New Cytotoxic Salinosporamides from the Marine Actinomycete Salinisporatropica

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北玄参根中的一个新环烯醚萜衍生物_英文_

A new iridoid derivative from the roots ofScrophularia buergerianaWU Xi-min 1†, ZHANG Liu-qiang 1†, CHEN Xiao-chong 1, FENG Li 1,XING Wang-xing 2*, LI Yi-ming 1*(1. School of Pharmacy, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China;2. School of Medicine, Hangzhou Normal University, Hangzhou 310036, China )Abstract : Phytochemical investigation of the roots of Scrophularia buergeriana Miq. (Scrophulariaceae), resulted in the isolation of a new iridoid derivative named as buergerinin (1). Its structure was elucidated as rel-(1R , 5R , 6R )-(2-oxa-bicyclo[3.3.0]oct-7-en-6, 7-diyl)dimethoxypropane based mainly on MS and 1D and 2D NMR spectroscopic analyses.Key words : Scrophularia buergeriana ; iridoid derivative; buergerinin CLC number : R284 Document code : A Article ID : 0513-4870 (2014) 07-1019-03北玄参根中的一个新环烯醚萜衍生物吴喜民1†, 张刘强1†, 陈小冲1, 冯丽1, 邢旺兴2*, 李医明1*(1. 上海中医药大学中药学院, 上海 201203; 2. 杭州师范大学医学院, 浙江杭州 310036)摘要: 从北玄参块根中发现了一个新的环烯醚萜衍生物, 命名为buergerinin (1)。

Anthocyanidin reductases from Medicago truncatula

Anthocyanidin reductases from Medicago truncatulaand Arabidopsis thalianaDe-Yu Xie,Shashi B.Sharma,and Richard A.Dixon *Plant Biology Division,Samuel Roberts Noble Foundation,2510Sam Noble Parkway,Ardmore,OK 73401,USAReceived 19November 2003,and in revised form 5December 2003AbstractAnthocyanidin reductase (ANR),encoded by the BANYULS gene,is a newly discovered enzyme of the ﬂavonoid pathway involved in the biosynthesis of condensed tannins.ANR functions immediately downstream of anthocyanidin synthase to convert anthocyanidins into the corresponding 2,3-cis -ﬂavan-3-ols.We report the biochemical properties of ANRs from the model legume Medicago truncatula (MtANR)and the model crucifer Arabidopsis thaliana (AtANR).Both enzymes have high temperature optima.MtANR uses both NADPH and NADH as reductant with slight preference for NADPH over NADH.In contrast,AtANR only uses NADPH and exhibits positive cooperativity for the co-substrate.MtANR shows preference for potential anthocyanidin substrates in the order cyanidin >pelargonidin >delphinidin,with typical Michaelis–Menten kinetics for each substrate.In contrast,AtANR exhibits the reverse preference,with substrate inhibition at high concentrations of cyanidin and pelargonidin.(+)-Catechin and (Æ)-dihydroquercetin inhibit AtANR but not MtANR,whereas quercetin inhibits both enzymes.Possible catalytic reaction sequences for ANRs are discussed.Ó2003Elsevier Inc.All rights reserved.Keywords:Condensed tannins;Flavonoids;Anthocyanidin;Kinetics;Model legumeCondensed tannins (CTs)1are oligomers or poly-mers of ﬂavonoid units (ﬂavan-3-ols,also known as catechins)linked by single C 4–C 8or C 4–C 6carbon–carbon bonds (Fig.1A).CTs occur widely throughout the plant kingdom,often in seed coats but also in other tissues such as leaves,ﬂowers,and stems.They play protective functions in the plant,particularly against herbivores [1,2],and have recently attracted consider-able attention in view of their potentially beneﬁcial eﬀects on human and animal health [1–3].CTs bind reversibly to proteins,and the tannins present in the leaves of some forage legumes such as Desmodium uncinatum and Lotus corniculatus can thereby slow the rate of protein degradation in the rumen and thus protect cattle and sheep from pasture bloat [1,3].CTsare also powerful antioxidants,and this property may explain their beneﬁcial eﬀects on cardiac health and immunity [4,5].The ﬂavan-3-ol building blocks of CTs are also antioxidants [6,7],and clinical studies have shown protective eﬀects of catechin,epicatechin,gal-locatechin,and epigallocatechin against stomach can-cer [8].It has recently been shown that epicatechin present in dark chocolate has potential to promote cardiovascular health in humans [9].CTs share the same upstream biosynthetic pathway as the anthocyanin ﬂower pigments (Fig.1A),and this pathway has been well deﬁned at both the biochemical and molecular genetic levels [10,11].It was previously believed that the branch point between the CT and an-thocyanin pathways occurred at the level of leucoanth-ocyanidin,which is converted to anthocyanidin by the action of leucoanthocyanidin dioxygenase (also known as anthocyanidin synthase,ANS)and,hypothetically,to ﬂavan-3-ol by a leucoanthocyanidin reductase (LAR).However,this model,which did not explain the origin of the 2,3-cis stereochemistry predominant in the building blocks of many CTs,has recently undergone revision as*Corresponding author.Fax:1-580-224-6692.E-mail address:radixon@ (R.A.Dixon).1Abbreviations used:CT,condensed tannin;Mt,Medicago trun-catula ;At,Arabidopsis thaliana ;ANR,anthocyanidin reductase;ANS,anthocyanidin synthase;LAR,leucoanthocyanidin reductase;DHQ,dihydroquercetin;MBP,maltose-binding protein;MDAR,monode-hydroascorbate reductase;DFR,dihydroﬂavonol reductase.0003-9861/$-see front matter Ó2003Elsevier Inc.All rights reserved.doi:10.1016/j.abb.2003.12.011Archives of Biochemistry and Biophysics 422(2004)91–102ABB/locate/yabbia result of the biochemical characterization of the BANYULS gene[12].The BANYULS gene was named after the color of a French red wine for a mutant of Arabidopsis thaliana that lacked CTs in the seed coat endothelium and pre-cociously accumulated anthocyanins[13,14].It was originally proposed that BANYULS might encode LAR [14].However,when expressed in Escherichia coli,the product of a BANYULS ortholog from the model le-gume Medicago truncatula eﬃciently convertedantho-cyanidins into their correspondingﬂavan-3-ols in the presence of NADPH[12].This resulted in the proposal of a new pathway to CTs in which anthocyanidins were key intermediates,as substrates for the BANYULS gene product anthocyanidin reductase(ANR).Chemical characterization of the reaction products of ANR identiﬁed two isomers ofﬂavan-3-ol,namely2R,3R-2, 3-cis-and2S,3R-2,3-trans[12].With cyanidin as sub-strate,these products are())-epicatechin and())-cate-chin,respectively.The2R,3R-2,3-cis-ﬂavan-3-ol,which was the major reaction product,is the common building block of CTs in many plants including Arabidopsis[15] and alfalfa(Medicago sativa)[16].Recently,a LAR gene has been cloned following puriﬁcation of the corresponding enzyme from D.un-cinatum leaf tissue and shown to be a member of the isoﬂavone reductase(IFR)-like gene family.The enzyme converted radiolabeled leucocyanidin to(+)-catechin (2R,3S-2,3-trans-ﬂavan-3-ol)[17].(+)-Catechin is often the starter unit for CT polymerization.Together,ANR and LAR would therefore appear to be suﬃcient for formation of most monomeric building blocks of CTs. However,still very little is understood concerning the biological processes resulting in CT polymerization,or the reaction mechanisms whereby the building blocks are produced via ANR and LAR.The basic anthocyanidin structure is generally con-sidered to be the coloredﬂavylium cation or salts thereof (Fig.1B)[18–22].However,anthocyanidins are not sta-ble in aqueous solution and undergo rapid,pH-depen-dent,reversible transformations(Fig.1B)[18,22,23].The colors fade rapidly in weakly acidic solutions(pH3–5), presumably due to formation of an equilibrium mixture of theﬂavylium cation and the colorless chromen-2-ol pseudobase(Fig.1B)[22].At pH5–8,anthocyanidins can also rapidly transform to the anhydro base or quinoidal base(Fig.1B)[22,23].These transformations complicate the assignment of a reaction mechanism to ANR.We here present a comparative study of the bio-chemical properties of ANRs from M.truncatula and A. thaliana.A full understanding of the mode of catalysis by ANR will facilitate future attempts to engineer con-densed tannins or catechins in a variety of plant species for improving nutritional and health-related traits.In-terestingly,the ANR enzymes from these two sources have quite diﬀerent substrate speciﬁcities and kinetic properties.We present hypothetical mechanisms for the ANR reaction.Materials and methodsChemicalsCyanidin chloride,pelargonidin chloride,and delphinidin chloride were purchased from Indoﬁne (Hillsborough,NJ,USA).Ten mM methanol stock so-lutions were stored at)20°C.NADPH and NADH were from Sigma(St.Louis,MO)and solutions were freshly prepared in cold reaction buﬀer when used for enzymatic assay.(+)-Catechin,(Æ)-catechin,())-epi-catechin,())-gallocatechin,())-epigallocatechin,(Æ)-di-hydroquercetin(DHQ),and quercetin were from Sigma, and methanolic stock solutions were stored at)20°C.Expression of MtANR and AtANR in E.coliThe open reading frame of the MtANR gene was ampliﬁed by polymerase chain reaction(PCR)using pSE380-BAN plasmid as template[12].The amino terminal primer,50-GCGGCGGAATTCATGGCTAG TATCAAACAAATAG-30containing the ATG start codon,introduced an Eco RI restriction site(under-lined),and the carboxyl terminal primer50-GCGGCG TCTAGATCACTTGA TCCCCTGAGTCTTC-30,in-cluding the stop codon,introduced an Xba I restriction site.PCR was carried out using Taq DNA polymerase (Promega,Madison,WI)at94°C for2min,35cycles of 94°C for45s,60°C for45s,and72°C for1.5min; followed by a10min extension at72°C.The PCR fragment was gel puriﬁed using a Sephaglas Bandprep kit(New England Biolabs,Beverly,MA)and ligated into pGEM-T-easy vector(Promega).The pGEM-T-MtANR plasmid was transformed into E.coli DH5a competent cells for sequence analysis.The open reading frame was then excised by digestion with Eco RI and Xba I and re-ligated into Eco RI and Xba I digested pMAL-c2X vector as an N-terminal fusion to the maltose-binding protein(MBP)gene to generate the protein expression construct pMMtANR.To clone AtANR for protein expression,total RNA from young siliques of A.thaliana cv Columbia was isolated using the Tri-Reagent kit(Molecular Re-search Center,Cincinnati,OH).First strand cDNA was synthesized from4l g total RNA from siliques by Su-perscriptII reverse transcriptase(Invitrogen,Carlsbad, CA)according to the manufacturerÕs instructions.The ANR gene was ampliﬁed from10l l of theﬁrst strand cDNA by highﬁdelity Pfu DNA polymerase-mediated PCR using forward primer50-GGGCCCATGGAC CAGA CTCTTACACACACCGGA-30and reverse primer50-CCCAGATCTAGAATGAGACCAAAG ACTCATAT ACT-30at94°C for5min,30cycles of 94°C for1min,55°C for1min,and72°C for1.5min, followed by a further10min extension at72°C.The PCR fragment was digested with Nco I and Xba I,and cloned into Nco I–Xba I digested pRTL2to give the construct pSBP77,which was then sequenced.For ex-pression of AtANR in E.coli,pSBP77was digested with Nco I and then blunted with mung bean nuclease(New England BioLabs).The ANR coding region fragment was puriﬁed after digestion with Xba I,and ligated intoD.-Y.Xie et al./Archives of Biochemistry and Biophysics422(2004)91–10293Xmn I and Xba I digested pMAL-c2X as an N-terminal fusion to the MBP gene to generate the protein expression construct pMAtANR.pMMtANR,pMAtANR,and pMAL-c2X(as control vector)were transformed into NovaBlue(DE3) competent cells(Novagen,Madison,WI).A single col-ony harboring either construct or control vector was inoculated into1liter of LB broth containing100mg/ liter ampicillin and cells were grown to an OD600of0.3 at37°C and250rpm.The cells were then transferred to 16°C and grown to an OD600of0.6–0.7,at which point isopropyl-1-thio-b-D-galactopyranoside(IPTG)was added to aﬁnal concentration of1mM.The cells were harvested24h later by centrifugation at3000rpm at 4°C for15min.Pellets were either used directly for enzyme extraction or stored at)80°C.Enzyme extraction and puriﬁcationAll procedures were performed at4°C.Crude en-zyme was extracted as described previously[12]and further puriﬁed on an amylose column.Three to4ml amylose homogenate(New England Biolabs)was loa-ded into a2.5Â16cm column,and then washed and equilibrated with at least10volumes of washing buﬀer (20mM Tris–HCl,pH7.0,and10mM b-mercap-toethanol).The crude extract was dilutedﬁve times with washing buﬀer and then loaded onto the column at aﬂow rate of1ml/min.The column was washed with at least15column volumes of washing buﬀer and the MBP-ANR fusion protein eluted with washing buﬀer supplemented with10mM maltose.The fusion protein was concentrated to 1.5–10l g/l l by passing through a10,000molecular weight cut-oﬀAmicon Ultra(Millipore)membrane and then analyzed by electrophoresis on a12%SDS–polyacrylamide gel stained with0.25%Coomassie brilliant blue(R250). ANR protein was separated from MBP by protease cleavage with Factor Xa(New England Biolabs)fol-lowing the manufacturerÕs protocol.Enzyme assays and determination of kinetic parameters All enzyme assays were performed by measuring ap-pearance ofﬂavan3-ol products by HPLC after stop-ping reactions after the required time periods.For determination of temperature optimum,enzyme assays were carried out for30min in a total volume of200l l containing100mM Tris–HCl,pH7.0,100l M cyanidin chloride,2mM NADPH,and50–100l g MBP-ANR or 30l g puriﬁed cleaved ANR.Determination of pH de-pendence and reaction kinetics was performed with MBP-ANR fusion proteins.pH proﬁles for MtANR and AtANR were determined at their respective temperature optima of45°C(MtANR) and55°C(AtANR)with30min incubations in aﬁnal volume of200l l consisting of50mM citrate/phosphate buﬀer,pH4.0–7.0,50mM Mes buﬀer,pH5.0–7.0or 100mM Tris–HCl buﬀer,pH7.0–8.8,100l M cyanidin, 2mM NADPH,and50–100l g puriﬁed fusion protein.Kinetic constants for NADPH or NADH were de-termined at the respective temperature optima of MtANR and AtANR in a total volume of200l l con-taining50mM Mes buﬀer,pH6.0(for MtANR)or 100mM Tris–HCl,pH8.0(for AtANR),100l M cyanidin,0–12mM NADPH or NADH,and100l g MBP-fusion protein,with30min incubation.Kinetic constants for anthocyanidin substrates were likewise determined in a total volume of200l l containing100l g MBP-fusion protein,0–300l M cyanidin chloride or delphinidin chloride,or0–500l M pelargonidin chlo-ride,and2mM NADPH.To determine the eﬀects ofﬂavonoids and Naþions, enzyme assays were carried out at the respective tem-perature and pH optima in a total volume of200l l containing100l g MBP-fusion protein,100l M cyanidin chloride,2mM NADPH,and either(Æ)-DHQ(0–1mM),(+)-catechin(0–1mM),quercetin(0–0.1mM)or NaCl(0–400mM).To determine whether MtANR or AtANR can epi-merize or racemize theﬂavan-3-ol products,assays were carried out at the respective temperature and pH optima in a total volume of200l l containing100l g fusion protein,2mM NADPH,and35–70l M())-epicatechin or50–100l M(Æ)-catechin in the presence or absence of 100l M cyanidin.Control experiments were performed under the same conditions with buﬀer or boiled enzyme instead of active enzyme.All enzyme reactions were initiated by adding enzyme and stopped by adding1ml ethyl acetate with vigorous vortexing for1min,and then centrifuged for1min at 10,000rpm.The ethyl acetate supernatant phase(0.9ml) was transferred to a new1.5ml microcentrifuge tube and dried under nitrogen gas at room temperature. Residues were dissolved in50l l HPLC grade methanol for HPLC analysis.HPLC analysis and data integrationHPLC analysis used a HP1100machine and C18 reversed phase column with UV detection at280nm for ())-epicatechin,(Æ)-catechin,(+)-catechin,())-epiafz-elechin,and())-afzelechin,and at270nm for())-gal-locatechin and())-epigallocatechin.The solvent system consisted of water(solvent A)and acetonitrile(solvent B).The gradient program consisted of ratios of solvent A to solvent B of95:5(0–5min),95:5to93:7(5–7min), 93:7(7–25min),93:7to60:40(25–40min),60:40to5:95 (40–40.5min),and5:95(40.5–49.5min).Forty l l sam-ples was injected with aﬂow rate of1.5ml per min. Product peaks were identiﬁed by retention time and UV spectrum as previously described[12].Authentic94 D.-Y.Xie et al./Archives of Biochemistry and Biophysics422(2004)91–102standards of(Æ)-catechin,())-epicatechin,())-gallo-catechin,and())-epigallocatechin were injected for reference.Each experiment was performed in triplicate and re-peated at least once.Product formation was estimated by integrating peak area using HP Chemstation Soft-ware.Amounts of reaction products were calculated based on established standard curves using authentic samples.Due to lack of available standards of())-epi-afzelechin and())-afzelechin,the products produced from pelargonidin were simply estimated by integrating peak areas.Values could not be calculated with pe-largonidin as substrate.The enzymatic products con-sisted of two isomers.Initial reaction velocity was calculated as the total sum of the products,i.e., ())-catechin and())-epicatechin,())-afzelechin and ())-epiafzelechin,or())-gallocatechin and())-epigallo-catechin.K m,V max,½S0:5,and n H values were calculatedthrough various plots of initial reaction velocity versus substrate concentration.V max values were not calculated for pelargonidin due to lack of authentic standards of (epi)-afzelechin for absolute quantitation.ResultsSequence comparison of MtANR and AtANR MtANR and AtANR(from A.thaliana ecotype Columbia)share only60%amino acid sequence identity. MtANR consists of338amino acids with a molecular weight(MW)of36,977Da,whereas AtANR consists of 340amino acids,with an MW of37,904Da.Both MtANR and AtANR contain the classical Rossmann dinucleotide-binding domain[24]character-ized by a conserved glycine rich amino acid sequence ﬁngerprint of either GXGXXA(such as found in monodehydroascorbate reductases,MDAR)[25]or GXXGXXG(such as found in plant dihydroﬂavonol reductases,DFR)(Fig.2).Within the GXXGXXG consensus,MtANR has the F22conserved in DFR homologs but AtANR has N at this position(Fig.2). Expression and puriﬁcation of ANR-MBP fusion protein The MtANR and AtANR open reading frames were ampliﬁed by PCR and cloned into the protein expression vector pMAL-c2X immediately downstream of the MBP to facilitate protein puriﬁcation.MBP fusion proteins have often been used for studying enzyme ki-netics,three-dimensional structures,and catalytic mechanisms[25–27].The constructs were transformed into E.coli NovaBlue(DE3).MtANR and AtANR MBP fusion proteins localized to inclusion bodies when expressed in E.coli at37°C,but were recovered as soluble enzymes following expression at16°C.This ex-pression system allowed for the production of from10 to20mg ANR protein per liter of culture,suitable amounts for crystallization studies.Fusion proteins were puriﬁed by amylose aﬃnity chromatography re-sulting in a single major band of approximatly70kDa by denaturing SDS–PAGE(Fig.3A).To ensure that the presence of the MBP did not ad-versely aﬀect the properties of ANR,we compared the speciﬁc activities and temperature optima of puriﬁed recombinant MBP-MtANR and cleaved‘‘native’’MtANR;the temperature optima were identical(45°C), and the speciﬁc activity of the native protein was re-duced by30%compared to that of the MBP fusion. Signiﬁcant protein losses occurred during separation of ANR from MBP following cleavage with Factor Xa at 4°C,although there is no Factor Xa cleavage site in the ANR protein,and this may explain the small reduction in speciﬁc activity.Eﬀects of temperature,pH,and NaCl on ANR activities The ANR reaction was linear over the30min time period used for standard assays.The temperature optima (reﬂecting the composite of reaction rate andproteinstability)of both MBP-MtANR and MBP-AtANR re-actions in vitro were quite similar (Table 1).However,dramatic diﬀerences were observed in pH dependence,with MtANR having a weakly acidic pH optimum and AtANR showing maximum activity at weakly basic pH (Figs.3B and C).The pH optimum of MtANR was 5.5in 50mM citrate/phosphate but 6.0in 50mM Mes buﬀer (Fig.3B).Addition of increasing concentrations of Na þto 50mM Mes buﬀer (pH 6.0)indicated that MtANR activity is inhibited by Na þconcentrations above200mM.This may explain the discrepancy between pH optima in citrate/phosphate and Mes buﬀer.NaCl con-centrations up to 400mM did not inhibit AtANR.Co-enzyme requirements of MtANR and AtANR Signiﬁcant diﬀerences between MtANR and AtANR were observed in their co-enzyme requirements.MtANR used either NADPH or NADH as co-sub-strates (Figs.4A and B).Double reciprocal plots of 1=VFig.3.ANR fusion protein expression in E.coli and determination of pH optima.(A)SDS–polyacrylamide (12%)gel electrophoresis of E.coli -expressed MBP-MtANR and MBP-AtANR fusion proteins stained with Commassie brilliant blue ne 1,prestained protein mass markers (New England Biolabs);lane 2,20l g crude extract from E.coli harboring pMMtANR;lane 3,20l g crude extract from E.coli harboring pMA-tANR;lane 4,2l g puriﬁed MBP-MtBAN;and lane 5,2l g puriﬁed MBP-AtBAN.(B)The eﬀect of pH on MtANR activity.(C)The eﬀect of pH on AtANR activity.In (B)and (C),curve 1is for 50mM citrate-phosphate,curve 2for 50mM Mes,and curve 3for 100mM Tris–HCl.Table 1Kinetic properties of MtANR and AtANRMtANR AtANRV max (nmol/min mg)K m (l M)k cat(min À1)k cat =K m (M À1s À1)V max (nmol/min mg)K m (l M)k cat(min À1)k cat =K m (M À1s À1)Substrates Cyanidin 1.2Æ0.712.9Æ0.410.8 1.4Â10410.0Æ1.873.9Æ5.962.7 1.4Â104Pelargonidin 14.552.8Æ3.9Delphinidin 60.649.8Æ10.880.32.7Â1040.9Æ0.0217.8Æ1.53.50.3Â104Cofactors NADPH 0.4Æ0.01450Æ120n H ¼3:3Æ0:3,½S 0:5¼125:5Æ0:7l MNADH 0.3Æ0.1940Æ155NR ÃTemperature:45°C55°C*NR,no reaction.96 D.-Y.Xie et al./Archives of Biochemistry and Biophysics 422(2004)91–102versus1/[NADPH]or1/[NADH]for MtANR indicated classic Michaelis–Menten steady-state kinetics(Figs.4A and B),with a lower K m and higher V max=K m for NADPH than for NADH(Table1).In contrast, AtANR did not use NADH as co-substrate and the saturation curve for NADPH was sigmoid rather than hyperbolic,suggesting positive cooperativity(Fig.4C).A plot of log(V=V maxÀV)versus log[NADPH]was a straight line(Fig.4D),from which a Hill co-eﬃcient (n H)of3.3and½S0:5of125l M were calculated(Table1).Properties of MtANR and AtANR towards anthocyanidin substratesMtANR and AtANR showed signiﬁcant diﬀerence in their steady-state kinetics with anthocyanidin substrates. At optimum pH,temperature,and NADPH concen-tration,MtANR exhibited Michaelis–Menten kinetics with cyanidin,pelargonidin,and delphinidin(Figs.5A–C).The double reciprocal plots indicated the highest K m for delphinidin,followed by pelargonidin and then cy-anidin(Table1).However,MtANR had its highest V max value for delphinidin(Table1),resulting in a twofold higher k cat=K m ratio for delphinidin compared to cyanidin.AtANR diﬀered markedly from MtANR in its ki-netic properties towards cyanidin and pelargonidin (Figs.5E–H),with the reaction velocity decreasing with anthocyanidin concentrations above100–150l M(Figs. 5E and G).At lower substrate concentrations,the re-action followed classic Michaelis–Menten kinetics(Figs. 5E and G).The upturn in the plots of1=V versus1/ [cyanidin]or1/[pelargonidin]on approaching the1=V axis(Figs.5F and H)is indicative of substrate inhibi-tion.This could result from low levels of a non-com-petitive inhibitor(perhaps a speciﬁc ionization product of the anthocyanidin,as depicted in Fig.1B)present in the substrate sample.The kinetics towards cyanidin and pelargonidin were thereforeﬁtted to the following ve-locity equation for a substrate plus non-competitive inhibitor:v=V max¼½S =f K mþ½S ð1þxK m=K iþx½S =K iÞg;where K m is the substrate concentration at1=2V max,x represents a coeﬃcient for[I]where½I ¼x½S in the substrate solution,and K i is the inhibitor constant de-ﬁned as f½E ½I g=½EI .This analysis facilitated calcula-tion of apparent K m values for cyanidin and pelargonidin as well as a V max for cyanidin(Table1).In contrast to the above observations with cyanidin and pelargonidin,AtANR displayed classical Micha-elis–Menten kinetics towards delphinidin(Fig.5D).The K m of AtANR was highest for cyanidin,followed by pelargonidin and then delphinidin(Table1).Neverthe-less,the k cat=K m ratio was highest with cyanidin,sug-gesting that this may be the preferred substrate in vivo.Inhibitors of MtANR and AtANRThe purity of the commercial cyanidin and pelarg-onidin substrates was checked by HPLC and was97–99%.Minor contamination byﬂavonols(kaempferol and quercetin)and dihydroﬂavonols(dihydroquercetin) was observed.(Æ)-DHQ,quercetin,and(+)-catechin (2R,3S-2,3-trans-ﬂavan-3-ol)(Fig.6A)were tested for their eﬀects on ANR activity in vitro.(+)-Catechindidnot inhibit MtANR activity(Fig.6B),but inhibited AtANR by50%at0.5mM(Fig.6C).(Æ)-DHQ did not inhibit MtANR(Fig.6D),but inhibited AtANR at concentrations as low as25l M(Fig.6E).Quercetin strongly inhibited both MtANR and AtANR(Figs.6F and G).The origin of the epimerization products of the ANR reactionWe have previously shown that ANR converts cyanidin into())-epicatechin as the major product,with ())-catechin as a minor product[12].Likewise,())-epi-afzelechin is the major product and())-afzelechin the minor product from pelargonidin.Equal amounts of ())-gallocatechin and())-epigallocatechin are formed from delphinidin[12].The question therefore arises as to whether())-catechin,())-afzelechin,and())-gallocate-chin are formed by non-enzymatic epimerization at C2 of the major products())-epicatechin,())-epiafzelechin, and())-epigallocatechin,respectively,or whether they are separate reaction products formed by the enzyme itself.To address this question,MtANR or AtANR was incubated with())-epicatechin or(Æ)-catechin in the presence of NADPH,and parallel controls were in-cluded in which active enzyme was replaced with boiled enzyme or buﬀer.No epimerization or racemization of ())-epicatechin or())-catechin into thecorresponding())-catechin or())-epicatechin,respectively,was ob-served under any of these conditions if Mes buﬀer was used.However,incubation of())-epicatechin in100mM Tris–HCl,pH7,at temperatures above30°C even without enzyme resulted in epimerization to catechin, suggesting that the())-catechin observed as a minor product in the enzyme assays might result from chemical epimerization rather than an ability of ANR to produce both2R,3R-2,3-cis-and2S,3R-2,3-trans-ﬂavan-3-ol isomers.DiscussionAmino acid sequence comparison and biochemical diﬀer-ences between MtANR and AtANRMtANR and AtANR share only60%amino acid sequence identity.Both enzymes contain the conserved Rossmann dinucleotide-binding domain at their amino-termini[24,28,29].To date,there are few available ANR gene sequences from diﬀerent plant species,and,to the best of our knowledge,no other reports of ANR sub-strate/co-substrate speciﬁcity.However,it seems likely that the two amino acid diﬀerences between the dinu-cleotide-binding domains of MtANR and AtANR(F22 and V23in MtANR corresponding to N21and L22in AtANR)may be related to the diﬀerent speciﬁcities of the two enzymes for NADPH and NADH.Examples exist where only a single amino acid change can alter co-factor binding speciﬁcity.For example,rat NADPH-dependent cytochrome P-450reductase cannot use NADH as cofactor,but mutation of Trp677to Ala generates an enzyme with quite good NADH turnover [30].Detailed structural studies of MtANR and AtANR will be necessary to deﬁnitively explain their diﬀerent co-factor speciﬁcities.The kinetics of MtANR for NADPH followed the usual Michaelis–Menten behavior observed with many reductases,whereas those for AtANR were sigmoidal and indicative of positive cooperativity,with NADPH acting as co-substrate and modulator.Otherreductases,such as ribonucleotide reductase[31],also show allo-steric behavior,and very diﬀerent kinetics of enzymes with NADPH do not necessarily require extensive dif-ferences in amino acid sequence[32,33].The non-linear kinetics of AtBAN could reﬂect the operation of the two successive hydride transfers in the overall ANR reac-tion.The turnover(k cat)of both MtANR and AtANR was quite slow.Both enzymes reduce cyanidin,pelargonidin, and delphinidin,indicating that the B-ring hydroxyl-ation pattern is not critical for catalysis.However,the relative binding aﬃnities for the three substrates diﬀer, with MtBAN exhibiting decreasing K m values in the order delphinidin>pelargonidin>cyanidin,and At-BAN exhibiting the inverse relationship.Nevertheless, both enzymes had the same k cat=K m ratio for cyanidin, which may be the major in vivo substrate for both en-zymes,consistent with the presence of())-epicatechin residues as the major building blocks in the CTs of both A.thaliana and M.truncatula.Similar to ANR,diﬀerent dihydroﬂavonol reductases(DFR)from M.truncatula, Malus domestica,and Pyrus communis showed signiﬁ-cantly diﬀerent kinetics towards DHQ and dihydroka-empferol,which diﬀer in B-ring hydroxylation pattern [34,35].High cyanidin or pelargonidin concentrations inhibit AtANR but not MtANR.This inhibition may result from the presence of non-competitive inhibitor(s)in the substrate preparations.Quercetin inhibited both MtANR and AtANR activities,whereas(+)-catechin and(Æ)-DHQ inhibited AtANR but not MtANR.M. truncatula produces(+)-catechin as a component of the seed coat CT,whereas the Arabidopsis CT does not contain(+)-catechin[15].())-Epicatechin did not in-hibit MtANR or AtANR over the tested concentration range.In the absence of information on the pool sizes of dihydroﬂavonols andﬂavan3-ols in the seed coat endothelium,it is not possible to conclude whether the above inhibitory eﬀects are of physiological signiﬁ-cance.Reaction mechanisms for reduction of anthocyanidins to ﬂavan-3-olsThe discovery of ANR explains the inversion of ste-reochemistry at C3during the biosynthesis of())-epi-catechin from leucoanthocyanidin.The heterocyclic C-ring of anthocyanidins,formed from chiral leuco-anthocyanidins,has two double bonds,at O1–C2and C3–C4,and is therefore achiral.ANR reduces anthocy-anidins toﬂavan-3-ols through NADPH-mediated re-duction at C2and C3,allowing for the inversion of stereochemistry at C3.The())-catechin(minor product) observed in enzymatic reactions may be produced by non-enzymatic epimerization at C2of())-epicatechin under the reaction conditions used.())-Catechin has recently been shown to exert strong alleopathic eﬀects and to be a major factor for colonization by the de-structive non-indigenous spotted knapweed in North America[36].Analysis of the properties of ANR from this weedy species may reveal alternative routes for the biosynthesis of())-catechin.Although anthocyanidins are often depicted as hav-ing a positive charge associated with the oxygen of the heterocyclic ring,it is recognized that the charge may be delocalized[19–21].Assessment of charge distributions on theﬂavylium cation through molecular orbital cal-culations suggests that electrophilic attack can occur at positions C8,C6and various B-ring sites,and nucleo-philic attack principally at C2and C4[16,19–21,37,38]. This is consistent with the facile in vitro chemical syn-thesis ofﬂavan-3-ols through reduction of anthocyani-dins with PtO2in a hydrogen atmosphere[38].Complete hydrogenation of cyanidin or its pentamethyl ether,and ﬁsetinidin and butinidin,resulted in the corresponding reduced products(Æ)-epicatechin and its pentamethyl ether,(Æ)-2,3-cis-3,30,40,70-tetrahydroxyﬂavan,and (Æ)-2,3-cis-30,40,7-trihydroxyﬂavan,respectively[38]. Hrazdina also synthesized(ÆÞ-30-O-methylepicatechin, (Æ)-30,50-di-O-methylepigallocatechin and(Æ)-30-O-methylepigallocatechin from peonidin(30-O-methylcy-anidin),malvidin(30,50-di-O-methyldelphinidin),and petunidin(30-O-methyldelphinidin),respectively,with sodium borohydride in ethanol or methanol[38].The intermediates of the in vitro reduction were theﬂav-3-en-3-ols andﬂav-2-en-3-ols(Fig.7),which are stable on exposure to air and in aqueous acidic solution.The initial reducedﬂavenes are in equilibrium with the cor-responding3-keto form(ﬂavan-3-one tautomer)(Fig.7), which are then further reduced to the corresponding ﬂavan-3-ols[39,40].Based on the above considerations,it is possible to postulate four possible enzymatic reduction sequences for the ANR reaction(Fig.7).In mechanism I-a,the ﬁrst hydride ion provided by NADPH/NADH nucleo-philically attacks C2to form a stableﬂav-3-en-3-ol,the conﬁguration of the C2-aryl group being2R with hydride addition by way of the b-face(addition to the a-face would be necessary to account for enzymatic production of the2S conﬁguration of the minor product ())-catechin).NADPH/NADH then provides a second hydride ion to attack C4,and a proton from the medium then quenches the charge at C3,thereby reducing the C3–C4double bond.The conﬁguration of the C3-hy-droxyl group as3R or3S is also dependent upon the direction of hydride ion addition.This will again be from the b-face to result in the formation of2R,3R-2, 3-cis-ﬂavan-3-ols.In mechanism I-b,theﬁrst step is the same as in I-a. In the second step,ANR transforms theﬂav-3-en-3-ol intermediate into its tautomericﬂavan-3-one(Fig.7) followed by reduction by NADPH/NADH at C3and100 D.-Y.Xie et al./Archives of Biochemistry and Biophysics422(2004)91–102。

甘西鼠尾草中三个新的萜类化合物

甘西鼠尾草为常用中药丹参（．ｍｉｉｒｉｇ．的同属植物，其主要成分为脂溶性二萜醌ＳｌｏｈａＢｅ）ｔｒｚ
类和水溶性酚酸类化合物 ’ ．已报道的药理活性主要有醛糖还原酶抑制活性、抑制超氧自由綦及抗氧化、保护心肌缺血及心肌缺血再灌注损伤、炎及抑菌。抗等．本课题组在对甘两鼠尾草活性部位的筛选中，证实了甘西鼠尾草对血清病性肾小球肾炎模型大鼠具有较好的治疗作用，可显著降低大鼠尿蛋白含量、减轻肾小球肿胀，还可降低正常大鼠的血液黏度，并具有显著且温和的利尿作用，以调整肾脏疾病中的电解质紊乱，对于治疗和缓解肾脏疾病的发展有积极的意可义．为了进一步考察其活性物质，本文对其活性部位的化学成分进行了深入研究，从甘西鼠尾草根和根茎的５％乙醇提取物中分离鉴定了２个新的二萜化合物和１个新的单萜苷化合物，０
１实验部分
１１试剂与仪器．
柱色谱硅胶与薄层色谱硅胶板为烟台江友硅胶开发公司产品；Ｓｐａｅ０凝胶为ＰａｍｃｅｈｄｘＩ２Ｈｈｒａｉａ
公司产品；ＤＩＯＳＣ相硅胶为Ｍｅｋ公司产品；ＩｅＣＰ０８反ｒｃＭＣｌＨ２Ｐ凝胶和ＨＰ２ｇ－０大孔吸附树脂为 ■菱
杨阳，吴志军’ ，杨颖博，来威，孙连娜，陈万生

翻译

天然产物杂志从对细胞死亡敏感和抗癌细胞系列中的海绵生物中提取抗恶性细胞增值活性的二萜异腈的评价人名和通讯地址等等暂不翻译背景信息摘要：一个新的在1位的和在已知2,4位的腈基的二萜，是从加勒比海海绵Pseudoaxinella在人类体外癌症细胞使用的线检MTT比色法检测和定量和电子扫描显微镜中分离出来的。

化合物14显示的为人类的PC3前列腺凋亡敏感的肿瘤细胞系的活动，。

化合物3和4表现出类似的增长抑制个人为三个APOP凋亡敏感和3个抗凋亡的肿瘤细胞株的定量电子显微镜分析表明，化合物1和2施加他们的活动，通过细胞毒性个人ECTS 通过抑制细胞生长的个人ECTS化合物3和4。

这些结果确定潜在的铅化合物对海洋中二萜异腈抗癌药物的发现。

萜类化合物，包含异氰和异硫氰团体往往在海洋无脊椎动物中发现的次生代谢产物，例如海绵等在过去的15年,海洋天然产物因为他们的生物学活性乎寻常的异类功能化，在科学界很多成员引起了广泛的兴趣，事实上，一个最有力的海洋抗疟药化合物是最初分离的二萜异腈，从热带海绵状物，特点是由amphilectane骨架。

在我们的研究项目的框架中, 被认为是新颖的先导化合物可以视作典型的抗癌试验，加勒比海的海绵化学海绵伪黄色生物进行了研究。

本研究引发了分离了一个二萜异腈化合物(1)和三个已知的类似的(24), 因为不同的数目和位置的异腈官能团和双键，所有这一切都是紧密的联系的。

用比色MTT检测评价生长抑菌浓度IC50(即全球经济增长的一个给定的细胞参与增殖培养了三天的化合物减少50%的浓度）的不同双萜的浓度体外测试。

定量的电子显微镜（即计算捷尔- 辅助相衬显微镜）将被用来检测是否影响细胞性通过细胞毒性或抑制细胞生长的途径。

我们已经成功地完成例如其他类型的化合物的特点包括真菌次级代谢产物, 类固醇治疗，Amar——和yllidaceae生物碱2007 Pawlik在大巴哈马海岸考察的海绵Pseudoaxinella蔺沿的（甜味剂礁）的样品被切成被冻结的小块样本然后运送到实验室用MeOH和CHCl3分别萃取提纯。

不同酶消化法提取猪原代肝细胞的效果比较

532024.4·试验研究0 引言猪圆环病毒（PCV ）是Circoviridae 科Circovirus 属的一种无囊膜的单链环状DNA 病毒。

在已知的4个血清型中，PCV2为猪易感的致病性病毒[1]。

PCV2感染会诱导宿主免疫抑制引起猪圆环病毒病（PCVD ），包括断奶仔猪多系统衰竭综合征、新生仔猪先天性脑震颤、皮炎与肾病综合征、猪呼吸道病综合征、母猪繁殖障碍等，给全世界养猪业带来较大的经济损失，是世界各国的兽医与养猪业者公认的造成重大影响的猪传染病[2]。

PCV2的感染在猪生长发育的不同阶段有不同的组织嗜性。

但无论是胎儿阶段还是出生后，肝细胞都是PCV2感染和复制的靶细胞。

因此，PCV2也被视为一种能够诱导猪肝炎的病毒[3]。

且PCV2诱导的肝细胞凋亡在PCV2引发的相关病变和疾病的发病机制中具有关键性作用[4]。

因此，方便、快捷地获取大量有活性的猪肝细胞对于研究PCVD 的致病机制具有重大意义。

目前获取肝细胞常用的方法主要包括机械分离细胞法、非酶分离细胞法、离体酶消化法和酶灌流法等[5]。

因此，本试验采用简便、经济、无需特殊设备、仅需部分肝组织的离体酶消化法，比较不同酶消化分离猪原代肝细胞的效果，为一般实验室提取分离大量有活性的猪肝细胞提供参考。

1 材料与方法1.1 材料1.1.1 主要试剂新鲜猪肝组织，Hank's 平衡盐溶液（HBSS ），磷酸盐缓冲液（无菌PBS ），4%多聚甲醛（PFA ），收稿日期：2024-01-27基金项目：国家自然科学基金项目：复杂器官与组织在脾脏内的功能性再生（32230056）作者简介：周徐倩（1999-），女，汉族，浙江温州人，硕士在读，研究方向：组织工程与再生医学。

*通信作者简介：董磊（1978-），男，汉族，安徽阜阳人，博士，教授，研究方向：组织工程与再生医学、生物材料。

周徐倩，董磊．不同酶消化法提取猪原代肝细胞的效果比较[J]．现代畜牧科技，2024，107（4）：53-55．　doi ：10.19369/ki.2095-9737.2024.04.014．　ZHOU Xuqian ，DONG Lei ．Comparison of the Eﬀect of Diﬀerent Enzyme Digestion Methods on Extraction of Porcine Primary Hepatocytes[J]．Modern Animal Husbandry Science & Technology ，2024，107（4）：53-55．不同酶消化法提取猪原代肝细胞的效果比较周徐倩，董磊*（南京大学，江苏南京 210023）摘要：猪肝细胞是猪圆环病毒的靶细胞，简单快速地提取猪原代肝细胞对于研究猪圆环病毒病的致病机制具有重要意义。

化妆品活性成分

Millicapsules A2 Millicapsules is a proprietary technology which utilizes two marine derived polymers, Alginic Acid and Agar, to produce visually distinctive skin and hair care products without the use of conventional emulsifiers. Spheres (1mm, 2mm or 4mm)are produced from these natural polymers. They can contain up to 20% emollient phase and be colored or pearlized. A2Millicapsules offer a unique alternative to conventional emulsions, serums or gels. Recommended concentration: 1-5%.INCI Name: Water (Aqua), Glycerin, Agar, Alginic Acid.Aldenine® Aldenine® is a complex of a tripeptide and hydrolyzed wheat and soy protein that boosts Collagen III synthesis while protecting cells from photo damage. Aldenine® detoxifies the skin from harmful RCS (Reactive Carbonyl Species).Recommended concentration: 2-5%.INCI Name: Hydrolyzed Wheat Protein, Hydrolyzed Soy Protein, Xanthan gum,Tripeptide-1.Alpha-Arbutin Alpha-Arbutin is a pure, water soluble, biosynthetic active ingredient. It is the more effective, faster and safer approach to promoting skin-brightening and an even skin tone on all skin types. Alpha-Arbutin also minimizes liver spots and meetsall the requirements of a modern skin-brightening and skin depigmentation product. Recommended concentration: 0.2%when formulated with an exfoliant or penetration enhancer, otherwise up to 2%.INCI Name: Alpha-Arbutin.Antarcticine® Antarcticine® is a novel high molecular weight glycoprotein obtained via biotechnology from an Antarctic strain of bacteria. Antarcticine® has cryoprotective effects providing stability to proteins and lipid membranes exposed toextremes of cold or dryness. It has also been shown to reduce the roughness of skin. Recommended concentration: 3-5%.INCI Name: Pseudoalteromonas Ferment Extract.Apt® Apt® is bio-engineered from a selected strain of red marine algae (Ahnfeltia concinna). Apt® increases cell turnover, firmness, elasticity and smoothness and brings about fine line reduction. It also contributes excellent skin feel andmoisturization properties to creams, lotions, gels and serums.INCI Name: Butylene Glycol, Ahnfeltia Concinna Extract.Argireline® Argireline® offers superior anti-wrinkle efficacy. Studies show it reduces the depth of expression wrinkles, especially in the forehead and around the eyes. It is a safer, cheaper and milder alternative to Botulinum Toxin, topically targeting thesame wrinkle-formation mechanism in a very different way. Argireline® is believed to function through modulation of theSNARE complex formulation. Recommended concentration: 3-10%.INCI Name: Acetyl Hexapeptide-8.Artemisia AO Artemisia AO, from the rare plant White Genepi, is cultivated by 100% organic methods. It is often compared to Vitamin C, and has been tested positively regarding anti-oxidative properties. It protects the skin with anti-oxidant, radicalscavenging properties, in addition to acting as an anti-inflammatory and anti-bacterial. Formulation applications includeskin-protection, anti-aging, cleansing and sun-care preparations. In addition, it is effective for reducing spotty skin.Recommended concentration: 1-3%.INCI Name: Glycerin, Water, Artemisia Umbelliformis Extract, Sodium Benzoate,Potassium Sorbate.Buddleja AO Buddleja AO, cultivated by 100% organic methods, protects the skin cells from UV damage. It has the capacity to reduce the DNA fragmentation of the keratinocytes induced by UVB radiations from 39% to 71% depending on the concentrationused. It is DNA-protective and an anti-oxidant as well. It also acts to heal skin wounds, enhancing effectiveness by itsanti-bacterial properties. Applications include anti-aging formulations, skin repair/healing and cleansing creams andlotions. Recommended concentration: 1-3%.INCI Name: Glycerin, Water, Buddleja Davidii Extract, Sodium Benzoate, PotassiumSorbate.Calendula GC Calendula GC is largely used in the care of irritated and reddened skin, against sunburn, erythema and superficial burns thanks to its anti-inflammatory, healing, soothing, hydrating, decongesting and purifying properties. Recommendedconcentration: 3-5%.INCI Name: Calendula Officinalis Flower Extract.Calendula-ECO Calendula-ECO extract is an aromatic plant of which the rich extract is produced from the flower heads. It is truly multi-functional. Due to the presence of polysaccharides, flavonoids, triterpenes and carotenes, this ECO extract is highlyeffective in tissue regeneration action. The saponins and polysaccharides are useful in moisturizing and soothingapplications. The triterpenes are also used for sensitive and irritated skin. Calendula-ECO extract also helps to preventphoto aging. Recommended concentration: 0.5-5%.INCI Name: Glycerin, Water, Calendula Officinalis Flower Extract.Cephalipin® Cephalipin® is an aqueous solution of botanically-derived lipids isolated and purified from wheat grains. These lipids help restore and normalize the natural barrier function of the stratum corneum. Cephalipin® is an ideal addition to skintreatment products. It is also compatible for after-shaving specialties, with or without ethanol. Recommendedconcentration: 2%.INCI Name: Cephalins, Disodium Cocoamphodiacetate.Ceramide I Ceramide I is a composition of C-18 Phytosphingosine base coupled to an amide-linked long-chain omega-OH fatty acid.When topically applied, Phytoceramide I optimizes dermal water retention to effect smoother, glossier and softer skin. Itis particularly suitable for treating aged or extra dry skin. This class of ceramides is believed to function as a 'molecularrivet,' cross linking lamellar structures in the barrier layer of the stratum corneum. Recommended concentration:0.01-1%.INCI Name: Ceramide 1.Ceramide III Ceramide III is a Phytosphingosine backbone acylated with stearic acid having correct 3-D human identical structure.Topical application of Ceramide III has been shown to significantly increase skin water content, reduce roughness, restoredetergent-damaged skin and protect healthy skin against detergent-induced dermatitis. Recommended concentration:0.05-1.0%.INCI Name: Ceramide 3.Ceramide IIIA Ceramide IIIA is a Phytosphingosine based human identical skin lipid in combination with the EFA, linoleic acid. Ceramide IIIA has barrier repair properties. It has additionally been shown to inhibit melanin synthesis in vitro without cytotoxicitymaking it an interesting constituent in skin lightening formulations. Recommended concentration: 0.05-1.0%.INCI Name: Ceramide 3.Ceramide IIIB Ceramide IIIB is a Phytosphingosine backbone acylated with oleic acid. Similar to Ceramide III, Ceramide IIIB differs in that it is more soluble in cosmetic emollients such as isostearic acid and isocetyl alcohol. Ceramide IIIB is recommendedin situations where its higher solubility may be required. Recommended concentration: 0.05-1.0%.INCI Name: Ceramide 3.Ceramide VI Ceramide VI is a member of the Ceramide 6 family whose chemical name is alpha-OH-stearoylphytosphingosine. It contains a long chain AHA (Alpha Hydroxyacid) found naturally in the skin which contrasts with short chain AHA's currentlyin use in skin care preparations. Efficacy studies have shown Ceramide VI to have a potent ability to smooth rough skin viamodulation of desquamation. Recommended concentration: 0.05-1.0%.INCI Name: Ceramide 6 II.Cerasol Cerasol is a 3% solution of Ceramide 3A (N-Linoleoyl-Phytosphingosine) in hexyldeconal. These ceramides are solubilized and thus bioavailable, leading to regeneration of the lipidic barrier and reduction of transepidermal water loss (TEWL). 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This activity can help delay early skin aging characterized by wrinkle formation, reduced elasticity, skin dryness and age spots. Colhibin isan ideal component for anti-aging products and well suited for sun and environmental stress protection formulations.Recommended concentration: 2-5%.INCI Name: Hydrolyzed Rice Protein.Collagen Collagen is a 1% solution of natural soluble porcine collagen. It is one of the most important scleroproteins of the connective tissue occurring as fibres. Collagen is the main structure element of the skin and is therefore largelyresponsible for its characteristic properties. It provides excellent smoothing and hydrating qualities with superb skin feel.Recommended concentration: 3-5%.INCI Name: Soluble Collagen.Complex Gel Complex Gel is a proprietary gel specially formulated to stabilize either free Vitamin A (Retinol) or Vitamin C. Complex Gel was designed to supply in dual compartment packaging with the end user freshly activating the final product at the timeof first use.INCI Name: Water, Ascorbic Acid, Glycerin, Propylene Glycol, Stearic Acid,Dimethylsilanol Hyaluronate, Hydrolyzed Wheat Protein, Carrageenans, Agar,Hydroxyethyl Cellulose, Ferrum Chloride, Zinc Chloride, EDTA, BHT.Cosmoferm® Mix III Cosmoferm® Mix III is a ready-to-use solution of Ceramide III (oil-soluble). Ceramide III and Ceramide IIIB support the renewal of the skin's natural protective layer and form an effective barrier against moisture loss. Thesehuman-skin-identical molecules are particularly suitable for long term protection and repair of sensitive and dry skin.Recommended concentration: 0.25-5.0%.INCI Name: Ceramide 3, Isocetyl Alcohol, Cetyl Alcohol.Decorinyl™Decorinyl™ is a synthesized tetrapeptide whic h mimics the sequences of decorin that bind to collagen fibrils. It has been shown to regulate fibrillogenesis and control fibril growth, improving firmness and elasticity of the skin, ensuringuniformity of fibril diameter and spacing of collagen fibrils. In effect, it replaces the non-functional decorin encountered asthe skin ages, and also sustains a superior moisture profile. Decorinyl™ has been incorporated into a liposomal system forenhanced penetration, stability and efficacy. It may be used in emulsions, gels and other formulations. Recommendedconcentration: 5%.INCI Name: Water (Aqua), Lecithin, Tipeptide-10 Citrulline, Carbomer,Triethanolamine, Phenoxyethanol, Caprylyl Glycol.DHA Film Fluid DHA (Dihydroxyacetone) Film Fluid is one of the widest used actives for sunless or self-tanning treatments. A film fluid, to stabilize DHA and enhance functionality, is created to result in a submicrodispersion, which is a stable mono-layerMolecular Film with high moisturizing effect, stabilized due to interaction with polymeric polysaccharides. The resultanttanning applications are without risks of skin cancer and aging and form a protective moisture film. DHA Film Fluid is easilyincorporated into O/W, W/O emulsions or gels. Recommended concentration: 3-5%.INCI Name: Dihydroxyacetone, Water (Aqua), Xanthan Gum, Citric Acid, SodiumHydroxide, Phenoxyethanol.Dismutin®-BT Dismutin®-BT is an aqueous solution of biotechnologically produced, highly purified superoxide dismutase from a natural yeast strain of Saccharomyces cerevisiae. It protects the skin from oxidative stress by restoring the anti-oxidant balancein the outer skin layer, thereby helping to delay early skin aging. Recommended concentration: 0.2-0.5%.INCI Name: Superoxide Dismutase.Eashave Eashave is a biopolymer complex composed of plant and biotechnologically derived materials. Eashave has proven moisturizing and soothing efficacy (supported by thermosgraphs) to skin damaged by shaving. Enhanced regeneration ofprotective lipids has also been shown.Recommended concentration: 2-5%.INCI Name: Triticum Vulgare (Wheat) Germ Extract, Saccharomyces CerevisiaeExtract, Sodium Hyaluronate.Echinacea GC Echinacia GC is a perennial, cultivated in the Swiss Alps under the highest standards of agronomic practice. A profusion of Caffeic Acid Derivatives protects skin against UV radiation damage by inhibiting degradation of Type III Collagen. ThePolysaccharide component prevents moisture loss by inhibiting Hyaluronidase enzyme actions. Essential Oils purify anddetoxify the skin and complete the smoothing, healing and firming properties of this botanical. Recommendedconcentration: 3-5%.INCI Name: Echinacea Purpura Extract.Edelweiss GC® Edelweiss GC® is a botanical extract derived from organically cultivated Edelweiss flowers grown at high altitudes in the Swiss Alps. There is environmental impact on wild strands of the rare protected plant. Plant derived glycerin is used as thecarrier which provides a well defined HPLC. Information regarding free radical scavenging, enzyme inhibitory propertiesand soothing benefits are available. Recommended concentration: 3-5%.INCI Name:Leontopodium Alpinum Extract.Edelweiss SP Edelweiss SP, an Alpine Extract, has high performing anti-oxidant and radical scavenging properties. Phenolic acids and tannins lead to increased type III collagen protection. Other compounds inhibit hyaluronidase and have anti-inflammatoryeffects, making Edelweiss SP ideal for sensitive skin in anti-aging and soothing preparations. Recommendedconcentration: 3-5%.INCI Name: Leontopodium Alpinum Extract.Elhibin® Elhibin® is a plant-derived treatment specialty which specifically inhibits skin enzymes (human leukocyte, elastase and tryptase) to minimize damage to skin from their unscheduled release. It has been clinically shown to have a soothingbenefit to skin and to improve firmness with regular use. Elhibin® normalizes the skin's enzyme/inhibitor balance. It iswell suited for anti-aging products. Recommended concentration: 3-7%.INCI Name: Glycine Soja(Soybean) Protein.Erythrulose Erythrulose, easily formulated as a high performance self-tanning product, is a natural keto-sugar, which reacts with free primary or secondary amino acid groups to form the polymer, Melanoidin. For optimal results Erythrulose is used incombination with DHA (Dihydroxyacetone). This combination is World Wide Patent Protected. It achieves a natural,long-lasting, non-streaking radiant glow, be it January or July. The skin is protected from UV Radiation, and remains moistand supple. Recommended concentrations for self-tanning products: 1-3% of Erythrulose should be combined with 2-4%DHA.INCI Name: Erythrulose.Eyeseryl® Eyeseryl® is an acetyl tetrapeptide with anti-oedema properties and a proven efficacy in reducing puffy eyebags. It also enhances skin elasticity, skin smoothness and shows a decongesting effect. Eyeseryl® can be incorporated in cosmeticformulations such as emulsions, gels, sera, etc., where a reduction of puffiness under the eye is desired. Recommendedconcentration: 1-10%.INCI Name: Water (Aqua), Acetyl Tetrapeptide-5.Fitobroside™Fitobroside™ is a lipid dispersion derived from botanical sources. It replaces key st ratum corneum lipids (glycolipids, phospholipids and sterols), helping to restore the skin's barrier function as well as providing moisturizing benefits.Recommended concentration: 3-6%.INCI Name: Triticum Vulgare (Wheat) Germ Extract.Fitoderm®: Pure, Botanically Derived Squalane... Fitoderm® is pure, botanically derived squalane, produced by the total hydrogenation of the squalene source in olive oil. It is a non-animal alternative to shark-derived squalane. Fitoderm® is highly resistant to oxidation, has a high affinity to skin and its natural lipids and is neither irritating nor allergenic. It is considered a specialty emollient and is soluble in a vast array of cosmetic solvents. Fitoderm® is perfect for moisturizing milks, creams and lotions, massage oils and a carrier for fragrance compounds.INCI Name: Squalane.Gigawhite™Gigawhite™ is a skin brightening complex of botanical origin, which was developed with the help of 'in-vitro' assays, human cell cultures and a skin model with barrier effect. Gigawhite™ at 5% has been proven to have a significant skinbrightening effect with regular topical use. It was additionally shown to reduce the color and size of age spots in a twelveweek 'in-vivo' study. Gigawhite™ is appropriate in topical emulsions, gels and aserums. Recommended concentration:3-5%.INCI Name: Malva Sylvestris (Mallow) Extract, Mentha Piperita (Peppermint) LeafExtract, Primula Veris Extract, Alchemilla Vulgaris Extract, Veronica OfficinalisExtract, Melissa Officinalis Leaf Extract, Achillea Millefolium Extract.Ginger-ECO Ginger-ECO, one of the best known botanicals since ancient times, has excellent anti-aging properties due to its ability to almost totally inhibit collagen degradation by collagenase enzyme inhibition. It allows the maintenance of protein levels inthe dermis and skin flexibility. It also acts as an anti-oxidant and anti-inflammatory. Ginger-ECO is used in formulationsfor skin and hair products, including refirming, massage and anti-dandruff preparations. Recommended concentration is0.1-2%.INCI Name: Glycerin, Water, Zingiber Officinate (Ginger) Root Extract.Ginkgo-ECO Ginkgo-ECO extract is produced from the leaves of the Ginkgo Biloba tree, and is multi-functional. The basis for its cosmetic benefits are two groups of compounds: Flavonoids and terpenes. The flavonoids indicate several properties:Antioxidant, anti-inflammatory, vessel protection, lipolysis stimulation and cell regeneration. Ginkgolides, the terpenes,have been demonstated to reduce platelet aggregation, allergic reactions and general inflammatory responses due to theirPAF antagonistic activity. These two groups promote anti-cellulite, anti-aging, photo-protection, re-epithelizing and othercosmetic advantages in formulation. Recommended concentration: 0.1-3%.INCI Name: Glycerin, Water, Ginkgo Biloba Leaf Extract.Glucare™ S 2%Glucare™ S 2% is an aqueous solution containing 2% of carboxymethylated derivative of 1, 3-Beta Glucans from yeast.The degree of carboxymethylation is carefully controlled to achieve good solubility while maintaining optimal biologicalactivity. Studies demonstrating soothing and immunoprotective effects are available. Glucare™ S 2% also has anexcellent silky skin feel. Recommended concentration: 2.5-10%.INCI Name: Sodium Carboxymethyl Betaglucan.Glucare™ S Powder Glucare™ S Powder is a form of Glucare™ S (2%). The degree of carboxymethylation is carefully controlled to achieve good solubility with optimal biological activity. Studies demonstrating soothing and immunoprotective effects areavailable. Recommended concentration: 0.05-0.40%.INCI Name:Sodium Carboxymethyl Betaglucan.Green Tea-ECO Green Tea-ECO is an active of diverse advantages, primarily due to the content of Polyphenols and Methylxanthines. The Polyphenols are responsible for the anti-oxidant and anti-inflammatory effects. In addition, they have the ability to inhibitcollagenase, the enzyme causing degradation of collagen and the vascular endothelium. The Methylxanthines promotevasodilator response, and prevent the lipid accumulation into the adipocytes. Green Tea-ECO also protects against UVBdamage. Applications include anti-cellulite treatments, sun-protectors, anti-wrinkling formulations, irritated and sensitiveskin products, and hair care products that improve strength and gloss. Recommended concentration: 0.1-2%.INCI Name: Camellia Sinensis.Happy Skin Happy Skin is a novel, natural active ingredient, derived from the Roseroot plant. It demonstrates the important dynamic interactions between the skin and the nervous system. Because of the stimulation of neuro-peptide production, and its antioxidantproperties, Happy Skin promotes inner wellness, relaxation and stress reduction that becomes manifest in the external appearanceof the body and facial skin and hair. Recommended concentration: 0.2-2%.INCI Name: Glycerin, Water (Aqua), Rhodiola Rosea Root Extract.Hibiscus-ECO The flowers of this plant have anti-oxidant properties and inhibit elastin degradation, resulting in maintaining the skin's elasticity and consequent anti-aging effects. It is useful for formulations targeting skin tone, anti-cellulite and refirmingproducts. Recommended concentration: 0.1-4%.INCI Name: Hibiscus Sabdariffa.Hispagel® 100 Hispagel® 100 is a hydrated polymeric clathrate containing vegetally-derived glycerin that is readily soluble in water, forming permeable films on the skin with excellent moisture retention and lubricating properties. It has excellentthickening and stabilizing properties for use in all types of personal care products.INCI Name: Glycerin, Glyceryl Polyacrylate.Hispagel® 200 Hispagel® 200 is a hydrated polymeric clathrate containing vegetally-derived glycerin that is readily soluble in water, forming permeable films on the skin with excellent moisture retention and lubricating properties. Excellent thickening andstabilizing properties for use in all types of personal care products.INCI Name: Glycerin, Glyceryl Polyacrylate.Hispagel® Oil LV Hispagel® Oil LV is a new, low viscosity hydrogel developed for topical use with an excellent skin feel and moisturizing properties. Hispagel® Oil is an oil-free hydrophilic system that delivers lubricity and emolliency in topical formulations.The LV form has a viscosity range of 500-1200 cps.INCI Name: Glycerin, Glyceryl Polyacrylate.Homeostatine® Homeostatine® is an active complex of two natural botanical ingredients, which through an innovative process, prevent and reduce wrinkles by recovering and maintaining homeostasis in the dermal extra-cellular matrix. Homeostatine®increases the production of dermal collagen and other ECM components in fibroblasts. It inhibits the synthesis ofMetalloproteinases (MMP) as well as the synthesis of pro-inflammatory mediators. The result is less wrinkled, more elasticand firmer skin. Recommended concentrations: 2-3% for early wrinkles and 2-5% for aged skin.INCI Name:Enteromorpha Compressa Extract, Caesalpinia Spinosa Gum.Houseleek-ECO Houseleek-ECO is an extract produced from the aerial part of Houseleek that contains multi-functional benefits for cosmetic uses. Astringent activity is due to the tannin content-regulating sebum secretions and promoting tissueregeneration, especially on irritated skin. Antioxidant and lipid level-decreasing properties help guard against damagingoxidative processes to skin and hair. Antimicrobial activity of phenols in Houseleek inhibits growth of microorganisms,enabling formulations for purifying and antiseptic products. The mucilage and organic acids found in the extract regulateTEWL and are especially gentle for sensitive skin. Recommended concentration: 0.1-3%.INCI Name: Glycerin, Water, Sempervivum Tectorum Extract.HSC Hydrophobic Sphingolipid Complex (HSC) contains ceramides, incorporated into an organo-compatible silicone/emollientmatrix. This matrix includes cyclomethicone, dimethiconol, phenyl trimethicone, octyl cocoate and phytosterols. HSC helps restore, replenish and restructure lipids within the stratum corneum. Extraordinary skin feel. Recommended concentration: 5-10%.INCI Name: Cyclopentasiloxane, Cyclotetrasiloxane, Dimethiconol, Dimethicone, Ethtylhexyl Cocoate, Phenyl Trimethicone, Lecithin, Glycolipids.Hyaluronic Acid Powder-BT Hyaluronic Acid Powder-BT has water storing properties and is an ideal swelling agent and lubricant, enabling its incorporation into cosmetics leading to a perceptible and visible improvement of skin condition. It forms a thin transparent visco elastic surface film. The film helps to preserve characteristics of youthful and healthy skin, suppleness, elasticity and tone. Recommended concentration: 0.01-1%.INCI Name: Sodium Hyaluronate.Hyaluronic Acid Solution-BT (Hyasol®-BT) Hyaluronic Acid Solution-BT (Hyasol®-BT) is a 1% solution of Hyaluronic Acid-BT. This naturally-derived (biofermentation) material is produced with special attention to maintaining its high relative molecular weight. Its excellent purity is assured though an innovative biofermentation process. Recommended concentration: 3-5%. INCI Name: Sodium Hyaluronate.Hydrolastan® Hydrolastan® is a natural scleroprotein and aqueous solution containing 10% of high molecular weight elastin hydrolysate. With high affinity to skin and hair, Hydrolastan® provides a natural protein layer of protection againstenvironmental factors. It has excellent smoothing properties and substantivity to skin. Recommended concentration:5-10%.INCI Name: Hydrolyzed Elastin.Hydromanil Hydromanil is a unique, three dimensional glycol-matrix delivery system that releases moisturizing molecules sequentially into the stratum corneum, resulting in a highly significant improvement of both immediate and long term skinhydration. Solubilized in water and propylene glycol. Recommended concentration: 2-10%.INCI Name: Propylene Glycol, Glycerin, Caesalpinia Spinosa Oligosaccharides,Caesalpinia Spinosa Gum (Pending).Hydromanil H.GL Hydromanil H.GL is a unique, three dimensional glycol-matrix delivery system that releases moisturizing molecules sequentially into the stratum corneum, resulting in a highly significant improvement of both immediate and long term skinhydration. Solubilized in water and glycerin. Recommended concentration: 2-10%.INCI Name: Glycerin, Hydrolyzed Caesalpinia Spinosa Gum, Caesalpinia SpinosaGum (Pending).Hypericum PG Hypericum PG extract is widely used for irritated and reddened skin, against sunburn, erythema and superficial burns.Hypericum PG is therefore well suited for after-sun agents as well as for healing products and acne-prone skin.Recommended concentration: 3-5%.INCI Name: Hypericum Perforatum Extract.Hyssopus AO The Hyssop plant is organically cultivated in the Alps for its diverse skincare and hair care properties. Its special acids, tannins, flavenoids and essential oils give this ingredient a range of functionalities. It may be used for products targetingacne prone skin, cleaning formulations requiring anti-bacterial properties, and anti-oxidant skincare protection, as well asrinse-off bath/shower and shampoo formulations. Recommended concentration: 1-3%.INCI Name: Glycerin, Water, Hyssopus Officinalis Extract, Sodium Benzoate, PotassiumSorbate.Immucell® Immucell® is a biotechnologically-derived fraction which is rich in oligopeptides. Immucell® is highly stimulatory to cell respiration. It has been shown to boost the immune system of skin and increase cell turnover. Recommendedconcentration: 2-6%.INCI Name: Glycoproteins.Imperatoria AO Imperatoria AO is cultivated by Alpaflor using 100% organic methods in the Alps. The Masterwort plant extract is composed of beneficial flavenoids, phenolic acids, tannins, sugars and essential oils. Its properties includere-epithelialization, anti-inflammatory functions, moisturization, antioxidant radical scavenging and astringency. It can beapplied in formulations for after-sun preparations, stretch mark treatments and skin repair products. Recommendedconcentration is 1-3%.INCI Name: Glycerin, Water, Peucedanum Osthruthium Leaf Extract, Sodium Benzoate,Potassium Sorbate.Ion-Moist 4Men Ion-Moist 4Men is a molecular film, specifically formulated for men, that provides natural hydration due to its NMF ingredient content (amino acids, sugars, lactate, urea, PCA and several ions). Ion-Moist 4Men restores the levels ofmoisture after shaving and preserves the natural barrier function of the epidermis. It stimulates moisture retention in thestratum corneum and enhances smoothness of the skin. Men tend to have more collagen than women, but less hydration.This unique ingredient can be used in O/W emulsions, body milks, sera, aftershave gels and other preparations. At least3-10% of the ingredient should be added to the final formulation.INCI Name: Water (Aqua), Glycerin, Calcium Pantothenate, Xanthan Gum, Urea,Caprylyl Glycol, Magnesium Chloride, Postassium Chloride, Postassium Lactate,Magnesium Lactate, Sodium Citrate, Glucose, Citric Acid, Ethylhexylglycerin.Iricalmin Iricalmin is a plant-derived, multi-functional biopolymer complex for use in skin care products. Iricalmin helps regenerate the protective lipid layer of damaged skin while delivering moisturization. It has also been shown to have soothingbenefits. Iricalmin is suitable for after-sun, after-depilation and after-shave products. Recommended concentration:2-5%.INCI Name: Triticum Vulgare (Wheat) Germ Extract, Saccharomyces CerevisiaeExtract, Sodium Hyaluronate.Lactomide Lactomide is an aqueous liposomal dispersion containing a naturally derived ceramide (ceramide-3) as well as phosphatidylethanolamine and phosphatidylcholine (milk lipids). The regenerating and moisture-regulating effect ofLactomide is due to the fact that its active substances are central components of the epidermal permeability barrier.Recommended concentration: 2-5%.INCI Name: Milk Lipids, Ceramide 3.Legactif Legactif is a complex of plant active ingredients. It is rich in flavonoids, such as rutin, hesperidin and naringin. Steroidal saponins are also major components for personal care applications. Legactif aims at relieving 'tired leg syndrome' and legheaviness, which affects over 1/2 of the adult population. Its cosmetic properties include blood circulation stimulation and。

Salinomycin_53003-10-4_DataSheet_MedChemExpress

P d D Sh Product Name:Salinomycin CAS No.:53003-10-4Cat. No.:HY-15597Product Data SheetMWt:751.00Formula:C42H70O11Purity :>98%Solubility:DMSOMechanisms:Biological Activity:Salinomycin is an antibacterial and coccidiostat ionophore therapeutic drug.Pathways:Anti-infection; Target:Antibacterial y p p gIC50 Value:Target: Antibacterial; Anticancer Salinomycin, a polyether ionophore antibiotic isolated from Streptomyces albus, has been shown to kill CSCs in different types of human cancers, most likely by interfering with ABC drug transporters,the Wnt/β-catenin signaling pathway, and other CSC pathways[1].in vitro: Salinomycin acts as a potassium ionophore and thereby interferes with transmembrane potassium potential, leading to mitochondrial and cellular potassium efflux. low concentrations of Salinomycin of 1 μM and 2 μM resulted in haggard and less confluent grown cells, increasing t ti f S li i f 5M d 10M lt d i l b l d d f t d ll l References:[1]. Naujokat C, Steinhart R. Salinomycin as a drug for targeting human cancer stem cells. J BiomedBiotechnol. 2012;2012:950658.[2]Thorsten Lieke Wolf Ramackers Sabine Bergmann Impact of Salinomycin on human concentrations of Salinomycin of 5 μM and 10 μM resulted in a globular and defragmented cellular phenotype[2]. Treatment of 10.2 uM Sal for 48 h caused G0/G1 cell cycle arrest in HepG2 cells,whereas 15.3 uM...[2]. Thorsten Lieke, Wolf Ramackers, Sabine Bergmann, Impact of Salinomycin on humancholangiocarcinoma: induction of apoptosis and impairment of tumor cell proliferation in vitro. BMCCancer. 2012; 12: 466.[3]. Fan Wang, Lei He, Wei-Qi Dai, Salinomycin Inhibits Proliferation and Induces Apoptosis ofHuman Hepatocellular Carcinoma Cells In Vitro and In Vivo. PLoS One. 2012; 7(12): e50638.Caution: Not fully tested. For research purposes onlyMedchemexpress LLC18 W i l k i n s o n W a y , P r i n c e t o n , N J 08540,U S AE m a i l : i n f o @m e d c h e m e x p r e s s .c o m W e b : w w w .m e d c h e m e x p r e s s .c om。

乳鼠原代心肌细胞的英语

乳鼠原代心肌细胞的英语英文回答：Neonatal Rat Primary Cardiomyocytes Isolation and Culture.Neonatal rat primary cardiomyocytes (NRCMs) areisolated from the hearts of newborn rats and cultured in vitro as a model system for studying cardiac biology and function. These cells are highly differentiated and exhibit many of the characteristics of adult cardiomyocytes, including the ability to contract spontaneously and respond to pharmacological agents. NRCMs have been used extensively in research to investigate a variety of cardiovascular diseases, including heart failure, arrhythmias, and ischemic injury.Isolation of NRCMs.NRCMs are typically isolated from 1to 3-day-oldSprague-Dawley rats. The rats are euthanized and the hearts are removed and placed in sterile phosphate-buffered saline (PBS). The hearts are then minced into small pieces and digested with a collagenase solution. The resulting cell suspension is filtered and centrifuged to separate the cardiomyocytes from other cell types.Culture of NRCMs.The isolated NRCMs are resuspended in a culture medium supplemented with serum and antibiotics and plated onto culture dishes. The cells are allowed to adhere to the dishes for 24 hours, after which the medium is replaced with a serum-free medium. NRCMs can be cultured for up to 4 weeks, although they typically begin to lose their differentiated characteristics after 2-3 weeks.Characterization of NRCMs.NRCMs can be characterized by their morphology, electrophysiological properties, and contractile function. Morphologically, NRCMs are polygonal in shape and have acentral nucleus. They exhibit spontaneous contractions and respond to electrical stimulation. NRCMs express a varietyof cardiac-specific proteins, including sarcomeric proteins, ion channels, and calcium-handling proteins.Applications of NRCMs.NRCMs have been used in a wide range of research applications, including:Investigation of cardiac diseases: NRCMs have beenused to study the mechanisms underlying a variety of cardiovascular diseases, including heart failure, arrhythmias, and ischemic injury.Development of new drugs: NRCMs have been used to screen for new drugs that may be effective in treating cardiovascular diseases.Toxicology testing: NRCMs have been used to test the toxicity of new drugs and chemicals.Gene therapy: NRCMs have been used to study the effects of gene therapy on cardiac function.Advantages of NRCMs.NRCMs offer a number of advantages over other cell types for studying cardiac biology and function. These advantages include:High degree of differentiation: NRCMs are highly differentiated and exhibit many of the characteristics of adult cardiomyocytes.Spontaneous contractility: NRCMs exhibit spontaneous contractions, which makes it possible to study cardiac function without the use of electrical stimulation.Response to pharmacological agents: NRCMs respond to pharmacological agents in a manner similar to adult cardiomyocytes, which makes them a good model system for studying the effects of drugs on cardiac function.Easy to isolate and culture: NRCMs are relatively easy to isolate and culture, which makes them a cost-effective and convenient model system.Disadvantages of NRCMs.NRCMs also have some disadvantages, including:Limited lifespan: NRCMs can only be cultured for up to 4 weeks, which limits their use for long-term studies.Loss of differentiated characteristics: NRCMs begin to lose their differentiated characteristics after 2-3 weeks in culture, which limits their use for studying chronic cardiac diseases.Variability between preparations: The isolation and culture conditions can affect the properties of NRCMs, which can lead to variability between preparations.中文回答：新生大鼠原代心肌细胞——分离与培养。

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New Cytotoxic Salinosporamides from the Marine ActinomyceteSalinispora tropicaPhilip G.Williams,Greg O.Buchanan,Robert H.Feling,Christopher A.Kauffman,Paul R.Jensen,and William Fenical*Center for Marine Biotechnology and Biomedicine,Scripps Institution of Oceanography,University ofCalifornia -San Diego,La Jolla,California 92093-0204wfenical@Received March 14,2005An extensive study of the secondary metabolites produced by the obligate marine actinomycete Salinispora tropica (strain CNB-392),the producing microbe of the potent proteasome inhibitor salinosporamide A (1),has led to the isolation of seven related γ-lactams.The most important of these compounds were salinosporamide B (3),which is the deschloro-analogue of 1,and salino-sporamide C (4),which is a decarboxylated pyrrole analogue.New SAR data for all eight compounds,derived from extensive testing against the human colon carcinoma HCT-116and the 60-cell-line panel at the NCI,indicate that the chloroethyl moiety plays a major role in the enhanced activity of 1.Bacteria belonging to the order Actinomycetales (com-monly called actinomycetes)are one of the most prolific resources for lead compounds in the development of new pharmaceuticals.1Unfortunately,after more then 50years of intense scrutiny,the rate that new biologically active metabolites are being discovered from terrestrial actinomycetes has been steadily diminishing.2If these chemically prolific microorganisms are to continue to provide new structures that are of medicinal relevance,then new strategies that lead to the isolation of geneti-cally novel strains must be found.Investigations target-ing actinomycetes have traditionally focused on soil-derived strains,3but recent evidence has unequivocally shown that the marine environment is host to a wide range of taxonomically diverse actinomycetes.4In 1991,we cultivated an unusual group of actino-mycetes from marine sediments.5Further examination showed that this group was unique among the actino-mycetes,as all members required seawater for growth.Phylogenetic characterization revealed that these strains represented a new genus for which the name “Salino-spora”was originally proposed.6This name was subse-quently changed to Salinispora in the formal taxonomic description.7Examination of more than 120distinct Salinispora strains from the two currently recognized species S.tropica and S.arenicola 7showed that greater than 80%of these organisms produced culture extracts that inhibited the in vitro growth of human colon carcinoma HCT-116.Examination of the culture broth of S.tropica strain CNB-392led to the isolation of salinosporamide A (1),which has an unusual fused γ-lactam- -lactone ring structure.8,9Salinosporamide A (1)is structurally related to clasto-lactacystin -lactone (2,omuralide),10a trans-(1)Berdy,J.J.Antibiot.2005,58,1-26.(2)Bull,A.T.;Ward,A.C.;Goodfellow,M.Microbiol.Mol.Bol.Rev.2000,64,573-604.(3)Okami,Y.;Hotta,K.In Actinomycetes in Biotechnology ;Good-fellow,M.,Williams,S.T.,Mordarski,M.;Academic Press:New York,1988;pp 33-67.(4)(a)Jensen,P.R.;Gontang,E.;Mafnas,C.;Mincer,T.J.;Fenical,W.Environ.Microbiol .2005,7,1039-1048.(b)Maldonado,L.A.;Stach,J.E.M.;Pathom-aree,W.;Ward,A.C.;Bull,A.T.;Goodfellow,M.Antonie van Leeuwenhoek 2005,87,11-18.(c)Montalvo,N.F.;Mohamed,N.M.;Enticknap,J.J.;Hill,R.T.Antonie van Leeuwenhoek 2005,87,27-36.(5)Jensen,P.R.;Dwight,R.;Fenical,W.Appl.Environ.Microbiol .1991,57,1102-1108.(6)Mincer,T.J.;Jensen,P.R.;Kauffman,C.A.;Fenical,W.Appl.Environ.Microbiol .2002,68,5005-5011.(7)Maldonado,L.A.;Fenical,W.;Jensen,P.R.;Kauffman,C.A.;Mincer,T.J.;Ward,A.C.;Bull,A.T.;Goodfellow,M.Int.J.Syst.Evol.Micr.,published on-line April 15,2005..Chem.2005,70,6196-620310.1021/jo050511+CCC:$30.25©2005American Chemical SocietyPublished on Web 07/01/2005formation product of the microbial metabolite lactacys-tin.11Compounds 1and 2are unique because they specifically inhibit theproteolytic activity of the 20S subunit of the proteasome without affecting any other protease activity.The proteasome is a multicatalytic complex that regulates intracellular protein degradation through three distinct proteolytic activities (chymo-trypsin-like,trypsin-like,and caspase-like).12One protein that is regulated by the proteasome is the transcription factor NF-κB.12This transcription factor promotes cell survival by regulating genes encoding cell-adhesion molecules,proinflammatory cytokines,and antiapoptotic proteins.13NF-κB is constitutively active in many ma-lignancies,including multiple myeloma (MM),and in-terfering with its activity through the use of proteasome inhibitors is the basis of the FDA-approved antitumor drug Velcade.14Interestingly,salinosporamide A is not only a nanomolar inhibitor of the 20S subunit of the proteasome but also active against Velcade-resistant multiple myeloma cells.15This combination of potent biological activity and structural novelty has attracted considerable interest in the synthetic community,which has culminated in the synthesis of 116and 2.17It has also prompted a closer examination of the extract of the culture broth of S.tropica strain CNB-392.We reporthere the results of that study which has led to the isolation of salinosporamide B (3)and C (4),along with five other related compounds,and the evaluation of their biological activities.Salinosporamide B (3),which was obtained from ethyl acetate as amorphous crystals,was the second most abundant component in the culture extract.High-resolu-tion mass spectral analysis suggested a molecular for-mula of C 15H 20NO 4,which was in accord with the structural information provided by the 13C NMR spec-trum (Table 1).The IR data of 3exhibited absorptions at 1700and 1820cm -1indicative of amide and -lactone functionalities,respectively,suggesting that 3was a structural analogue of 1.This was supported by the 1H(8)Feling,R.H.;Buchanan,G.O.;Mincer,T.J.;Kauffman,C.A.;Jensen,P.R.;Fenical,W.Angew.Chem .,Int.Ed.2003,42,355-357.(9)Recently,compounds analogous to 1that lacked the chlorine substituent were reported from a terrestrial actinomycete of the genus Streptomyces .Stadler,M.;Seip,S.;Mueller,H.;Mayer-Bartschmid,A.;Bruening,M.-A.;Benet-Buchholz,J.;Togame,H.;Dodo,R.;Reinemer,P.;Bacon,K.;Fuchikami,K.;Matsukawa,S.;Urbahns,K.U.S.Patent 2004071382,2004.(10)(a)Dick,L.R.;Cruikshank,A.A.;Grenier,L.;Melandri,F.D.;Nunes,S.L.;Stein,R.L.J.Biol.Chem .1996,271,7273-7276.(b)Corey,E.J.;Li,W.-D.Z.Chem.Pharm.Bull .1999,47,1-10and references therein.(11)Omura,S.;Fujimoto,T.;Otoguro,K.;Matsuzaki,K.;Moriguchi,R.;Tanaka,H.;Sasaki,Y.J.Antibiot.1991,44,113-116and references therein.(12)Adams,J.Proteasome Inhibitors in Cancer Therapy.Humana Press:Totowa,New Jersey,2004.(13)Wang,C.Y.;Mayo,M.W.;Korneluk,R.G.;Goeddel,D.V.;Baldwin,A.S.,Jr.Science 1998,281,1680-1683.(14)Richardson,P.G.;Barogie, B.;Berenson,J.;Singhal,S.;Jagannath,S.;Irwin,D.;Rajkumar,S.V.;Srkalovic,G.;Alsina,M.;Alexanian,R.;Siegel,D.;Orlowski,R.Z.;Kuter,D.;Limentani,S.A.;Lee,S.;Hideshima,T.;Esseltine,D.L.;Kauffman,M.;Adams,J.;Schenkein,D.P.;Anderson,K.C.N.Engl.J.Med.2003,348,2609-2617.(15)Chauhan,D.;Catley,L.;Li,G.;Podar,K.;Hideshima,T.;Mitsiades,C.;Ovaa,H.;Berkers,C.;Munshi,N.;Chao,T.;Nicholson,B.;Neuteboom,S.T.;Richardson,P.;Palladino,M.;Anderson,K.C.Cancer Cell ,submitted.(16)(a)Reddy,L.R.;Saravanan,P.;Corey,E.J.J.Am.Chem.Soc.2004,126,6230-6231.(b)Endo,A.;Danishefsky,S.J.J.Am.Chem.Soc.2005,127,8298-8299.(c)Reddy,L.R.;Fournier,J.-F.;Reddy,B.V.S.;Corey,.Lett.2005,7,2699-2701.(17)(a)Crane,S.N.;Corey,.Lett.2001,3,1395-1397.(b)Corey,E.J.;Reichard,G.A.;Kania,R.Tetrahedron Lett.1993,34,6977-80.(c)Nagamitsu,T.;Sunazuka,T.;Tanaka,H.;Omura,S.;Sprengeler,P.A.;Smith,A.B.,III J.Am.Chem.Soc.1996,118,3584-90.(d)Corey,E.J.;Li,W.;Reichard,G.A.J.Am.Chem.Soc.1998,120,2230-2236.(e)Corey,E.J.;Li,W.;Nagamitsu,T.Angew.Chem.,Int.Ed.1998,37,1676-1679.(f)Corey,E.J.;Li,W.Tetrahedron Lett.1998,39,8043-8046.(g)Corey,E.J.;Li,W.Chem.Pharm.Bull.1999,47,1-10.(h)Panek,J.S.;Masse,C.E Angew.Chem.,Int.Ed.1999,38,1093-1095.(i)Soucy,F.;Grenier,L.;Behnke,M.L.;Destree,A.T.;McCormack,T.A.;Adams,J.;Plamondon,L.J.Am.Chem.Soc.1999,121,9967-9976.(j)Crane,S.N.;Corey,E.J.J .Org.Chem.2003,68,2760-2764.(k)Brennan,C.J.;Pattenden,G.;Rescourio,G.Tetrahedron Lett.2003,44,8757-8760.(l)Ooi,H.;Ishibashi,N.;Iwabuchi,Y.;Ishihara,J.;Hatakeyama,.Chem.2004,69,7765-7768.(m)Corey,E.J.;Li,W.Tetrahedron Lett.1998,39,7475-7478.(n)Reddy,L.R.;Saravanan,P.;Fournier,J.-F.;Reddy,B.V.S.;Corey,.Lett.2005,7,2703-2705.T ABLE 1.NMR Spectral Data for 1and 3at 300MHzC/H 1δH a multiplicity(J in Hz)1δCb3δH a multiplicity(J in Hz)3δC b 1176.4,C 176.9,C 2 3.17,t (7.1)46.2,CH 2.72,dd (8.8,5.8)49.8,CH 386.1,C 86.3,C 480.2,C 79.3,C 5 4.24,d (9.2)70.9,CH 4.24,t (8.8)70.4,CH 6 2.85,br m 39.2,CH 2.90,m38.7,CH 7 6.42,d (10.1)128.4,CH 6.44,d (10.3)128.2,CH 8 5.88,m 128.8,CH 5.88,m 128.4,CH 9 1.91,m 25.3,CH 2 1.92,m 24.7,CH 210a 1.38,m 21.7,CH 2 1.39,m 21.1,CH 210b 1.66,m 1.72,m 11a 1.66,m 26.5,CH 2 1.72,m 25.9,CH 211b 2.37,m 2.32,m 12a 2.32,m 29.0,CH 2 1.90,m 18.5,CH 212b 2.48,m 2.14,m 13a 4.01,m 43.2,CH 2 1.21,t (7.3)12.3,CH 213b 4.14,m 14 2.07,s 20.0,CH 3 2.05,s 20.2,CH 315169.0,C169.1,CNH 10.60,br s 10.42,br s OH7.60,br s7.49,d (8.8)aRecorded in C 5D 5N.b Number of attached protons determined by DEPT experiments.New Cytotoxic Salinosporamides.Chem ,Vol .70,No .16,20056197NMR spectrum of3that displayed most of the diagnostic resonances present in1,including an isolated methyl group(δH2.05,s),a cis-alkene(δH6.44,d and5.88,m), and an amide proton signal(δH10.42,br s).There were noticeable differences,though,in the1H NMR spectrum of3,which included an upfield shift of the H-12meth-ylene proton signal and the presence of a methyl triplet (δH1.21,t).All this information,including differences in the molecular formulae for1and3,suggested that3was an analogue of1that lacked the chlorine substituent. This was confirmed by analysis of the gCOSY and gHMQC data(Table S1,Supporting Information)which revealed all the spin systems in3and established the planar structure of3as13-deschlorosalinosporamide A. The relative stereochemistry of3was determined to be identical to that of1on the basis of analysis of the NOESY spectral data and the proton-proton coupling constants.Specifically,enhancements between H-14and H-5and from H-14to H-2established their syn relation-ship and thus the relative stereochemistry around the bicyclic ring,while the similarity of the proton-proton coupling constant between H-5and H-6(1;3J5H,6H)9.2, 2;3J5H,6H)8.8)suggests the same configuration at thesecenters.Finally,since the literature18suggests that the replacement of a halide by a hydrogen atom,if it is distant from a chiral center,has only a small effect(10-20%)on the magnitude of the optical rotation,and since both1and3are levorotatory{1;[R]D25-72.9°(c0.55, MeOH);2[R]D25-54.5°(c0.286,MeOH)},the latter has been assigned the same absolute configuration (2R,3S,4R,5S,6S)as was determined by X-ray analysis of1.8,19,20Examination of the other HPLC fractions led to the isolation of a more polar compound salinosporamide C (4).The elemental composition ofthis compound,as determined by HRMALDI-FTMS,was C14H19ClNO3onthe basis of the observance of a pseudomolecular ion peak at284.1059(MH+,+0.6mDa).While this molecular formula required the same number of degrees of unsat-uration(6)that was required by the elemental composi-tion of1,it was clear from the other spectral data(Table 2)that the structural features of1and4differed significantly.Analysis of the13C NMR spectrum of4,recorded in pyridine-d5,allowed all of the degrees of unsaturation to be assigned to an amide(δC176.9),a ketone(δC210.1),a carbon-carbon double bond(δC157.2,128.5),and,by elimination,three rings.On the basis of the chemical shifts of the olefinic carbons,it was clear that this carbon-carbon double bond was in conjugation with either the ketone or the amide carbonyl.The former was the case,as HMBC correlations from an allylic methyl proton signal atδH2.09(H-14)and a methylene signal atδH2.72(H-12)to C-1,C-2,and C-3established the amide carbonyl as part of an R, -unsaturated system(C-1 through C-3)(Figure1).The second carbonyl(C-9)was assigned as part of a substituted cyclohexanone ring(C-6 through C-11)on the basis of a network of COSY and HMBC correlations(Table2),which could be extended at C-6to include two downfield methine signals(δH4.05 and4.23)on the basis of COSY correlations between H-6/ H-5and H-5/H-4.This substructure was then joined to the R, -unsaturated amide on the basis of HMBC cor-relations between H-4and C-3to form a tricyclic system containing all the carbons(Figure1).The final ring connectivities were established by HMBC correlations, specifically from H-7and H-4to the amide carbonyl(C-1)and between H-7and C-4,to give the hexahydro-3H-pyrrolo-[1,2a]-indol-3,6-dione ring structure depicted. The relative stereochemistry of4was established by a two-dimensional ROESY NMR experiment(Figure2).A ROESY correlation between H-7and H-6established a cis junction in the cyclohexanone ring,while ROESY correlations between H-7and H-5and H-5and H-6(18)Jacque,J.;Gros,C.;Mourcier,S.Absolute Configurations of 6000Selected Compounds with One Asymmetric Carbon Atom;Georg Thieme Publishers:Stuttgart,Germany,1997.Specifically,see refer-ences for data on1-chloro-4-methylhex-1-en-3-one,1-chloro-4-methyl-hexane,1-chloro-5-methylheptane,1-bromo-3-methylnonane,1-bromo-4-methylnonane,and the corresponding deshalogenated compounds.(19)All of our attempts to convert1into3resulted in cleavage of the -lactone and subsequent decarboxylation.Dechlorination of1was attempted using(a)Mg in i-PrOH with sonication for4days;(b) hydrogenation with10%Pd/C in i-PrOH;and(c)NaBH4reduction in i-PrOH and also in DMSO.An attempt was also made to convert1to the iodo derivative using the Finkelstein reaction(NaI in refluxing acetone),which under these conditions was not successful.(20)It should be noted that the structural representation of1used in this manuscript is different than that used in the original paper8 where the structure was originally disclosed.During the review process on this manuscript,it was brought to our attention that the original figure was in violation of the IUPAC rules because a hashed line between two adjacent stereocenters(C-4and C-5)is not allowed under this system.The stereochemical representations used in this paper for1-9are in accord with these IUPAC recommendations.See:Moss, G.P.Pure Appl.Chem.1996,68,2193-2222.T ABLE2.NMR Spectral Data for4at300MHzC/HδH a multiplicity(J in Hz)δc b,c COSY HMBC d,e ROESY 1176.9,C4,7,12,142128.5,C4,12,13,143157.2,C4,12,144 4.23,d(8.2)71.4,CH5,145,6,7,1410a,145 4.05,br t(8.2)74.4,CH OH,4,64,6,76,11b6 2.85,m45.8,CH7,117 4.41,dt(6.4,5.9)53.7,CH6,88,115,6,8a,8b 8a 3.00,m45.5,CH27,8b8b 2.89,m7,8a9210.1,C7,8,10,1110a 2.57,m38.5,CH210b,11b6,8,1110b 2.34,m10a11a 2.35,m20.5,CH211b5,6,7,1011b 2.12,m10a,11a12 2.72,m27.8,CH21313,1413 3.83,m43.6,CH212121214 2.09,br s13.1,CH34OH7.47,br d5a Recorded in C5D5N.b Recorded at100MHz.c Number of attached protons determined from an edited HSQC spectrum.d Protons showing long-range correlation with indicated carbon.e Correlations were observed for n J CH)8Hz.F IGURE1.Key HMBC correlations used to establish the structure of4.Williams et al..Chem.,Vol.70,No.16,2005established the configuration of the proton at H-5.Finally,ROESY cross-peaks between H-4and H-10aestablished the rest of the relative stereochemistry.LC-MS analysis of the extract from the large-scale fermentation of this S.tropica strain identified five more minor analogues of 1that were subsequently isolated to provide compounds 5-9.These compounds were pro-duced in comparable yields to salinosporamide B (3)and C (4),but only in trace amounts when compared to 1.The structures of these compounds were deduced as follows (See Tables S2-S6in Supporting Information for tabulated spectral data).The 1H NMR spectrum of compound 5was essentially identical to that of 1,except that it contained an additional methyl resonance at δH 3.69.This suggested that 5was the methyl ester ana-logue of the seco-acid of 1.This conclusion was consistent with both the molecular formula of C 16H 24ClNO 5,which was determined by HRMS analysis in conjunction with the NMR data (See Tables S2and S3).Compound 6also possessed a methyoxy singlet in its 1H NMR spectrum.This resonance again suggested that the -lactone ring of 1had been opened to the methyl ester,but the molecular formula of 6(C 16H 24NO 5)still required the same number of rings as in 1.The other important conclusion that was clear from the molecular formula of 6was that this derivative did not incorporate prehensive analysis of the two-dimensional NMR data,recorded in CDCl 3(Table S4),allowed the gross structure of 6to be assigned.Specifically,the cyclohexene ring was established on the basis of HMBC correlations from the vinyl protons H-7and H-8to the carbons of this ring (H-7to C-5,C-6,C-8,C-9,and C-11;H-8to C-6,C-7,C-9,and C-10),while HMBC correlations from H-2and H-14to C-1,C3,and C-4established the presence of the -lactam ring functionality.Finally,HMBC correlations from H-2to C-12and C-13indicated that the two-carbon side chain of 1was still intact but revealed that the chemical shift of C-13in 6was significantly further downfield than the corresponding carbon signal in 1.This,in conjunction with HMBC correlations from H-13a and H-13b to C-3,established that 6was a tetrahydro-furan analogue of 1.The relative configuration of the -lactam ring in 6was established on the basis of interpretation of the NOESY NMR data.Cross-peaks were observed from H-2to H-14and between H-14and H-5,indicating that these three protons were in a syn orientation around the -lactam ring (Figure 3).The relative configuration of the vicinal centers C-5and C-6was assigned as 5R *,6R *on the basis of comparison of the 3J H,H values (in pyridine-d 5)between these centers in 1,5,and 6(See Tables 1and S2).The molecular formula of 7was established as C 14H 18-ClNO by HRMALDI -FTMS,indicating that 7contained six double-bond equivalents.Some of these degrees of unsaturation were reflected in the UV chromophore of 7,which showed a significant bathochromic shift as compared to 1and 3.This suggested extended conjuga-tion,which was also reflected in the IR spectrum of 7,which showed an infrared carbonyl absorption at a lower wavenumber (1684cm -1)than seen in the other com-pounds.Analysis of the proton and carbon NMR data indicated that 7was structurally related to 1.For example,the NMR data clearly showed that the chloro-ethyl and cyclohexene moieties were present in 7on the basis of the characteristic proton resonances for H-12,H-7,and H-8(δH 3.55,5.66,and 5.48),but it also revealed some differences.Specifically,the NMR data indicated the presence of additional trisubstituted (δH 4.89;δc 114.7,CH;δc 138.2,C)and tetrasubstituted alkenes (δc 128.4,C;δc 143.3,C)as well as the loss of the signal for the proton adjacent to the amide carbonyl (H-2).These data,in conjunction with the rest of the NMR information (Tables S2and S3)established the structure of 7as the R , ,γ,δ-unsaturated lactam shown.The geometry of the exocyclic double bond in 7was assigned as Z on the basis of an NOE correlation between the vinyl methine proton and the methyl signal at C-14.Compound 8was isolated as a colorless oil.The HRMALDI -FTMS data defined the molecular formula of 8as C 14H 20ClNO 2,which was 44amu less than salinosporamide A (1).This difference was reflected in the 13C NMR spectrum of 8in which only 14resonances were observed (Table S3).The most prominent difference,as compared to 1,was the absence of the ester carbonyl signal at δC 169.0,which implied that the -lactone moiety was not present in 8.In contrast,the -lactam ring structure was present in 8,as suggested by HMBC correlations (Table S5)from the allylic methyl proton signal (H-14)to C-1,C-2,C-3,and C-4.Interestingly,C-4,which had been a quaternary carbon in 1,3and 5-7,now showed a 1J CH to a proton signal at δH 3.70,which was coupled to the amide proton signal at δH 8.55.This signal at δH 3.70also displayed a COSY correlation to the oxygenated methine proton signal at 3.27ppm.This in turn could be connected to the cyclohexene ring on the basis of a series of COSY and HMBC correlations (See Table S5).These signals defined the R , -unsaturated γ-lactam structure of 8.F IGURE 2.ROESY correlations used to establish the relativeconfiguration of 4.F IGURE 3.NOESY correlations used to establish the relativeconfiguration of 6.New Cytotoxic Salinosporamides.Chem ,Vol .70,No .16,20056199The low-resolution mass spectral data for compounds 8and9showed that they were isomers,since both gave the same pseudomolecular ion at270.1(MH+),which corresponded to the molecular formula C14H20ClNO2, which was determined by HRMS.Analysis of the two-dimensional NMR data established that9(Table S6)had the same planar structure as8(Table S5),indicating that they were not constitutional isomers.A detailed com-parison of the1H NMR spectra for the two compounds revealed that they were configurational isomers.This was based on the multiplicity of the H-4proton signal;in compound8,H-4was a broad singlet when the proton NMR spectrum was recorded in C6D6,while in9this signal was a doublet.Of the possible sites of epimeriza-tion that would explain this difference in the multiplicity of the H-4proton signal,C-4and C-5,the former is more likely based on mechanistic considerations(vide infra). While the relative configurations of8and9were not rigorously established,a tentative assignment for C-4/ C-5was postulated on the basis of comparison of the proton-proton coupling constants between H-4and H-5of8and9with that of model compounds recorded in the appropriate parison of the literature cou-pling constants between H-4and H-521for the anti and syn diastereomers of the model compounds10and11 suggest that the latter has the larger3J4H,5H value(2.1 vs5.7Hz respectively;CDCl3).A5Hz proton-proton coupling constant was also observed in the related compound12that also has a syn configuration at these two stereogenic centers(Figure4).22This suggests that the C-4/C-5junction is anti in8(3J H-4/H-51.3Hz)and syn in9(3J H-4/H-5)6.7Hz,CDCl3;the rest of the data in this solvent are not shown).The presence of the methyl esters in5and6raises suspicion that these compounds were artifacts of the isolation procedure and not produced in the fermentation process.To test this hypothesis,1(1.8mg)was stirred in a1:1mixture of DCM/MeOH(1mL total volume)for 45h at27°C.These specific conditions were chosen since they mimic the resin extraction procedure of the initial fermentation broth.LC-MS analysis of this reaction mixture showed the gradual appearance and growth of peaks that had identical retention times and mass fragmentations as5and6.Presumably,these compounds are produced by initial methanolysis of1to form5and then subsequent intramolecular nucleophilic displace-ment of the chloride by the adjacent hydroxyl group to form6(Figure5).The finding that5and6could be produced from1 called into question the origin of compounds7-9.It was possible that7-9were produced from1through a decarboxylation mechanism.There is ample precedence for the decarboxylation of -lactones,but this reaction usually requires elevated temperatures in excess of those experienced by1during the isolation process.23To explore this possibility a sample of1(1mg)was stirred at27°C in a solution of0.5M NaOH,which had been buffered to pH8with HCl,and acetonitrile(0.5mL each of the CH3-CN and NaOH).This was the same pH as the fermenta-tion broth initially.Aliquots of this mixture,which were analyzed by LC-MS over the course of17h,showed that 1was being converted into a suite of related compounds during this time period,as shown by the UV chromato-grams of the reaction mixture monitored at210and254 pounds7-9could be clearly identified in the reaction mixture on the basis of comparison of the retention times and the corresponding molecular ions with those of the samples of7-9that had been charac-terized spectroscopically.Co-injection of authentic samples of7-9provided further proof for the degradation of1 into these compounds.Given these overall observations, it was clear that compounds5-9were produced during the isolation process and are not natural products(Figure 5).It should be noted that the conversion of1into5-9 established the absolute configurations of all these products since the configuration of C-6is preserved as S.24The finding that7-9can be formed from1necessitates a few comments.Conceptually,there are two general pathways for the conversion of1into7-9.The most important difference,as it relates to the following discus-sion,between the two possible pathways is that in route A the S absolute configuration of1at C-5is retained in the products8and9,while in route B the absolute configuration of1at C-5is not retained in the products 8and9.While the exact mechanistic details of this conversion are not important for the following discussion, one possible mechanism for each route will still be discussed to illustrate this point about the different stereochemical outcomes.One hypothetical route A reac-tion pathway involves,after saponification,decarboxy-lation of the resulting vinylogous-δ-amide acid25to produce an aromatic pyran ring.Subsequent tautomer-ization of13produces a mixture of two diastereomers that are epimeric at C-4(8and9)but that retain the S configuration at C-5(Figure6,route A).Dehydration of these compounds8and9then produces7.By compari-son,route B(Figure6)might proceed through a concerted mechanism involving a decarboxylation/dehydration se-quence that initially gives rise to7in which the5S configuration of1is lost.Nucleophilic addition of water to7would then give rise to a mixture of diastereomers (8and9).The most important consequence of this pathway is that theoretically four diastereomers26should be produced by any route B reaction pathway.The(21)Casiraghi,G.;Spanu,P.;Rassu,G.;Pinna,L.;Ulgheri,F.J. Org.Chem.1994,59,2906-2909.(22)Schiehser,G.A.;White,J.D.Tetrahedron Lett.1986,46,5587-5590.(23)(a)Lowe,C.;Vederas,.Prep.Proced.Int.1995,27, 305-346.(b)Pommier,A.;Pons,J.M.Synthesis1993,5,441-459.(24)Attempts to determine the absolute configuration of C-5in8 and9by preparation of the MTPA derivatives resulted in elimination to form7.(25)For an example of decarboxylation of a -amide ester under basic conditions,see:Shehata,I.A.;Glennon,R.A.J.Heterocycl. Chem.1987,24,1291-1295.F IGURE4.Model compounds used to establish the relativeconfiguration of8and9.Williams et al. .Chem.,Vol.70,No.16,2005isolation of just two diastereomers,8and 9,from the fermentation mixture and,much more importantly,only those same two diastereomers from the degradation studies on 1is evidence against the route B general mechanism.This evidence suggests that 8and 9are epimeric at C-4,as depicted,rather than at the alterna-tive C-5center.It should be noted that the above discussion also implies that 8and 9have an absolute configuration at C-5of S analogous to salinosporamide A (1).The finding that 5-9can be produced from 1also calls into question the origin of salinosporamide C (4).Clearly,on the basis of a number of factors,4is not a direct degradation product of salinosporamide A (1).Any “deg-radation”of 1into 4would require a number of trans-formations that would include decarboxylation of the -lactone,oxidation of C-9to a ketone,and formation of the pyrrole ring by the attachment of the nitrogen to C-7.This complexity makes it highly unlikely that 4is a degradation product of salinosporamide A (1),but nev-ertheless it is still possible that 4is an artifact caused by decarboxylation of the -lactone compound 14(Figure 7).A detailed analysis of the culture broth by LC-MS over the duration of the fermentation has not led to the identification of this putative metabolite;thus,we must(26)Molecular modeling of the four potential diastereomers (4R ,5R ,6S ;4R ,5S ,6S ;4S ,4R ,6S ;4R ,5R ,6S )indicates that the lowest energy conformers of these diastereomers are all within 2kcal/mol.This suggests that all four diastereomers should be produced,albeit in unequalamounts.F IGURE 5.Reactivity of 1toward MeOH and diluteNaOH.F IGURE 6.Possible degradation mechanisms.New Cytotoxic Salinosporamides.Chem ,Vol .70,No .16,20056201。