R,3'R)- or (2'S,3'S)-cis-N-diphenylphosphinyl aziridinoyl sultams of high de. (c) 2006 Elsevier Ltd. All rights reserved.

The 'glial' glutamate transporter, EAAT2 (Glt-1) accounts for high affinity glutamate uptake into adult rodent nerve endings

The excitatory amino acid transporters (EAAT) removes neurotransmitters glutamate and aspartate from the synaptic cleft. Most CNS glutamate uptake is mediated by EAAT2 into glia, though nerve terminals show evidence for uptake, through an unknown transporter. Reverse-transcriptase PCR identified the expression of EAAT1, EAAT2, EAAT3 and EAAT4 mRNAs in primary cultures of mouse cortical or striatal neurones. We have used synaptosomes and glial plasmalemmal vesicles (GPV) from adult mouse and rat CNS to identify the nerve terminal transporter. Western blotting showed detectable levels of the transporters EAAT1 (GLAST) and EAAT2 (Glt-1) in both synaptosomes and GPVs. Uptake of [3H]D-aspartate or [3H]L-glutamate into these preparations revealed sodium-dependent uptake in GPV and synaptosomes which was inhibited by a range of EAAT blockers: dihydrokainate, serine-o-sulfate, l-trans-2,4-pyrrolidine dicarboxylate (PDC) (+/-)-threo-3-methylglutamate and (2S,4R )-4-methylglutamate. The IC50 values found for these compounds suggested functional expression of the 'glial, transporter, EAAT2 in nerve terminals. Additionally blockade of the majority EAAT2 uptake sites with 100 micro m dihydrokainate, failed to unmask any functional non-EAAT2 uptake sites. The data presented in this study indicate that EAAT2 is the predominant nerve terminal glutamate transporter in the adult rodent CNS.

Asymmetric synthesis of cyclic β-amino acids and cyclic amines via sequential diastereoselective conjugate addition and ring closing metathesis

Diastereoselective conjugate addition of lithium (S)-N-allyl-N-alpha-methylbenzylamide to a range of alpha,beta-unsaturated esters followed by ring closing metathesis is used to afford efficiently a range of substituted cyclic beta-amino esters in high d.e. Alternatively, conjugate addition to alpha,beta-unsaturated Weinreb amides, functional group conversion and ring closing metathesis affords cyclic amines in high d.e. The further application of this methodology to the synthesis of a range of carbocyclic beta-amino esters via conjugate addition, enolate alkylation and ring closing metathesis is also described. Application of this methodology affords, after deprotection, (S)-homoproline, (S)-homopipecolic acid, (S)-coniine and (1S,2S)-trans-pentacin.

New half-sandwich heterobimetallic CpMPt (M=Rh, Ir) dithiolato bridged complexes

Cationic heterobimetallic complexes 5–7 [(PPh3)2Pt(μ-edt)MClCp′)]BF4 (edt=−S(CH2)2S−; 5: M=Rh and Cp′=η5-C5H5; 6: M=Rh and Cp′=η5-C5Me5 and 7: M=Ir and Cp′=η5-C5Me5) were prepared by reaction of [Pt(edt)(PPh3)2] with [Cp′ClM(μ-Cl)2MClCp′] in THF in the presence of two equivalents of AgBF4. The crystalline structure of 5 was determined by X-ray diffraction methods. Cationic heterobimetallic complexes [(PPh3)2Pt(μ-S(CH2)2S)MClCp′)]BF4 (M=Rh, Ir) were prepared. The crystalline structure of [(PPh3)2Pt(μ-edt)RhClCp)]BF4 was determined by X-ray diffraction methods.

Formation of novel photoadducts from irradiation of 5(S)-5-O-tert-butyldimethylsiloxymethylfuran-2(5H)-one

Irradiation of 5S-5-O-tert-butyldimethylsiloxymethylfuran-2(5H)-one 1 in acetonitrile yields the C2-symmetric bis(lactone), 1S,2S,6S,7S-[3S,10S]-bis-tert-butyldimethylsiloxymethyl-4,9-dioxatricyclo[5.3.0.02,6]deca-5,8-dione 6, and a 3-substituted intramolecular product resulting from an apparent 8-endo-trig cyclisation.

Structures, reactivity, and catalytic activity of dithiolato-bridged heterobimetallic MRh (M = Pt, Pd) complexes

Heterobimetallic complexes [(P−P)Pt(μ-S−S)Rh(cod)]ClO4 (P−P = (PPh3)2, Ph2P(CH2)3PPh2 (dppp), and Ph2P(CH2)4PPh2 (dppb); S−S = -S(CH2)2S- (EDT), -S(CH2)3S- (PDT), -S(CH2)4S- (BDT), cod = 1,5-cyclooctadiene) reacted with CO to form the carbonyl complexes [(P−P)Pt(μ-S−S)Rh(CO)2]ClO4 and then with PR3 ligands to give [(P−P)Pt(μ-S−S)Rh(CO)(PR3)]ClO4. The binuclear framework of these cod complexes was maintained in the reactions reported. The cod complexes were tested as catalyst precursors in the hydroformylation of styrene. HPNMR in situ studies showed that mononuclear species formed under catalytic conditions.

Profiling dehydrin gene sequence and physiological parameters in drought tolerant and susceptible spring wheat cultivars.

Physiological and yield traits such as stomatal conductance (mmol m-2s-1), Leaf relative water content (RWC %) and grain yield per plant were studied in a separate experiment. Results revealed that five out of sixteen cultivars viz. Anmol, Moomal, Sarsabz, Bhitai and Pavan, appeared to be relatively more drought tolerant. Based on morphophysiological results, studies were continued to look at these cultivars for drought tolerance at molecular level. Initially, four well recognized primers for dehydrin genes (DHNs) responsible for drought induction in T. durum L., T. aestivum L. and O. sativa L. were used for profiling gene sequence of sixteen wheat cultivars. The primers amplified the DHN genes variably like Primer WDHN13 (T. aestivum L.) amplified the DHN gene in only seven cultivars whereas primer TdDHN15 (T. durum L.) amplified all the sixteen cultivars with even different DNA banding patterns some showing second weaker DNA bands. Third primer TdDHN16 (T. durum L.) has shown entirely different PCR amplification prototype, specially showing two strong DNA bands while fourth primer RAB16C (O. sativa L.) failed to amplify DHN gene in any of the cultivars. Examination of DNA sequences revealed several interesting features. First, it identified the two exon/one intron structure of this gene (complete sequences were not shown), a feature not previously described in the two database cDNA sequences available from T. aestivum L. (gi|21850). Secondly, the analysis identified several single nucleotide polymorphisms (SNPs), positions in gene sequence. Although complete gene sequence was not obtained for all the cultivars, yet there were a total of 38 variable positions in exonic (coding region) sequence, from a total gene length of 453 nucleotides. Matrix of SNP shows these 37 positions with individual sequence at positions given for each of the 14 cultivars (sequence of two cultivars was not obtained) included in this analysis. It demonstrated a considerable diversity for this gene with only three cultivars i.e. TJ-83, Marvi and TD-1 being similar to the consensus sequence. All other cultivars showed a unique combination of SNPs. In order to prove a functional link between these polymorphisms and drought tolerance in wheat, it would be necessary to conduct a more detailed study involving directed mutation of this gene and DHN gene expression.

The formation of ice in a long-lived supercooled layer cloud

This article focuses on the characteristics of persistent thin single-layer mixed-phase clouds. We seek to answer two important questions: (i) how does ice continually nucleate and precipitate from these clouds, without the available ice nuclei becoming depleted? (ii) how do the supercooled liquid droplets persist in spite of the net flux of water vapour to the growing ice crystals? These questions are answered quantitatively using in situ and radar observations of a long-lived mixed-phase cloud layer over the Chilbolton Observatory. Doppler radar measurements show that the top 500 m of cloud (the top 250 m of which is mixed-phase, with ice virga beneath) is turbulent and well-mixed, and the liquid water content is adiabatic. This well-mixed layer is bounded above and below by stable layers. This inhibits entrainment of fresh ice nuclei into the cloud layer, yet our in situ and radar observations show that a steady flux of ≈100 m−2s−1 ice crystals fell from the cloud over the course of ∼1 day. Comparing this flux to the concentration of conventional ice nuclei expected to be present within the well-mixed layer, we find that these nuclei would be depleted within less than 1 h. We therefore argue that nucleation in these persistent supercooled clouds is strongly time-dependent in nature, with droplets freezing slowly over many hours, significantly longer than the few seconds residence time of an ice nucleus counter. Once nucleated, the ice crystals are observed to grow primarily by vapour deposition, because of the low liquid water path (21 g m−2) yet vapour-rich environment. Evidence for this comes from high differential reflectivity in the radar observations, and in situ imaging of the crystals. The flux of vapour from liquid to ice is quantified from in situ measurements, and we show that this modest flux (3.3 g m−2h−1) can be readily offset by slow radiative cooling of the layer to space.

Infrared Observations of XTE J1118+480

Infrared observations of the outbursting black hole XTE J1118+480 (ATEL #383) were performed using SQIID on the Kitt Peak National Observatory 2.1m telescope. Observations spanning 2005 January 15.42-15.58 found it somewhat fainter than the previous outburst (IAUC # 7394 , # 7407 ), at average brightness J=12.91+/-0.03, H=12.50+/-0.03, K=11.95+/-0.03. The colors again correspond to an approximately flat spectrum in F_nu. No orbital variation is apparent, but there is substantial unresolved rapid variability with rms amplitude 22% in K (between 2s exposures). Further observations are planned nightly until Jan 21.