Carbohydrate Research, Volume 339, Issue 2, 22 January 2004, Pages 429-433

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1-s2.0-S0008621513002139-maingood

NoteChemical structures of the secondary cell wall polymers (SCWPs)isolated from bovine mastitis StreptococcusuberisAnna Czaban´ska,Otto Holst,Katarzyna A.Duda ⇑Division of Structural Biochemistry,Research Center Borstel,Leibniz-Center for Medicine and Biosciences,Airway Research Center North (ARCN),Member of the German Center for Lung Research (DZL),Parkallee 4a/c,D-23845Borstel,Germanya r t i c l e i n f o Article history:Received 23April 2013Received in revised form 22May 2013Accepted 23May 2013Available online 1June 2013Keywords:MastitisStreptococcus uberis SCWPsWTA structure Rhamnan NMR analysisa b s t r a c tThe cell envelope of Gram-positive bacteria is decorated with a variety of polysaccharides.In this study wall teichoic acid (WTA)and neutral polysaccharides were isolated from the cell envelope of bovine mas-titis Streptococcus uberis .The polysaccharides were released by lysozyme treatment,and puriﬁed by hydrophobic interaction chromatography.Further separation was achieved utilizing anion-exchange chromatography which yielded two products,that is,a neutral polysaccharide with a high content of Rha and less Glc (rhamnan)and an anionic phosphate-rich one containing glycerol and Glc (WTA).The structures of these molecules were elucidated applying 1D and 2D nuclear magnetic resonance experi-ments as well as chemical analyses.In the rhamnan sample two independent molecules were identiﬁed,that is,a glucorhamnan with the structure ?2)-a -L -Rha p -(1?3)-[a -D -Glcp-(1?2)-]a -L -Rha p -(1?,and a homopolymeric rhamnan ?2)-a -L -Rha p -(1?3)-a -L -Rha p -(1?.The WTA comprised a polyphosphoglyc-erol chain substituted nonstoichiometrically with b -Glc p .Ó2013Elsevier Ltd.All rights reserved.1.IntroductionBovine mastitis,namely inﬂammation of the udder,is a result of intramammary infection by bacteria.1The course of the disease may be either sub-clinical or clinical depending on the factors in the host and the invading pathogen.Sub-clinical mastitis shows no obvious signs of disease,goes often unnoticed and untreated,resulting in long duration of the infection.2The clinical form of the disease is characterized by visible abnormalities in the milk (protein aggregates or clots)accompanied by pain and swelling in the affected gland and sometimes production of a secretion composed solely of aggregated protein in a serous ﬂuid.In severe cases there may be systemic signs such as elevated temperature and loss of appetite which may lead to bacteremia,septicemia,and death of the animal.1Over 135infectious agents were reported to cause mastitis 3but most infections are due to one of the three bacterial species Strep-tococcus uberis ,4Escherichia coli,4and Staphylococcus aureus.5The cell envelope of Gram-positive bacteria is decorated with a variety of polysaccharides.Based on their structural character,they can be classiﬁed into the three groups,(i)teichoic acids,(ii)teichu-ronic acids,and (iii)other polysaccharides (neutral or acidic)which do not belong to any of the other groups.6Teichoic acids [TAs,lipoteichoic acid (LTA),and wall teichoic acid (WTA)]are phosphate-rich polyalditols which were shown to contribute to resistance to environmental stresses,7,8antimicro-bial peptides,9cationic antibiotics,and lytic enzymes produced by the host.10Non classical polysaccharides were reported to play an important role in non-covalent attachment of the S-layer to the underlying peptidoglycan (PG).11The structure of the S.uberis LTA has been published recently.12A body of literature reveals a wide structural diversity of Gram-positive bacterial WTAs which are covalently bound to O -6of muramic acid (MurNAc)of PG via a phosphodiester bond.13Their repeating unit often possesses a similar structure as that of LTA,comprising a polyglycerol-or poly-ribitolphosphate nonstoichiometrically substituted with alanine or glycosyl residues,14,15but may also show considerable differences.Structural analyses of non-classical SCWPs revealed that they are either charged 16or neutral 17heteropolysaccharides covalently bound to PG via a phosphodiester 18or pyrophosphate linkage.19The present study deals with the structures of WTA and a neutral polysaccharide isolated from the cell envelope of one of the most important mastitis pathogens,S.uberis.2.Results and discussionThe polysaccharides were released from disrupted bacterial cells of S.uberis 233by lysozyme treatment,followed by incuba-tion with RNase,DNase,and proteinase K.Hydrophobic interaction0008-6215/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.carres.2013.05.015Corresponding author.Tel.:+4945371882490;fax:+4945371887450.E-mail address:kduda@fz-borstel.de (K.A.Duda).chromatography(HIC)was performed in order to remove possible remains of LTA.Separation of polysaccharides could be achieved by anion-exchange chromatography which yielded three fractions.Of these,theﬁrst and second contained the same material according to the anomeric regions of1H NMR spectra,a rhamnan,due to the high content of Rha residues.Since the quality of the2D spectra re-corded for fraction1was not sufﬁcient for a successful assignment of the signals,further analyses were performed on fraction2.The third fraction,phosphate-rich,was identiﬁed as WTA.Compositional analysis of the rhamnan identiﬁed Rha and Glc in an approx.molecular ratio of4:1.Their absolute conﬁgurations were determined as L and D,respectively.Methylation analysis revealed the presence of2-substituted,3-substituted,and2,3-disubstituted Rha,and terminal Glc by identifying1,2,5-tri-O-acetyl-6-deoxy-3,4-di-O-methyl-[1-2H]mannitol,1,3,5-tri-O-acetyl-6-deoxy-2, 4-di-O-methyl-[1-2H]mannitol,1,2,3,5-tetra-O-acetyl-6-deoxy-4-O-methyl-[1-2H]mannitol,and1,5-di-O-acetyl-2,3,4,6-tetra-O-methyl-[1-2H]glucitol.The WTA consisted of Gro,phosphate,and Glc.The absolute conﬁguration of Glc was determined as D.Both,rhamnan and WTA were investigated by1D and2D NMR experiments.The1D1H NMR spectrum of the rhamnan revealed5 major signals in the anomeric region[4originating from Rha p(A–C,E)and1from Glc p(D)residues],several characteristic signals at d1.28–1.24(methyl groups of6-deoxyhexoses)and2signals at d 2.06and d2.02(methyl group of N-acetyl function)(Fig.1a).Since the latter signals were greatly diminished after further puriﬁcation of the sample on TSK40(not shown)they were assigned as impu-rities.The anomeric conﬁgurations were determined on the basis of1J C-1,H-1values ranging from173to178Hz(derived from a cou-pled heteronuclear single quantum coherence1H,13C-HSQC spec-from PG constituents,namely GlcNAc and MurNAc as the samples were treated with the lysozyme.However,the complete spin sys-tems of GlcNAc and MurNAc could not be identiﬁed.Detailed analyses applying1H,1H correlation spectroscopy (COSY),total correlation spectroscopy(TOCSY),rotating frame Overhauser effect spectroscopy(ROESY),and1H,13C heteronuclear single quantum coherence(HSQC),and heteronuclear multiple bond correlation(HMBC)experiments were performed in order to establish structures of both examined molecules.The2D spectra of the rhamnan identiﬁed several minor signals in the anomeric region and those originating from short phospho-glycerol chains which were not detected by the1H NMR experi-ment.After further puriﬁcation of the sample by size exclusion chromatography on TSK-40these signals could be assigned to impurities.Due to better quality of spectra,chemical shift assign-ments of strong signals corresponding to all peaks of1H spectrum were made on the non-puriﬁed sample(Table2)and only those cross-peaks are depicted in Figure2.In the HMBC experiment,con-nectivities between A,H-1/C,C-3;B,H-1/E,C-3;C,H-1/A,C-2;E, H-1/B,C-2and C,H-2/D,C-1(for A-E,see Table1)were found which were conﬁrmed by a ROESY experiment(inter-residual NOE contacts between A H-1/C H-3,B H-1/E H-3,C H-1/A H-2,D H-1/C H-2,and E H-1/B H-2).These data suggested the existence of two rhamnan polymers,ﬁrst possessing the structure?2)-B-(1?3)-E-(1?,and the second?2)-A-(1?3)-[D-(1?2)-]C-(1?] (Fig.3).No indication was obtained that both partial structures were linked(no cross connectivity between E-1and A-2was ob-served,and NOE contacts between A H-3/D H-4and A H-6/D H-2 found in the ROESY spectrum were not observed between B and D).2D NMR experiments of the WTA allowed the assignment of allFigure1.1H NMR spectra(700MHz)of(a)rhamnan and(b)WTA isolated from uberis233.The capital letters refer to the residues deﬁned in Tables1and2.Spectra were recorded in D2O at27°C relative to external acetone(d H2.225;d C31.45).2.Excerpt of1H,13C HSQC-DEPT spectrum(700MHz)of the rhamnan isolated from S.uberis233.The spectrum was recorded in D2O at27°C relative to external acetone(d H2.225;d C31.45).A.Czaban´ska et al./Carbohydrate Research377(2013)58–6259nals originating from unsubstituted phosphoglycerol were ob-served(E0).The presence of Gro signals linked to the phosphate at O-1and at O-3free(G0)indicated that WTA chains were short. The proposed structure of the WTA is shown in Figure5.For isolation of the described polysaccharides lysozyme was used as we aimed at the elucidation of polymer structures together with their linkers to PG.This could not be achieved as we were not able to determine spin systems of PG constituents(MurNAc and GlcNAc)and thus identify the linking ually,WTAs are bound covalently to O-6of MurNAc of PG via a phosphodiester bond.13The non-classical SCWPs were reported to be either cova-lently bound to PG via a phosphodiester18or pyrophosphate linkage.19To our knowledge all published structures of non classical SCWPs indicated that these were heteropolymers.Here,novel het-ero-and homopolymers(glucorhamnan and rhamnan,respec-tively)of S.uberis were isolated and characterized.WTA of S.uberis isolated in this study was shown to be com-posed of polyphosphoglycerol substituted nonstoichiometrically A nonstoichiometrical substitution of Gro with b-Glc has been re-ported for Staphylococcus epidermidis I2,22however here the posi-tion O-2of phosphoglycerol was alternatively substituted by D-Ala.3.Experimental3.1.Bacterial growthS.uberis233was isolated from a mastitis-diseased cow in New Zealand and was cultivated as standing cultures to OD6000.8in Brain Heart Infusion(BHI)medium with and without addition of 1%agar at37°C for12h.3.2.Extraction and puriﬁcation of polysaccharidesBacterial cells were harvested after12h,washed once with citric buffer(0.1M,pH4.7)and disrupted with a cell homogenizer (Vibrogen—Zellmühle Edmunt Bühler GmbH)and glass beads (0.1mM,Roth,Karlsruhe).Supernatant was separated from the glass beads by centrifugation(1000Âg,RT,5min).Then the glass beads were washed3–5Âwith citrate buffer.In order to release polysaccharides bound to the PG,an enzymatic digestion was per-formed.First,10Âbuffer for enzymatic treatment was added to the resuspended cell particles,followed by lysozyme(0.2mg/ml), DNase I and RNase(each0.1mg/ml).The sample was shaken at 37°C for24h.The insoluble cell wall fragments were removed by centrifugation(4°C,30min,10,000Âg),and the supernatant was then treated with proteinase K(0.1mg/ml)at37°C for24h. After digestion,the solution was dialyzed(3.5ku cut off)against water and lyophilized.The sample was puriﬁed by hydrophobic interaction chromatography(HIC)as described previously12in or-der to remove the remaining LTA.Fractions4–14were combined and the sample was lyophilized,and then resuspended in water,ﬁltered,(0.45l m,PALL Life Sciences,HPLC certiﬁed)and applied on HiTrep Q Sepharose column and puriﬁed by anion-exchange chromatography.The bound material was eluted with a gradient from0to1M of NaCl of36columns volume(1column volume was5ml)with aﬂow rate of3ml/min.After elution the columnwas washed,ﬁrst with20columns volume of1M NaCl,then with5 columns volume of2M NaCl.Fractions1–80were examined in re-gard to the phosphate content.In order to desalt the obtained sam-ples,separation on a column(2.5Â100cm)of Sephadex G-10was performed.The elution was carried out without a pump and with a buffer containing4ml pyridine and10ml glacial acetic acid in1l of water.The pyridine–acetate buffer was removed by repeated evaporation of the solvent on a rotary evaporator.Further puriﬁca-tion of the rhamnan was performed by size exclusion chromatog-raphy on a column(1Â100cm)of TSK-40utilizing the same conditions as for Sephadex G10.positional analysesThe components of the isolated molecules were determined after methanolysis(0.5M HCl/MeOH,85°C,45min),acetylation (85°C,10min),and detection by GLC–MS[Hewlett Packard HP 5890(series II)gas chromatograph equipped with a fused-silica SPB-5column(Supleco,30mÂ0.25mmÂ0.25l mﬁlm thick-ness),FID and MS5989A mass spectrometer with vacuum gauge controller59827A].The temperature program was150°C for 3min,then5°C minÀ1to330°C.The hexoses were detected as alditol acetates[2M triﬂuoroacetic acid,100°C,2h,then reduction (NaBH4,16h in the dark)and acetylation(85°C,10min)]by GLC [HP5890(series II)gas chromatograph with FID and a column (Agilent Technologies,30mÂ0.25mmÂ0.25l mﬁlm thickness)]. The temperature program was150°C for3min,then3°C minÀ1to 320°C.The absolute conﬁguration of Rha and Glc was determined by GLC,by comparison with authentic standards of the acetylated (S)-2-butanol derivative after methanolysis(2M HCl/MeOH,85°C, 45min),butanolysis(2M HCl/(S)-2-BuOH,65°C,4h),and acetyla-tion(85°C,10min).Rhamnan was methylated with CH3I after dis-solving in dry DMSO with addition of dry NaOH(room temperature,stirring,1h)and puriﬁed by chloroform–water extraction.The product was hydrolyzed(4M triﬂuoroacetic acid, 100°C,4h),reduced(NaBD4in MeOH/H2O1:1),acetylated D2O and27°C.All1D and2D NMR H,H COSY,TOCSY,and ROESY as well as1H,13C HSQC,coupled HSQC,and1H,31P HMQC experi-ments were recorded by a Bruker DRX Avance III700MHz spec-trometer(operating frequencies of700.75MHz for1H NMR, 176.2MHz for13C NMR and283.7MHz for31P)and standard Bru-ker software.COSY,TOCSY,and ROESY experiments were recorded using data sets(t1by t2)of4096by512points,with16scans for COSY,and32for TOCSY and ROESY.The TOCSY experiments were carried out in the phase-sensitive mode with mixing times of60 and180ms and ROESY of300ms.The1H,13C correlations mea-sured in the1H-detected mode via HSQC-DEPT with proton decou-pling in the13C domain and HMBC spectra were acquired using data sets of4096by512points and80scans for each t1value. HMBC spectra were adjusted to J coupling constant value of 145Hz and long range proton carbon coupling constant of10Hz.Chemical shifts were reported relative to external acetone(d H 2.225,d C31.45)or external phosphoric acid(d P0.0). AcknowledgmentsWe thank Pﬁzer Animal Health forﬁnancial support,Dr.Uwe Mamat for anion-exchange chromatography,Hermann Moll for help with GLC–MS,and Heiko Käßner and Dr.Nicolas Gisch for NMR recordings.References1.Leigh,J.A.Vet.J.1999,157,225–238.2.Zadoks,R.N.;Gillespie,B.E.;Barkema,H.W.;Sampimon,O.C.;Oliver,S.P.;Schukken,Y.H.Epidemiol.Infect.2003,130,335–349.3.Watts,J.L.Vet.Microbiol.1988,16,41–66.4.Leigh,J.A.;Egan,S.A.;Ward,P.N.;Field,T.R.;Coffey,T.J.Vet.Res.2010,41,63.5.Schukken,Y.H.;Günther,J.;Fitzpatrick,J.;Fontaine,M.C.;Goetze,L.;Holst,O.;Leigh,J.;Petzl,W.;Schuberth,H.-J.;Sipka,A.;Smith,D.G.E.;Quesnell,R.;Watts,J.;Yancey,R.;Zerbe,H.;Gurjar,A.;Zadoks,R.N.;Seyfert,H.-M.Vet.Immunol.Immunopathol.2011,144,270–289.6.Schäffer,C.;Messner,P.Microbiology2005,151,643–651.7.Vergara-Irigaray,M.;Maira-Litran,T.;Merino,N.;Pier,G.B.;Penades,J.R.;Lasa,I.Microbiology2008,154,865–877.8.Oku,Y.;Kurokawa,K.;Matsuo,M.;Yamada,S.;Lee, B.-L.;Sekimizu,K.J.Bacteriol.2009,191,141–151.Table21H and13C NMR data of the WTA and deacylated LTA isolated from S.uberis233Residue1(a)1b23(a)3b456a6bA01H 5.16 3.68 3.86 3.49 3.69 3.71 3.81 2-a-D-Glc p13C97.176.673.171.073.061.8B01H 5.09 3.60 3.77 3.46 3.93 4.11 4.05 t-a-D-Glc p13C97.372.373.970.773.166.0C01H 4.47 3.31 3.51 3.40 3.46 3.90 3.77 t-b-D-Glc p13C103.374.476.770.876.961.9D01H 3.83 3.51nd 3.67 3.60Gro-A013C70.0nd63.1E01H 3.93 4.02 4.06 3.93 4.02–P-Gro-P–13C67.570.367.5F01H 4.11 4.03 4.22 4.11 4.03–P-Gro-(C0)P–13C66.178.566.1G01H 3.90 3.96 3.91 3.60 3.685.Proposed structure of WTA of S.uberisA.Czaban´ska et al./Carbohydrate Research377(2013)58–62619.Peschel,A.J.Biol.Chem.1999,274,8405–8410.10.Peschel,A.;Vuong,C.;Otto,M.;Götz,F.Antimicrob.Agents Chemother.2000,44,2845–2847.11.Schäffer,C.;Messner,P.Biochimie2001,83,591–599.12.Czaban´ska, A.;Neiwert,O.;Lindner, B.;Leigh,J.;Holst,O.;Duda,K. A.Carbohydr.Res.2012,361,200–205.13.Araki,Y.;Ito,E.Crit.Rev.Microbiol.1989,17,121–135.14.Sánchez Carballo,P.M.;Vilen,H.;Palva,A.;Holst,O.Carbohydr.Res.2010,345,538–542.15.Endl,J.;Seidl,H.P.;Fiedler,F.;Schleider,K.H.Arch.Microbiol.1983,135,215–223.16.Schäffer, C.;Kählig,H.;Christian,R.;Schulz,G.;Zayni,S.;Messner,P.Microbiology1999,145,1575–1583.17.Steindl,C.;Schäffer,C.;Wugeditsch,T.;Graninger,M.;Matecko,I.;Müller,N.;Messner,P.Biochem.J.2002,368,483–494.18.Steindl,C.;Schäffer,C.;Smrecki,V.;Messner,P.;Müller,N.Carbohydr.Res.2005,340,2290–2296.19.Schäffer,C.;Müller,N.;Mandal,P.K.;Christian,R.;Zayni,S.;Messner,P.Glycoconjugate J.2000,17,681–690.20.Kessler,R.E.;van de Rijn,I.;McCarty,M.J.Exp.Med.1979,150,1498–1509.21.Kurokawa,K.;Gong,J.-H.;Ryu,K.-H.;Zheng,L.;Chae,J.-H.;Kim,M.-S.;Lee,B.L.p.Immunol.2011,35,835–839.22.Archibald,A.R.;Baddiley,J.;Shaukat,G.A.Biochem.J.1968,110,583–588.62 A.Czaban´ska et al./Carbohydrate Research377(2013)58–62。

Strasser_2001_Carbohydrate-Polymers

Pectic substances from red beet(Beta vulgaris conditiva).Part I.Structural analysis of rhamnogalacturonan I usingenzymic degradation and methylation analysisG.R.Strasser,R.Amado`*Swiss Federal Institute of Technology,Institute of Food Science,ETH-Zentrum,Schmelzbergstrasse9,CH-8092Zurich,SwitzerlandAccepted2March2000AbstractCell wall material from ripe red beet(Beta vulgaris L.var.conditiva)was isolated as alcohol insoluble residue(AIR).The chelator-soluble pectin obtained by cyclohexane-trans-1,2-diaminotetraacetate(CDTA)extraction of the AIR was fractionated by anion exchange chromatography(AEC).The main fraction was further fractionated by gelﬁltration chromatography(GFC).Fractions from both chromatographic systems were stepwise degraded by endo-polygalacturonase,endo-b-(134)-d-galactanase,endo-a-(135)-l-arabinanase and a-l-arabinofuranosidase.Degradation products were fractionated by GFC or by AEC.Polymeric fractions were investigated by methylation analysis after carbodiimide-activated reduction with NaBD4.Selected fractions were additionally methylated with trideuteromethyliodide to enable the detection of O-methyl substituted sugars.The results indicate that the CDTA-soluble pectins of red beet cell walls are composed of three different sub-units:a homogalacturonan,which accounts for about75%,a highly ramiﬁed rhamnogalacturonan I(RG-I)and a typical rhamnogalacturonan II(RG-II).RG-I consists of a highly ramiﬁed backbone composed of nearly equal amounts of rhamnose and galacturonic acid.Side chains,mainly arabinans,galactans and type-II arabino-galactans are attached to the RG-I backbone.Some arabinans are connected via short galactan chains directly or indirectly to this backbone.Type-II arabinogalactans are formed by“inner”chains consisting of(133)-linked galactans and short“outer”chains composed of an average number of one to three(136)-linked galactose residues.Terminal arabinofuranoses are linked via the O-3-position to galactose residues.Nearly all non-reducing ends consist of glucuronic acid.Approximately65%of the glucuronic acid residues are substituted by a methyl ether group and approximately10%,most probably,by a terminally linked rhamnose.᭧2001 Elsevier Science Ltd.All rights reserved.Keywords:Red beet pectic substances;Rhamnogalacturonan I;Characterisation1.IntroductionPectins are a group of polysaccharides from the primary cell wall and the middle lamella of higher plants(Carpita& Gibeaut,1993;John&Dey,1986;McCann et al.,1995). Changes in the texture of fruits and vegetables and in the properties of their products are related to changes in pectic components.Pectins comprise a family of acidic polymers, like homogalacturonans and rhamnogalacturonans with several neutral polymers such as arabinans,galactans and arabinogalactans attached to them.They have been exten-sively investigated for their structure and functions within the plant cell wall using chemical analysis and enzymic degradation.Rhamnogalacturonan I(RG-I)is a poly-saccharide solubilised from plant cell walls after treatment with polygalacturonase(PG).The RG-I polymer is composed of alternating l-rhamnose and d-galacturonic acid residues.l-Arabinosyl-and d-galactosyl-rich side chains are attached to this backbone.Occasionally the side chains are terminated by l-fucosyl,d-glucuronosyl or4-O-methyl-d-glucuronosyl residues(Albersheim,Darvill, O’Neill,Schols&Voragen,1996).The aim of our work was the characterisation of the chelator-soluble pectic substances from ripe red beet(Beta vulgaris L.var.conditiva).For this purpose the alcohol-insoluble residue(AIR)was extracted with cyclohexane-trans-1,2-diaminotetraacetate(CDTA),the extracts were fractionated and characterised using enzymic degradation and methylation analysis.The isolation procedure and the characterisation of an RG-I are described below. The characterisation of an RG-II from ripe red beet willCarbohydrate Polymers44(2001)63–70 0144-8617/01/$-see front matter᭧2001Elsevier Science Ltd.All rights reserved.PII:S0144-8617(00)/locate/carbpol*Corresponding author.Tel.:ϩ41-1632-32-91;fax:ϩ41-1632-11-23. E-mail address:renato.amado@ilw.agrl.ethz.ch(R.Amado`).be described in part II of this paper (Strasser &Amado`,2000).2.Experimental2.1.Isolation of pectic materialCell wall material from ripe red beet of the variety Red Ace F1was isolated as AIR.15kg of red beets were peeled,cut into small pieces and blended in 90%boiling ethanol for 10min.The residue was homogenised with a commercial Waring blendor,ﬁltered (Polyestergaze Polynom,Schweiz.Seidenfabrik AG,Zurich,CH),and washed 11times with 70%ethanol.AIR (30g dry weight)was stirred in 3.0l of 50mM 1,2-diaminocyclohexane-N ,N ,N H ,N H -tetraacetic acid (CDTA)solution (pH 6.5)at 20ЊC for 6h.The residue was removed by ﬁltration through a D3glass ﬁlter funnel and washed with water.The residue was re-extracted under the same conditions for 2h.The washings from both extractions were combined,ﬁltered (0.45m m,Millipore)and dialysed (Servapor 44146,Serva &Co,Heidelberg,G)ﬁrst against tap water (3days),and then against de-ionised water (4days).After exhaustive dialysis the extract wasconcentrated under vacuum and freeze-dried (fraction:CDTAS,Fig.1).2.2.Fractionation of CDTASAnion exchange chromatography (AEC)and gel ﬁltration chromatography (GFC)were performed as described earlier(Strasser,Wechsler &Amado`,1996).Fractions IE0.0M,IE0.1M,IE0.2M,IE1.0M,and GF1,GF2,GF3,respec-tively,were pooled (Fig.1).2.3.Enzymic degradation with polygalacturonase The methyl ester and O-acetyl groups were saponiﬁed prior to enzymic degradation with 150ml 0.05M NaOH at 0ЊC for 14h.The pH was corrected to 4.5with 0.1M acetic acid containing 0.01%NaN 3.Fraction IE0.2M and the GF x -fractions (1.0g)were incubated separately with a PG (90IU,pH 4.5,35ЊC,6h),heated to inactivate the enzyme (100ЊC,10min),ﬁltered (0.45m m,Millipore)and fractionated by GFC (Sephacryl S-300HR,Pharmacia,95×2:6cm :A pH 5.0sodium acetate buffer (0.05M,containing 0.01%NaN 3)was used as eluent at a ﬂow-rate of 1.8ml/min.The separations were monitored with a HP 1037A RI-Detector (30ЊC).The corresponding fractions were pooled.High molecular weight fractions were dialysed and freeze-dried (fractions:PG1,PG2and GF x PG1,GF x PG2,respectively,Fig.1).The PG used for these degra-dation experiments had been puriﬁed and characterised by Elgorriaga (1994).2.4.Enzymic degradation by an endo-a -(135)-l -arabinanase or an endo-b -(134)-d -galactanase PG1-and PG1GF x -samples (100mg in 30ml 0.02M sodium acetate buffer,containing 0.01%NaN 3)were either incubated with endo-arabinanase (gift from Novo Nordisk A/S,Bagsvaerd,DK;50IU,pH 5.5,35ЊC,12h)or endo-galactanase (Megazyme Ltd,Boronia,AUS;60IU,pH 4.5,45ЊC,5h).The solutions were ﬁltered (0.45m m,Millipore)after inactivation of the enzyme (100ЊC,10min)and frac-tionated by AEC (DEAE-Sepharose CL-6B,Pharmacia,35×2:6cm using 170ml 0.02M and 200ml 0.8M sodium acetate buffer (pH 5.0M,0.01%NaN 3)for elution.Two fractions were collected,dialysed and freeze-dried (neutral fractions:An or Gn,ionic fractions:Ai or Gi,Fig.1).2.5.Enzymic degradation by a -l -arabinofuranosidase or by a combination of endo-arabinanase and a -l -arabinofuranosidasePG1Gi-and GF x PG1Ai-samples (90mg,30ml in 0.05M sodium acetate buffer,containing 0.01%NaN 3)were incu-bated with arabinofuranosidase (gift from Novo Nordisk A/S,Bagsvaerd,DK;60IU,pH 4.5,40ЊC,5h,additional 40IU,40ЊC,48h)or by arabinanase and arabinofuranosidase (50mg GF x PG1AiAF1-sample in 30ml 0.05M sodiumG.R.Strasser,R.Amado`/Carbohydrate Polymers 44(2001)63–7064Fig.1.Scheme for the isolation and fractionation of pectic substances from red beet cell wall material by CDTA-extraction,AEC,GFC and treatment with different pectin-degrading enzymes.acetate buffer containing0.01%NaN3,33IU and40IU,pH 5.0,40ЊC,48h).The solutions wereﬁltered(0.45m m, Millipore)after inactivation of the enzymes(100ЊC, 10min)and fractionated by GFC(Sephacryl S-200, Pharmacia,95×2:6cm using0.05M sodium acetate buffer(pH5.0containing0.01%NaN3)at aﬂow-rate of 1.8ml/min.The separations were monitored using a HP 1037A RI-Detector(30ЊC).The corresponding fractions were pooled.High molecular weight fractions were dialysed (SpectraPor3Membranes,Socochim SA,Lausanne,CH) and freeze-dried(fractions:AF1,Fig.1).2.6.Analytical methodsNeutral sugars and uronic acids of enzymatically non-treated samples were determined by GC as alditol–acetates (Blakeney,Harris,Henry&Stone,1983)and photo-metrically by the m-hydroxy-diphenyl method(Blumen-krantz&Asboe-Hansen,1973),respectively.Analysis of the glycosidic linkages in polysaccharides was performed by methylation analysis after carbodiimide-activated reduc-tion with NaBD4.Carbodiimide-activated reduction was carried out by a method developed by Kim and Carpita (1992)and modiﬁed by Wechsler(1997).Methylation analysis was performed based on Harris,Henry,Blakeney and Stone(1984)and Kvernheim(1987),as modiﬁed by Wechsler(1997).The fraction PG1GiAF1was additionally methylated using CD3I instead of CH3I.2.7.Partial acid hydrolysis of RG-IA solution of fraction PG1Ai(40mg)in2M triﬂuoroacetic acid(TFA,3ml)was treated at100ЊC for1h(An,O’Neill, Albersheim&Darvill,1994).The solvent was removed by co-distillation with2-propanol.A solution of the residue in water (1ml)was applied to a DEAE-Sepharose CL-6B column (Pharmacia,35×2:6cm :The column was eluted with water(100ml),and then with1.5%formic acid(200ml). The solvent of the acidic fraction was removed by co-distillation with2-propanol.2.8.Puriﬁcation of the acidic oligosaccharides obtained by partial acid hydrolysisThe acidic oligosaccharides were puriﬁed using a semi-preparative CarboPac PA-100column(9×250mm; Dionex).The column was eluted at4ml/min with a gradient of NaOAc in100mM NaOH as follows:0–50mM NaOAc (0–5min),50–150mM NaOAc(5–20min),150–200mM NaOAc(20–30min),350–400mM NaOAc(30–35min), 0.9mM NaOAc(35–40min).The column was re-equili-brated in100mM NaOH for15min prior to the next injec-tion.The column eluent was split,25%going through a pulsed electrochemical detector(gold working electrode) and75%collected by an automatic fraction collector (LKB Superrac2211,Pharmacia).Fractions were desalted by an anion self-regenerating supressor(ASRS-I,Dionex) and dried by co-distillation with2-propanol.2.9.Glycosyl-residue composition analysisThe sample was hydrolysed in2M TFA(1ml)at120ЊC for1h.The solvent was removed by co-distillation with2-propanol.A solution of the residue in water(1ml)was analysed using a CarboPac PA-100column(4×250mm; Dionex)equipped with a pulsed electrochemical detec-tor(gold working electrode).The column was eluted at 1ml/min with a gradient as follows:100–0mM NaOH (0–0.1min),0–100mM NaOH(0.1–20min),0–250mM NaOAc in100mM NaOH(20–50min),0.9mM NaOAc in 0.1M NaOH(50–55min).The column was re-equilibrated in100mM NaOH for15min prior to the next injection.G.R.Strasser,R.Amado`/Carbohydrate Polymers44(2001)63–7065Fig.2.GFC(Sephacryl S-300HR)of CDTA-soluble red beet pectin fractions after degradation with a puriﬁed PG.3.Results and discussion3.1.CDTA-soluble pectinsRed beet pectins isolated by CDTA from the AIR were fractionated by AEC and GFC.Besides galacturonic acid, arabinose and galactose,typical residues known to be present in side chains of pectins(arabinan,galactan,and type-II arabinogalactan)were detected in the CDTA extract and in the fractions obtained by AEC and GFC.In addition, sugars indicating the presence of RG-II such as1,3H-Api,T-Fuc,1,3,4-Fuc and1,3-Rha p were detected as well.The results of these investigations are summarised in the paper by Strasser et al.(1996).Incubation of the different chro-matographic fractions with various enzymes followed by chromatographic fractionation of the degradation products (Fig.1)led to a more detailed insight into the structure of CDTA-soluble pectic substances.3.2.Degradation by PG and fractionation by GFC Samples degraded by PG yielded three fractions each (PG1,PG2and PG3,Fig.2).PG3-fractions were analysed by HPAEC-PAD,and were shown to be composed of mono-, di-and trigalacturonic acid.This fraction was quantitatively predominant and indicated the presence of large homoga-lacturonan domains in the CDTA-soluble pectic substances of red beet.PG2-fractions contained high amounts of1,3H-Api p,T-2-O-Me-Fuc p,T-2-O-Me-Xyl p,1,3,4-Fuc p and 1,2-GlcA p,indicating the presence of an RG-II in the inter-mediate molecular weight fraction obtained by GFC(results are presented in part II of this study,Strasser&Amado`, 2000).Results of the methylation analyses of the PG1-frac-tions are presented in Table1.All fractions contain high amounts of arabinose.The high molecular weight fraction GF1PG1contains more and considerably more branched arabinans than the two fractions with lower molecular weights,GF2PG1and GF3PG1.1,3-Gal p,1,6-Gal p and 1,3,6-Gal p residues indicate the presence of type-II arabino-galactan.Smaller molecules seem to contain more type-II arabinogalactan than larger molecules.Furthermore, surprisingly high values of terminally linked glucuronic acid were detected.The results obtained with the PG1-frac-tions strongly suggest the presence of RG-I-like structures in the not or only slightly PG-sensitive part of CDTA-solu-ble pectins from red beet.The absence of1,3H-Api p,1,3,4-Fuc p and1,2-GlcA p in the PG1-fractions clearly indicate that they are not contaminated with RG-II.3.3.Degradation of PG1-fractions by arabinanase or galactanaseTreatment of the different PG1-fractions by arabinanase or by galactanase led to a release of neutral polysaccharides. Methylation analysis of fractions obtained by AEC showed similar results for the ionic fractions(Ai and Gi)compared to their educts.Only slightly smaller amounts of arabinose could be detected(results not presented).The neutral frac-tions removed by either arabinanase(An)or galactanase (Gn)showed the presence of an arabinan in all the PG1-fractions(Table1).This result suggests that some arabinans are linked directly or indirectly through a galactan to the pectic backbone.The arabinan removed by arabinanase was much more branched(resistant against further degradation) than the arabinan removed by galactanase.These results are consistent with the results published by Sakamoto and Sakai (1995),who found a fraction similar to our Gn-fraction in sugar beet pectin containing95%of arabinose.Furthermore de Vries,den Uijl,Voragen,Rombouts and Pilnik(1983) have isolated fragments of arabinogalactans with side-chains consisting of arabinans with a degree of polymerisa-tion of about25from apple pectins by treatment with a galactanase.Yamada,Kiyohara,Cyong and Otsuka(1987) found some arabinans directly attached to a1,4-linked galactan chain.For CDTA-soluble pectins from red beet the amount of galactose remained nearly unchanged.There-fore,it can be concluded that the linking galactans must be relatively short.Arabinans liberated by the arabinanase treatment contained about97mol%arabinose,thus indicat-ing that essentially no other sugars are attached to the outer regions(non reducing ends)of the arabinans.3.4.Treatment of PG1Gi-and GFxPG1Ai-fractions by an arabinofuranosidase and by a combination of arabinanase and arabinofuranosidaseDegradation by arabinofuranosidase removed most term-inally linked arabinose(Table2),whereas the linear1,5-linked arabinofuranose remained nearly unchanged.This might be because of sterical inaccessibility.Similar results are described in the literature(Cheetham,Cheung&Evans, 1993;McCleary,1989;Renard,Voragen,Thibault&Pilnik, 1991).A dramatic change was observed for the patterns of the galactose residues.Since the arabinofuranosidase removed only single arabinose residues(as shown by HPAEC-PAD)this result indicates that arabinose residues were originally linked to galactans.The sum of galactose residues remained constant and is calculated to100mol% (Table3).From Table3it can easily be concluded that the removal of arabinose leads to a decrease of1,3,6-Gal p,1,4-Gal p and1,3-Gal p residues,which became1,6-Gal p and T-Gal p,respectively.These results suggest the arabinose resi-dues in type-II arabinogalactans to be linked to the position O-3in galactans.Moreover the proportion of1,6-Gal p (outer side-chains of type-II arabinogalactan)to1,3,6-Gal p(branching points of type-II arabinogalactan)allows the estimation of an average number of residues within an outer side-chain of type-II arabinogalactan.In CDTA-solu-ble pectins of red beet approx.1.5–2.11,6-linked galactose residues are linked to approx.50%of the O-6-position of a 1,3-linked galactan chain(inner chain).The decrease of the residue1,4-Gal p is an additional clue to the existence of arabinans linked through galactans.G.R.Strasser,R.Amado`/Carbohydrate Polymers44(2001)63–70 66Most of the remaining arabinose residues were removed by simultaneous incubation with arabinanase and arabino-furanosidase(Table2).The removal of arabinose led to an enrichment of the other sugars.However,fucose,xylose, glucose and mannose were not affected,and are thus believed not to be part of the RG-I present in red beet. Rhamnose was mainly present as terminal-,1,2-or1,2,4-linked residue.The ratio of1,2,4-Rha p to arabinose was much higher than one suggesting that most O-4-positions of rhamnose are substituted by galactose.During the differ-ent enzymatic degradation steps the ratio of the backbone sugars1,2-Rha p and1,2,4-Rha p remained nearly unchanged in all samples,indicating that sugars attached to the O-4-position of rhamnose were not removed.In all samples T-Rha p was found in almost equal amounts to1,4-GlcA p suggesting a terminal disaccharide Rha p-(134)-GlcA p-(13as was described to be present in type-II arabinoga-lactan(Mollard&Joseleau,1994;Pellerin,Vidal,Williams &Brillouet,1995).Remarkably high amounts of terminally linked glucuronic acid residues were present in the enriched samples,probably attached to galactans(see below).Link-age analysis of the fraction PG1GiAF1using CD3I showed that two-thirds of the glucuronic acid residues were originally methylated at position O-4.Galacturonic acid was found in approx.equal amounts to the sum of 1,2-Rha p and1,2,4-Rha p,thus indicating the presence of an alternating rhamnogalacturonan backbone.Considerable amounts of1,3,4-GalA p were shown to be present in all samples.The almost total absence of xylose suggests that the1,3,4-GalA p-residues are not part of a xylogalacturonan. Glucuronic acid does not seem to be a substituent either(see below).Because of the remaining residues,galactose as well as arabinose might possibly be attached to the O-3-position of galacturonic acid.Both linkages are already described to be present within the plant kingdom(Guillon&Thibault, 1989;Samuelson et al.,1996).3.5.Semipreparative HPAEC-PAD fractionation and structural characterisation of the acidic material released by partial acid hydrolysis of fraction PG1GiAF1The acidic material released by partial acid hydrolysis of PG1GiAF1was isolated by AEC and then fractionated by HPAEC using a semipreparative CarboPac PA-100column (Fig.3).Some fractions were pooled,desalted using a supressor and analysed for their glycosyl residues by analy-tical HPAEC-PAD(Table4).The values for each sugar and for each pool were calculated to the corresponding concen-tration of the respective sugar at a speciﬁc retention time. These results were graphically compared with the original HPAEC-PAD-chromatogram(Fig.3).Peak A is composed of rhamnose and galacturonic acid.G.R.Strasser,R.Amado`/Carbohydrate Polymers44(2001)63–7067 Table1Glycosyl-linkage composition(mol%)of red beet RG-I-fractions obtained by enzymic treatment with PG,arabinanase and galactanase(indication of the different fractions,see text and Fig.1)PG1GF1PG1GF2PG1GF3PG1PG1Gn PG1An GF1PG1An GF2PG1n GF3PG1An T-Ara f24.326.724.724.225.638.438.838.939.3T-Ara p0.30.30.30.40.20.20.30.50.31,2-Ara f0.50.30.60.70.40.3 1.00.40.51,3-Ara f 2.2 2.5 2.1 2.0 2.1 3.2 1.6 3.4 3.51,5-Ara f18.620.819.318.944.222.022.822.622.11,2,5-Ara f 2.2 1.7 2.6 2.7 2.5 1.1 1.1 1.2 1.31,3,5-Ara f12.215.411.810.318.830.529.928.628.71,2,3,5-Ara f 2.1 2.0 1.5 1.8 1.8 3.5 3.3 2.9 3.1T-Gal p 1.8 2.7 2.6 2.10.40.10.10.10.01,3-Gal p 3.0 1.7 2.6 3.40.80.00.00.50.11,4-Gal p 3.9 5.4 4.1 3.00.40.50.20.60.51,6-Gal p 1.8 1.7 2.2 2.20.30.00.00.00.01,3,4-Gal p0.00.20.00.10.00.00.00.00.01,3,6-Gal p9.4 3.510.413.5 1.30.10.30.10.01,4-Glc p0.60.30.30.30.70.20.30.30.21,4-Man p0.00.00.00.00.00.00.30.20.3T-Rha p0.50.8 1.0 1.00.00.00.00.00.01,2-Rha p 1.5 1.5 1.3 1.30.00.00.00.00.01,2,4-Rha p 2.7 2.3 1.9 2.10.20.00.00.00.01,4-Xyl p0.20.00.10.00.10.00.00.00.01,4-GalA p 5.7 4.4 2.9 2.10.20.00.00.00.01,2,4-GalA p0.20.20.20.20.00.00.00.00.01,3,4-GalA p 1.2 1.5 1.40.90.00.00.00.00.0T-GlcA p 4.2 3.3 5.6 6.20.00.00.00.00.01,4-GlcA p0.60.40.60.70.00.00.00.00.0Total100100100100100100100100100Yield(%)13111515424201610This peak is assumed to consist of the dimer a -d -GalA p –(132)-a -Rha p ,which was conﬁrmed by the formation of peak A during hydrolysis of peak F (see below).Peaks B and C contain more or less exclusively galactose and 4-O-methylglucuronic acid.Considering the results obtained by the linkage analysis,the measured retention time and the comparison with results by An et al.(1994),led to the conclusion that these peaks contain dimers composed of 4-O-Me–GlcA–Gal.The type of linkage of these dimers remains unknown.Peaks D and E contain galactose and glucuronic acid and suggest the presence of dimers of GlcA–Gal.In addition,peak D contains some monomeric galacturonic acid,which elutes at exactly the same retention time.The surpris-ingly high amounts of glucuronic acid (peak E)and 4-O-methylglucuronic acid (peak C)can also be explained by the presence of free monomeric uronic acids that co-elute with the dimers.Peak F contains predominantly rhamnose and galacturonic paring with the results obtained by An et al.(1994),peak F couldcorrespond to the tetramer a -d -GalA p –(132)-a -Rha p –(134)-a -d -GalA p –(132)-a -Rha p .In summary,these results demonstrate that 4-O-methylglucuronic and glucuronic acid are exclusively attached to galactose.G.R.Strasser,R.Amado`/Carbohydrate Polymers 44(2001)63–7068Table 3Galactose residues of PG-treated red beet RG-I-fractions before and after arabinofuranosidase degradation (indication of the different fractions,see text and Fig.1)GF1PG1GF2PG1GF3PG1AiAF1Differ.Ai AF1Differ.Ai AF1Differ.T-Gal p 172371014491241,3-Gal p 107Ϫ31210Ϫ21812Ϫ51,4-Gal p 3533Ϫ21815Ϫ3129Ϫ31,6-Gal p 11251493828940311,2,4-Gal p 0000000001,3,4-Gal p 10Ϫ10000001,3,6-Gal p 2612Ϫ145123Ϫ285327Ϫ26Total100100100100100100Table 2Glycosyl-linkage composition (mol%)of red beet RG-I-fractions obtained by enzymic treatment with PG ϩarabinofuranosidase and PG ϩarabinofuranosidase ϩarabinanase,respectively (indication of the different fractions,see text and Fig.1)GF1PG1AiAF1GF2PG1AiAF1GF3PG1AiAF1GF1PG1AAF1GF2PG1AAF1GF3PG1AAF1T-Ara f 4.8 3.9 3.9 2.3 2.5 2.9T-Ara p 0.30.30.30.20.20.31,2-Ara f 0.7 1.1 1.70.7 1.0 1.11,3-Ara f 4.4 3.1 2.1 1.6 1.2 1.91,5-Ara f 17.410.5 4.4 1.4 1.4 3.91,2,5-Ara f 1.6 1.70.00.00.30.31,3,5-Ara f 2.2 1.60.90.40.00.81,2,3,5-Ara f 0.00.10.10.00.00.1T-Fuc p 0.00.10.00.20.10.0T-Gal p 8.0 6.67.210.88.28.31,3-Gal p 2.4 4.67.1 3.9 6.1 6.91,4-Gal p 11.17.0 5.214.58.0 5.41,6-Gal p 8.517.322.912.120.323.21,2,4-Gal p 0.00.00.00.00.00.71,3,6-Gal p 3.910.715.5 5.512.515.91,4-Glc p 0.60.40.30.30.70.51,6-Man p 0.00.00.00.00.00.2T-Rha p 1.1 1.5 1.7 1.2 1.9 1.91,2-Rha p 2.8 2.9 1.7 4.6 2.9 1.41,3-Rha p 0.30.00.00.00.00.01,2,3-Rha p 0.20.00.00.00.00.01,2,4-Rha p 8.1 6.1 4.710.48.1 4.41,2,3,4-Rha p 0.20.10.10.60.20.1T-Xyl p 0.20.10.00.00.10.01,4-Xyl p 0.20.10.10.20.00.0T-GalA p 0.00.00.00.50.00.01,4-GalA p 8.6 5.0 3.411.1 5.4 3.21,2,4-GalA p 0.50.40.20.60.50.21,3,4-GalA p 3.7 2.6 1.9 5.1 3.2 2.01,4,6-GalA p 0.00.00.00.50.00.0T-GlcA p 7.310.912.810.013.512.91,4-GlcA p 0.8 1.4 1.7 1.1 1.7 1.7Total 100.0100.0100.0100.0100.0100.0Yield (%)555460878079Oligosaccharides containing 4-O-methylglucuronic acid or glucuronic acid do not contain galacturonic acid or rhamnose.4.ConclusionIncubation of CDTA-soluble pectic substances of red beet with a PG yielded an enzyme-resistant fraction.Linkage analyses indicated the presence of an RG-I.The ratio of rhamnose to galacturonic acid and the results obtained using partial hydrolysis and semipreparative HPAEC-PAD suggest the presence of an alternating rhamnogalacturonan backbone.This backbone is highly ramiﬁed,since two-thirds of the rhamnose residues are branched.Side chains consisting of arabinans,galactans and type-II arabinogalac-tans are attached to the RG-backbone.Arabinans have been shown to have an a -(135)-linked backbone.Single arabinose residues or small oligoarabinans are attached to the backbone at the O-3-position,and to a lower extent at the O-2-position.Some of the arabinans are indirectly attached through small galactans to the pectic backbone.Short b -(134)galactan chains have been shown to occur in red beet RG-I as well.Most of them are attached directly to the pectic backbone.Type-II arabinogalactans are the most complex side chains within red beet RG-I pectins.They consist of anG.R.Strasser,R.Amado `/Carbohydrate Polymers 44(2001)63–7069Table 4Monosaccharide composition of acidic material released from a PG ϩgalactanase ϩarabinofuranosidase-treated red beet RG-I-fraction after partial acid hydrolysis and HPAEC-PAD fractionation (indication of the different fractions,see text)Pool number P1P2P3P4P5P6P7P8P9P10P11P12P13P14Rha 6550010000000072Gal142828183166641410141937166Me-GluA 081634330164011111GalA 18420010035100048GlcA 00112111471559101Total391324422789781212022359727129Total/fract.1)204444117849277571232522Num./fract.2)23221233433356Fig.3.HPAEC-PAD chromatograms of acidic material released from a (PG ϩgalactanase ϩarabinofuranosidase)-treated red beet RG-I-fraction after partialacid hydrolysis (bold line),compared to calculated sugar concentrations at speciﬁc retention times (thin lines).inner chain composed of b-(133)-linked galactose residues.Approximately50%of the galactose residues are substitutedat the O-6-position by short outer side chains consisting of b-(136)-linked galactose residues(average:1–3resi-dues).Some of these outer side chains are substituted byterminally linked glucuronic acid.Further work is needed toﬁgure out the type and position of the glycosidic linkagebetween glucuronic acid and galactose(a-or b-,(133)-,(134)-or(136)-linked,respectively).The resultsobtained in this study suggest the presence of at least twodifferently linked glucuronic acid residues in red beet RG-I.Approximately65%of the glucuronic acid residues aresubstituted by a methyl ether group and approx.10%aremost probably substituted by terminally linked rhamnoseresidues.Further work is needed to elucidate theﬁne struc-ture of red beet RG-I.AcknowledgementsWe thank Novo-Nordisk A/S,(Bagsvaerd,DK)for thegenerous gift of enzymes.ReferencesAlbersheim,P.,Darvill,A.G.,O’Neill,M.A.,Schols,H.A.,&Voragen,A.G.J.(1996).An hypothesis:the same six polysaccharides arecomponents of the primary cell walls of all higher plants.In J.Visser &A.G.J.Voragen,Pectins and pectinases(pp.47–55).Amsterdam: Elsevier.An,J.,O’Neill,M.A.,Albersheim,P.,&Darvill,A.G.(1994).Isolation and structural 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McCleary,B.V.(1989).Novel and selective substrates for the assay of endo-arabinanase.In G.O.Phillips,P.A.Williams&D.J.Wedlock Gums and stabilisers for the food industry(pp.291–298).vol.5.Oxford:Oxford University Press.Mollard,A.,&Joseleau,J.-P.(1994).Acacia senegal cells cultured in suspension secrete a hydroxyproline-deﬁcient arabinogalactan-protein.Plant Physiology and Biochemistry,32,703–709.Pellerin,P.,Vidal,S.,Williams,P.,&Brillouet,J.-M.(1995).Character-ization ofﬁve type II arabinogalactan-protein fractions from red wine of increasing uronic acid content.Carbohydrate Research,277,135–143. Renard,C.M.G.C.,Voragen,A.G.J.,Thibault,J.-F.,&Pilnik,W.(1991).Comparison between enzymatically and chemically extracted pectins from apple cell walls.Animal Feed Science and Technology,32,69–75. Sakamoto,T.,&Sakai,T.(1995).Analysis of structure of sugar-beet pectin by enzymatic methods.Phytochemistry,39,821–823. Samuelson,A.B.,Paulsen,B.S.,Wold,J.K.,Otsuka,H.,Kiyohara,H., Yamada,H.,&Knutsen,S.H.(1996).Characterisation of a biologically active pectin from Plantago major L.Carbohydrate Polymers,30,37–44.Strasser,G.R.,&Amado`,R.(2000).Pectic substances from Red Beet(Beta vulgaris conditiva).Part II.Structural characterisation of rhamnogalac-turonan II(in preparation).Strasser,G.R.,Wechsler,D.E.,&Amado`,R.(1996).Structural features of pectic polysaccharides of red beet(Beta vulgaris conditiva).In J.Visser &A.G.J.Voragen,Pectins and pectinases(pp.631–636).Amsterdam: Elsevier.de Vries,J.A.,den Uijl,C.H.,Voragen,A.G.J.,Rombouts,F.M.,& Pilnik,W.(1983).Structural features of the neutral sugar side chains of apple pectic substances.Carbohydrate Polymers,3,193–205. Wechsler D.E.(1997).Charakterisierung der Struktur von Pektinen wa¨hrend der Reifung und Lagerung von A¨pfeln.PhD thesis.ETH No.12044,Zurich.Yamada,H.,Kiyohara,H.,Cyong,J.-C.,&Otsuka,Y.(1987).Structural characterization of an anti-complementary arabinogalactan from the roots of Angelica acutiloba Kitagawa.Carbohydrate Research,159, 275–291.G.R.Strasser,R.Amado`/Carbohydrate Polymers44(2001)63–70 70。

超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床价值

·临床研究·超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床价值韩小军袁理郭道宁摘要目的探讨超声引导下微波消融联合贝伐珠单抗治疗晚期结肠癌伴肝转移的临床应用价值。

方法选取在我院就诊的102例晚期结肠癌伴肝转移患者，按随机数字表法分为观察组和对照组各51例，对照组采用贝伐珠单抗联合常规化疗治疗，观察组在此基础上采用超声引导下微波消融治疗；比较两组患者治疗后疗效、免疫功能、不良反应及预后情况。

结果治疗后，观察组客观缓解率（ORR）、疾病控制率（DCR）均高于对照组（均P<0.05）；两组CD3+、CD4+、CD8+均较治疗前下降，且观察组CD3+、CD4+、CD4+/CD8+均高于对照组，CD8+低于对照组，差异均有统计学意义（均P<0.05）。

治疗后，两组胃肠道反应、食欲减退、疲劳乏力等不良反应比较差异均无统计学意义；观察组累积无复发生存率及累积总生存率分别为78.77%、57.45%，均高于对照组（49.32%、34.23%），差异均有统计学意义（χ2=10.086、4.536，P=0.001、0.033）。

结论超声引导下微波消融联合贝伐珠单抗能提高晚期结肠癌伴肝转移患者的治疗效果，缓解免疫功能抑制，改善生存状况，具有较好的临床应用价值。

关键词超声引导；微波消融；结肠癌，晚期；肝转移；贝伐珠单抗［中图法分类号］R445.1［文献标识码］AClinical value of ultrasound-guided microwave ablation combined withbevacizumab in the treatment of advanced colonadenocarcinoma with liver metastasisHAN Xiaojun，YUAN Li，GUO DaoningDepartment of Ultrasound Medicine，Mianyang Hospital Affiliated to School of Medicine，University of Electronic Science andTechnology of China，Sichuan621000，ChinaABSTRACT Objective To explore the application clinical value of ultrasound-guided microwave ablation combined with bevacizumab in the treatment of advanced colon adenocarcinoma（COAD）with liver metastasis.Methods A total of102 patients with advanced COAD with liver metastasis treated in our hospital were selected，and divided into the observation group and the control group by random number table method，with51cases in each group.The control group was treated with bevacizumab combined with conventional chemotherapy.On this basis，the observation group was treated with ultrasound-guided microwave thermal ablation.The curative effect，immune function，adverse reactions and prognosis after treatment of the two groups were compared.Results After treatment，the objective remission rate（ORR）and disease control rate（DCR）in the observation group were higher than those in the control group（both P<0.05）.After treatment，the CD3+，CD4+and CD4/CD8+in the observation group were higher than those in the control group，and CD8+was lower than that in the control group，the differences were statistically significant（all P<0.05）.After treatment，there were no statistically significant difference in the incidence rates of adverse reactions such as gastrointestinal reactions，loss of appetite and fatigue between the two groups.The cumulative recurrence-free survival rate and cumulative overall survival rate in observation group were78.77%and57.45% respectively，which were significantly higher than those in control group（49.32%and34.23%），the differences were statistically significant（χ2=10.086，4.536，P=0.001，0.033）.Conclusion Ultrasound-guided microwave ablation combined with作者单位：621000四川省绵阳市，电子科技大学医学院附属绵阳医院绵阳市中心医院超声医学科（韩小军、郭道宁），肿瘤科（袁理）通讯作者：郭道宁，Email：******************结肠癌是常见的消化道肿瘤，近年来其发病率和死亡率均逐渐升高。

Carbohydrate Research 371(2013)32-39

NoteSynthesis of Fondaparinux:modular synthesis investigation for heparinsynthesisFeng Lin a ,b ,⇑,Gaoyan Lian b ,Ying Zhou ba State Key Laboratory of New Drugs &Pharmaceutical Process,Shanghai Institute of Pharmaceutical Industry,1111Zhongshanbeiyi Road,Shanghai 200437,China bState Key Laboratory of Bio-organic and Natural Products Chemistry,Shanghai Institute of Organic Chemistry,CAS,345Linlin Road,Shanghai 200032,Chinaa r t i c l e i n f o Article history:Received 22November 2012Received in revised form 4January 2013Accepted 6January 2013Available online 16January 2013Keywords:FondaparinuxModular synthesis L -Idopyranosyl donora b s t r a c tThe anti-thromboembolic pentasaccharide Fondaparinux was synthesized in 36steps for the longest lin-ear route,with 0.017%overall yield from D -glucose.Only three kinds of protecting groups were used for hydroxyl protection,Bn,Ac,and Bz,to accomplish this complex synthesis without decreasing the syn-thetic efﬁciency.Three L -idopyranosyl donors were investigated.Thioethyl glycoside is an efﬁcient donor for L -idopyranosyl glycosylation with full a -selectivity,while L -idopyranosyl trichloroacetimidate resulted in poor a /b selectivity.A practical synthesis of key intermediate 1,6-anhydro-L -idopyranose 17by H +/b -CD catalyst was developed.Ó2013Elsevier Ltd.All rights reserved.1.IntroductionHeparin and heparin sulfate (HS)are linear polysaccharides consisting of uronic acid-(1,4)-a -glucosamine repeating disaccha-ride subunits.Variable patterns of substitution (O -sulfate,N -sul-fate,and N -acetyl)of the disaccharide subunits give rise to a large number of complex sequences,rendering them the most complex nature-occurring biopolymers.Heparin and HS play cru-cial roles in numerous biological systems through their interaction with diverse proteins.1Heparin is used in clinic as an anticoagulant drug for over 70years owing to its high afﬁnity binding with antithrombin III.2Its synthetic derivative,Fondaparinux (1,trade name:Arixtra Ò)is the ﬁrst in a new class of selective factor Xa inhibitors.By selec-tively binding to antithrombin,it induces a conformational change that speciﬁcally increases by about 340-fold the natural neutraliza-tion of factor Xa by antithrombin,resulting in dose-dependent inhibition of factor Xa but no effect on thrombin.3Unlike unfrac-tionalized heparin,Fondaparinux does not bind to platelets or platelet factor 4and less likely to induce thrombocytopenia.4The synthetic route of Fondaparinux developed by Sanoﬁ-Aven-tis compromised more than 50steps,5and the annual global sales of Arixtra Òwere £2.54m in 2009.6Concerning this complex and synthetic-challenging molecule,chemical community has devel-oped some interesting strategies,such as modular synthesis 7and late-stage oxidation to introduce uronic acid structure.8This synthesis of Fondaparinux was investigated as model study for modular synthesis of heparin/HS.We investigated some strate-gies and developed an efﬁcient synthesis of GH disaccharide unit,using ethylthio–idosyl donor with superior a -stereoselectivity than that of corresponding imidate donor;we also applied TEM-PO–Ca(ClO)2selective oxidation method 9to introduce uronic acid structure at disaccharide stage.Our work was reported herein.2.Results and discussionProtection strategy plays an important role in heparin-HS syn-thesis.Though some new protecting groups were introduced for modular synthesis strategy,we found that the simplest way worked well and was proved effective in several heparin-HS syn-theses:3acyl groups were used for those hydroxyl groups which will be sulfonated,while benzyl groups for those will not and azido group was the protecting form for amino group.This simple and uniﬁed design seemed quite limited in applications,but we proved here that combining sophisticated selective protection–deprotec-tion techniques,this protection strategy was of great efﬁciency and ﬂexible in heparin synthesis,and is valuable in modular syn-thesis researches.For the strategy of building blocks,we preferred using disaccha-rides with glucosamino unit on the reducing end.10The retro-syn-thetic analysis is shown in Figure 1.Uronic units were introduced at the disaccharide level to avoid the glycosylation with the usually inactive uronate derivatives.Compounds 511and 1212were prepared starting from D -glu-cose,and disaccharide 6was synthesized from cellobiose in 13steps with 12%yield (Scheme 1).130008-6215/$-see front matter Ó2013Elsevier Ltd.All rights reserved./10.1016/j.carres.2013.01.003Corresponding author.Tel.:+862165310547;fax:+862165169893.E-mail address:linfeng@ (F.Lin).The epoxide15was prepared from D-glucose in5steps with 56%yield.14Hung and co-workers reported the key intermediate 1,6-anhydro-L-idopyranose17,8a but its large scale preparation was difﬁcult.Because the preparation of expensive L-idopyranose intermediates was the bottle-neck for heparin synthesis,we tried to develop a practical preparation method for1,6-anhydro-idose 17from epoxide15in one step.Hydrolysis of15in various acids(HCl,TsOH,HOAc,or H2SO4) and solvents(EtOH,MeOH,or H2O)at reﬂuxing temperature was screened.With0.4M H2SO4and EtOH/H2O=1/5(v/v)mixed solvent,the best yield(17%)of17was obtained,while the major product was idopyranose16,accompanied with some black solid which might come from carbonization of the substrate.Then sev-eral phase transfer catalysts were screened to avoid the formation of black solid,in order to improve the yield of17.Quaternary ammonium salt(TBAB,Aliquat336)gave no improvement,while addition of0.1equiv of b-CD improved the yield of17to37%. The H2SO4adding process affected the substrate solubility greatly, which in turn affected the yield of17.At60°C,b-CD in hot0.4M H2SO4solution was added into the17/EtOH solution,then the reac-tion temperature was raised rapidly to90°C.By this process,the black solid did not form,the system kept a clear solution to theF.Lin et al./Carbohydrate Research371(2013)32–3933end,and the yield of17raised to65%.The by-product16can be transferred to17under a second run of the same condition.Thus, the total yield of17with two runs was improved to80%.This method is a good supplement for Hung’s preparation for17 (Scheme2).The catalysis mechanism of b-CD was not clear yet.The yield of 17was around20%in the absence of b-CD,which must serve more than PTC reagent,because other PTC reagent(such as quaternary ammonium salt)showed no improvement.If H2SO4was absent while all other conditions were the same,only50%epoxide open-ing product19was obtained,which indicated that hydrogen bond (HB)network in the b-CD cage could catalyze epoxide opening.Be-cause17could be prepared from16,the HB network of b-CD may induce the conformation change of idosyl ring16and stabilize its 4C1conformer which beneﬁts the formation of1,6-anhydro-idose.After selective benzoylation of the2-OH,the product18was acetolyzed with catalyst TESOTf to provide the ring-opening ace-tates9(a/b mixture,72%two steps).2.1.Investigation of three L-idopyranosyl donorsOwing to Petitou and co-workers’systematic study of various L-iduronic acid glycosyl donors,most of the heparin synthesis used L-iduronate donors.Because L-idosyl donor is more active than corre-sponding L-iduronate donor,we studied on L-idosyl donors to eval-uate their application in modular synthesis.Three donors were investigated:trichloroacetimidate,thioglycoside,and acetate.The glycosylation of idopyranosyl acetate9with glucoside12 required0.5equiv of TMSOTf,(Scheme3)and the optimized yield of21a is only60%.Although the a-selectivity is great,the strong acidic condition caused a remarkable decomposition of the sub-strate.This indicated that the idopyranosyl acetate is not an active donor,the strong acidic reaction condition limits its application in modular synthesis.Then trichloroacetimidate10was investigated.The yields of selective anomeric Ac deprotecting by benzylamine or hydra-zine–HOAc were moderate,because6-O-Ac was likely cleaved un-der these conditions.By changing THF/MeOH solvent ratio,which in turn changed the saturated concentration of ammonia in the mixed solvent at0°C,we selectively deprotected the anomeric Ac to give20in90%yields.Further treatment with CCl3CN and K2CO3provided trichloroacetimidate10.Glycosylation of10with 12under the promotion of0.1equiv TMSOTf at0°C provided 21a/b in96%yield,which was inseparable by TLC or column chro-matography;and the1H NMR was complicated to tell a/b ratio.With0.2M anhydrous HCl/methanol,40and60-O-Ac was selec-tively cleaved in94%yield,with6-O-Bz intact.After deacetylation, 7a/b could be separated by column chromatography,and a/b ratio was3:2.By this two-step determination method,we found that when the glycosylation temperature wasÀ20°C,the ratio of a/b was2:1.We proposed that the idosyl trichloroacetimidate10 was an active donor,so that some pathway without NGP(neighbor group participation)effect occurred,which resulted in b-isomer.By lowering the reaction temperature,the reactivity of donor de-ceased and NGP effect increased,resulting in improved a-selectiv-ity.Hung and co-workers reported good a-selectivity of idosyl trichloroacetimidate glycosylation atÀ78°C,8a,14which was in accordance with our observation.Then donor thioglycoside11was investigated,which was ex-pected to be moderate and undergo NGP pathway to ensure high a-selectivity.Thioglycoside11was prepared from9in85%yields, and the glycosylation of11with12gave85%of21a with full a paring with iduronate thioglycosides,which were poor donors12b due to the electron-withdrawn effect of carboxyl group at5-position,L-idopyranosyl thioglycoside11was a donor with good synthetic efﬁciency and great a-glycosylation selectivity (Table1).To test its donor property,11was also reacted with22and24, the good yield and a-glycosylation selectivity proved thioethyl gly-coside11is an excellent L-idopyranosyl donor.2.2.Preparation of uronate at disaccharide stageby TEMPO–Ca(OCl)2selective oxidationWe employed TEMPO(2,2,6,6-tetramethylpiperidine-1-oxyl)–KBr–Ca(OCl)2system as an effective selective oxidation method to prepare the uronate disaccharide synthons.The reaction was very sensitive to pH value.The traditional TEMPO–KBr–NaOCl method requires strictly in situ pH control of the reaction mixture, which makes the procedure complex and sometimes less repro-ducible.We made a change that Ca(OCl)2was added to Na2CO3–NaHCO3buffer to produce NaOCl as a terminal ing solid Ca(OCl)2could avoid the dilution of the reaction system and Na2CO3–NaHCO3buffer also help to maintain pH value at8–9.5. This practical improvement showed its convenience in our synthe-sis.After methylation by CH2N2,uronate26was obtained in73% yield.The trisaccharide3was prepared according to the literature in68%yield(three steps)13(Scheme4).Selective oxidation of7a with the same oxidation condition with Bu4NBr as PTC reagent,provided54%of4.When Bu4NBr was replaced by Aliquat336(C8H17)3NCl),the yield was increased to93%.This result showed that the choice of PTC reagent should be considered in TEMPO selective oxidation.Coupling of3with4under the promotion of TMSOTf provided2 with a selectivity in78%yield(H-1000at5.10ppm with J value of 3.6Hz,C-1000at97.49ppm).Then the acyl group and the methyl es-ter of2were deprotected to afford28in65%yields.The free OH group of28was sulfated with triethylamine-sulfur trioxide com-plex.The azido and the OBn groups were hydrogenolysed with Pd/C and then the resulting amino groups were selectively sulfated34 F.Lin et al./Carbohydrate Research371(2013)32–39with pyridine–sulfur trioxide complex to furnish functionalized Fondaparinux (1)in 56%yield (Scheme 5).3.ConclusionThe anti-thromboembolic pentasaccharide Fondaparinux (1)was synthesized in 36steps for the longest linear route,with 0.017%overall yield from D -glucose and cellobiose.Only three kinds of protecting groups were used for hydroxyl protection,Bn,Ac,and Bz,to accomplish this complex synthesis without decreas-ing the synthetic efﬁciency.This work proved that this simple and uniﬁed protection strategy,when combined with sophisticated selective protection–deprotection techniques,was valuable for heparin modular synthesis.We also studied the modular synthesis of disaccharide building blocks,especially L -idopyranosides.The synthesis of key intermediate 1,6-anhydro-idopyranose 17was im-proved by H +/b -CD catalyst.Three L -idopyranosyl donors were investigated.Thioethyl glycoside is an efﬁcient donor for L -idopyr-anosyl glycosylation with full a -selectivity,while the a /b selectiv-ity from L -idopyranosyl trichloroacetimidate glycosylation should be carefully investigated.TEMPO–Ca(ClO)2selective oxidation is a practical method for disaccharide uronate building blocks,and the choice of PTC should be concerned.Thus we investigated and developed some synthetic methods for heparin modular synthesis.4.Experimental 4.1.General methodsReagents and solvents were of reagent grade and used without further puriﬁcation.Methylene dichloride was distilled from cal-cium hydride.All reactions involving air or moisture sensitive re-agents or intermediates were performed under a nitrogen or argon atmosphere.Analytical TLC was visualized by irradiation (254nm)or by staining with H 2SO 4/methanol solution.1H and 13C NMR spectra were obtained using 300MHz Brucker AM300.High resolution mass spectra (MALDI/DHB)were performed on IonSpec 4.7Tesla FTMS from Varian.4.2.3-O -Benzyl-1,6-anhydro-b -L -idopyranose (17)At 60°C,b -CD (1.1g,0.1equiv)in 0.4M sulfuric acid solution (50mL)was added rapidly into 10mL ethanol solution of compound 158a (2.74g,9.4mmol),then heated to 90°C for 9h.Then it was cooled to 0°C and neutralized with Na 2CO 3,concentrated in vacuo,and puriﬁed by column chromatography (CH 2Cl 2/EA =2:1).Compound 17(1.543g,65%yield)and 16(0.75g,30%)as syrup were obtained.By applying this reaction condition to compound 16,additional 0.349g of 17could be obtained.The combined yield of 17is 80%.R f =0.71(PE/EA =1:2).1H NMR (300MHz,CDCl 3):d 7.39–7.26(m,5H,ArH),5.30(d,1H,J =2.1Hz,H-1),4.96(d,1H,J =11.7Hz,PhCH 2),4.74(d,1H,J =11.7Hz,PhCH 2),4.43(t,1H,J =4.5Hz,H-5),4.03(d,1H,J =7.2Hz,H-6a),3.87(m,1H,H-4),3.73(m,1H,H-6b),3.66(dd,1H,J =1.8,7.5Hz,H-2),3.39(t,1H,J =7.8Hz,H-3).13C NMR (75MHz,CDCl 3):d 138.4,128.7,128.0,127.9,101.8,84.2,75.4,75.0,74.5,71.1,65.1.ESI/MS:275.1(M+Na +).Anal.Calcd for C 13H 16O 5:C,61.90;H,6.39.Found:C,61.73;H,6.47.4.3.2-O -Benzoyl-3-O -benzyl-1,6-anhydro-b -L -idopyranose (18)At 0°C,to 17(690mg, 2.7mmol)and pyridine (0.65mL,8.2mmol)in 10mL CH 2Cl 2,was added BzCl (0.4mL,3.56mmol).After 4h,concentrated and puriﬁed by column chromatography (hexane/EtOAc =2:1)to obtain 18(0.89g,91%yield)as white solid.R f =0.36(PE/EA =2:1).1H NMR (300MHz,CDCl 3):d 8.06(dd,2H,Table 1Glycosylation with L -idopyranosyl thioglycoside 11AcceptorProductYield92%,a only95%,a only 15F.Lin et al./Carbohydrate Research 371(2013)32–3935J=1.3,7.4Hz,BzH),7.57(t,1H,J=7.4Hz,BzH),7.46(t,2H,J=7.6Hz, BzH),7.26(m,5H,ArH),5.53(d,1H,J=1.4Hz,H-1),5.07(dd,1H, J=1.5,8.1Hz,H-2),4.80(d,1H,J=11.5Hz,PhCH2),4.65(d,1H, J=11.5Hz,PhCH2), 4.51(t,1H,J=4.6Hz,H-5), 4.15(d,1H, J=7.5Hz,H-6a),3.87(t,1H,J=8.2Hz,H-3),3.76(dd,1H,J=4.6, 7.5Hz,H-6b).13C NMR(75MHz,CDCl3):d165.58,137.78,133.24, 129.75,129.26,128.25,128.20,127.78,127.64,99.37,80.20,76.68, 75.03,74.55,71.28,65.20.ESI-MS(m/z):379.25[M+Na+].Anal.Calcd for C20H20O6:C,67.41;H,5.66.Found:C,67.18;H,5.74.4.4.1,4,6-Tri-O-acetyl-2-O-benzoyl-3-O-benzyl-L-idopyranose(9)Under N2at0°C,TMSOTf(0.15mL,0.1equiv)was added into 18(2.3g, 6.5mmol)in14mL Ac2O.After30min,the reaction was quenched with NEt3at0°C,then20mL MeOH was added and stirred for30min,concentrated and puriﬁed by column chro-matography(hexane/EtOAc=4:1)to obtain a/b mixture9(2.86g, 88%yield)as white syrup.9a:R f=0.79(PE/EA=2:1)1H NMR(300MHz,CDCl3):d8.12(m, 2H,BzH),7.60(t,1H,J=7.5Hz,BzH),7.46(t,2H,J=7.8Hz,BzH), 7.37–7.26(m,5H,ArH),6.20(d,1H,J=1.2Hz,H-1),5.30(m,1H, H-2), 4.93(t,1H,J=2.1Hz,H-4), 4.78(dd,2H,J=12.3Hz, PhCH2–), 4.47(m,1H,H-5), 4.27(m,2H,H-6), 4.00(t,1H, J=3.0Hz,H-3),2.07,1.92(s each,3H each for acetyl).9b:R f=0.82(PE/EA=2:1)1H NMR(300MHz,CDCl3):d8.07(m, 2H,BzH),7.59(t,1H,J=7.5Hz,BzH),7.46(t,2H,J=7.8Hz,BzH), 6.22(s,1H,H-1),5.21(m,1H,H-2),4.99(br,1H,H-4),4.81(dd, 2H,J=12.3Hz,PhCH2–),4.61(m,1H,H-5),4.31(m,2H,H-6), 3.90(br,1H,H-3),2.09(s,6H,Ac),1.94(s,3H,Ac).13C NMR(/mixture)(75MHz,CDCl3):d170.6,170.0,168.9,168.8, 165.5,136.9,136.6,130.0,129.9,128.6,128.5,127.8,127.5,91.5, 90.5,73.7,73.2,72.5,72.0,71.8,66.9,66.5,66.2,66.0,65.7,62.4, 62.3,21.0,20.9,20.8,20.7.ESI-MS(m/z):518.4[M+NH4+],523.3 [M+Na+].Anal.Calcd for C26H28O10:C,62.39;H,5.64.Found:C, 62.33;H,5.74.IR(cmÀ1)m max=1745,1371,1224,1114,715.4.5.Ethyl4,6-di-O-acetyl-2-O-benzoyl-3-O-benzyl-1-thio-a/b-L-idopyranoside(11)Under N2at0°C,BF3ÁEt2O(0.09mL,1.1equiv)was added into9 (281mg,0.56mmol)and EtSH(0.063mL,0.84mmol)in dry4mL CH2Cl2,after3h,quenched with NEt3at0°C.The solution was diluted with CH2Cl2and washed with aq NaHCO3and brine,dried and con-centrated and puriﬁed by column chromatography(hexane/ EtOAc=4:1)to obtain a/b mixture11(232mg,81%yield)as white solid.R f=0.78(PE/EA=2:1),1H NMR(a/b=1:1.5)(300MHz,CDCl3): d8.18–8.07(m,5H,BzH),7.57(m,2.5H,BzH),7.47–7.32(m,17.5H, ArH),5.44(s,1H),5.27(t,1H,J=1.5Hz),5.22(m,1.5H),5.13(d, 1.5H,J=1.8Hz),4.92–4.68(m,10H),4.25(m,5H),3.97(t,1.5H, J=3.0Hz),3.82(m,1H),2.78(m,3H),2.71–2.66(m,2H),2.06(s, 7.5H,Ac),1.97(s,3H,Ac),1.79(s,4.5H,Ac),1.34(m,7.5H,CH3).13C NMR(a/b mixture)(75MHz,CDCl3):d170.6,170.1,170.0,165.7, 165.5,165.1,137.1,133.5,129.9,129.8,129.5,128.4,128.3,128.2, 127.9,127.8,127.6,82.6,81.4,72.9,72.7,72.6,72.5,72.0,71.9,69.4, 68.9,67.2,66.0,64.0,62.8,62.7,26.7,25.9,20.7,20.5,15.1,15.0.HRMS (ESI)Calcd for C26H30O8SNa525.1554.Found525.1561;IR(cmÀ1) m max=2929,1743,1724,1453,1267,1111,1070,1027,713.4.6.4,6-Di-O-acetyl-2-O-benzoyl-3-O-benzyl-a/b-L-idopyranose(20)At0°C,9(472mg)in THF–MeOH mixed solvent(8:2,v/v)was bubbled with ammonia till saturation,and stirred for30min at 0°C.TLC showed most of9converted.Concentrated and puriﬁed by column chromatography(hexane/EtOAc=4:1)to obtain a/b mix-ture20(389mg,90%yield)as white syrup.1H NMR(a/b mixture, 300MHz,CDCl3):d8.17–7.27(m,10H,Ar),5.28(s,1H),5.13and 4.95(AB,2H,C H2Ph),4.82(br s,1H),4.78(m,1H),4.62(m,1H), 4.35(m,1H),4.30–4.19(m,2H),4.05(br,1H),2.09(s,3H,OMe), 1.89and1.83(2s,3H total,OMe).13C NMR(a/b mixture,75MHz, CDCl3)anomer A:d92.69,73.64,71.07,66.41,65.78,62.90,62.73; anomer B:d91.81,73.02,72.0,68.53,66.01,62.65;170.75,170.10, 165.60,165.12,20.80,20.60.ESI-MS(m/z):481.30[M+Na+].HRMS (ESI)Calcd for C24H26O9Na481.1469.Found481.1467;IR(cmÀ1)m 3345,1745,1365,1227,1111,1070,715.4.7.Methyl O-(2-O-benzoyl-3-O-benzyl-a-L-idopyranosyl)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-D-glucopyranoside(7a)and methyl O-(2-O-benzoyl-3-O-benzyl-b-L-idopyranosyl)-(1?4)-2-azido-6-O-benzoyl-2-O-benzyl-2-deoxy-a-D-glucopyranoside(7b)100mg K2CO3,20(156mg,0.34mmol),and Cl3CCN(0.1mL, 1mmol)in1mL CH2Cl2were stirred for2h.Concentrated and puriﬁed by column chromatography(hexane/EtOAc=4:1)to36 F.Lin et al./Carbohydrate Research371(2013)32–39obtain a/b mixture10(166mg,81%yield)as white syrup.R f=0.5 (hexane/EtOAc=3:1).Under Argon,4ÅMS,donor10(190mg,0.32mmol,1.2equiv) and acceptor12(108mg,0.26mmol)in2mL dry CH2Cl2were cooled toÀ20°C and0.09M TMSOTf/CH2Cl2(0.3mL,0.1equiv) was added.After30min,moved to RT and stirred till the reaction completed.Quenched with NEt3,ﬁltered and concentrated,and puriﬁed by column chromatography(hexane/EtOAc=4:1)to ob-tain a/b mixture21(216mg,96%yield)as white syrup.Under Argon at0°C,a/b mixture21(94mg,0.11mmol)in3mL MeOH was added AcCl0.07mL(to prepare0.2M HCl/MeOH),then stirred at RT for3h.Neutralized with NaHCO3,ﬁltered and concen-trated,and puriﬁed by column chromatography(hexane/ EtOAc=2:1)to obtain7a(53mg,63%yield)and7b(26mg,31% yield)as white syrup.7a R f=0.48(hexane/EtOAc/CH2Cl2=1:1:1).½a 25D+45.8(c1.0, CHCl3),1H NMR(300MHz,CDCl3):d7.97–7.25(m,20H,Ar),5.54 (d,J=2.1Hz,1H),5.40(br s,1H),5.02(t,J=3Hz,1H),4.90–4.75 (m,4H), 4.59–4.46(m,2H), 4.16(m,1H), 4.03–3.97(m,4H), 3.57–3.44(m,5H),3.25(m,1H),3.05(m,1H).13C NMR(75MHz, CDCl3),d165.86,165.22,137.50,137.37,133.47,133.06,129.74–127.83(Ar),98.34(C-1),97.76(C-10a),79.09,75.57,75.32,73.87, 72.43,69.40,68.21,67.83,67.21,64.08,62.75,62.55,55.34. ESI-MS(m/z):792.274[M+Na+](Calcd792.274).Anal.Calcd for C41H43N3O12:C,63.97;H,5.63;N,5.46.Found:C,63.74;H, 5.39;N, 5.25.IR(cmÀ1)m max=3480,2103,1722,1270,1101, 1027.7b R f=0.26(hexane/EtOAc/CH2Cl2=1:1:1).13C NMR(75MHz, CDCl3),d166.30,165.16,137.81$127.75(Ar),107.78(C-10b), 98.35(C-1),81.58,80.40,80.26,79.08,76.37,75.85,75.07,71.73, 70.28,68.79,63.54,60.31,55.22.ESI-MS(m/z):792.27[M+Na+]. IR(cmÀ1)m max3465,2101,1722,1270,1101,1027.4.8.Methyl O-(4,6-di-O-acetyl-2-O-benzoyl-3-O-benzyl-a-L-idopyranosyl)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-D-glucopyranoside(21a)Under Argon,4ÅMS,donor11(0.74g,1.46mmol,1.2equiv) and acceptor12(0.50g,1.22mmol)in20mL dry CH2Cl2were cooled toÀ15°C,NIS(384mg,1.2equiv)and AgOTf/dry toluene (43mg,0.14equiv)solution(2mL)were added successively.After 30min,moved to RT and stirred till the reaction completed. Quenched with NEt3,ﬁltered and concentrated,and puriﬁed by column chromatography(hexane/EtOAc=4:1)to obtain21a (910mg,86%yield)as colorless oil.½a 25D+49.1(c1.1,CHCl3),1H NMR(300MHz,CDCl3):d8.10–7.97(m,4H),7.57–7.25(m,16H, Ar),5.28(d,J=2.5Hz,1H),5.19(br s,1H),4.91–4.67(m,7H), 4.49(dd,1H),4.03–3.91(m,4H),3.85(m,1H),3.53–3.47(2s,3H, OMe),3.47–3.41(m,1H),1.99,1.92(s,3H each,OAc).13C NMR (75MHz,CDCl3),d170.33,169.97,165.82,165.07,137.72–127.49(Ar),98.32,97.10,78.99,75.02,74.43,72.70,72.57,69.08, 67.64,66.68,64.32,64.14,62.65,62.17,55.27,20.65,20.63.ESI-MS(m/z):876.296[M+Na+](Calcd876.295).Anal.Calcd for C45H47N3O14:C,63.30;H,5.55;N,4.92.Found:C,63.49;H,5.35; N,4.72.IR(cmÀ1)m max=2110,1744,1732,1272,1052.4.9.O-(Methyl2,3-di-O-benzyl-b-D-glucopyranosyluronate)-(1?4)-2-O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-D-glucopyranose(26)24mg KBr(0.2mmol)and24mg Bu4NBr(0.07mmol)in2.2mL NaHCO3–Na2CO3buffer(pH9.5)were added into6(0.59g, 1.0mmol)and TEMPO(3mg,0.01mmol)in3.3mL CH2Cl2solu-tion,then at0°C with rapid stirring,Ca(ClO)2(263mg,1.7equiv) was added in portions.The mixture was stirred at0°C for1h,TLC showed the reaction was completed(CH2Cl2/MeOH=20:1). Then at0°C,104mg NaHSO3(1mmol)was added,and HOAc was added to adjust the pH value to3.After diluting with20mL EtOAc,the aqueous phase was extracted with EtOAc,and the com-bined organic phase was washed with brine,dried,ﬁltered,and concentrated.The syrup dissolved in3mL ether,and CH2N2in ether was added till no bubble came out.Then a few drops of HOAc were added to consume the excess CH2N2,concentrated and puri-ﬁed by column chromatography(PE/EA/CH2Cl2=2:1:1).0.451g syrup was obtained(73%yields).R f=0.33(CH2Cl2/ace=18:1).1H NMR(300MHz,CDCl3):d 5.49(H-1,br), 5.29(H-3,dd,J=1.5Hz), 4.99(1H,AB, J=10.8Hz),4.87(2H,AB,J=11.4Hz),4.77(1H,AB,J=10.8Hz), 4.64(H-10,d,J=7.5Hz),4.57(H-5,d,J=5.1Hz),4.02(H-4,dd, J=7.5Hz,0.9Hz), 3.88(1H,dd,J=8Hz), 3.84(H-50,d, J=9.6Hz),3.79(s,3H,OMe),3.76(m,1H),3.65(1H,br),3.55 (m,2H),3.20(H-2,br),2.12(s,3H,OAc);ESI-MS(m/z):622.2 [M+Na+].Anal.Calcd for C29H33N3O11:C,58.09;H, 5.55;N, 7.01.Found:C,58.19;H,5.35;N,6.88.IR(cmÀ1)m3485,2107, 1748,1228,1070.4.10.O-(6-O-Acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-(1?4)-O-(methyl2,3-di-O-benzyl-b-D-glucopyranosyluronate)-(1?4)-2-O-acetyl-1,6-anhydro-2-azido-2-deoxy-b-D-glucopyranose(27)Under Argon,4ÅMS,donor5(386mg,0.68mmol)and accep-tor26(270mg,0.45mmol)in6mL dry toluene were cooled to À20°C and0.5M TMSOTf/CH2Cl2(0.3mL)was added.After 30min,moved to RT and stirred till the reaction completed. Quenched with NEt3,ﬁltered and concentrated,and puriﬁed by column chromatography(hexane/EtOAc=4:1)to obtain27 (432mg,95%yield)as white syrup.1H NMR(400MHz,CDCl3):d7.34–7.25(20H,Ar),5.53(d,1H, J=3.6Hz,H-100),5.47(s,1H,H-1),5.22(m,1H,H-3),5.00(m, 1H),4.84–4.73(m,4H),4.67(d,1H,J=7.57Hz,H-10),4.56(m, 2H),4.15(t,1H,J=8.79Hz,H-40),3.96(d,1H,J=6.86Hz,H-6a), 3.88(dd,1H,J=10.3and8.79Hz,H-300),3.75(s,3H,CO2Me),3.64 (m,1H,H-4),3.51(t,1H,J=8.79Hz,H-400),3.26(dd,1H,J=10.5 and3.60Hz,H-200),3.21(s,1H,H-2),2.10(s,3H,Ac),2.03(s,3H, Ac).13C NMR(100MHz,CDCl3):d170.66,168.16,164.76, 138.19–127.39(Ar),103.34,100.28,97.69,83.77,81.52,80.03, 77.52,75.84,75.44,75.13,74.91,74.48,73.84,70.58,69.61, 65.00,63.32,62.28,58.88,52.72,20.80.ESI-MS(m/z):1031.9 [M+Na+].4.11.Methyl O-(methyl2-O-benzoyl-3-O-benzyl-a-L-idopyranosyluronate)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-D-glucopyranoside(4)With the similar procedure of26,except PCT reagent was Ali-quat336(0.2equiv),0.327g white foam4was obtained(93% yields).R f=0.34(hexane/EtOAc=2:1)½a 25D+31.8(c0.98,CHCl3),1H NMR(300MHz,CDCl3):d8.10–7.25(20H,aromatic),5.37(d, 1H,J=2.5Hz,H-10),5.21(br s,1H,H-20),4.97(d,H-50,J=3Hz), 4.76(AB,2H,J=11.4Hz), 4.81–4.67(m,3H), 4.78(d,1H, J=3.5Hz,H-1),4.48(AB,1H,J=12Hz),4.02(d,1H,J=7.8Hz), 4.12–3.96(m,2H),3.92–3.86(m,2H),3.48(s,CO2C H3),3.45(s, 3H,OMe), 3.44(m,1H), 2.66(d,OH,J=9Hz);13C NMR (100MHz,CDCl3):d169.44,165.84,164.95,137.60–127.32, 98.31,97.93,78.59,75.25,74.97,74.73,72.56,69.14,68.82,68.08, 67.83,63.65,62.59,55.29,51.92.ESI-MS(m/z):820.3[M+Na+]; HRMS(ESI)Calcd for C42H43N3O13Na820.2688.Found 820.2691.IR(cmÀ1)m3488,3071,1728,1648,1602,1452,1278, 1109,711.F.Lin et al./Carbohydrate Research371(2013)32–39374.12.Methyl O-(6-O-acetyl-2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-(1?4)-O-(methyl2,3-di-O-benzyl-b-D-glucopyranosyluronate)-(1?4)-O-(3,6-di-O-acetyl-2-azido-2-deoxy-a-D-glucopyranosyl)-(1?4)-O-(Methyl2-O-benzoyl-3-O-benzyl-a-L-idopyranosyluronate)-(1?4)-2-azido-6-O-benzoyl-3-O-benzyl-2-deoxy-a-D-glucopyranoside(2)Under Argon,4ÅMS,donor3(80mg,0.1mmol)and acceptor4 (158mg,0.12mmol)in2mL dry CH2Cl2were cooled toÀ20°C and 0.1M TMSOTf/CH2Cl2(0.07mL,0.06equiv)was added.After 30min,moved to RT and stirred till the reaction completed. Quenched with NEt3,ﬁltered and concentrated,and puriﬁed by column chromatography(hexane/EtOAc/CH2Cl2=4:1:1)to obtain pentasaccharide2(135mg,70%yield)as white syrup.1H NMR (400MHz,CDCl3):d D5.36(d,1H,J=3.6Hz,H-1),3.23(dd,1H, J=7.6and3.6Hz,H-2);E4.40(d,1H,J=7.8Hz,H-1),3.33(H-2); F5.10(d,1H,J=3.6Hz,H-1),3.64(H-2);G5.47(d,1H,J=4.4Hz, H-1),5.00(H-2);H4.76(d,1H,J=3.2Hz,H-1),3.42(H-2).13C NMR(75MHz,CDCl3):d170.55,170.03,170.0,169.62,168.27, 165.98,165.27,137.95,137.47,137.27,133.37,132.91,129.80–125.45(Ar),103.14,98.34,98.19,97.49(2C),83.65,81.63,80.04, 78.40,76.77,75.41,74.89,72.95,71.40,69.62,69.05,62.20, 61.26,60.62,55.30,52.16.HRMS(MALDI/DHB)Calcd for C95H101N9O30Na1870.6547.Found1870.6578.4.13.Methyl O-(2-azido-3,4-di-O-benzyl-2-deoxy-a-D-glucopyranosyl)-(1?4)-O-(2,3-di-O-benzyl-b-D-glucopyranosyluronic acid)-(1?4)-O-(2-azido-2-deoxy-a-D-glucopyranosyl)-(1?4)-O-(3-O-benzyl-a-L-idopyranosyluronic acid)-(1?4)-2-azido-3-O-benzyl-2-deoxy-a-D-glucopyranoside (28)AtÀ5°C,2mL1.25M LiOH solution and4.5mL30%H2O2were added into the THF solution of2(162mg)and stirred for6h at 0°C,then5mL MeOH and2.2mL4N NaOH were added and stir-red for6h.Quenched with4mL5%Na2SO3,adjusted pH to2–3 with6M HCl.Extracted with CH2Cl23times,combined the organic phase and dried over Na2SO4ﬁltered and concentrated,and puri-ﬁed by reverse phase C18column chromatography(CH3CN/H2O 1:9to7:3)to obtain pentasaccharide28(84mg,65%yield).1H NMR(400MHz,CD3OD):d D5.55(d,1H,J=3.6Hz,H-1),3.27 (H-2);E 4.74(d,1H,J=8Hz,H-1), 3.50(H-2);F 5.13(d,1H, J=3.6Hz,H-1),3.29(H-2);G5.31(d,1H,J=4.4Hz,H-1),3.65 (H-2);H 4.78(d,1H,J=3.2Hz,H-1), 3.36(H-2).13C NMR (75MHz,CD3OD):d172.50,171.97,139.66–139.31(Ar),129.56–128.78(Ar),103.93,102.27,99.99,99.12,98.86,85.20,83.09, 80.87,79.68,79.06,78.38,77.35,76.23,75.89,75.41,74.80, 73.13,72.72,71.84,64.67,64.49,61.58,60.58,55.54.ESI-MS: 1508.70(M+Na+),1524.60(M+K+).4.14.Methyl O-(2-deoxy-2-sulfamido-6-O-sulfo-a-D-glucopyranosyl)-(1?4)-O-(b-D-glucopyranosyluronic acid)-(1?4)-O-(2-deoxy-2-sulfamido-3,6-di-O-sulfo-a-D-glucopyranosyl)-(1?4)-O-(2-O-sulfo-a-L-idopyranosyluronic acid)-(1?4)-2-deoxy-2-sulfamido-6-O-sulfo-a-D-glucopyranoside decasodium salt(1)40mg28and188mg SO3ÁNMe3in1.7mL DMF were stirred at 50°C for10h,concentrated in vacuo,exchanged to sodium salt with DOWEX50-WX4-Na,then puriﬁed by reverse phase C18col-umn chromatography(CH3CN/H2O1:9to9:1)to obtain pentasac-charide29(48mg,91%yield).1H NMR(400MHz,CD3OD):d D5.53 (H-1);E4.96(H-1);F5.32(H-1);G5.44(H-1);H4.78(H-1),3.21 (H-2).13C NMR(75MHz,CD3OD):d D103.71(C-1),81.23(C-3), 65.14(C-2);E98.58(C-1);F98.98(C-1),78.52(C-3),64.99(C-2);G99.92(C-1);H94.19(C-1),79.78(C-3),64.70(C-2).ESI-MS (m/z):(negative)627.0[(MÀ3H)/3](Calcd627.4).48mg pentasaccharide29in1mL H2O and2mL MeOH,was hydrogenated in the presence of10%Pd/C(30mg)at1bar.After 10h,Pd/C wasﬁltered and theﬁltrate was concentrated and puri-ﬁed by reverse phase C18column chromatography(MeOH/H2O1:9 to9:1)to obtain pentasaccharide30(26mg,98%yield).ESI-MS(m/ z):(negative)601.5[(MÀ3H)/3](Calcd601.5);913.9[(M+NaÀ3H)/ 2](Calcd913.7).14mg SO3Ápy and14mg NaHCO3were added into pentasaccha-ride30(26mg)in2mL H2O,the same amount of SO3Ápy and NaH-CO3was added at the end of2,4,and6h.then stirred for another 2h,concentrated and exchanged to sodium salt with DOWEX50-WX4-Na,then puriﬁed by reverse phase C18column chromatogra-phy(CH3CN/H2O1:9to4:1).The fraction contained1was com-bined and desalted on a Sephadex G-25column to obtain pentasaccharide1(24mg,65%yield).1H NMR(600MHz,CDCl3):d D5.60(H-1),3.24(H-2),3.61(H-3),3.88(H-4),3.56(H-5);E4.62(H-1),3.40(H-2),3.82(H-3),3.76 (H-4),4.16(H-5);F5.50(H-1),3.44(H-2),4.36(H-3),3.96(H-4), 4.14(H-5);G5.19(H-1),4.30(H-2),4.14(H-3);H5.00(H-1), 3.27(H-2),3.66(H-3),3.94(H-4),3.77(H-5).13C NMR(150MHz, CDCl3):d D100.98(C-1),69.53(C-6),60.71(C-2);E103.89(C-1); F98.77(C-1),68.86(C-6),59.39(C-2);G102.45(C-1);H100.3 (C-1),69.06(C-6),60.42(C-2),58.42(OMe).HiRes ESI-MS(m/z): (negative)818.46[(M+6NaÀ8H)/2](Calcd818.42),829.45 [(M+7NaÀ9H)/2](Calcd829.41),840.44[(M+8NaÀ10H)/2](Calcd 840.40).AcknowledgmentsThis work was supported by the Committee of Science and Technology of Shanghai(10QB1403900),the National Natural Sci-ence Foundation of China(20902059),and the National Science Grand Projects of New Drug R&D(2011ZX09202-101-01),we wish to thank Professor Biao Yu for his instruction of this work.Supplementary dataSupplementary data associated with this article can be found,in the online version,at /10.1016/j.carres.2013.01. 003.References1.(a)Conrad,H.E.Heparin-Binding Proteins;Academic Press:San Diego,1998;(b)Casu,B.;Lindahl,U.Adv.Carbohydr.Biochem.2001,57,159–206;(c)Capila,I.;Linhardt,R.J.Angew.Chem.,Int.Ed.2002,41,390–412.2.Balasa,Vinod.V.Pediatr.Blood Cancer2005,45,741–752.3.(a)van Boeckel,C.A.A.;Petitou,M.Angew.Chem.,Int.Ed.1993,32,1671–1690;(b)Petitou,M.;He´rault,J.-P.;Bernat, A.;Driguez,P.-A.;Duchaussoy,P.;Lormeau,J.-C.;Herbert,J.-M.Nature1999,398,417–422.4.Savi,P.;Chong,B.H.;Greinacher,A.;Gruel,Y.;Kelton,J.G.;Warkentin,T.E.;Eichler,P.;Meuleman,D.;Petitou,M.;Herault,J.P.;Cariou,R.;Herbert,J.M.Blood2005,105,139–144.5.Petitou,M.;Jacquinet,J.C.;Sinay,P.;Choay,J.U.S.Patent4,818,816,1989.6.GSK annual report2009.7.a)Haller,M.;Boons,.Chem.2002,2033–2038;(b)Orgueira,H.A.;Bartolozzi,A.;Schell,P.;Litjens,R.;Palmacci,E.R.;Seeberger,P.H.Chem.Eur.J.2003,9,140–169.8.(a)Lee,J.-C.;Lu,X.-A.;Kulkarni,S.S.;Wen,Y.S.;Hung,S.-C.J.Am.Chem.Soc.2004,126,476–477;b)Haller,M.;Boons,G.-J.J.Chem.Soc.,Perkin Trans.12001, 814–822.9.Lin, F.;Peng,W.;Xu,W.;Han,X.;Yu, B.Carbohydr.Res.2004,339,1219–1223.10.Recent review:Poletti,L.;Lay,.Chem.2003,16,2999–3024.11.Czernecki,S.;Leteux,C.;Veyrieres,A.Tetrahedron Lett.1992,33,221–224.12.(a)Fernandez-Mayoralas,Alfonso;Marra,Alberto;Trumtel,Michel;Veyrieres,Alain;Sinay,P.Carbohydr.Res.1989,188,81–95;(b)Tabeur,C.;Machetto,F.;Mallet,Jean-M.;Duchaussoy,P.;Petitou,M.;Sinay,P.Carbohydr.Res.1996,281, 252–276.38 F.Lin et al./Carbohydrate Research371(2013)32–39。

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多糖结构分析(英文)

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0008-6215/95/$09.50 © 1995 ElsevierScienceB.V. All rights reserved 0008-6215(94)00355-6
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R. Falshaw, R.H. Furneaux / Carbohydrate Research 269 (1995) 183-189
Ruth Falshaw *, Richard H. Furneaux
Industrial Research Ltd, P.O. Box 31-310, Lower Hun, New Zealand
Received15 March 1994;revised21 October1994; accepted21 October1994
particularly suitable when one of the glycosidic bonds is more susceptible to cleavage than the others. Most red algal galactans have a linear backbone comprised of alternating 3-1inked fl-D-galactopyranosyl and 4-1inked a-D- or L-galactopyranosyl residues. In the case of the commercially important gel-forming agars and carrageenans, the 4-1inked galactosyl residue is largely present as the 3,6-anhydride. The 3,6-anhydrogalactosidic bond is particularly labile to acid hydrolysis. Under relatively mild hydrolysis conditions virtually all the 3,6-anhydrogalactosidic bonds can be cleaved while most of the galactosidic bonds remain intact. In the presence of the borane reducing agent, this leads to the production of 'biitols', namely 3,6-anhydro-4-O-/3-D-galactosylgalactitols [3]. (Strictly these should be named as 1,4-anhydrogalactitol derivatives, but we have retained the 3,6-anhydrogalactitol nomenclature throughout for clarity.) In agars the 3,6-anhydrogalactosyl units are of the L-configuration whilst in carrageenans they are in the D-fOrm. The derived agarobiitol and carrabiitol species, respectively, are thus diastereomers when they have the same substituents. After acetylation to improve volatility, they can be separated and identified by GLC-MS. This method has been developed and used by Usov and co-workers [4-6] to classify algal biomass into agarophytes or carrageenophytes. One limitation of this method is that, under the acetylation conditions used (acetic anhydride/pyridine, 100°C, 1 h), if the biitol contains a sulfate ester substituent, this will not necessarily be removed. Even if the resulting derivative is organic soluble and volatile enough to be eluted by GLC it will have a different retention time from the peracetylated product. In this note we describe a modified reductive partial-hydrolysis procedure for preparing disaccharide alditol derivatives from naturally and chemically methylated red algal galactans containing 3,6-anhydrogalactosyl residues. Because the procedure incorporates an acetylative desulfation step, it is equally applicable to neutral or sulfated galactans. We have applied this procedure to chemically methylated samples, in particular, because GLC-MS analysis of the resulting partially methylated, partially acetylated biitols enables substitution patterns of adjacent residues to be determined. A range of partially methylated, partially acetylated biitols has been produced from polysaccharides of known composition and characterised by GLC and GLCCI(NHa)MS. Each biitol derivative had a unique GLC retention time (Tables 1 and 2). Each species gave an (M + NH4) + ion from which the total number of O-methyl groups could be determined, and a major fragment ion corresponding to the /3-D-galacTable 1 GLC Retentiontimesof partiallymethylated,partiallyacetylatedcarrabiitols Retention time(min) Position of O-methylgroups Source desulfated,methylatedr-carrageenan 7.5 2,2',4',6'permethylatedK-carrageenan 7.9 2,2',6'base-treated,permethylatedA-carrageenan 8.1 2',4',6'permethylated~carrageenan 8.6 2',6'base-treated,permethylatedA-carrageenan 9.0 4',6'partiallymethylated~-carrageenan 9.1 6'partiallymethylatedr-carragecnan 9.8 2'r-/r-carrageenan 10.5

细胞壁多糖海洋绿藻石莼“杜松”(ulvales,绿藻)。提取和化学成分

Keywords: Ulva; Chlorophyceae; Algae; Cell wall; Ulvan; Xyloglucan; Glucuronan
1. Introduction The marine green seaweed genus Ulva is poorly utilized and only a small part of its biomass is used as edible seaweeds [1]. Periodically, particular species overgrow as "green-tides" in eutrophicated areas (Brittany, Venice lagoon) and create ecological and economical problems [2]. One particularly interesting feature of Ulva is their richness in cell-wall polysaccharides, which can be used for their functional a n d / o r biological
2. Experimental
MateriaL--Algae were collected at Piriac (Pointe du Castelli, Loire-Atlantique, France) in May 1993 and were tentatively identified as Ulva "rigida" from the morphology and collection site. The seaweeds were freed from attached impurities, washed thoroughly with tap water, dried in a current of air at 60°C and ground to a flour in a Waring Blender. Extraction ofpolysaccharides.--The algal flour was extracted sequentially with hot sodium oxalate solution, hot water, 1 M and 4 M KOH solution at 20°C. Sodium oxalate extraction.--The dried weed (372 g) was suspended in 3.8 L of 0.05 M sodium oxalate solution (pH 6) and stirred for 1.5 h in a boiling water bath. The suspension was diluted by the addition of 3 L of sodium oxalate solution prior to centrifugation (20 min, 14,740g). The residue was re-extracted with 3.8 L of boiling sodium oxalate for a further 1 h and then with 5 L of boiling deionized water for 2 h. The last slurry was filtered through a fritted-glass G-4 filter (porosity 5 - 1 5 / z m ) and the residue washed thoroughly with deionized water. The combined extracts (sodium oxalate and water) and water washings were ultrafiltered (Mw cutoff 30 kDa, Amicon hollow fibre cartridges) and the retentate (3 L) was precipitated in 3 vol 95% ethanol. The precipitate was recovered by centrifugation (20 min, 14740g), washed thoroughly with 80% ethanol, 95% ethanol, and acetone, and finally dried over P205 in vacuo (A; yield 76.4 g). The residue was extracted sequentially at room temperature with 3 L of 60%, 80%, 95% ethanol and acetone. The pale-yellow solid (B) was air-dried and then dried in vacuo over P205 (yield 142 g). Alkaline extract.--Extraction with 1 M and 4 M KOH solution were performed

美洲大蠊多肽PAP—2对H22荷瘤小鼠的抑瘤作用研究

美洲大蠊多肽PAP—2对H22荷瘤小鼠的抑瘤作用研究【摘要】本研究旨在探讨美洲大蠊多肽PAP—2对H22荷瘤小鼠的抑瘤作用。

首先进行了美洲大蠊多肽PAP—2的提取和纯化工作，然后建立了荷瘤小鼠模型。

随后观察了PAP—2对H22荷瘤小鼠的抑瘤作用，并进行了机制探讨。

实验结果表明，PAP—2对H22荷瘤小鼠具有显著的抑制作用。

结论指出美洲大蠊多肽PAP—2可能具有潜在的抗肿瘤活性，展望未来研究方向为进一步探究其具体作用机制和临床应用前景。

这项研究的重要性在于提供了新的抗肿瘤治疗思路，具有较大的临床应用前景和意义。

【关键词】美洲大蠊多肽PAP—2、H22荷瘤小鼠、抑瘤作用、提取和纯化、荷瘤小鼠模型、机制探讨、实验结果分析、展望未来研究方向、重要性和意义1. 引言1.1 研究背景肿瘤是一种危害人类健康的严重疾病，世界卫生组织统计数据显示，肿瘤已经成为影响人类寿命的主要因素之一。

肿瘤的发生与生长涉及复杂的细胞信号传导通路和调控机制，当前临床治疗肿瘤的方法主要是手术切除、放射治疗和化疗，然而这些治疗方法往往伴随着严重的副作用和复发率较高的问题。

美洲大蠊是一种常见的害虫，在传统中医药中被广泛应用。

研究表明，美洲大蠊体内含有多种具有抗菌、抗炎和抗肿瘤活性的生物活性物质，具有很好的药用价值。

美洲大蠊多肽PAP—2是一种具有抗肿瘤活性的生物活性物质，已经引起了科学家们的广泛关注。

本研究旨在探究美洲大蠊多肽PAP—2对H22荷瘤小鼠的抑瘤作用及其可能的机制，为发展新的肿瘤治疗方法提供理论和实验依据。

1.2 研究目的本研究旨在探究美洲大蠊多肽PAP-2对H22荷瘤小鼠的抑瘤作用机制，从而为开发新的抗肿瘤药物提供理论依据和临床应用价值。

具体目的包括：1. 研究美洲大蠊多肽PAP-2的提取和纯化方法，确保实验所用样品的纯度和有效性；2. 建立H22荷瘤小鼠模型，为后续的抗肿瘤实验奠定基础；3. 观察美洲大蠊多肽PAP-2对H22荷瘤小鼠的抑瘤作用，包括肿瘤体积、生长速度和生存周期等指标的变化；4. 探讨美洲大蠊多肽PAP-2的抗肿瘤机制，为其临床应用提供理论支持；5. 对实验结果进行详细的数据分析，评估美洲大蠊多肽PAP-2在抗肿瘤治疗中的潜力和优势。

Cao_2006_Carbohydrate-Polymers

Structure of an anti-tumor polysaccharide from Angelica sinensis(Oliv.)DielsWei Cao,Xiao-Qiang Li,Li Liu,Tie-Hong Yang,Chen Li,Hui-Ting Fan,Min Jia,Zheng-Guang Lv,Qi-Bing Mei *Department of Pharmacology,School of Pharmacy,The Fourth Military Medical University,Xi’an,Shannxi 710032,ChinaReceived 20November 2005;received in revised form 26January 2006;accepted 24February 2006Available online 17April 2006AbstractAn arabinoglucan,named APS-1d with a molecular weight of 5.1kDa determined by high-performance gel-permeation chromatog-raphy,was extracted from the roots of Angelica sinensis (Oliv.)Diels and further puriﬁed by DEAE-Sephadex A-25and Sephadex G-100columns.The monosaccharides in the APS-1d,determined by GC,consisted of Glc and Ara in molar ratio of 13.8:ing methylation analysis,partial acid hydrolysis,FT-IR,1D and 2D NMR (H/H-COSY,HSQC,and HMBC)experiments,the structure of APS-1d was elucidated.APS-1d had a backbone composed of 1,4-a -D -glucopyranosyl residues,with branches attached to O-6of some residues.The branches were composed of 1,6-a -D -Glc p residues,and terminated with b -L -arabinofuranose residues.The anti-tumor activities of APS-1d were investigated both in vitro and in vivo.MTT assay revealed that APS-1d signiﬁcantly inhibited the proliferation of human cervix carcinoma HeLa cells and lung carcinoma A549cells in vitro.Furthermore,APS-1d inhibited the growth of the tumors on the mice transplanted S180in a dose dependent manner.The inhibitory rate in mice treated with 100mg/kg APS-1d reached to 50.7%.Ó2006Elsevier Ltd.All rights reserved.Keywords:Angelica sinensis ;Polysaccharide;Structure;Anti-tumor1.IntroductionChinese Danggui is the root of Angelica sinensis (Oliv.)Diels,which is one of the most widely used herbs of all traditional Chinese medicines.While A.sin-ensis has been used historically in gynecology as early as over 2000years ago (Sarker &Nahar,2004;Zhao et al.,2003),recent scientiﬁc investigations have focused on its cardiovascular,antioxidant,anti-tumor,and immunomodulatory activities (Cheng et al.,2004;Hou et al.,2004;Wang,Ouyang,Liu,Wei,&Yang,2001;Tsai et al.,2005;Weng,Zhang,Gong,&Xiai,1987;Wilasrusmee et al.,2002;Wu,Ng,&Lin,2004;Yim et al.,2000).Low-molecular weight compounds such as essential oil,phenylpropanoids,benzenoids,and cou-marins have for a long time been considered as the active principles of this plant (Mei,Tao,&Cui,1991;Zhao et al.,2003),but they cannot account for all the eﬀects mentioned above.In recent years,as more and more polysaccharides have been reported to exhibit a variety of biological activities,including anti-tumor (Wasser,2002),immunostimulation (Wasser,2002;Yamada,1994),anti-oxidation (Li et al.,2003;Liu,Ooi,&Chang,1997),etc.,the nonstarchy poly-saccharides have emerged as an important class of bioactive natural products.To elucidate the medical mech-anism of Chinese Danggui,the polysaccharides have also attracted much attention.The previous study showed that the crude polysaccharide from A.sinensis displayed anti-tu-mor,immunostimulation,blood coagulation,platelet aggregation,and gastrointestinal protection activities (Cho et al.,2000;Choy,Leung,Cho,Wong,&Pang,1994;Shang et al.,2003;Yang,Jia,Mei,&Shang,2002;0144-8617/$-see front matter Ó2006Elsevier Ltd.All rights reserved.doi:10.1016/j.carbpol.2006.02.034*Corresponding author.Tel.:+862984774555;fax:+862984774552.E-mail address:qbmei@ (Q.-B.Mei)./locate/carbpolCarbohydrate Polymers 66(2006)149–159Ye,So,Liu,Shin,&Cho,2003).So,the presence of such compounds could partly be responsible for the clinical eﬀects of this plant.In the last few years,the structures of several polysac-charides isolated from the other herbs of the same genus, such as Angelica acutiloba have been reported(Kiyohara, Yamada,&Otsuka,1987;Yamada,Kiyohara,Cyong,& Otsuka,1985;Yamada et al.,1990;Yamada,Kiyohara,& Otsuka,1984).For example,the water-soluble polysac-charide from A.acutiloba was a1,4-linked a-D-glucan having side chains at O-6of the glucosyl residues of the main chain(Yamada et al.,1984).However,to our knowledge,few studies on the structural features and linkage composition of polysaccharide from A.sinensis have been undertaken.Wang,Ding,Zhu,He,and Fang (2003)reported that polysaccharide from A.sinensis was composed of fucose,galactose,glucose,arabinose,rham-nose,and xylose(mole ratio: 1.0:13.6:15.0:8.7:21.3:3.7), which was similar to the results reported by others(Chen, Wang,Xu,Xu,&Chang,2001).Furthermore,a neutral polysaccharide(ASP1)and two kinds of acidic polysac-charides(ASP2,ASP3)were isolated from A.sinensis recently(Sun,Tang,Gu,&Li,2005).The composition analysis displayed that ASP1was rich in glucose,galac-tose,and arabinose.Although these structural investiga-tions shed light on the monosaccharide compositions of polysaccharides from A.sinensis,detailed studies of the structures were lacking.Therefore,we fractionated the polysaccharide from A.sinensis and obtained several distinctive polysaccharides. Among them,a low molecular weight arabinoglucan exert-ed anti-tumor activity.So,the aim of this research was to investigate the complete structure of the unique arabino-glucan and its anti-tumor eﬀects both in vitro and in vivo.2.Experimental2.1.MaterialsThe roots of A.sinensis were collected in Minxian County, Gansu Province,China in October2002and identiﬁed by Professor Niu X.F.in the department of pharmacy,Xi’an Jiaotong University(Xi’an,China),by comparison with a voucher specimen deposited at the herbarium in the depart-ment of pharmacy,Xi’an Jiaotong University.The coarse powder of the roots was air-dried in the shade and stored in a well-closed vessel for use.T-series Dextran,DEAE-Sephadex A-25,Sephadex G-100,and Sephadex G-75were purchased from Amer-sham biosciences(Uppsala,Sweden).Triﬂuoroacetic acid (TFA),3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazoli-um bromide(MTT),and dimethyl sulfoxide(DMSO)were purchased from Sigma(St.Louis,MO,USA).Roswell Park Memorial Institute(RPMI)-1640medium,phos-phate-buﬀered saline(PBS),and fetal bovine serum were purchased from Gibco(Grand Island,NY,USA).All other chemical reagents were analytical reagent grade.2.2.Extraction and fractionation of polysaccharideThe powdered roots(1.8kg)of A.sinensis(Oliv.)Diels were extracted three times with ethanol(3.6L)at80°C for3h,in order to remove the pigments.The residue was decocted three times with water(7.2L)at80°C for2h,ﬁl-tered through the gauze and centrifuged to remove water-insoluble materials.The aqueous extract was concentrated at50°C in vacuum and treated with3volumes of ethanol for precipitation at4°C overnight.The gel-like precipitate was suspended in water and dialyzed against distilled water (exclusion limit3.5kDa).The nondialyzable portion was frozen atÀ20°C,then thawed and centrifuged again to remove insoluble materials.After the freeze–thaw process was repeated six times,the supernatant was lyophilized and the brown product was obtained(APS-0,yield:39.6g).APS-0(13.2g)was dissolved in distilled water,ﬁltered through a0.65l m membraneﬁlter and loaded onto a DEAE-Sephadex A-25column(90cm·5cm).The col-umn wasﬁrst eluted with distilled water followed by 0.3M and0.5M NaCl,respectively.Fractions of10ml were collected and monitored for the presence of carbohy-drate using phenol–sulfuric acid assay(Dubois,Gilles, Hamilton,Rebers,&Smith,1956).The largest water-elut-ed fraction was pooled,dialyzed,lyophilized and designat-ed as APS-1(yield:4.8g).APS-1was further fractionated on a column(100cm·5cm)of Sephadex G-100,eluted with0.1M NaCl and separated into four sub-fractions. The forth fraction,which molecular weight was lower than the other fractions,was pooled according to the elution proﬁle.After concentration,the solution was applied onto the column of Sephadex G-100once more,to get a puriﬁed polysaccharide.The relevant fraction was concentrated, dialyzed and lyophilized to get a white powder polysaccha-ride(APS-1d,225mg).2.3.General methodsCarbohydrate content was measured by the phenol-sulfu-ric acid method,using D-glucose as the standard(Dubois et al.,1956).Protein was measured by the Bradford(1976) method using bovine serum albumin as standard.The uronic acid content of the sample was measured by spectrophotom-etry according to the colorimetric method reported by Eng-lyst,Quigley,and Hudson(1994).Optical rotation was measured at20°C using a Perkin-Elmer343polarimeter. Each sample was analyzed four times by the methods described above.IR spectra(KBr disc)were recorded with a Bruker-Equinox55spectrophotometer for detecting func-tional groups.The homogeneity and molecular weight of the polysaccharide were evaluated and determined by high-per-formance gel-permeation chromatography(HPGPC)using a Waters Alliance2690instrument equipped with a tandem of a Shodex sb-803HQ(Showa Denkko,8mm·30cm) and a Biosep SEC-S3000(Phenomenex,7.8mm·30cm) column,eluted with0.05M Na2SO4at aﬂow rate of 0.8ml/min.The elution was monitored by Waters Alliance150W.Cao et al./Carbohydrate Polymers66(2006)149–1592414RI detector and the data were analyzed with Millennium32(Waters Alliance)software.The columns were calibrated with standard T-series Dextran T-130, T-80,T-40,T-20,and T-10(Wei&Fang,1989).2.4.Monosaccharide analysisAPS-1d was hydrolyzed with2M TFA at110°C for4h (Honda,Suzuki,Kakehi,Honda,&Takai,1981).Paper chromatography(PC)and gas chromatography(GC)were used for identiﬁcation and quantiﬁcation of the monosac-charide composition.PC was performed on Xinhua (Hangzhou,China)No.1paper in the following solvent system:EtOAc-n-butyl alcohol–isopropyl alcohol–acetic acid–water–pyridine(20:7:12:7:6:6)and visualized by spraying with phthalic acid reagent and heating at100°C for15min(Shang et al.,2001).The sugars in the hydroly-sate were converted to their alditol acetates as described (Johnes&Albersheim,1972;Oades,1967)and analyzed by GC on an Agilent6890N instrumentﬁtted with FID and equipped with SE-54column(30m·0.32mm ·0.25l m)at a temperature program as follows:160°C (10min)–280°C with a rate of5°C/min.The injector and detector heater temperatures were250and300°C,respec-tively.The rate of N2carrier gas was1.2ml/min.2.5.Methylation analysisMethylation of APS-1d was carried out three times using the method described early(Needs&Selvandran, 1993).The methylated polysaccharide was examined by IR plete methylation was conﬁrmed by the lack of a hydroxyl peak.The methylated products were hydrolyzed,reduced,acetylated as described by Sweet,Shapiro,and Albersheim(1975).The resulting aldi-tol acetates were subjected to GC and GC–MS analysis. Analyses were performed on an Agilent6890N GC inter-faced with an Agilent5973N mass selective detector at 70eV ionization energy.The GC column was a HP-1 (30m·0.2mm·0.33l m)at a temperature program from 140to280°C with a rate of3°C/min.The rate of helium carrier gas was at a rate of3.0ml/min.The quantiﬁcation for molar ratio for each sugar was calibrated using the peak area and response factor of the FID in GC.2.6.Nuclear magnetic resonance(NMR)spectroscopyThe polysaccharide(20mg)was dissolved in D2O (0.5ml).The13C NMR,13C DEPT(distortionless enhance-ment by polarization transfer)and1H NMR were recorded at30°C with a Bruker Avance500MHz spectrometer (Germany).The13C NMR spectrum was recorded using a Bruker5mm broadband probe at125.76MHz,and the chemical shifts were expressed in ppm relative to the resonance of the internal standard,TMS.The DEPT exper-iment was carried out using a polarization transfer pulse of 135.Two-dimensional1H–1H COSY(homonuclear shift correlation)spectrum was recorded in the phase-sensitive mode.Spectral width was1707Hz in both dimensions.In the1H–13C heteronuclear single quantum coherence (HSQC)experiment,echo/antiecho gradient selection with decoupling was used.Spectral widths were1707and 11,261Hz for the proton and the carbon dimensions, respectively.The heteronuclear multiple quantum coher-ence(HMBC)experiment was recorded using gradient pulses for selection.The repetition time was2.1s.2.7.Partial acid hydrolysisPartial acid hydrolysis of APS-1d(80mg)was per-formed as described by Ishurd and Kennedy(2005)using 0.2mol/L TFA at100°C for2h.The mixture was evapo-rated to dryness using methanol,and the residue was dis-solved with a small amount of water,placed in a dialysis tubing(molecular weight cut-oﬀof2kDa)and dialyzed against distilled water(3·2000ml).The dialyzable frac-tion was concentrated,hydrolyzed and its monosaccha-ride composition was analyzed by PC.The nondialyzate was concentrated and puriﬁed by chromatography on Sephadex G-75(90·1.6cm)column to give a pure frag-ment HAPS-1d(40mg)as a single and symmetric peak eluted with distilled water.To establish the sequence of linkages in HAPS-1d,it was subjected to monosaccharide composition analysis and methylation analysis as described above.2.8.Cell lines and animalsHuman cervix carcinoma HeLa cells and human lung carcinoma A549cells were obtained from Xi’an Cell Engineering Center(Xi’an,China).HeLa and A549cells were cultured in RPMI-1640medium sup-plemented with10%heat-inactivated fetal bovine serum (FBS),penicillin(100U/ml),and streptomycin(100mg/ L)(Hsu,Kuo,&Lin,2004;Tomatsu,Ohnishi-Kameyama,&Shibamoto,2003)in a humidiﬁed5% CO2atmosphere at37°C.S180tumor cells were maintained in peritoneal cavities of ICR mice obtained from Shannxi Academy of Traditional Chinese Medicine(SATCM)(Shannxi,China). Male ICR mice,weighed20.0±2.0g,purchased from SATCM,were housedﬁve per plastic cages with wood chip bedding in an animal room with a12h light and12h dark cycle at room temperature(25±2°C)and allowed free access to standard laboratory diet(purchased from the Laboratory Animal Center of the Forth Military Medical University).The animal experiments were conducted according to the‘Guidelines for Animal Experimentation’of the Forth Military Medical University.2.9.Cell proliferation assayThe proliferation of HeLa and A549cells was deter-mined using the colorimetric MTT assay as describedW.Cao et al./Carbohydrate Polymers66(2006)149–159151previously(Mosmann,1983).Brieﬂy,cells were seeded at a density of3·103cells/well in a100l l volume of the medium in96-well plates and allowed to attach24h. The dosages of APS-1d on the selected cell lines were in the range of3–100l g/ml while the negative controls were treated with the medium only.MTT(5g/L)20l l was added48h later.After incubated at37°C for4h, the supernatant was aspirated,and150l l DMSO was added to each well.Absorbance was measured at 570nm by a96-well microplate reader(Mode680,Bio-Rad,Tokyo,Japan).2.10.Assay of anti-tumor activity in vivoS180tumor cells(3·106)were implanted subcuta-neously into right hind groin of the ICR mice(Zhou et al.,2004).Mice were randomly divided intoﬁve groups(n=10).One day after inoculation,APS-1d was dissolved in distilled water and administered intraperito-neally(i.p.)to the mice at the doses of20,50,and 100mg/kg.Positive and negative controls were set for comparison.The positive control was given with0.2ml cyclophosphamide(20mg/kg)and negative one with physiological saline instead of the test solution.Animals were sacriﬁced after2weeks.The body weights were measured.Tumors and spleens were excised and the tumor inhibitory ratio were calculated by following for-mula:Inhibition ratio(%)=[(AÀB)/A]·100,where A and B were the average tumor weights of the negative control and treated groups,respectively.2.11.Statistical analysisData were expressed as means±SD.Data in all the bio-assays were statistically evaluated by Student’s t test and P<0.05was considered signiﬁcant.3.Results and discussion3.1.Isolation and puriﬁcation of polysaccharideThe crude polysaccharide,APS-0,was obtained by precipitation with ethanol and dialysis from the water extract of the roots of A.sinensis.APS-0which contained86%of neutral carbohydrate and4.1%of pro-tein was fractionated on a DEAE-Sephadex A-25anion-exchange column.The unabsorbed fraction(APS-1) obtained in water eluate,was fractioned by gelﬁltration on Sephadex G-100column to get four fractions as shown in Fig. 1.As APS-1d displayedsigniﬁcant Fig.1.Isolation and puriﬁcation of the APS-1d on a column of Sephadex G-100.The inserted graph showed the elution proﬁle of APS-1d on HPSECusing a RI detector.Table1The results of methylation analysis of APS-1d and HAPS-1dMethylated sugar Retention time(min)Molar ratios Mass fragments(m/z)Linkage typeAPS-1d HAPS-1d2,3,5-Me3-Ara8.73 1.0743,73,87,101,117,129,161,189b-Ara f-(1ﬁ2,3,4,6-Me4-Glc p11.00 1.10143,71,87,101,113,117,129,145,161a-Glc p-(1ﬁ2,3,6-Me3-Glc p13.687.1894.0843,87,99,113,117,129,161,191,203,233ﬁ4)-a-Glc p-(1ﬁ2,3,4-Me3-Glc p14.19 4.9743,87,101,117,129,143,161,173,189,203,233ﬁ6)-a-Glc p-(1ﬁ2,3-Me2-Glc p16.59143,85,101,117,142,159,187,201,207,261ﬁ4,6)-a-Glc p-(1ﬁ152W.Cao et al./Carbohydrate Polymers66(2006)149–159anti-tumor activity among four fractions,it was further puriﬁed by gel ﬁltration and gave a homogeneous poly-saccharide,which was conﬁrmed by HPSEC.The mole-cular weight of APS-1d was estimated to be 5.1kDa from the calibration curve in reference to standard dex-trans.APS-1d contained 91.5%of carbohydrate,0.5%of uronic acid and only trace (less than 2%)of protein.The component sugars of APS-1d were determinedtoFig.2.FT-IR spectrum ofAPS-1d.Fig.3.13C NMR and DEPT spectra of APS-1d.(A)DEPT,(B)13C NMR.W.Cao et al./Carbohydrate Polymers 66(2006)149–159153be glucose and arabinose with a molar ratio of13.8:1. Moreover,the positive value of optical rotation(½a 20D +45.9(c0.2,H2O))suggested the dominating presence of a-form glycosidic linkages in APS-1d.3.2.Determination of the structure of APS-1dA modiﬁed methylation analysis(Needs&Selvandran, 1993)of the APS-1d gaveﬁve homogeneous peaks on the bined with the corresponding MS spec-tra,they were identiﬁed as2,3,5-Me3-Ara,2,3,4,6-Me4-Glc p,2,3,6-Me3-Glc p,2,3,4-Me3-Glc p,and2,3-Me2-Glc p, in the molar ratio of1:1:7:5:1(Table1).The analysis of the methylated sugars was conducted by GC–MS of their alditol acetates(Sweet et al.,1975).So they were identiﬁed as terminal arabinose,terminal,1,4-linked,1,6-linked,and 1,4,6-linked glucose,respectively.A relatively good agree-ment was found between molar ratios of methylated alditol acetates and that of their parent sugars measured by direct analysis.Methylation analysis displayed around6.7%of glucose was branched,and all of the arabinose residues were present as terminal arabinose.Furthermore,the pro-portion of terminal glucose units was7.2%indicating a degree of polymerization about2,according to the mean molecular weight(5.1kDa)of APS-1d.The FT-IR spectrum of APS-1d was shown in Fig.2. The band in the region of3411.19cmÀ1was due to the hydroxyl stretching vibration of the polysaccharide.The band in the region of2930.27cmÀ1was due to C–H stretching vibration and the band in the region of 1639.47cmÀ1was owing to associated water.The positive speciﬁc rotation and the characteristic absorption at 848.18cmÀ1in the IR spectrum indicated a-conﬁguration of D-glucan(Barker,Bourne,Stacey,&Whiﬀen,1954).The13C NMR spectrum of APS-1d showed in Fig.3. Based on the data available in the literature,it was possible to identify that the resonances in the region of97–100ppm were attributed to the anomeric carbon atoms of glucopyranose(Glc p)and arabofuranose(Ara f),respec-tively.Two main peaks at d99.6and97.7ppm correspond-ed to C-1of the1,4-D-Glc p and1,6-D-Glc p residues, respectively.The signals at d98.5and98.0ppm correspond to C-1of1,4,6-D-Glc p,and T-D-Glc p residues,respectively (Funane et al.,2001;Seymour,Knapp,&Bishop,1976; Tylianakis,Spyros,Dais,Taravel,&Perico,1999; Uzochukwu,Balogh,Loeﬂer,&Ngoddy,2002;Wang,Table2Chemical shifts of resonances in the13C and1H NMR spectra of APS-1dSugar residues Chemical shifts(d,ppm)C1/H1C2/H2C3/H3C4/H4C5/H5;H50C6/H6;H60b-Ara f-(1ﬁ100.0/5.3376.2/4.1175.2/4.0180.3/3.8759.9/3.70;3.61a-Glc p-(1ﬁ98.0/4.9071.8/3.6273.1/3.6269.5/3.3570.2/3.6263.4/3.70;3.60ﬁ4)-a-Glc p-(1ﬁ99.6/5.3371.2/3.5573.4/3.8976.7/3.5771.5/3.6260.4/3.78;3.76ﬁ6)-a-Glc p-(1ﬁ97.7/4.9071.2/3.4873.1/3.6269.5/3.4470.2/3.8365.5/3.90;3.67ﬁ4,6)-a-Glc p-(1ﬁ98.5/4.9071.2/3.5573.1/3.7076.7/3.5770.2/3.9465.9/3.90;3.67Fig.4.500MHz HSQC spectrum of APS-1d in D2O solutions at30°C.(A)ﬁ4)-a-Glc p-(1ﬁ,(B)ﬁ6)-a-Glc p-(1ﬁ,(C)ﬁ4,6)-a-Glc p-(1ﬁ,(D)a-Glc p-(1ﬁ,and(E)b-Ara f-(1ﬁ.154W.Cao et al./Carbohydrate Polymers66(2006)149–159Peng,Huang,Peng,&Tian,2001).The appearance of the respective carbon signals indicated that the Glc p moieties both were the a-anomeric conﬁguration(Uzochukwu et al.,2002).While the resonance due to C-1of T-Ara f was observed at100.0ppm which revealed a b-anomeric conﬁg-uration(Cardoso,Silva,&Coimbra,2002;Ryden,Colqu-houn,&Selvendran,1989;Swamy&Salimath,1991).The conﬁgurations were conﬁrmed by the1H NMR spectrum, which displayed signals forﬁve anomeric protons at5.33 (H-1of the1,4-D-Glc p and T-Ara f)and4.90ppm(H-1of the1,6-D-Glc p,T-D-Glc p,and1,4,6-D-Glc p).Furthermore, the signals in the highﬁeld of59–66ppm were investigated by DEPT135(Fig.3).In this experiment,the methylene carbons show opposite amplitude to the methyl and the methyne carbons.So the well-deﬁned signals at59.9,60.4, 63.4,65.5and65.9,could be attributed to C-5of T-L-Ara f,C-6of1,4-D-Glc p,T-D-Glc p,1,6-D-Glc p,and 1,4,6-D-Glc p,respectively,referred to the previous reports (Cardoso et al.,2002;Wang et al.,2001).The carbon signal at d65.5ppm should be C-6of the1,6-D-Glc p,whichwas Fig.5.500MHz H/H-COSY and HMBC spectra of APS-1d in D2O solutions at30°C.(A)ﬁ4)-a-Glc p-(1ﬁ,(B)ﬁ6)-a-Glc p-(1ﬁ,(C)ﬁ4,6)-a-Glc p-(1ﬁ,(D)a-Glc p-(1ﬁ,and(E)b-Ara f-(1ﬁ.W.Cao et al./Carbohydrate Polymers66(2006)149–159155shifted about4ppm downﬁeld compared with the resonance of standard methyl glycoside due to the eﬀect of glycosyla-tion(Seymour,Knapp,Bishop,&Jeans,1979).Similarly, the C-4signal at76.7ppm of1,4-D-Glc p and1,4,6-D-Glc p appeared5.25ppm downﬁeld compared with that of the standard methyl glycoside(Agarwal,1992).Other signals in13C NMR and1H NMR spectra,which were summarized in Table2,were assigned on the basis of the correlation of H-H-COSY,HSQC,and HMBC exper-iments and referred to the previous reports(Cardoso et al., 2002;Seymour et al.,1976;Tylianakis et al.,1999; Uzochukwu et al.,2002;Wang et al.,2001).The HSQC spectrum of APS-1d was shown in Fig. 4.There were several pairs of H-6–C-6cross peaks presenting in the high ﬁeld.These H-6resonances appeared as doublets due to coupling each other.The C-6signals of1,4-D-Glc p, 1,6-D-Glc p,1,4,6-D-Glc p,and T-D-Glc p were correlated with the resonances at d H-63.78and3.76,3.90and3.67, 3.90and3.67,3.70and3.60,respectively.Similarly,The C-5signal of T-L-Ara f was correlated with the resonances at d H-53.70and3.61.From the COSY spectrum(Fig.5),it was possible to correlate H-1of1,6-D-Glc p(d4.90)with H-2(d3.48),H-2with H-3(d3.62),H-3with H-4(d3.44),and H-4with H-5(d3.83).These results and the analysis of the HSQC spectrum could help to assign C-2,C-3,C-4and C-5of 1,6-D-Glc p to d C-271.2,d C-373.1,d C-469.5and d C-570.2, respectively.C-2,C-3,C-4,and C-5of other residues were assigned by a similar procedure.d71.2,d73.4,d76.7,and d 71.5were due to C-2,C-3,C-4,and C-5of1,4-D-Glc p. However,clear assignments of the H-2s of T-D-Glc p and 1,4,6-D-Glc p were unobtainable due to a high degree of overlap between the H-2,3.Referred to the reports men-tioned above(Seymour et al.,1976;Wang et al.,2001), the C-2signals of T-D-Glc p and1,4,6-D-Glc p were correlat-ed with the resonances at d71.8and d71.2.So C-3,C-4, and C-5of T-D-Glc p were assigned to d73.1,d69.5,and d70.2,C-3,C-4,and C-5of1,4,6-D-Glc p were assigned to d73.1,d76.7,and d70.2,respectively,according to the cross peaks in the COSY and HSQC spectra.Further-more,as there was no clear H-2–C-1cross peak of T-L-Ara f in the COSY spectrum,the signal at d4.11was assigned to H-2according to the literature(Cardoso et al.,2002).With the assistant of HSQC spectrum,cross peaks of H-2of T-L-Ara f with H-3(d4.01)and H-3with H-4(d3.87)could found in the COSY spectrum.To deduce more information about the structure of APS-1d and to conﬁrm the assignments made from HSQC and COSY spectra,a HMBC spectrum was recorded (Fig.5).In the HMBC spectrum,cross peaks H-1–C-6 (linkages of two1,6-D-Glc p residues),H-1–C-4(linkages of two1,4-D-Glc p residues),and similarly,H-6–C-1(link-ages of1,6-D-Glc p and T-L-Ara f residue)were identiﬁed. Moreover,there was no cross peak between H-1of1,4-D-Glc p and C-6of1,6-D-Glc p or cross peak between H-1of 1,6-D-Glc p and C-4of1,4-D-Glc p founded.These results did not support the occurrence of the direct linkage of 1,4-D-Glc p to1,6-D-Glc p,but suggested that the T-b-L-Ara f should be linked directly to a1,6-D-Glc p residue.The cross peaks at d H-15.33and the13C resonances at d70–74in HMBC spectrum allowed their identiﬁcation as C-3(d 73.4)and C-2(d71.2)of1,4-D-Glc p residues.These,along with the additional intra-residue correlations observed between the resolved H-2s of each residue and their respec-tive C-1and C-3resonances,or the resolved H-3s of each residue and their respective C-2and C-4resonances,served to conﬁrm the assignments within each glucosyl residue.APS-1d was partially hydrolyzed with0.2mol/L TFA, the major dialyzable fraction contained glucose and arabi-nose,indicating that they were present at outer chains.The nondialyzable fraction was further fractionated by Sepha-dex G-75column and a pure fraction HAPS-1d with a molecular weight of2.8kDa was obtained.HAPS-1d was per-methylated and analyzed on GC–MS.The result (Table1)showed all the Ara residues and1,6-Glc p were lost,while the proportion of1,4-Glc p residues increased, compared with methylation analysis of APS-1d.It suggest-ed that1,6-Glc p existed as side chain and attached directly to the1,4-Glc p main chain.These results were in agreement with the data above.Since the molecular weight of APS-1d was5.1kDa,the possible structure was shown in Fig.6.3.3.Anti-tumor activity of APS-1dThe anti-tumor activity of the polysaccharide was usual-ly believed to be a consequence of the stimulation of the cell-mediated immune response(Ooi&Liu,2000).For instance,immunostimulatory activities were found inthe Fig.6.Predicted structure ofAPS-1d.Fig.7.The MTT assay of HeLa and A549cells induced with diﬀerent concentrations of APS-1d.Signiﬁcant diﬀerences from negative control group were evaluated using Student’s t test:*P<0.05,**P<0.01.156W.Cao et al./Carbohydrate Polymers66(2006)149–159polysaccharides from Panax ginseng,Ganoderma lucidum, Coriolus versicolor,etc.,which suggested that immunostim-ulatory eﬀects might be the main mechanism of polysac-charides’anti-tumor activities(Shin et al.,2002;Cao& Lin,2004;Ho,Konerding,Gaumann,Groth,&Liu, 2004).But some polysaccharides,such as polysaccharides from Phellinus linteus(Li et al.,2004)and Cordyceps sinensis(Chen,Shiao,Lee,&Wang,1997),could directly inhibit the proliferation of cancer cell in vitro.In this study, we investigated the anti-tumor activities of APS-1d against two kinds of human solid cancer cell lines,HeLa and A549 in vitro.Fig.7showed the eﬀects of the polysaccharide on the growth of HeLa and A549cells.At the concentrations from3to100l g/ml,APS-1d signiﬁcantly inhibited the proliferation of HeLa cells(P<0.01)and the eﬀects were in a concentration-dependent manner.At the highest con-centration of100l g/ml,APS-1d had the inhibition ratio of23.0±5.5%.However,although APS-1d also had signif-icant suppressing activity on A549cells,there was no clear relationship between the concentrations and the eﬀects. The highest inhibition ratio on A549cells was6.6±3.6% between four dosages,which was far lower than the ratio (17.0±5.8%)on HeLa cells at the same concentration (10l g/ml).From above,APS-1d displayed signiﬁcant anti-tumor activity,especially in human HeLa cells.To conﬁrm the anti-tumor activity of APS-1d in vivo, we used the mice transplanted S180to evaluate the eﬀects and the results were summarized in Table3.APS-1d could inhibit the growth of the tumors(P<0.01)in a dose-de-pendent manner.The inhibitory rate in mice treated with 100mg/kg APS-1d was50.7%,being the highest in the three doses.Furthermore,during the experiments,the appetite,activity and coat luster of each animal in APS-1d groups were better than the mice treated with cyclophosphamide.On the14th day,the average increased body weight of negative control mice was4.22g,whereas the weight of mice in APS-1d group at dose of100mg/kg was6.92g.The increased body weights of the most test groups were signiﬁcantly greater than those in the cyclo-phosphamide group(P<0.01),even the negative control mice(P<0.05).The average weights of the spleens in test groups were signiﬁcantly greater than that in the cyclo-phosphamide mice(P<0.01),and even that of the negative control mice(P<0.05),indicating that APS-1d could increase the weights of immune organs.These results suggested that activating immune responses in the host might be one of the mechanisms of anti-tumor activity of APS-1d,as many anti-tumor polysaccharides found in the world(Ooi&Liu,2000;Wasser,2002).4.ConclusionAPS-1d,ﬁrst isolated from the roots of A.sinensis,was a heteropolysaccharide,having a backbone composed of 1,4-a-D-Glc p,with branches attached to O-6of some resi-dues.The branches were composed of1,6-a-D-Glc p resi-dues,and terminated with b-L-Ara f residues.APS-1d exhibited signiﬁcant anti-tumor activates both in vitro and in vivo.The structural and pharmacological results obtained might help enlarge the knowledge of structural correlation to anti-tumor eﬀects of polysaccharides. AcknowledgmentsThis study was supported by the Research Fund of the Fourth Military Medical University.The authors are thankful to Dr.Xiaoyong Li at Xi’an Jiaotong University, for the GC–MS analysis and Mr.Minchang Wang at Xi’an Modern Chemistry Research Institute,for recording the NMR spectra.ReferencesAgarwal,P.K.(1992).NMR spectroscopy in the structural elucidation of oligosaccharides and glycosides.Phytochemistry,31,3307–3330. Barker,S.A.,Bourne,E.J.,Stacey,M.,&Whiﬀen,D.H.(1954).Infra-red spectra of carbohydrates.Part I.Some derivatives of D-glucopyr-anose.Journal of the Chemical Society,171–176.Bradford,M.M.(1976).A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.Analytical Biochemistry,72,248–254.Cao,Q.Z.,&Lin,Z.B.(2004).Antitumor and anti-angiogenic activity of Ganoderma lucidum polysaccharides peptide.Acta Pharmacologica Sinica,25,833–838.Cardoso,S.M.,Silva,A.M.S.,&Coimbra,M.A.(2002).Structural characterisation of the olive pomace pectic polysaccharide arabinan side chains.Carbohydrate Research,337,917–924.Chen,R.,Wang,H.,Xu,H.,Xu,G.,&Chang,L.(2001).Isolation, puriﬁcation and determination of polysaccharides X-C-3-III and X-C-3-IV from Angelica sinensis(Oliv)Diels.Zhong Yao Cai,24(1),36–37. Chen,Y.J.,Shiao,M.S.,Lee,S.S.,&Wang,S.Y.(1997).Eﬀect of Cordyceps sinensis on the proliferation and diﬀerentiation of human leukemic U937cells.Life Sciences,60,2349–2359.Table3Anti-tumor activities of APS-1d on S180tumorSample Dose(mg/kg)Increase of body weight(g)Spleen weight(g)Tumor weight(g)Inhibitory rate of tumor(%) Negative control 4.22±0.980.18±0.03 1.42±0.43Positive control20 1.10±0.940.09±0.030.45±0.2068.3120 4.04±2.42d0.20±0.04ad0.78±0.26bd45.07APS-1d50 5.83±1.74ad0.21±0.04ad0.71±0.19bd50.00100 6.92±1.31bd0.21±0.03bd0.70±0.24bc50.70Signiﬁcant diﬀerences from negative control group were evaluated using Student’s t test:a P<0.05,b P<0.01.Signiﬁcant diﬀerences from positive control group(spleen weight):c P<0.05,d P<0.01.W.Cao et al./Carbohydrate Polymers66(2006)149–159157。

异氰酸酯聚氨酯英文

Carbohydrate Polymers 101 (2014) 1043–1060Contents lists available at ScienceDirectCarbohydratePolymersj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r b p olReviewFunctionalized bacterial cellulose derivatives and nanocompositesWeili Hu,Shiyan Chen ∗,Jingxuan Yang,Zhe Li,Huaping Wang ∗State Key Laboratory for Modiﬁcation of Chemical Fibers and Polymer Materials,Key Laboratory of High-performance Fibers and Products,Ministry of Education College of Materials Science and Engineering,Donghua University,Shanghai 201620,Chinaa r t i c l ei n f oArticle history:Received 9August 2013Received in revised form 23September 2013Accepted 29September 2013Available online 6 October 2013Keywords:Bacterial cellulose Modiﬁcation Nanocomposites Functionalizationa b s t r a c tBacterial cellulose (BC)is a fascinating and renewable natural nanomaterial characterized by favor-able properties such as remarkable mechanical properties,porosity,water absorbency,moldability,biodegradability and excellent biological afﬁnity.Intensive research and exploration in the past few decades on BC nanomaterials mainly focused on their biosynthetic process to achieve the low-cost preparation and application in medical,food,advanced acoustic diaphragms,and other ﬁelds.These investigations have led to the emergence of more diverse potential applications exploiting the function-ality of BC nanomaterials.This review gives a summary of construction strategies including biosynthetic modiﬁcation,chemical modiﬁcation,and different in situ and ex situ patterns of functionalization for the preparation of advanced BC-based functional nanomaterials.The major studies being directed toward elaborate designs of highly functionalized material systems for many-faceted prospective applications.Simple biosynthetic or chemical modiﬁcation on BC surface can improve its compatibility with differ-ent matrix and expand its utilization in nano-related applications.Moreover,based on the construction strategies of functional nanomaterial system,different guest substrates including small molecules,inor-ganic nanoparticles or nanowires,and polymers can be incorporated onto the surfaces of BC nanoﬁbers to prepare various functional nanocomposites with outstanding properties,or signiﬁcantly improved physicochemical,catalytic,optoelectronic,as well as magnetic properties.We focus on the preparation methods,formation mechanisms,and unique performances of the different BC derivatives or BC-based nanocomposites.The special applications of the advanced BC-based functional nanomaterials,such as sensors,photocatalytic nanomaterials,optoelectronic devices,and magnetically responsive membranes are also critically and comprehensively reviewed.Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved.Contents 1.Introduction ..........................................................................................................................................10442.Construction strategies of BC-based functional nanomaterials .....................................................................................10452.1.Biosynthetic modiﬁcation .. (1045)2.1.1.Altered BC structure ................................................................................................................10452.1.2.Nanocomposites .. (1046)2.2.Chemical surface modiﬁcation ...............................................................................................................10462.3.In situ formation of nanostructures . (1047)2.3.1.In situ formation of nanostructures through reduction reaction ..................................................................10482.3.2.In situ formation of nanostructures through precipitation reaction...............................................................10482.3.3.In situ formation of nanostructures through sol-gel reaction (1048)2.4.Ex situ introduction of components..........................................................................................................10492.5.Other combined strategies ...................................................................................................................10493.Applications of BC-based functional nanomaterials .................................................................................................10533.1.Sensors........................................................................................................................................10533.2.Photocatalytic nanomaterials . (1053)∗Corresponding authors.Tel.:+862167792950;fax:+862167792726.E-mail addresses:chensy@ (S.Chen),wanghp@ (H.Wang).0144-8617/$–see front matter.Crown Copyright © 2013 Published by Elsevier Ltd. All rights reserved./10.1016/j.carbpol.2013.09.1021044W.Hu et al./Carbohydrate Polymers101 (2014) 1043–10603.3.Optoelectronics (1054)3.3.1.Electrically conductiveﬁlms (1054)3.3.2.Optically transparentﬁlms (1055)3.3.3.Flexible displays (1056)3.3.4.Photoluminescent and photochromicﬁlms (1056)3.4.Magnetically responsiveﬁlms (1057)4.Concluding remarks (1058)Acknowledgements (1059)References (1059)1.IntroductionIt is well known that cellulose is a very important and fasci-nating biopolymer and an almost inexhaustible and sustainable natural polymeric raw material,which is of special importance both in industries and in daily lives.In the past decade,the design and development of renewable resources and innovative prod-ucts for science,medicine and technology have led to a global revival of interdisciplinary research and utilization of this abun-dant natural polymer.Formed by repeated connection of glucose building blocks,cellulose possesses abundant surface hydroxyl groups forming plentiful inter-and intra-molecular hydrogen bonds,characterized by its hydrophilicity,chirality,biodegrad-ability,and broad chemical-modifying capacity(Klemm,Heublein, Fink,&Bohn,2005).The properties of cellulose largely depend on the speciﬁc assembling and supramolecular order controlled by the origin and treatment of cellulose(Eichhorn et al.,2010).Bacterial cellulose(BC)has the same molecular formula as plant cellulose, but with unique and sophisticated three-dimensional porous net-work structures.Intrinsically originated from the unique structure, BC demonstrates a serious of distinguished structural features and properties such as high purity,high degree of polymerization(up to8000),high crystallinity(of70–80%),high water content to99%, and high mechanical stability,which is quite different from the natural cellulose(Barud et al.,2011).These speciﬁc parameters are determined by the biofabrication approach of BC(Fig.1)and the controllable shape and supramolecular structure through the alter-ation of cultivation conditions during fermentation.These amazing physicochemical properties have attracted signiﬁcant interest from both research scientists and industrialists.So far,BC has wide appli-cation in variousﬁelds including medical,food,advanced acoustic diaphragms,and so on(Klemm et al.,2011).These research and exploration have led to the emergence of more diverse potential applications exploiting the functionality of BC nanomaterials.In view of expanding the scope of BC applications,it is important to take full advantage of the unique structure and properties of BC nanomaterials to develop novel BC-based nanomaterials with ground-breaking new features.Various modiﬁcation methods have been explored to open up possibilities for endowing BC with new functionalities(Klemm et al.,2011).Simple biosynthetic or chemical modiﬁcation on BC surface can improve its compatibility with different matrixes and expand its utilization in nano-related applications.Another notable feature of BC is its high aspect ratio and abundant active functional hydroxyl groups,which makes it suitable for combination with different nanostructures by provid-ing powerful interaction of BC with surrounding species,such as inorganic and polymeric nanoparticles and nanowires(Huang& Gu,2011).This novel concept breaks new ground to make optimum use of the speciﬁc chemical properties of the guest substrates,in association with the unique features of renewable BC resources. As a promising template in the synthesis of a great variety of nanostructures with designed properties and functionalities,BC can play the role of reducing agent,structure-directing agent and stabilizer(Yang,Xie,Deng,Bian,&Hong,2012).The polymeric or inorganic ingredients can be incorporated and cooperatively inter-act with BC nanoﬁbers to attain some functional products.Such new high-value materials are the subject of continuing research and are commercially interesting in terms of new products from the inexhaustible and sustainable natural polymeric raw material.In the last few years,growing worldwide activity can be observed regarding extensive scientiﬁc investigation and increas-ing efforts for the practical use of the BC materials.There is an increasing annual publication activity on BC(also known as micro-bial cellulose or bacterial nanocellulose)since2000as presented in Fig.2.In recent years,the investigation and utilization of BC in functional materials have been the focus of research,and a growing number of works have been included in thisﬁeld.Functional BC-based nanomaterials are especially an attractive topic because they enable the creation of materials with improved or new properties by mixing multiple constituents and exploiting synergistic effects, such as electronic,optical,magnetic,catalytic properties and bioac-tivity.With a special property or several remarkable functions, functional BC-based nanomaterials are a type of high value-added materials possessing potential applications in speciﬁcﬁelds.In this review,recent developments on BC-based advanced functional nanomaterials including modiﬁed BC nanomaterials,functional BC-based nanocomposites and their applications will be discussed and reviewed.A variety of surface functionalization through biosyn-thetic or chemical modiﬁcation will be considered,which can improve the functionality of BC nanomaterials and expandits Fig.1.The illustration of biofabrication process of BC.W.Hu et al./Carbohydrate Polymers101 (2014) 1043–10601045Fig.2.The illustration of the annual number of publications on BC since2000 (SciFinder Scholar search system,search term“bacterial cellulose”).potential applicationﬁelds.Various approaches to the preparation of functional BC-based nanocomposites by incorporating different guest substrates including small molecules,inorganic nanoparti-cles or nanowires,and polymers on the surfaces of BC nanoﬁbers are summarized,which mainly focus on the preparation methods,per-formances and some formation mechanisms of speciﬁc functional nanomaterials.2.Construction strategies of BC-based functional nanomaterialsAlthough BC nanomaterial has unique physical and chemical characteristics,its high degree of crystallinity and sole functional group lead to its poor dissolubility and processability,thereby limiting the applicationﬁelds.BC possesses an abundance of hydroxyl groups on the surface,where modiﬁcation can be easily achieved.It can be modiﬁed to achieve alternative functional groups and patterns of functionalization using in situ and ex situ modiﬁcation methods as shown in Fig.3.The properties of BC derivatives are primarily determined by the type of the functional groups.In particular,modiﬁed BC with more than one functional group possessing different surface characteristics such as lipophilic–hydrophilic properties,magnetic and optical properties combined with a controlled pre-set functionalization pattern is in the center of interest.2.1.Biosynthetic modiﬁcationThe shape and supramolecular structure of BC can be controlled by the change of cultivation conditions such as the type of strain, carbon source,and additives(Heßler&Klemm,2009;Klemm et al., 2006).Studies have demonstrated the potential for manipulating the biogenesis of BC in order to produce modiﬁed BC nanoﬁbers with a controlled composition,morphology,and properties.The inclusion of additives in the nutrient media components during biosynthesis can inﬂuence the assembly and microstructure of BC including the crystallinity,crystalline polymorphism,crystal-lite size and ribbon width.The presence of additives in the media may interfere with the bacterial cells or bind directly to the cel-lulose during production,thereby affecting the yield,structure, morphology and physical properties of BC.The in situ generation of composites can also be effectively regulated during biosynthesis by the inclusion of additives and dispersed particles.2.1.1.Altered BC structureAs a remarkable beneﬁt of BC,the ultrastructure and morphol-ogy of BC can be altered by introducing additives not speciﬁcally required for bacterial cell growth in the media.The effects of the water-soluble agents in culture media on the aggregation and crystallization of BC microﬁbrils were intensively studied.Adding various water-soluble chemical reagents can modify the microﬁb-rillar features of the cellulosic ribbons.Adding nalidixic acid or chloramphenicol produced ribbons with an apparently larger width,probably because several ribbons from a cluster of cells whose dividing process was inhibited,combined or intertwined. While adding dithiothreitol produced ribbons with only45%width of the control ribbons on average(Yamanaka&Sugiyama,2000). Fig.3.Schematic illustration of the generalized synthetic routes to modiﬁed BC nanomaterials(Chen et al.,2010;Geng et al.,2011;Heßler&Klemm,2009;Hu,Chen,Xu, et al.,2011;Ifuku et al.,2009;Oshima et al.,2008;Shen et al.,2009;Yamanaka&Sugiyama,2000).1046W.Hu et al./Carbohydrate Polymers101 (2014) 1043–1060The pore system and water content of BC can be controlled in situ by the incorporation of water soluble polymers such as carboxymethylcellulose(CMC),hydroxypropylmethyl cellu-lose(HPMC),methylcellulose(MC),poly(vinyl alcohol)(PVA)and polyethylene glycol(PEG)to the culture medium(Seifert,Hesse, Kabrelian,&Klemm,2004).In the presence of these additives,the pore system and elasticity of BC,as well as the water adsorption and water holding capacity can be controlled.It has been claimed the addition of HPMC,CMC and MC can cause decreased crystallinity and crystal size,as well as greater thermal stability and pore size (Chen,Chen,Huang,&Lin,2011).Along with the formation of porelike network structure,the water retention ability and the ion absorption capacity increase.The functionalized BC produced with CMC also showed good adsorption performances for copper and lead ions(Chen et al.,2009).Nevertheless the presence of PVA in the culture medium results in a reduced water absorption ability and a slightly higher copper ion capacity in comparison with original BC.The addition of␤-cyclodextrin or PEG400causes a remarkable pore size increase(Heßler&Klemm,2009).Surprisingly,these co-substrates act as removable auxiliaries not incorporated in the BC samples.Other polymers such as Tween80,urea,ﬂuorescent bright-ener and sodium alginate(NaAlg)have also been incorporated into the BC fermentation medium,with the observed differences in pore size,degree of polymerization,crystallinity,ﬁber widths and mechanical strength(Huang,Chen,Lin,Hsu,&Chen,2010; Ruka,Simon,&Dean,2013;Zhou,Sun,Hu,Li,&Yang,2007). The results revealed that the addition of urea can increase the mechanical strength.The bundle widths of BC produced withﬂuo-rescent brightener increased and the cellulose network void grew. BC produced with NaAlg added has a lower crystallinity,a smaller crystalline size and an enhanced yield.2.1.2.NanocompositesIn particular,the additives can be in situ incorporated into the growing BCﬁbrils to create a novel type of nanocomposites,which represents a very speciﬁc and important modiﬁcation method of BC.This in situ technique can combine the properties of altered supramolecular structure of BC with those of the incorporated com-ponents.So far,researchers have put different efforts to obtain BC nanocomposites by applying various additives such as organic com-pounds,polymers and inorganic substances(Klemm et al.,2011).The biological fermentation of BC in presence of cationic starch leads to the formation of double-network BC composites by incor-poration of the starch derivative in the BC network(Heßler& Klemm,2009).This double-stage structure consists of an opaque upper and a transparent under part.As shown in Fig.4a,in case of the upper layer,the additive adsorbs at the celluloseﬁbers and causes an irregular distribution of the pore sizes.The under layer indicates the incorporation of the starch into the solvate shells of the BC prepolymer,forming an exciting skinnyﬁlm structure (Fig.4b).Other characteristic examples include the additives of poly(ethylene oxide)(Brown&Laborie,2008),PVA(Gea,Bilotti, Reynolds,Soykeabkeaw,&Peijs,2010)and starch(Grande et al., 2009)in the media for the formation of nanocomposites with the incorporation of these additives into the network of BC.Along with the increase of the additive content,the cellulose crystallized into smaller nanoﬁbers,which further bonded together into bundles. The BC nanoﬁbers were well dispersed in the composites and the nanocomposites typically show signiﬁcantly improved mechanical properties.The inorganic additives can drastically modify the performance of BC.BC/multiwalled carbon nanotubes(MWCNTs)composites can be obtained in the presence of MWCNTs in an agitated cul-ture(Park,Kim,Kwon,Hong,&Jin,2009;Yan,Chen,Wang,Wang, &Jiang,2008).Interestingly,a core–shell structure model was demonstrated,with the packed MWNTs attached nascent sub-elementaryﬁbrils as the core of the cellulose assemblies as shown in Fig.4c.While in the static culture method,band-like assemblies with sharp bends and rigidness were produced in the presence of MWCNTs.Similarly,the crystallinity index,crystallite size,and cellulose I␣content also changed,which may be attributed to the interaction between the hydroxyl groups of treated MWCNTs and the sub-elementary BCﬁbrils,interfering with the aggre-gation and crystallization of BC microﬁbrils.By adding silica or titanium precursor into the static growth medium,the SiO2and TiO2nanoparticles about several tens of nanometers in size can be incorporated onto BC microﬁbrils(Geng et al.,2011;Yano,Maeda, Nakajima,Hagiwara,&Sawaguchi,2008)(Fig.4d).It is inferred that the basic proteins in the outer membrane of bacterium cell act as the catalyst for the hydrolysis and condensation of inorganic precur-sor,the surface of both outer membrane and BC nanoﬁbers renders the nucleation and growth sites for inorganic nanoparticles.The space-temporal effect endows bacteria the delicate control ability over formation of the nanocomposites.2.2.Chemical surface modiﬁcationAlthough the biosynthetic modiﬁcation of BC is a kind of green sustainable technology,the strict microbial fermentation environ-ment restricts the introduction of some additives.Other questions involving the interaction mechanism between the additives and microﬁbrils growth,as well as the structure control of BC nanoﬁbers still need to be addressed.While chemically modiﬁed methods are not limited by the required types of pared to the biosynthesis method,its more clearly deﬁned objectives and prin-ciples make it a feasible modiﬁed method for BC material.Since the chemical composition of BC is similar to plantﬁbers,it can also be carboxymethylated,acetylated,phosphorylated,and mod-iﬁed by other graft copolymerization and crosslinking reaction to obtain a series of BC derivatives.The introduction of new functional groups to the BC structure can endow BC with various features such as hydrophobicity,ions adsorption capacity and optical proper-ties while maintaining the unique three-dimensional nano network and excellent mechanical properties of BC.In most cases,the modiﬁcation of BC focuses on the improve-ment of its applicability and performance in different application ﬁelds.Several methods have been employed to achieve BC derivatives with improved metal ions absorption capacity.The functionalized diethylenetriamine-BC(EABC),amidoximated BC (Am-BC)and phosphorylated BC have been prepared as new adsor-bents for metal ions(Chen,Shen,Yu,Hu,&Wang,2010;Oshima, Kondo,Ohto,Inoue,&Baba,2008;Shen et al.,2009).The experi-mental data showed that the microporous network structure of BC was maintained after the modiﬁcation and these novel adsorbent showed good adsorption performances for different metal ions.To anchor metallic ions on BC nanoﬁbers,the carboxylate groups have been introduced onto the BC nanoﬁber surface using the2,2,6,6-tetramethylpiperidine-1-oxyradical(TEMPO)-mediated oxidation system(Ifuku,Tsuji,Morimoto,Saimoto,&Yano,2009).The oxida-tion can proceed under mild aqueous conditions maintaining the crystallinity and crystal size of BC nanoﬁbers.In order to improve the dispersability and compatibility in dif-ferent solvents or matrices that are suitable in the production of nanocomposites,the acetylation of BC based on a non-swelling reaction mechanism was reported recently.BC could be partially acetylated by theﬁbrous acetylation method to modify its physical properties,while preserving the microﬁbrillar morphology(Kim, Nishiyama,&Kuga,2002).The hydrophobicity of the acetylated surface is advantageous for maintaining a large surface area on drying from water and would make the microﬁbrils compatible with other hydrophobic materials.While most of the chemicallyW.Hu et al./Carbohydrate Polymers101 (2014) 1043–10601047Fig.4.SEM images of freeze-dried upper part of BC/starch composite(a),under part of BC/starch composite(b),middle layer of BC/MWCNTs composites(c),and BC/SiO2 nanocomposites(d)(Geng et al.,2011;Heßler&Klemm,2009;Park et al.,2009).modiﬁed techniques require tedious solvent exchanges that strongly diminish the environmental beneﬁt of the use of BC.A major thrust of recent BC modiﬁcation research focused on the design of more economical and environmentally friendly method to obtain novel BC derivatives.Thus,a solvent-free derivatization system appears as an important goal to get BC derivative prod-ucts with hydrophobic surfaces with a minimum environment impact.In line with the solvent-free BC derivatization concept,BC preserving the microﬁbrillar morphology has been partially acety-lated using acetic anhydride in the presence of iodine as a catalyst (Hu,Chen,Xu,&Wang,2011).Another example is the reported vapor-phase technique which has been applied recently for the surface esteriﬁcation of BC microﬁbrils with the help of gaseous triﬂuoroacetic acid mixed with acetylating agents(Berlioz,Molina-Boisseau,Nishiyama,&Heux,2009).Experimental results have shown the acetylation proceeded from the surface to the interior crystalline core of BC nanﬁbers.Hence,for moderate degree of sub-stitution,the surface was fully grafted whereas the cellulose core remained unmodiﬁed and the originalﬁbrous morphology was maintained.The obtained acetylated BC membrane shows more hydrophobic surface and good mechanical properties as shown in Fig.5,which is in favor of enhancing the hydrophobic non-polar polymeric matrix.2.3.In situ formation of nanostructuresBC has unique micro-nano porous three-dimensional network, which can facilitate the penetration of various metal ions into the interior.It also possesses a great deal of hydroxyl and ether bonds,forming the effective reactive sites to anchor metallic ions on the surface of the nanoﬁbers.Then a variety of inorganic nanoparticles or nanowires can be formed through precipitation, oxidation–reduction and sol–gel reaction as shown in Fig.6.Dif-ferent from the doped nanoparticles into BC matrix,the size and morphology of the nanoparticles can be regulated by adjusting the structure of BC template and the in situ preparation condi-tions.At the same time,the nanospace in the BCﬁbers can behave as an effective nanoreactor to prevent the unwanted agglomera-tion phenomenon,ensuring the effective dispersion of the formed nanoparticles in the BC matrix.In the process of in situ preparation of BC-based nanocompos-ites,the second components such as metal and polymer in the form of particles orﬁbers can be introduced into the BC matrix,while retaining the unique three-dimensional nanoporous network of BC.BC can be viewed as a soft template to control the synthesis of desired nano-materials or nano-structure with speciﬁc size and shape.This can obtain a variety of novel functional materialswith Fig.5.(a)FE-SEM image of vacuum-dried acetylated BC,and the inset shows the proﬁle of water droplets on the membrane surface.(b)Tensile stress–strain behaviors of air dried BC and acetylated BC(Hu,Chen,Xu,et al.,2011).1048W.Hu et al./Carbohydrate Polymers 101 (2014) 1043–1060Fig.6.Schematic diagram of the in situ preparation of nanoparticles/BC nanocomposites.unique features and outstanding ing BC as a tem-plate in the in situ synthesis of nano-materials has the following advantages:BC is a kind of environment-friendly and renewable material which can be produced from a wide range of raw mate-rials.The shape,structure and properties can be easily adjusted during the biosynthesis,pretreatment and chemical modiﬁcation process.Furthermore,the BC matrix can be removed by calcination to obtain the pure inorganic nanoparticles and nanowires structure.Therefore,BC with controllable structure and pore size can provide restrictive environment to ensure a variety of nanostruc-tures effectively embedded in the matrix.The method does not require severe conditions,and is simple and easy to implement,which can obtain the nano-particles with narrow size distribution.So far,this method has been applied to synthesize different nano-materials such as metal,semiconductor and electrically conducting polymers through different preparation methods.2.3.1.In situ formation of nanostructures through reduction reactionThere have been some reports regarding the application of BC as a soft template to in situ form different metal nanoparticles by the reduction reaction as shown in Table 1.The size and mor-phology of the formed nanoparticles can be controlled by using different reducing agents including sodium borohydride (NaBH 4),triethanolamine,hydrazine (NH 2NH 2),hydroxylamine (NH 2OH),ascorbic acid,polyethylenimine (PEI)and so on (Barud et al.,2008;Yang,Xie,Hong,Cao,&Yang,2012;Zhang et al.,2010).These reducing agents can serve as an assistant material for stabilizing nanoparticles,preventing their aggregation.In addition,BC itself can be used as a reductant to produce metal nanoparticles (Yang,Xie,Deng,et al.,2012),without introducing additional reducing agent or stabilizing agent,thus avoiding the secondary pollutants and guaranteeing the feasibility of its applications in medical and catalytic ﬁeld.2.3.2.In situ formation of nanostructures through precipitation reactionWell-separated nanoﬁbrils of BC create an extensive surface area forming active sites for metal ion adsorption,and the sub-sequent introduction of precipitating agent will react with the immobilized metal ions to generate the initial nuclei of metal or oxide.Then the nuclei continue to grow,thereby further forming functional inorganic nanoparticles as shown in Fig.7.The growth of the nanoparticles was readily controlled by repeated alternating dipping of BC membranes in the metal ion and precipitating agent solution followed by a rinse step.So it is feasible for BC to serve as an excellent matrix in the synthesis of nanoparticles through the in situ precipitation reaction.CdS and CdSe nanoparticles have been synthesized and stabi-lized on BC nanoﬁbers using in situ precipitation method (Li et al.,2009a;Yang et al.,2012a ).At ﬁrst,hydroxyl and ether groups of BC anchored Cd 2+or thioglycolic acid capped Cd 2+,then the anchored Cd 2+reacted with S 2−or Se 2−to generate CdS or CdSe nanoparti-cles on the BC nanoﬁbers as shown in Fig.8.The results indicatedthat nanoparticles with the diameter of 20–30nm deposited on BC nanoﬁbres are well-dispersed in the BC nanoﬁbre-network.AgCl nanoparticles with a size of several tenths of nanometers have been in situ synthesized in the three-dimensional non-woven network of BC nanoﬁbrils (Hu et al.,2009).The growth of the nanoparticles was readily obtained by repeated alternating dipping of BC mem-branes in the solution of silver nitrate or sodium chloride followed by a rinse step.The nanopore is essential for introduction of silver ions and reaction with Cl −to form AgCl particles into BC ﬁbers and removal of the excess chemicals from BC ﬁbers.BC can serve as an excellent matrix in the in situ synthesis of the Fe 3O 4nanoparticles through the coprecipitation reaction (Zhang et al.,2011).The ultraﬁne network architecture of BC gives a good tunnel for Fe 2+/Fe 3+adsorption and ensures the effec-tive anchoring of absorbed ions onto the BC nanoﬁbers through ion–dipole interactions.After being rinsed with distilled water to remove the unanchored ions,the obtained ions/BC complexes were immersed into the excessive NaOH solution.The interaction between Fe 2+/Fe 3+and OH −can induce the formation of Fe 3O 4nanoparticles with the diameter of 80–100nm as shown in Fig.9.During the in situ formation process of different nanostruc-tures,the size and size distribution of the formed nanoparticles are controllable by adjusting synthetic parameters such as the con-centration of metal ions.When the concentration of metal ions is too high,the BC nanoﬁbers fails to fully immoblize and dis-perse the large number of metal ions due to the limited hydroxyl reactive sites shown in Fig.10.Meanwhile,the presence of excess metal ions would result in the agglomeration of the nanoparticles due to the higher aggregation speed than the orientation speed in the particle growth process.Therefore the concentration of the reaction solution needs to be reduced appropriately to ensure the metal ions and nanoparticles effectively dispersed and ﬁxed onto the surface of the nanoﬁbers.However,when the concentra-tion is too low,the loading amount of the nanoparticles will be signiﬁcantly reduced,which would degrade the performance of the ﬁnal products.The exploration of optimal reaction conditions would be particularly important to achieve the effective dispersion of nanoparticles with sufﬁcient loading amount,thereby obtain-ing the functional nanocomposite materials with excellent optical,electrical,magnetic and antibacterial properties.2.3.3.In situ formation of nanostructures through sol-gel reactionTaking advantage of the ultraﬁne nanoﬁbrous structure,as well as abundant porous channels inside the network,BC nanomateri-als can be used as the templates to in situ prepare metal oxides through the sol–gel reaction.The schematic diagram of the in situ preparation process is described in Fig.11.When immersing BC into the precursor solution,the inorganic ions can be immobilized onto the BC nanoﬁbers.Then it can be solidiﬁed to form the gel through hydrolysis and condensation,and subsequently heated to form the desired oxides.BC template can stabilize and disperse the formed oxides nanoparticles through van der Waals forces and hydrogen bonding interaction,prompting generated nanoparticles distributed along the surface of nanoﬁbers.The BC template can。

1H NMR relaxation study of a chitosan-cyclodextrin network

0008-62 15/97/$17.00
PIZ SOOO8-62
0 1997 Elsevier Science Ltd. All rights reserved
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78
G. Paradossi et al. / Carbohydrate Research 300 (1997) 77-84
CARBOHYDRATE RESEARCH
ELSEVIER
Carbohydrate
Research 300 (1997) 77-84
‘ NMR relaxation study of a H chitosan-cyclodextrin network
, Gaio Paradossi ‘ *, Francesca Cavalieri ‘ Vittorio Crescenzi b 7
’ Corresponding author. Fax: + 39-6-7259-4328.
investigated. A number of polysaccharides are principal components of these polymer networks because of their hydrophilicity, availability in nature, and biocompatibility. In addition, carbohydrate polymer hydrogels (physical gels) can form spontaneously in dilute aqueous media as a consequence of disorderto-order transitions, gellan gum being a well-known example in this context. To understand how water interacts with the network chains is a very demanding task, because many of the conventional techniques used in polymer science do not give information about the gel state.

Polyelectrolyte nanoparticles based on water-soluble chitosan–poly (1)

Polyelectrolyte nanoparticles based on water-soluble chitosan–poly (L -aspartic acid)–polyethylene glycol for controlled protein releaseShujun Shu,Xinge Zhang *,Dayong Teng,Zhen Wang,Chaoxing Li *The Key laboratory of Functional Polymer Material of Ministry of Education,Institute of Polymer Chemistry,Nankai University,94#Weijin Road,Tianjin 300071,PR Chinaa r t i c l e i n f o Article history:Received 26November 2008Received in revised form 12April 2009Accepted 16April 2009Available online 20April 2009Keywords:Water-soluble chitosan Poly(L -aspartic acid)Polyethylene glycolPolyelectrolyte nanoparticles Protein drug deliverya b s t r a c tWater-soluble chitosan (WSC)–poly(L -aspartic acid)(PASP)–polyethylene glycol (PEG)nanoparticles (CPP nanoparticles)were prepared spontaneously under quite mild conditions by polyelectrolyte complexa-tion.These nanoparticles were well dispersed and stable in aqueous solution,and their physicochemical properties were characterized by turbidity,FTIR spectroscopy,dynamic light scattering (DLS),transmis-sion electron microscope (TEM),and zeta potential.PEG was chosen to modify WSC–PASP nanoparticles to make a protein-protective agent.Investigation on the encapsulation efﬁciency and loading capacity of the bovine serum albumin (BSA)-loaded CPP nanoparticles was also conducted.Encapsulation efﬁciency was obviously decreased with the increase of initial BSA concentration.Furthermore,its in vitro release characteristics were evaluated at pH 1.2,2.5,and 7.4.In vitro release showed that these nanoparticles provided an initial burst release,followed by a slowly sustained release for more than 24h.The BSA released from CPP nanoparticles showed no signiﬁcant conformational change compared with native BSA,which is superior to the BSA released from nanoparticles without PEG.A cell viability study sug-gested that the nanoparticles had good biocompatibility.This nanoparticle system was considered prom-ising as an advanced drug delivery system for the peptide and protein drug delivery.Ó2009Elsevier Ltd.All rights reserved.1.IntroductionSigniﬁcant advances in biotechnology and genetic research have resulted in the discovery of a large number of proteins and pep-tides that are very effective in disease treatment.1,2Routinely,pep-tides and proteins are administered through the parenteral route,which has poor absorption efﬁciency in patients.Therefore,a large amount of work has focused on protein delivery by the oral route.3–5However,the bioavailability of peptide after oral admin-istration is low due to instability and poor absorption of proteins in the gastrointestinal (GI)tract under most circumstances.One pos-sible way to improve the GI uptake of peptides is to encapsulate them in colloidal nanoparticles that can protect the peptides from being degraded in the GI tract and facilitate their transportation into systemic circulation.6,7Polymeric nanoparticles have been widely investigated as carri-ers for drug delivery.7,8Among them,much attention has been paid to the nanoparticles that are made of synthetic biodegradable poly-mers such as poly(e -caprolactone)and polylactide due to their good biocompatibilities.9,10However,these nanoparticles are not ideal carriers for hydrophilic protein drugs because of their hydro-phobic properties.Owing to the special capability of a polyelectrolyte complex,many complex phenomena in an organism,such as the transfer of gene information,and the interaction between an antibody and its antigen,are related to these complexes,either directly or indirectly.These interactions could contribute to the simulation of the process in an organism and to the accomplishment of some special functions.11In particular,a polyelectrolyte complex is ob-tained when two polymers carrying opposite charges are mixed and interact via electrostatic interactions.Chitosan (CS),a weak cationic polysaccharide produced by deacetylation of the natural polymer chitin,has many useful bio-logical properties,such as biocompatibility,biodegradability,and bioactivity.Because of the existence of amine groups,CS is a polycation and is able to form intermolecular complexes with a wide variety of polyanions including hyaluronic acid,12–14poly(galacturonic acid),15alginate,16gelatin,17dextran sulfate,18and poly(acrylic acid).19–21Most commercially available CS has a quite large molecular weight (M r )and needs to be dissolved in an acetic acid solution at a pH value of approximately 4.0.How-ever,there are potential applications of CS in which a low M r would be essential.Given a low M r ,the polycationic characteristics of water-soluble chitosan can be used together with a good solubility at a pH value close to physiological ranges.Loading of peptide or protein drugs at physiological pH ranges may prevent their bioac-tivity from decreasing.22WSC can improve the transportation of hydrophilic drugs across the intestinal epithelium,and it is0008-6215/$-see front matter Ó2009Elsevier Ltd.All rights reserved.doi:10.1016/j.carres.2009.04.018*Corresponding authors.Tel.:+862223501645;fax:+862223505598(C.L.).E-mail addresses:zhangxinge@ (X.Zhang),lcx@ (C.Li).Carbohydrate Research 344(2009)1197–1204Contents lists available at ScienceDirectCarbohydrate Researchj o u r n a l h o m e p a g e :w w w.e l s e v i e r.c o m /l o c a t e /c a r r esbelieved that WSC can increase the permeability of epithelial tis-sues by disrupting intercellular tight junctions.23,24On this basis, water-soluble chitosan,was used to study the preparation of nanoparticles.PASP is a poly(a-amino acid),which can be classiﬁed as a weak synthetic polyelectrolyte.Poly(amino acids)would seem to have considerable advantages over other polymers owing to their pro-tein-like structure.PASP is of potential interest for being used as a biodegradable water-soluble poly(carboxylic acid).PEG is a nontoxic hydrophilic polymer which is widely used for protein conjugation and protein puriﬁcation.Segmentalﬂexibility and uncharged chemical composition of PEG help to easily adapt their conformations toﬁt the surface topology of proteins.25PEG is reported to abrogate immunogenicity of proteins while preserv-ing their biological activity,and PEG-conjugated peptides are widely investigated for the applications of drug delivery.26,27As these derivatives are uncharged in nature,incorporation of linear PEG chains enhances the mucoadhesive property of the nanoparti-cle systems by improving mobility andﬂexibility.28In this study,a novel nanoparticles system,composed of WSC, PEG,and PASP,was prepared by a simple coagulation method.Poly-electrolyte complexes consisting of WSC and PASP may possess merit of both WSC and PASP,and these have a broad range of uses in biomedical applications.However,there are only few reports concerning WSC–PASP–PEG complexes available in the literature.29 Based on the reasons mentioned above,the aim of this paper is to develop a new nanoparticulate system,composed of WSC,PASP, and PEG.Physicochemical characteristics of the prepared nanopar-ticles were characterized by Fourier-transform infrared(FTIR) spectroscopy,dynamic light scattering(DLS),transmission electron microscopy(TEM),and zeta potential.The potential of WSC–PASP–PEG nanoparticles,which are regarded as a carrier system for a model protein,bovine serum albumin(BSA),was also presented.2.Experimental2.1.MaterialsWSC with an M r=6kDa was purchased from Yuhuan Ocean Biochemical Co.,Ltd(Zhejiang,China),and the degree of deacetyla-tion was0.93(Fig.1).Polyethylene glycol(M r=4kDa)was ob-tained from Tianjin Ke Mi Ou Chemical Co.,Ltd.(Tianjin,China). L-Aspartic acid was purchased from Beijing Xing Jing Ke Biotech-nology Co.,Ltd BSA,with an M r of66.7kDa,was purchased from Beijing Jun Yao Wei Ye Biotechnology Co.,Ltd Coomassie brilliant blue G-250was obtained from Fiuka(Milwaukee,USA).All other reagents were of analytical grade and were used without further puriﬁcation.2.2.Preparation of PASPPoly(succinimide)(PSI)was synthesized by the acid-catalyzed thermal polycondensation of L-aspartic acid as previously re-ported.30,31Upon hydrolysis of PSI in aqueous NaOH,poly(L-aspar-tic acid)was obtained.This PASP consists of L-aspartic acid repeating units in the a and b forms(Scheme1).The ratio of a to b form,which was determined by the area ratio in the1H NMR spectrum,was26:74,indicating that the b form predomi-nated rather than a form.30Viscosity measurements were carried out using an Ubbelohde-type viscometer,with temperature control in the range of25±0.1°C,M v=23kDa.The weight-average molec-ular weights of PASP were measured to be22.9kDa by gel-perme-ation chromatography(Waters510,USA),M r=22.9kDa, PDI=1.402.2.3.Turbidimetric titration measurementThe interactions between PASP and WSC were investigated by turbidimetric titration according to the reported method.31A solution of2mg/mL PASP and2mg/mL WSC was prepared at pH6.8.Titrant was delivered to each sample with a microburette under gentle stirring,and the pH was monitored by a digital pH meter.Changes in turbidity were monitored at500nm by a UV–vis spectrophotometer,and the turbidity was estimated by theabsorbance.Figure1.Chemical structure of WSC(DD is the degree of deacetylation).1198S.Shu et al./Carbohydrate Research344(2009)1197–12042.4.Preparation of WSC–PASP–PEG nanoparticlesCPP nanoparticles were prepared by mixing negatively charged PASP and positively charged WSC and PEG by a dropping method. The procedure is as follows:200mg of chitosan was dissolved in 100mL of deionized water(2mg/mL,w/v)under magnetic stirring at room temperature without addition of acetic acid and the solu-tion was stirred for12h andﬁltered byﬁlter paper.PASP(200mg) was dissolved in100mL of deionized water,stirred for12h,and ﬁltered by theﬁlter paper.Afterwards,the WSC solution was dropped into PASP solution in different molar ratios under mag-netic stirring.And then,the resultant opalescent emulsion was stirred for30min to allow nanoparticles to form uniform particles. These nanoparticles were isolated by ultracentrifugation at 40,000rpm for30min at4°C.2.5.pH measurementWSC–PASP–PEG nanoparticles were prepared at pH6.8,and di-lute NH4OH or dilute HOAc was added to obtain different pH val-ues.The pH value was measured at20±0.1°C in a PHS-3TC digital pH meter with an error of0.01pH units.A combined glass electrode E-201was employed,and the pH-meter was calibrated with two buffer solutions supplied by Shanghai Hongbei Reagent Factory.2.6.Determination of process yieldFor the calculation of the nanoparticle production yields,the nanoparticle suspensions were centrifuged at40,000rpm for1h, and the supernatants were discarded.The tubes containing the sediments were freeze dried for24h,and the difference in the the-oretical solid weights and the actual freeze-dried nanoparticles weights was determined.The yield of the process was calculated as follows:32Process yield%¼nanoparticles weightÂ100%2.7.Evaluation of nanoparticles stabilitiesThe stabilities of the nanoparticles were investigated in phos-phate buffer solution(PBS)at pH7.4at room temperature.Aliquots of fresh suspensions of nanoparticles were diluted with the addi-tion of PBS,reaching a concentration1mg/mL,and the evolution of the size was assessed by photon correlation spectroscopy for several hours at room temperature.2.8.Preparation of drug-loaded WSC–PASP–PEG nanoparticlesThe drug-loaded nanoparticles were prepared by dropping a mixture of PASP and BSA into WSC–PEG solution.PASP was dis-solved in4mL of deionized water at a speciﬁed concentration (0.1,0.5,1.0,1.5,and2.0mg/mL),and2mL of BSA(2mg/mL) was added.Then this solution was dropped into a mixture solution of4mL of a speciﬁed concentration(0.1,0.5,1.0,1.5,and2.0mg/ mL)of WSC–PEG solution and2mL of deionized water,while son-icating with an ultrasonic sonicator at160W.2.9.Particles size and zeta potential measurementThe size of self-aggregates was measured by a dynamic light scattering method based on the particle size option in Zeta Plus (Brookhaven Instruments Co.,Holtsville,New York,He–Ne laser). The scattered intensity was registered at a scattering angle of90°at25°C.Zeta potentials were measured by a Zeta Plus instrument with Brookhaven electrodes coated by palladium.The zeta poten-tial was the average value of analyses in triplicate.2.10.Transmission electron microscope(TEM)observationsThe morphology of WSC–PASP–PEG nanoparticles was gained by using a Tecnai G220S—TWIN transmission electron microscope.A drop of nanoparticle,suspension was mounted on a carbonﬁlm coated on a copper grid for viewing.Observation was made at200 KV in a Tecnai G220S—TWIN transmission electron microscope.2.11.Fourier-transform infrared measurementThe Fourier-transform infrared(FTIR)spectra of the nanopartic-ulate samples were also identiﬁed to determine the interaction be-tween—NH3þof WSC and–COOÀof PASP.The WSC–PASP–PEG nanoparticles were lyophilized(Flexi-Dry)to obtain dried parti-cles.The WSC–PASP–PEG nanoparticles so obtained were mixed with KBr and pressed in a pellet for further measurement.2.12.Swelling testThe dry nanoparticles(100mg)were immersed in solutions of different pH for24h at room temperature until a swollen equilib-rium was reached.The swollen samples were collected byﬁltra-tion,blotted withﬁlter paper for the removal of the absorbed water on the surface,and then weighed immediately.The swelling ratio was calculated using the following equation:33Swelling ratioð%Þ¼W sÀW dW dÂ100where,W s and W d are the weights of swollen and dry samples, respectively.2.13.Determination of BSA-loading capacity and encapsulation efﬁciency of the nanoparticlesBound and unbound BSA were separated by ultracentrifugation of the nanosuspension at38,000rpm at4°C for60min(Optima LE-80k Ultracentrifuge,Beckman).The amount of free BSA in the clear supernatant was measured by a Bradford protein assay using a UV Spectrometer at595nm(Shimadzu UV2550,Japan).34The BSA encapsulation efﬁciency(EE)and loading capacity(LC)of the nanoparticles were calculated using the following equation: EE%¼total proteinÀfree proteintotal proteinÂ100%LC%¼total proteinÀfree proteinnanoparticles weightÂ100%All measurements were performed in triplicate and averaged.2.14.In vitro release studiesThe in vitro release proﬁles of BSA from WSC–PASP–PEG nano-particles were determined as follows:the BSA-loaded WSC–PASP–PEG nanoparticles separated from9mL of suspension were placed into test tubes with6mL of PBS at pH7.4and incubated at37°C under stirring at60rpm.At appropriate intervals samples were ultracentrifuged,and1mL of the supernatant was replaced by fresh medium.The amount of BSA released from the nanoparticles was evaluated by the Bradford method.34The calibration curve was made using non-loaded BSA nanoparticles as a correction.All re-lease tests were run in triplicate,and the error bars in the plot show the standard deviation.S.Shu et al./Carbohydrate Research344(2009)1197–120411992.15.Cell viabilityCell viability was evaluated by using the NIH3T3cell line.The cells were cultured in Dulbecco’s modiﬁed Eagle’s medium (DMEM)in a humidiﬁed atmosphere(5%CO2:95%O2).The cells were seeded into96-well plates at10,000cells per well.The plates were then returned to the incubator,and the cells were allowed to grow to conﬂuence for24h.Various nanoparticles were dissolved in DI water.Afterﬁltration through aﬁlter paper,the resulting solution was diluted with culture medium to give aﬁnal range of concentrations from0.1to1.5mg/mL.Then the media in the wells were replaced with the pre-prepared culture medium—sample mixture(200l L).The plates were then returned to the incubator and maintained in5%CO2at37°C for48h.Each sample was testedin six replicates per plate.After incubation the culture medium and 20l L of MTT solutions were used to replace the mixture in each well.The plates were then returned to the incubator and incubated for a further4h in5%CO2at37°C.Then,the culture medium and MTT were removed.DMSO(150l L)was then added to each well to dissolve the formazan crystals.The plate was placed in5%CO2at 37°C for10min and for15min at6°C before measurement.The optical density was read on a microplate reader at490nm.Cell via-bility was determined as a percentage of the negative control(un-treated cells).2.16.Circular dichroism(CD)measurementsCircular dichroism(CD)spectroscopy(Jascow715spectropolar-imeter)was used to measure the conformational change of the re-leased BSA with respect to the native one.Solution of the native BSA or the released BSA was diluted to0.1mg/mL and scanned over the wavelength range200–260nm,using a1-mm quartz cylindrical cell.353.Results and discussionIn this study,a novel nanoparticle system composed of WSC and PASP was prepared with a simple and mild coagulation method un-der magnetic stirring at room temperature.This technique is prom-ising because the nanoparticles can be prepared under mild conditions without using harmful solvents.It is well known that or-ganic solvents may cause denaturation of peptide or protein drugs that are unstable and sensitive to their environments.36The pre-pared nanoparticles modiﬁed by PEG are intended to be used to protect the peptide drugs from denaturalizing in the GI tract and facilitate their transportation into systemic circulation.37,383.1.The mechanism of the formation of the nanoparticlesIn particular,the polyelectrolyte complex is formed by the reac-tion of two oppositely charged polymers.The electrostatic interac-tion between the positive charge of—NH3þgroup and the negative charge of carboxyl group is one of the most important factors.The formation and properties of the polymer complex depend on the charge ratio of the anionic-to-cationic species.39When PASP is dropped into WSC solution,inter-and intramolecular electrostatic attractions occur between the anionic carboxyl group from PASP and the cationic amino groups of WSC.PEG was incorporated as a third polymer in the system.The carboxyl group in PASP can act as good proton donor,while the ether group of PEG serves as a proton acceptor,thus making interpolymer interactions stronger. These attractions could make the macromolecular chains of WSC and PASP curl up,which leads to the formation of an insoluble WSC–PASP complex.40,41A schematic representation of the com-plex is shown in Figure2.3.2.FTIR spectrum analysisThe interaction of WSC and PASP wasﬁrst investigated by using FTIR spectroscopy.As seen in Figure3,for the IR spectrum of WSC, the characteristic peak at1550cmÀ1corresponds to protonated amino groups.In the spectrum of PASP,the characteristic absorp-tion peaks appear at1597cmÀ1and1403cmÀ1.The two strong peaks are observed due to the asymmetrical and symmetrical COO-groups,respectively.These results indicate that some carbox-ylic groups of PASP have been dissociated into COOÀgroups,which will be able to complex with the protonated amino groups of WSC through electrostatic interactions to form the polyelectrolyte com-plex.Hence,in the IR spectrum of WSC–PASP nanoparticles,the peaks of COOÀin PASP become weak and shift to1557cmÀ1and 1397cmÀ1,respectively.3.3.Turbidimetric titration measurement and effect of the molar ratio of WSC and PASP on nanoparticlesThe formation of colloids based on polyelectrolyte complexes of WSC and PASP nanoparticles was studied as a function of the mix-ing molar ratio.Four kinds of phenomena were observed in turn during the addition of WSC solution into PASP solution:clear solu-tion,light opalescent,opalescent and precipitation(Table1).As shown in Figure4,the absorbance heightened along with the increasing molar ratio of WSC recorded.The absorbance increased sharply when the WSC:PASP(molar ratio)was up to the ratio of 1.43:1.Further increase in molar ratio led to system precipitation, at which point absorbance began to decrease.While the turbidity of WSC–PASP nanoparticles was obtained through the slow addi-tion of PASP solution into WSC solution,it would be out ofcontrol.Figure2.Interaction mechanism of WSC–PASP–PEGnanoparticles.Figure3.FTIR spectra of the WSC,PASP,and CPP nanoparticles.1200S.Shu et al./Carbohydrate Research344(2009)1197–1204It is probably due to the difference of M r .Because M r of PASP is much larger than that of WSC,the charge molar ratio was also large.It led to the turbidity increase sharply.This proved the for-mation of insoluble polyelectrolyte complexes since neither WSC nor PASP absorbed light at 500nm.It was found that only when the molar ratio of WSC to PASP was lower than 1.43:1,the nanoparticles could be formed.The nano-particles thus prepared carried a positive or negative charge and had a mean size in the range of 100–200nm.From the zeta poten-tial data,when the CS:DS molar ratio increased,the zeta potential varied from a negative charge to a positive charge.The magnitude of the zeta potential gives an indication of the potential stability of the nanoparticle system.All the nanoparticles have negative or po-sitive charge,they will repel each other,and there is dispersion sta-bility.This system can be stable for more than two weeks without aggregation.In Figure 5,the result showed that the colloidal dispersions were stable due to the electrostatic repulsion,which prevented further coagulation.Figure 6shows the morphological characteris-tics of the nanoparticles.CPP nanoparticles modiﬁed by PEG were spherical in shape and about 160nm in diameter.The chosen mo-lar ratio was equal to 1.25:1as the research sample for other characterizations.3.4.Effect of polyethylene glycol concentration on nanoparticlesFrom Figure 7,with the concentration of PEG increasing,the size of the nanoparticles decreased.The incorporation of PEG and WSC–PASP nanoparticles was through intermolecular hydrogen between the electropositive amino hydrogen of WSC and theelectronegative oxygen atom of PEG.Thus WSC–PEG–PASP semi-interpenetration network was formed.Additionally,PASP can form interpolymer complexes with PEG through cooperativehydrogenFigure 4.(A)The turbidity titration of WSC–PASP nanoparticles obtained by slow addition of a WSC solution into a PASP solution;(B)the turbidity titration of WSC–PASP nanoparticles obtained by slow addition of a PASP solution into a WSCsolution.Figure 5.Evaluation of nanoparticles size at different time (Rh is the ﬂuid dynamic radius).Table 1Effect of the molar ratio of WSC and PASP on the particle sizes of nanoparticles WSC/PASP (n/n)StatusMean size (nm)Zeta potential (mv)Process yield (%)1.78/1Precipitation ———1.43/1Opalescent 209.0±16.2+20.21±0.8345.3±41.25/1Opalescent182.8±10.5+19.82±1.2555.0±31.07/1Light opalescent 137.1±32.2À33.89±4.3329.1±30.71/1Light opalescent 99.00±23.7À20.38±3.67 1.20±50.36/1Clear solution———Figure 6.TEM of CPPnanoparticles.Figure 7.Effect of PEG concentration on the size of nanoparticles.S.Shu et al./Carbohydrate Research 344(2009)1197–12041201bonding amongst acid-ether groups.Carboxyl groups in PASP can act as proton donors,thus making interpolymer interactions stron-ger.When the concentration of PEG was up to40mg/mL,the mean size increased sharply.This may be because there were so many hydrogen bonds that two or more nanoparticles congregated.3.5.Effect of ionic strength on nanoparticlesThe effect of ionic strength on the size of nanoparticles is shown in Figure8.The size of the nanoparticles increases along with the addition of NaCl solution with the concentration in the range of 0.005–0.025mol/L.When C NaCl>0.025M,the size of the nanopar-ticles decreases along with the addition of NaCl solution.The ob-served phenomenon probably results from two competing effects.On one hand,the addition of salt favors the formation and the growth of nanoparticles,which is indicated by the increase in the number and size of nanoparticle aggregates.42On the other hand,the addition of salt interferes with the electrostatic attrac-tion between the polymer chains of opposite charge,which re-duces the interaction.Therefore,at low salt concentrations,theeffect on the formation and growth of nanoparticles may exceedthe screening of the interaction,which leads to the enhancement of theﬁnal interaction.At higher salt concentrations,the dominant screening of the interaction leads to the total reduction of the interaction.3.6.Effect of pH on nanoparticle swelling behaviorIn order to investigate the effect of pH values on CPP nanopar-ticles,a series of experiments were carried out.As shown in Figure9,the swelling ratio of various samples is plotted as a func-tion of pH.It is known that the p K a values of PASP and WSC are4.4 and6.5,respectively.43At pH1.2,the increased swelling may be caused by the dissociation of ionic bonds between WSC and PASP. As most of the carboxyl acid groups are protonated,and amino groups are in the—NH3þform,so the molecules have net positive charge.It is probably because the water-soluble chitosan assumes a randomly extended conformation in the low pH region,due to both hydration of the protonated amino group and a strong posi-tive charge repulsion among the—NH3þgroups,which will lead to the swelling of the WSC–PASP nanoparticles.At a pH near to 5.0,the amino groups in WSC are protonated and the carboxyl groups in a PASP are ionized;thus,the strong electrostatic attractions between WSC and PASP restrain the swelling of nano-particles.At high pH,most of the amine groups of WSC are in the –NH2form,and most of the carboxyl groups of PASP are in the–COOÀform,and so the electrostatic attractions were weak-ened in nanoparticles,thus increasing nanoparticle swelling.The nanoparticles show pH sensitivity,a property that can be used for the development of drug delivery systems.3.7.BSA encapsulation efﬁciency of the nanoparticles and release behavior in vitroBSA encapsulation efﬁciency and loading capacity of CPP nano-particles were studied under six different concentrations of BSA (0.5, 1.0, 1.5, 2.0, 2.5and 3.0mg/mL).Encapsulation efﬁciency and loading capacity of nanoparticles are shown in Figure10, which clearly indicates that encapsulation efﬁciency of these par-ticles was affected by initial concentration of the protein used.As the initial concentration of BSA was increasing,encapsulation efﬁ-ciency decreased slowly while loading capacity increased.An approximate90%encapsulation efﬁciency was observed for these particles when the initial concentration of BSA was0.5mg/mL. As the initial concentration of BSA increased up to3.0mg/mL, the encapsulation efﬁciency decreased by less than60%,while loading capacity increased by more than40%.The release of BSA from CPP nanoparticles was performed in the buffer solution of pH1.2,2.5and7.4,respectively,which was sim-ulation of the condition of pH in the GI tract.Figure11depicts the release proﬁles of BSA at pH1.2,2.5and7.4.Particles withcarbox-Figure8.Effect of salt concentration on the size ofnanoparticles.Figure9.Effect of pH on the swellingratio.Figure10.BSA encapsulation efﬁciency and loaded capacity of CPP nanoparticles. 1202S.Shu et al./Carbohydrate Research344(2009)1197–1204ylic acid groups swell in weakly alkaline or neutral pH,and protein diffuses into the matrix quite easily.In other words,particles con-taining carboxylic acid groups display pH-sensitive,swelling/deswelling behavior with the protonation/deprotonation of the carboxylic acid groups.At pH 1.2(simulating the pH in the stom-ach after a meal),most carboxylic groups of PASP were in the form of COOH,and there was little electrostatic interaction between WSC and PASP.In addition,the particles became unstable and sub-sequently broke apart.At pH 2.5(simulating the pH environments of a fasting stomach),the amount of BSA released from the nano-particles was about 20%within the ﬁrst 2h.Therefore,the nano-particles prepared in the study may be orally administered only before meals (pH 2.5–3.7).Similarly,at pH 7.4(simulating the pH oft the bloodstream),in the ﬁrst 2h,more than 60%of the BSA was released.WSC was deprotonated,which led to the col-lapse of the nanoparticles.Finally,it is noted that the nanoparticles present a pH-dependent release pattern,which can not only pro-tect protein drug loss in an acid environment but also control drug release in the GI tract.3.8.Cell viabilityCell viability was used to evaluate the biocompatibility of the nanoparticles.In order to further evaluate the role of WSC,thechitosan (CS)with high molecular weight was evaluated.The cells were exposed to CPP,CS–PEG–PASP nanoparticles,and the nanop-articulate dispersions at various concentrations were incubated for 48h (Fig.12).As shown in Figure 12,the CPP nanoparticle system has a better biocompatibility than the other one.The CS–PEG–PASP has lower cell viability than CPP nanoparticles.This may be due to chitosan which has a high molecular weight and needs to be dis-solved in an HOAc solution at a pH value of approximately 4.0.One possible explanation for this can be ascribed to the cell toxicity of the acid solution.Under these circumstances,the bioactivity of the cells are easily damaged.The CPP nanoparticles also have bet-ter biocompatibility than WSC–PASP.PEG is probably one of the best-known hydrophilic polymers,and incorporation of PEG can increase biocompatibility.The results suggest that CPP nanoparti-cles may be good biocompatible carriers for drug delivery and may have potential for in vivo use.3.9.Circular dichroism (CD)spectraCircular dichroism spectroscopy has been used to examine the conformation and self-association of BSA.There are three common secondary structures in BSA,namely a helices,b sheets,and turns.The native BSA has two extreme valleys at 208and 222nm.44Since a helices are one of the elements of secondary structure,the quan-titative analysis of the structural change of BSA can be evaluated by determining the amount of a helix preserved.The a helix content of a protein is estimated according to the following equation:35%a -helix content ¼h mrd À400033000À4000where,h mrd is the mean molar ellipticity per residue at 208nm (deg cm 2dmol À1).Usually the raw data from the experiment are ex-pressed in terms of h d (the ellipticity in the unit of mdeg).However,it can be converted to mean molar ellipticity per residue using the following equation:45h mrd ¼h d M 10CLNwhere,M is the BSA molecular weight (Da),C is the BSA concentra-tion (mg/mL),L is the sample cell path length (cm),and N is the number of amino residues.As indicated by the CD spectra (Fig.13),the native BSA has 50%a helix,which is close to the liter-ature value of 48%.46After release from CPP nanoparticles,the a helix content is 47%,which is very close to that of the native BSA.In other words,no signiﬁcant conformational change wasnotedFigure 11.In vitro release of BSA from CPP nanoparticles at different pHvalues.Figure 12.Viability of cells after incubation as a function of nanoparticle concentration by MTT assay,at 37°C for 48h.Figure 13.Circular dichroism (CD)spectra of BSA released from test nanoparticles at pH 7.4and native BSA.S.Shu et al./Carbohydrate Research 344(2009)1197–12041203。

卡拉胶低聚糖结构研究

Carbohydrate Research 337(2002)433–440 /locate /carresStructural studies on k -carrageenan derived oligosaccharidesGuangli Yu,a Huashi Guan,a Alexandra S.Ioanoviciu,b Sulthan A.Sikkander,bCharuwan Thanawiroon,b Joanne K.Tobacman,c Toshihiko Toida,dRobert J.Linhardt b,*aMarine Drug and Food Institute ,Ocean Uni 6ersity of Qingdao ,Qingdao 266003,People ’s Republic of ChinabDepartment of Medicinal and Natural Products Chemistry ,Chemistry ,and Chemical and Biochemical Engineering ,College of Pharmacy ,Uni 6ersity of Iowa ,Iowa City ,IA 52242,USAcDepartment of Internal Medicine ,Uni 6ersity of Iowa Health Care ,Iowa City ,IA 52242,USAdDepartment of Bioanalytical Chemistry ,Graduate School of Pharmaceutical Sciences ,Chiba Uni 6ersity ,Chiba 263-8522,JapanReceived 3September 2001;received in revised form 7January 2002;accepted 8January 2002AbstractOligosaccharides were prepared through mild hydrochloric acid hydrolysis of k -carrageenan from Kappaphycus striatum carrageenan.Three oligosaccharides were puriﬁed by strong-anion exchange high-performance chromatography.Their structure was elucidated using mass spectral and NMR data.Negative-ion electrospray ionization (ESI)mass spectra at different fragmentor voltages provided the molecular weight of the compounds and unraveled the fragmentation pattern of the k -carrageenan oligosaccharides.2D NMR techniques,including 1H–1H COSY,1H–1H TOCSY and 13C–1H HMQC,were performed to determine the structure of a trisulfated pentasaccharide.1D NMR and ESIMS were used to determine the structures of a k -carrageenan-derived pentasaccharide,heptasaccharide,and an undecasaccharide.All the oligosaccharides characterized have a 4-O -sulfo-D -galactopyranose residue at both the reducing and nonreducing ends.©2002Elsevier Science Ltd.All rights reserved.Keywords :k -Carrageenan;Kappaphycus striatum ;NMR data;Oligosaccharides1.IntroductionCarrageenans are a family of anionic polymers ex-tracted from certain marine red algae.These polysac-charides are used as additives to improve food texture,gelation,stability,and viscosity 1and are generally re-garded as safe (GRAS)by the Food and Drug Admin-istration in the US.2Breakdown products of molecular weight B 40,000,called poligeenans,have been impli-cated in gastrointestinal malignancy in animal mod-els.3,4Thus,additional biological evaluation of poligeenans is warranted.4–7Carrageenans consist of alternating (1 3)-linked b -D -galactopyranose (Gal p )and (1 4)-linked a -D -galactopyranose.The commercially important k -carrageenan (Scheme 1)contains a 3,6-anhydro-a -D -galactopyranose (AnGal p )in place of a -D -galactopy-ranose,giving it gelling properties.2The addition of a 2-O -sulfo group to the k -carrageenan sequence [ 3)-b -D -Gal p 4S-(1 4)-a -D -AnGal p -(1 ]n affords another commercially important carrageenan,i -carrageenan [ 3)-b -D -Gal p 4S-(1 4)-a -D -AnGal p 2S-(1 ]n .8Struc-tural studies on these intact polymers have primarily relied on 1H and 13C NMR spectroscopy.9Other stud-ies have prepared k -carrageenan oligosaccharides through depolymerization by acid hydrolysis,10,11methanolysis,12reductive hydrolysis,13,14and using k -Scheme 1.*Corresponding author.Tel.:+1-319-3358834;fax:+1-319-3356634.E -mail address :robert-linhardt@ (R.J.Linhardt).0008-6215/02/$-see front matter ©2002Elsevier Science Ltd.All rights reserved.PII:S 0008-6215(02)00009-5G .Yu et al ./Carbohydrate Research 337(2002)433–440434Fig.1.Gradient PAGE analysis to follow the acid hydrolysis of k nes a and b contain oligosaccharide standards prepared in our laboratory.The heparin oligosac-charide bands in lane b are labeled on the left of the gel with their molecular weights.24Lanes c and i contain k nes d,e,f,g,and h contain k -carrageenan hy-drolyzed with 0.1M HCl at 37°C for 24,36,48,60,and 72h,nes j and k contain k -carrageenan hy-drolyzed with 0.1M HCl at 60°C for 2and 4h,respectively.The gel was visualized by Alcian blue staining.This cationic dye binds to polyanions with af ﬁnity that increases with increased charge,thus oligosaccharides having a low-molecu-lar weight and /or a low level of charge are not detected well using this method.signi ﬁcant breakdown,as measured by viscosimetry and gel-permeation chromatography.On the basis of these studies,we decided to undertake a detailed exam-ination of controlled acid hydrolysis under relatively mild conditions to examine the stability of k -car-rageenan,to prepare oligosaccharide standards,and to test their biological activity.This paper reports the structure determination of three major oligosaccharides prepared from k -carrageenan by mild-acid hydrolysis,using NMR spectroscopy,and ESIMS.2.Results and discussionCarrageenan from Kappaphycus striatum was fractionated 23to obtain pure k -carrageenan in 66%d-acid hydrolytic depolymerization of k -car-rageenan affords poligeenan,a mixture of lower molec-ular-weight polysaccharides and oligosaccharide products.Depolymerization reactions using 0.1M hy-drochloric acid at 37and 60°C were monitored by discontinuous gradient polyacrylamide gel elec-trophoresis,24and visualized by staining with Alcian blue dye (Fig.1).At 60°C,the 2and 4h time points (Fig.1,lanes j and k)both showed a good distribution of low-molecular weight products.Thus,a 3-h time-point was selected for a larger scale (5g)preparation of carrageenan oligosaccharides of a size suitable for structural characterization.The poligeenan –oligosac-charide mixture was pressure ﬁltered to remove poligeenan components M W \5000from the desired carrageenan oligosaccharides.The ﬁltrate was then de-salted and fractionated by semi-preparative SAX-HPLC (Fig.2).The early peaks eluting at low-salt concentration between 0and 100mL,corresponding to low-molecular weight oligosaccharides (monosaccha-ride to tetrasaccharides),were dif ﬁcult to desalt and were not characterized.Gradient PAGE analysis (not shown)indicated that the late peaks eluting at high-salt concentrations between 200and 300mL contained inseparable,complex mixtures of higher oligosaccha-rides and thus,were not further characterized.The major peaks eluting between 100and 200mL (labeled 1–3in Fig.2)were collected,re-fractionated on SAX-HPLC,desalted,and analyzed.Gradient PAGE analysis using Alcian blue staining suggested that fractions 2and 3were suf ﬁciently pure to undertake their analyses (Fig.3(A)).Alcian blue staining visualizes highly sulfated oligosaccharides,but is relatively insensitive to oligosaccharides having a low content of sulfo groups.25Our failure to observe oligosaccharide 1suggested that it contained a rela-tively small number of sulfo groups.To further assess the purity of oligosaccharides 1–3,each was labeled by reductive amination with ANDS,a charged ﬂuorescent tag,fractionated by PAGE,and visualized byFig.2.SAX-HPLC fractionation of carrageenan oligosaccha-rides.The major peaks labeled 1–3eluting between 100and 200mL were characterized.carrageenanase.15–18The structures of the resulting oligosaccharides have been determined using 1D and 2D 1H and 13C spectroscopy 12,15,18and electrospray ionization –mass spectrometry (ESIMS).19There is interest in the possibility that k -carrageenans in foods might be hydrolyzed in the acidic environment of the stomach producing poligeenans.Strong mineral acids are known to hydrolyze carrageenans and have been useful in preparing monosaccharide components for derivatization and composition analysis by GC –MS.20Earlier work 21,22showed that k -carrageenan treated with simulated gastric ﬂuid (pH 1.0)showedG.Yu et al./Carbohydrate Research337(2002)433–440435Fig.3.PAGE analysis of carrageenan oligosaccharides1–3.(A)Gradient PAGE analysis of fractions2–3using Alcian blue staining.(B)Gradient PAGE analysis of ANDS-labeled oligosaccharides1,2,and3with visualization by UV transil-lumination.The lane on the left marked std.corresponds to an ANDS-labeled dermatan sulfate hexasaccharide stan-dard.29(data not shown)indicated1,2,and3were73,89,and 88%pure.The impurities observed corresponded to multiple,very minor peaks,suggesting that all three samples were suitably pure for spectral analysis.The structure of oligosaccharides1–3was next inves-tigated using mass spectrometry and NMR spec-troscopy.ESIMS gave molecular ions for the sodium salt forms of all three sulfated oligosaccharides.ESIMS analysis of compound1(Table1)showed a molecular ion[M−Na+]−at m/z1075corresponding to a mass of1098suggesting that it was a pentasaccharide con-taining three galactopyranose(Gal p)residues and two 3,6-anhydrogalactopyranose(AnGal p)residues and had three O-sulfo groups.Fragmentation was consis-tent with each of the three Gal residues carrying one O-sulfo group.Next,full structural characterization was undertaken using1H and13C NMR spectroscopy (Fig.4)and was compared to published values for carrageenan and carrageenen oligosaccharides(Tables2 and3).The1H–1H COSY spectrum of1demonstrated the presence of several spin systems.Theﬁrst spin system describes the reducing end(re)a-4-O-sulfogalactopy-ranose anomer(a-Gal p4S re).The correlations present in the COSY spectrum allowed the assignment of H-1, H-2,H-3,and H-4of this galactopyranose unit(Table 2).The coupling constant between H-4and H-5pro-tons in this galactopyranose residue was too small to give a correlation.The signal of H-1of the AnGal p residue,directly linked to the a-Gal p4S re unit,was slightly shifted downﬁeld.A distinct signal could betransillumination26(Fig.3(B)).The results of this anal-ysis showed that oligosaccharides1,2,and3eachcontained a major band in addition to minor contami-nating bands.High sensitivity,quantitative analysis ofoligosaccharide purity,relying on reversed-polarity cap-illary electrophoresis usingﬂuorescence detection27Table1Assignment of ESIMS of carrageenan oligosaccharides1–3Carrageenan oligosaccharide aIon1231075(0,70,140,210)[M−Na+]−11483(210)1138(70,210) [M−2Na+]−2730(70,210)526(0,70,140,210)751(70,210) [M−3Na+]−3343(0,70)479(70,210)557.5(70,210) [M−4Na+]−4353.5(70)364(70)[M−6Na+]−6[M−NaSO3−+H+−Na+]−1973(70)475(0,70)[M−NaSO3−+H+−2Na+]−2[M−Gal p4S−Na+]−1811(140,210)547(140,210)[M−2Gal p4S−Na+]−1[M−Gal p4SAnGal p−4Na+]−4455.5(70) [M−Gal p4SAnGal p Gal p4S−3Na+]−3527(70)[M−Gal p4SAnGal p Gal p4SAnGal p Gal p4S−H2O−Na+]−11201(210)667(0)[Gal p4SAnGal p Gal p4S−Na+]−1667(210)811(210)[Gal p4SAnGal p2SAnGal p−Na+]−1403(210)[Gal p4SAnGal p−Na+]−1403(210)403(210) [Gal p4SAnGal p−H2O−Na+]−1385(210)385(210)385(210) [Gal p4S−H2O−Na+]−1241(210)241(210)241(210)a Fragmentor voltages are shown in parentheses.G.Yu et al./Carbohydrate Research337(2002)433–440 436Fig. 4.NMR characterization of oligosaccharide1using two-dimensional spectroscopy.(A)COSY spectrum with cross-peaks labeled:(a),a Gal p re4S H-1/H-2;(b)AnGal p H-1/ H-2;(c)a Gal p re4S H-3/H-4;(d)b Gal p int4S H-3/H-4;(e) b Gal p re4S H-3/H-4;(f)b Gal p nre H-3/H-4;(g)b Gal p re4S and b Gal p int4S H-1/H-2;(h)b Gal p nre H-1/H-2;(i)AnGal p H-5/H-6a;(j)AnGal p H-2/H-3;(k)AnGal p H-6a/H-6b;(l) a Gal p re4S H-2/H-3;(m)b Gal p int4S H-2/H-3;(n)b Gal p re4S H-2/H-3;(o)b Gal p nre H-2/H-3.(B)HMQC spectrum with cross-peaks labeled:(a)a Gal p re4S H-1/C-1;(b)AnGal p adja-cent to a Gal p re4S H-1/C-1;(c)AnGal p H-1/C-1;(d) a Gal p re4S H-4/C-4;(e)b Gal p int4S H-4/C-4;(f)b Gal p re4S H-4/C-4;(g)b Gal p nre H-4/C-4;(h)AnGal p H-5/C-5;(i)An-Gal p H-4/C-4;(j)AnGal p H-3/C-3;(k)b Gal p re4S H-1/C-1;(l) b Gal p nre4S H-1/C-1;(m)b Gal p int4S H-1/C-1;(n)AnGal p H-6b/C-6;(o)a Gal p re4S H-3/C-3;(p)AnGal p H-2/C-2;(q) AnGal p H-6a/C-6;(r)b Gal p re4S and b Gal p int4S H-3/C-3;(s) a Gal p re4S H-2/C-2;(t,u and v)unidentiﬁed(b Gal p nre H-3/C-3and b,a Gal p H-5/C-5);(w)b Gal p int4S H-2/C-2; (x)b Gal p re4S H-2/C-2;(y)b Gal p nre H-2/C-2;(z)Gal p4S H-6/C-6.observed downﬁeld of the AnGal p H-1,having the same small coupling constant.The integral for this other AnGal p H-1at5.10ppm is equal to the integral of the anomeric proton signal a-Gal p4S re at5.29ppm, and from the comparison of the value of this integral to that of the other AnGal p H-1,the a,b mutarotation equilibrium in2H2O at25°C was estimated35–40%a and60–65%b.The anomeric proton at5.29ppm has a coupling constant of3.8Hz,typical of a Gal p4S re H-1 coupling with Gal p4S re H-2and clearly indicates that a galactopyranose residue is present at the reducing end. The a-Gal p4S re H-2proton is a doublet of doublets centered at3.89ppm having a small J1,2(3.8Hz)and a large J2,3value(10Hz).The a-Gal p4S re H-3,which resonates at4.15ppm,shows a correlation to H-4with a J3,4of3.0Hz and this proton in turn appears as a doublet at4.87ppm.These coupling constants corre-spond to a reducing-end unit galactose in which J1,2 and J3,4are small due to axial–equatorial coupling and J2,3is large reﬂecting the diaxial coupling.The most upﬁeld signal at3.49ppm is the entry point for the nonreducing end(nre)residue.The H-2signal, a doublet of doublets centered at 3.49ppm,shows correlations to H-1,a signal at4.60ppm and to H-3,a signal centered at3.71ppm.The correlation between H-3and H-4allows the identiﬁcation of the Gal p4S nre H-4as a signal centered at4.56ppm.The coupling constants for J1,2and J2,3are8.0and10Hz,res-pectively.The analysis of the next spin system begins with the next upﬁeld signal centered at3.56ppm,corresponding to the H-2of both the internal(int)Gal p4S int residue and the H-2of the b anomer of the Gal p4S re residue. This signal shows an additional splitting due to the overlap of these three different hydrogen atoms.The H-2signal corresponding to b-Gal4S re and Gal p4S int shows correlations to an H-1proton at4.63ppm.In addition,this also correlates to the H-3proton by showing two doublet of doublets at3.95and3.99ppm. The H-3coupling with H-4leads to the identiﬁcation of the H-4at4.82and4.84ppm.The coupling constant of the protons in b-Gal p4S re and Gal p4S int are identical to those of b-Gal p4S nre(described above).Theﬁnal spin system belongs to the two AnGal p int residues whose signals overlap completely.AnGal p int H-1at5.09ppm correlates to H-2,a doublet at4.12 ppm,which correlates to the doublet of doublets for H-3at4.51ppm.The connectivity from H-3to H-4 could not be established,due to the small J3,4.The two protons,H-6a at4.05ppm and H-6b at4.21ppm,were easily identiﬁed due to their strong correlation resulting from their geminal coupling of−10Hz.The H-6b signal is downﬁeld of H-6a,probably due to anisotropic effects of the pyranose ring oxygen atom.Only H-6a shows a correlation to H-5signal at4.63ppm,which overlaps with others located in the same region.G.Yu et al./Carbohydrate Research337(2002)433–440437The1H–1H TOCSY spectrum(not shown)conﬁrmed all the assignments made using1H–1H COSY.The 1H–13C HMQC spectrum allowed the transfer of the proton assignments to the corresponding carbon atoms and the carbon assignments of oligosaccharide1are given in Table3.The a,b equilibrium of the Gal p4S re residue affected not only the carbon atoms of the terminal residue,but also some of the carbon atoms ofTable21H NMR assignments for k-carrageenan oligosaccharides1–3Residue Proton Chemical shift(ppm)a3Lit.values b12a-Gal p4S re 5.325.215.215.29H-13.923.82H-2 3.89 3.82H-3 4.07 4.07 4.164.15H-4 4.87 4.804.80 4.864.65–4.66b-Gal p4S int and b-Gal p4S re 4.564.564.63H-13.49 3.59–3.60H-2 3.56 3.50H-3 3.92 3.91 3.98–4.013.95,3.99H-4 4.76 4.76 4.83–4.864.82,4.843.67–3.88H-5 3.60–3.80 3.60–3.78 3.77–3.813.67–3.88H-6a,6b 3.78–3.803.60–3.783.60–3.804.49b-Gal p4S nre H-1 4.60 4.52H-2 3.42 3.413.49H-3 3.60–3.80 3.60–3.783.784.574.564.65H-43.67–3.88 3.60–3.80 3.60–3.78H-5H-6a,6b 3.67–3.88 3.60–3.80 3.60–3.78H-1 5.01AnGal p adjacent to a-Gal p4S re 5.125.10 5.01AnGal p 5.00H-1 5.105.09 5.00H-2 4.12 4.04 4.03 4.144.534.43H-3 4.51 4.434.56 4.61H-4 4.60–465 4.564.54H-5 4.55 4.654.633.964.06H-6a 4.05 3.964.224.134.134.21H-6ba Chemical shift value of4.76ppm for HO2H was used in calculating the chemical shifts.b Literature values were taken from Ref.12.Table313C NMR assignments for k-carrageenan oligosaccharide1Chemical shift(ppm)ResidueC-6C-4C-3C-5C-2C-194.7(92.8)a69.5(67.6)77.5(75.6)77.1(75.3)63.6(61.9) a-Gal p4S re80.6(78.6)b-Gal p4S re76.1(74.2)98.9(96.9)63.8(61.8)72.9(71.0)Gal p4S int71.7(69.8)76.9(78.5)77.2(75.1)104.8(102.8)b-Gal p4S nre105.076.079.274.363.8 AnGal p96.9(94.9)Adjacent to a re97.0(95.0)AnGal pAdjacent to b reAnGal p71.7(69.4)78.9(77.8)80.7(78.8)81.5(81.3)97.0(95.0)71.9(69.9)a Literature values shown in parentheses are taken from Ref.12.G .Yu et al ./Carbohydrate Research 337(2002)433–440438Fig.5.One-dimensional 1H NMR spectrum of oligosaccha-ride 3.Signals:(a)a Gal p re 4S H-1;(b)AnGal p H-1;(c)b Gal p int 4S H-4;(d)b Gal p int 4S H-1,b Gal p re 4S H-1,AnGal p H-4and b Gal p nre H-4;(e)AnGal p H-5;(f)AnGal p H-3;(g)AnGal p H-6b;(h)AnGal p H-2;(i)AnGal p H-6a;(j)b Gal p int 4S H-3;(k)b Gal p int 4S H-2;(l)b Gal p nre H-2.to be a trisulfated pentasaccharide of the structure:b -D -Gal p 4S-(1 4)-a -D -AnGal p -(1 3)-b -D -Gal p 4S-(1 4)-a -D -AnGal p -(1 3)-a ,b -D -Gal4S.MS and NMR analysis (Tables 1and 2)of oligosac-charide 2showed it to be homologous to oligosaccha-ride 1,containing one additional repeat unit.Oligo-saccharide 2is a tetrasulfated heptasaccharide of molecular weight 1506.Its structure is b -D -Gal p 4S-(1[ 4)a -D -AnGal p -(1 3)-b -D -Gal p 4S-(1 ]24)-a -D -AnGal p -(1 3)-a ,b -D -Gal4S.Oligosaccharide 3was the largest and was among the purest of the three oligosaccharides prepared.One-di-mensional 1H NMR con ﬁrmed the high level of purity of oligosaccharide 3(Fig.5).Comparisons of the area for the H-2protons of the Gal p 4S residues were made to the area for H-1protons of the AnGal p residues afforded a ratio consistent with an oligomer containing 11saccharide units.Structural assignment of oligosac-charide 3also relied on ESIMS fragmentation (Figs.6and 7).ESIMS analysis had been previously used to successfully analyze k -carrageenan oligosaccharides,in-cluding a dodecasaccharide prepared using k -car-rageenanase.19Oligosaccharide 3is a hexasulfated undecasaccharide of molecular weight 2322and is ho-mologous in its structure to oligosaccharides 1and 2.It has the structure:b -D -Gal p 4S-(1[ 4)-a -D -AnGal p -(1 3)-b -D -Gal p 4S-(1]4 4)-a -D -AnGal p -(1 3)-a ,b -D -Gal4S.the adjacent AnGal p residue.This accounts for the unexpectedly large number of carbon peaks observed.Although the relative intensities of the peaks in the decoupled 13C spectrum do not provide an accurate quantitative assessment in the absence of quaternary carbon atoms (which would give low-intensity peaks),it was possible to identify some lower intensity signals as peaks arising from carbon atoms of the a Gal p 4S re residue.No peaks are present at 66.4ppm correspond-ing to C-4of unsulfated Gal p residues;thus,the 13C and the HMQC spectra con ﬁrm that all Gal p residues contained a 4-O -sulfo group.NMR and MS analysis de ﬁnitively establish the structure of oligosaccharide 1Fig.6.ESIMS analysis of oligosaccharide 3.(A)ESIMS spectrum obtained at a fragmentor voltage of 70V.(B)ESIMS spectrum obtained at a fragmentor voltage of 210V.Fig.7.Fragmentation analysis of oligosaccharide 3.G.Yu et al./Carbohydrate Research337(2002)433–440439Work is currently underway to prepare and charac-terize larger molecular-weight oligosaccharides for bio-logical evaluation.3.ExperimentalMaterials.—Carrageenan from K.striatum(Philip-pines)was purchased from Yantai Algae Industries (Shandong,China).Bio-Gel P2(superﬁne)was from Bio-Rad(Richmond,CA).SAX-HPLC was performed on a POROS HQ50column(10m m,22×100mm) column.Acrylamide(ultrapure)and Tris(ultrapure) were from Life Technologies Inc.(Gaithersburg,MD). Alcian blue dye,bromophenol blue dye,and ammo-nium persulfate were from Boehringer Mannheim Bio-chemicals(Indianapolis,IN).Glycine,sodium EDTA, boric acid,sucrose,N,N-methylene bisarylamide,and TEMED were from Fisher Chemical Co.(Fair Lawn, NJ).2H2O(99.96,atm.%)was from Aldrich Chemical Co.(Milwaukee,WI).Puriﬁcation and characterization of s-carrageenan.—The yield of carrageenan from K.striatum algae was approximately30%.The carrageenan was treated with NaOH and KCl to increase the content of AnGal p residues to enhance gel strength.Properties provided by the manufacturer include:a gel strength of800g/cm2at 1.5%water;mp50°C,and condensing point,41°C. The polymer is a random coil at temperatures of \50°C and is a double helix at rt.k-Carrageenan was puriﬁed by the method of Smith et al.23The polysaccharide was converted to the sodium salt,dialyzed,lyophilized,and analyzed as C,30.25;H, 5.53;S,7.35%.IR analysis shows an absorbance at848 cm−1indicative of the presence of4-O-sulfo esters.The 1H and13C NMR spectra were consistent with the structure of k-carrageenan.GPC–HLPC performed on PL Aquagel-OH-mixed and PL Aquagel-OH-30(8m m, 7.5×300cm)columns(Polymer Laboratories Ltd., Amherst,MA,USA)in series at aﬂow rate of1.0 mL/min(0.1M Na2SO4,pH7.0)at35°C utilized RI detection.Dextran and dextran sulfate standards of molecular weights10,000,21,400,41,000,84,000, 133,800,and500,000gave a standard curve of log M n=−0.435x+10.59(r2=0.997;x,min).Based on this equation,k-carrageenan gave an M n=310,400, M w=431,320,M z=553,400,and a polydispersity of 1.19.Mild-acid hydrolysis of s-carrageenan.—k-Car-rageenan(5g,10mg/mL)was dissolved at60°C in0.1 M hydrochloric acid and kept for3h.The degradation was terminated by neutralization with0.1M NaOH, thenﬁltered by Millipore membrane(GS,0.45m m),and then the supernatant was pressureﬁltered by a5000 MWCO membrane using a600mL,stirred pressure-ﬁltration cell with a nitrogen gas pressure of1.0kg/cm2.Theﬁltered sample was concentrated by rotaryevaporation.Desalting.—All desalting was performed on Bio-Gel-P2columns(2.6×80cm or1.5×60cm)monitoredusing a refractive index detector(Gilson132RI,France).Separation and puriﬁcation of s-carrageenan oligosac-charides.—The oligosaccharide mixture was separatedon a semi-preparative SAX-HPLC(POROS HQ50,10 m m,PE Perspective Biosystems Inc.)column.The column was pre-equilibrated with0.1M NaCl(pH7.0)at5.0mL/min,then in a linear gradient of NaCl from0.1to2.0M in120min.By using fraction collector(RediFrac,Pharmacia Biotech.),each fraction(5mL/tube)was automatically collected and the content ofeach fraction was tested by colorimetric assay.28Thepurity of each fraction was monitored by PAGE analy-sis.24The major peaks,were pooled,desalted on aBio-Gel-P2column,freeze-dried and again applied tothe semi-preparative SAX-HPLC column(using anewly optimized gradient),and the fractionation anddesalting were repeated.Finally,pure oligosaccharideswere obtained.PAGE analysis of s-carrageenan oligosaccharide.—Gradient(12–22%)discontinuous polyacrylamide gelelectrophoresis(PAGE)analysis was performed on avertical slab(0.1×16×20cm)gel system.The gel wasloaded with20–50m g of sample and subjected toelectrophoresis for4h at400V while cooling at5–10°C with a circulating bath.The gel was visualizedwith Alcian blue(0.5%in2%AcOH)staining.Fluorescent labeling of oligosaccharides.—Mono-potassium7-amino-1,3-napthalenedisulfonic acid(ANDS)(Aldrich)was recrystallized from deionizedwater for theﬂuorescent labeling of the oligosaccha-pounds1,2,and3(100m g of each)weredissolved in a solution containing ANDS(5mg in100 m L of15%AcOH)and incubated for1h at rt after which100m L of1.0M sodium cyanoborohydride in water was added,and the mixture was incubated for12 h at45°C.Excess ANDS was removed by gel-perme-ation chromatography on a Sephadex G-25column (45×1.5cm i.d.)with detection at247nm.Theﬂuores-cently labeled oligosaccharide fractions(eluting prior to the excess ANDS)were collected,pooled,and freeze-dried.Electrophoresis was performed in a Mini-Protean II electrophoresis system from Bio-Rad Laboratories (Hercules,CA).The gel was loaded with2–5m g of sample and subjected to electrophoresis for about1h at 200V(constant voltage).The gel was visualized in a UV–light chamber and photographed.Capillary electrophoresis ofﬂuorescently labeledoligosaccharides.—The labeled oligosaccharides wereanalyzed with a capillary electrophoresis system(Dionex,Sunnyvale,CA)at25kV byﬂuorescencedetection(u ex of250nm and u em of450nm).SeparationG.Yu et al./Carbohydrate Research337(2002)433–440 440and analysis were carried out in a reversed-polaritymode using a fused silica(external coated except wherethe tube passed through the detector)capillary tube(55-cm long×50m m i.d.).The separation buffer con-tained20mM of sodium phosphate,pH 3.5.Thesample was pressure injected(5s,5psi)resulting in aninjection volume of0.5m L.NMR analysis.—Each sample was dissolved in2H2O(approximately0.5–1.1mM)and freeze-dried twice toreplace all exchangeable protons with deuterium.The 1H NMR and1H–1H2D NMR were all acquired at 25°C using a VARIAN INOVA500instrument and VNMR 6.1C software.The water peak served as a reference(HO2H,4.76ppm).Shift values were conﬁ-rmed using acetone as an internal standard to give asignal at2.22ppm.In the1H–1H COSY and1H–1HTOCSY spectra,a12-ppm spectral width was used inboth dimensions.In F2,1024complex points werecollected while the resolution in the F1dimension was512complex points using States Habercorn phase cy-cling.The13C spectrum was recorded on a Bruker DPX400spectrometer;DSS(sodium4,4-dimethyl-4-silapen-tane-1-sulfonate)served as an external reference.The 1H–13C HMQC was run on a Bruker AMX600,using a1K×1K matrix(80scans per block,an acquisition time of0.213s).ESIMS analysis.—The low-resolution ESI massspectra of compounds1–3were acquired on a1100mass selective detector(Agilent Technologies)equippedwith a single quadrapole,at different fragmentorvoltages using a1:1mixture of1%NH4OH–MeCN.The fragmentor voltage was changed from70to2100Vin70V intervals to modify the degree of fragmentation.Nitrogen gas was used as a nebulizer gas at4.2kg/cm2and kept at350°C.Theﬂow rate was0.2mL/min andsamples of50ng were injected in a5m L loop. AcknowledgementsThe authors thank John Snyder of the NMR CentralResearch Facility,William Kerney of the College ofMedicine NMR facility,and Lynn Teesch of the High-Resolution Mass-Spectrometry Facility at the Univer-sity of Iowa.The authors are also grateful to ProfessorD.Loganathan of IIT Madras and Nur Sibel Gunay ofthe University of Iowa for their assistance in assigningspectra.This research was funded by a grant(GM38060)from the National Institutes of Health. 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Rheological characterisation of thermogellingchitosan/glycerol-phosphate solutionsA.Chenite a,*,M.Buschmann b ,D.Wang a ,C.Chaput a ,N.Kandani caBIOSYNTECH Limited,475Armand Frappier Bd.,Laval TechnoPark,Montreal (Laval),PQ,Canada H7V 4B3bDepartment of Chemical Engineering and Institute of Biomedical Engineering,Ecole Polytechnique,Montreal,PQ,Canada H3C 3A7cDeÂpartement de Chimie,Faculte Âdes Sciences Semlalia,Universite ÂKadi Ayad,Marrakech,Maroc Received 14June 2000;revised 25July 2000;accepted 28July 2000AbstractIn this study we demonstrate that chitosan solutions can be neutralised up to physiological pH (,7.2)using b -glycerol phosphate withoutcreating immediate gel-like precipitation and furthermore that subsequent heating of these solutions induces hydrogel formation.The addition of the particular basic salt,glycerol phosphate,provides the correct buffering and other physicochemical conditions including control of hydrophobic interactions and hydrogen bonding which are necessary to retain chitosan in solution at neutral pH near 48C and furthermore to allow gel formation upon heating to 378C.Rheological investigation evidenced the endothermic gelation of chitosan/b -glycerol phosphate solutions and allowed the establishment of a sol/gel diagram.The gelation process appears to be governed by delicate interplay between the pH and the temperature.The role of b -glycerol phosphate is discussed in the light of relevant literature particularly those indicating the role of glycerol and polyols in the stabilisation of proteins and polysaccharides.q 2001Elsevier Science Ltd.All rights reserved.Keywords :Chitosan;Glycerol phosphate;Rheological measurements1.IntroductionChitosan is an aminopolysaccharide obtained by alkaline deacetylation of chitin,a cellulose-like polymer present in fungal cell walls and exoskeletons of arthropods such as insects,crabs,shrimps,lobsters and other vertebrates (Muzzarelli,1977).Chitosan is a biodegradable (Struszczyk,Wawro,&Niekraszewicz,1991),bio-compatible (Chandy &Sharma,1990;Hirano,Seino,Akiyama,&Nonaka,1990a)and mucoadhesive (He,Davis,&Illum,1998;Henriksen,Green,Smart,Smistad,&Karlsen,1996;Lehr,Bouwstra,Schacht,&Junginger,1992)biopolymer,which is emerging to play a signi®cant role in biomedical applications (Felt,Buri,&Gurny,1998;Illum,1998;Madhavan,1992;Malette,Quigley,Gaines,Johnson,&Rainer,1983;Sandford,1988),due to its abundance and wide scope of use.Chitosan has been recommended as an appropriate material for many purposes in pharmaceutical,medical and food industries,wherenumerous international patents have claimed applications of chitosan in these areas (Nordquist,1998).The term chitosan is commonly used to describe a series of chitosan polymers with various weight average molecularweights 50kDa # Mw #2000kDa and degrees of deacetylation 40,DDA ,98% :Chitosan is typically not soluble in water,but chitosan solutions can be obtained in acidic aqueous media which protonate chitosan amino groups,rendering the polymer positively charged and thereby overcoming associative forces between chains.When adding a strong base (i.e.NaOH)to such solutions,chitosan remains in solution up to a pH in the vicinity of 6.2.Further basi®cation,to pH .6:2;systematically leads to the formation of a hydrated gel-like precipitate.This precipita-tion,or gel formation,is due to the neutralisation of chitosan amine groups and the consequent removal of repulsive inter-chain electrostatic forces which subsequently allows for extensive hydrogen bonding and hydrophobic interactions between chains.The inability to maintain chitosan in solution up to a physiological pH in the region of 7.0±7.4,has been the main obstacle to date in the development of certain biomedical applications of chitosan,for example as an encapsulating or delivery system for living cells or for pH-sensitive proteins.In the context of the current study,itCarbohydrate Polymers 46(2001)39±470144-8617/01/$-see front matter q 2001Elsevier Science Ltd.All rights reserved.PII:S0144-8617(00)00281-2/locate/carbpol*Corresponding author.Tel:11-450-686-2437,ext.232;fax:11-450-686-8952.E-mail address:chenite@ (A.Chenite).is important to note a signi®cant exception to the above general description of the solubility behaviour of chitosan Ðchitosans with a relatively low DDA,from40to60%, remain in solution up to a pH near9,and are therefore not the subject of investigation in the current work.Here we report the preparation and characterisation of thermogelling chitosan solutions formulated at conditions including physiological pH.The endothermically gelling chitosan solution is prepared by supplementing an aqueous solution of chitosan with glycerophosphate salt,an additive which plays three essential roles:(1)to increase the pH into the physiological range of7.0±7.4;(2)to prevent immediate precipitation or gelation;and(3)to allow for controlled hydrogel formation when an increase in temperature is imposed.Our results suggest that the addition of this particular basic salt provides the correct buffering and other physicochemical conditions including control of hydrophobic interactions and hydrogen bonding which are necessary to retain chitosan in solution at neutral pH and furthermore to allow gel formation upon heating to378C. This system is likely to receive considerable attention in the biomedical®eld,since such liquid polymer solutions can be loaded with therapeutic materials at low but non-freezing temperatures,and then injected into body sites to form degradable gel implants in situ(Chenite et al.,1999).An additional physicochemical characteristic of chitosan bodes well for its use as a scaffold or carrier system in tissue regeneration and repair and local drug and gene delivery. Namely the anionic nature of most human tissues due to the presence of glycosaminoglycans in the extracellular matrix, and the cationic character of chitosan,(at pH<7.2, approximately17%of amino groups are still protonated), allows for adherence of these thermally gelling solutions to tissue sites.Recently we have also shown that some formulations can be prepared to have physiological pH and osmotic pressure and to thereby offer a suitable micro-environment for living cells to maintain functional characteristics after injection and implant formation (Hoamann,Sun,Binette,McKee,&Buschmann,2000).2.MaterialsMedium molecular weight chitosan( M w<3:5£105 with a high degree of deacetylation DDA,91% ;was generously provided by Maypro(Purchase,NY,USA). The weight average molecular weight and the DDA of chitosan were determined by using size exclusion chroma-tography(SEC)and13C-NMR spectroscopy,respectively. For SEC we used a HP1100chromatograph equipped with WAT011545column connected to WAT011565guard column in series(both from Waters Inc.,Milford,MA), with an on-line detection obtained with G1362A differential refractometer.13C-NMR was performed using a CMX-300 NMR spectrometer operating at75.4MHz and room temperature.The DDA was determined following the previously described procedure(Pelletier,Lemire,Sygusch, Chornet,&Overend,1990).Hydrated b-glycerophosphate disodium salt(b-GP), (C3H7O3PO3Na25H2O;M W 306),was purchased from Sigma-Aldrich Cie,USA.2.1.Preparation of thermogelling solutionsClear solutions of chitosan were obtained by dissolving 200and400mg of chitosan in18ml of aqueous hydro-chloride solutions,0.05and0.1M,respectively.A series of b-GP solutions were prepared by dissolving from0to 1.6g of b-GP in,1ml deionised water and the®nal volume made up to2ml with deionised water.The chitosan solutions were cooled down to,48C and continuously stirred while adding drop by drop2ml of the b-GP solution. Thus the®nal20ml solutions contained1or2%(w/v)of chitosan corresponding to0.055or0.110M of amine groups,taking into account the DDA of91%,and a concen-tration of b-GP ranging from0to0.262M.2.2.Turbidity measurementsTurbidity of chitosan and chitosan/b-GP samples was monitored using an LP2000turbidity Meter(Hanna Instruments),covering a0±1000FTU range(FTU, Formazide Turbidity Unit).Aliquots(10ml)of chitosan and chitosan/b-GP solutions were poured into cuvets and incubated at378C.The turbidimeter uses an infrared beam with a wavelength peaking at890nm and performs accord-ing to the ISO7027International Standard.It was calibrated by using two AMCO AEPA-1standard solutions having0 and10FTU.2.3.Rheological analysisRheological measurements were performed on a Bohlin CVO rheometer(Bohlin Instruments,Inc.Grandbury,NJ) using C25concentric cylinders.Solution aliquots of12ml were introduced between the concentric cylinders and then covered with mineral oil in order to prevent evaporation during the measurements.The values of the strain amplitude were veri®ed in order to ensure that all measurements were performed within the linear viscoelastic region,such that the storage modulus(G0)and loss modulus(G00)were indepen-dent of the strain amplitude.Frequency dependent G0and G00of solutions and gels were measured in the frequency range between0.05and100Hz,at controlled constant temperatures of108C(solution)and378C(gel).To deter-mine gelation temperature,oscillatory measurements were performed at1Hz,while the temperature was increased at the rate of18C/min between4and708C.The gelation temperature was determined as the temperature at which both G0and G00followed a power law G0/v n and G00/ v n with the same exponent n.The value of n was found to be,0.48,in accordance with the analysis of Chambon and Winter(1987)and Winter and Chambon(1986).ToA.Chenite et al./Carbohydrate Polymers46(2001)39±47 40determine gelation time,oscillatory measurements at 1Hz were started just after introducing cold solutions,between 4and 108C,into the rheometer chamber which was pre-equilibrated at the desired temperature in the range 30±508C.The temporal evolution of G 0and G 00was thereby measured at constant temperature.Gelation time was also determined as the time when both G 0and G 00followed a power law G 0/v n and G 00/v n with the same exponent n .3.Results and discussionChitosan solutions can be neutralised to pH values between 6.5and 7.3via b -GP addition,without inducing immediate precipitation or gelation,provided the tempera-ture is maintained between 4and 158C.The pH of these chitosan solutions approached the physiological region when the molar concentration of b -GP exceeded the molar concentration of the amine groups of chitosan (0.110M in Fig.1).This ability to maintain chitosan in solution and prevent chain aggregation at neutral or nearly neutral pH is in part due to the mild alkalinity of b -GP p K a2 6:34 and possibly also due to the presence of the glycerol moiety in b -GP molecules,potentially coating the chitosan polymers and inhibiting chain to chain aggregation.Most importantly,chitosan/b -GP solutions were found to beA.Chenite et al./Carbohydrate Polymers 46(2001)39±4741Fig.1.pH Variation of chitosan solution (2%)as function of b -GP concentration,at roomtemperature.Fig.2.Turbidity changes with the time of chitosan solutions (2%)incubated at 378C:(a)in the absence of b -GP at pH ,5.4;and (b)in the presence of b -GP (0.262M)at pH ,7.2.thermosensitive since heating from 4to 378C and above induced gelation,visualised by an increase in turbidity which was only present when b -GP was added (Fig.2).Additionally,the chitosan used in this study was of a high degree of deacetylation (91%)which has been found previously to precipitate below a pH of 6(Skaugrud,Hagen,Borgersen,&Dornish,1996).3.1.Rheological characterisation of the temperature-dependent gelation processChitosan solutions with or without added b -GP displayed typical semi-dilute (NystroÈm,Walderhaug,Hansen,&Lindman,1995)solution rheological behaviour when measured at low temperature,,108C (Fig.3a).Notably,rheological properties measured at low temperature (,108C),following the addition of b -GP and attainment of a pH of ,7.15reduced both G 0and G 00compared to chitosan alone,potentially due to charge neutralisation and resulting increased ¯exibility of chitosan polymers,or due to b -GP impeding chitosan/chitosan chain to chain interactions (Fig.3a).Upon heating,however,from 5to 708C,a rapid increase of G 0indicated a temperature of incipient gelation near 378C (Fig.3b).After incubation atA.Chenite et al./Carbohydrate Polymers 46(2001)39±4742Fig.3.(a)Frequency-dependence of elastic (G 0)and viscous (G 00)modulus of chitosan/b -GP solution (chitosan,2%;b -GP,0.262M;pH ,7.2)(®lled and open circles)and of chitosan solution (chitosan,2%;pH ,5.4)(®lled and open triangles),both measured at a low temperature of 108C.(b)Temperature-dependence of elastic (G 0)and viscous (G 00)modulus for chitosan/b -GP solution [chitosan,2%;b -GP,0.262M;pH ,7.2],upon heating from 5to 708C.(c)Frequency dependence,at constant temperature (378C),of the elastic (G 0)and viscous (G 00)modulus for chitosan/b -GP solution (chitosan,2%;b -GP,0.262M)previously gelled by heating in (b).378C for at least 60min,rheological measurements indicated a nearly frequency independent G 0,while G 00increased slightly with the frequency as is characteristic for hydrogel materials (Clark,Richardson,Ross-Murphy,&Stubbs,1983;Nishinari,1997;NystroÈm et al.,1995)(Fig.3c).The strength of the gel can be appreciated by the magnitude of G 0in the range of several kPa,and by the great difference between G 0and G 00,also indicating a strong gel in the present case with G 0@G 00:The neutralisation behaviour of chitosan/b -GP solutions is evidently a central characteristic which determines their solubility and phase transition phenomena.Gelation temperature,for example,determined by rheological measurements,is greatly affected by the pH of a prepared chitosan/b -GP solution (Fig.4).A solution composed of 2%chitosan with a pH of 7.2gels at ,378C,while slight acidi®cation to pH 6.85increases the gelation temperature to near 508C.This monotonic decrease of gelation tempera-ture with increasing pH (Fig.4)suggests that the number of charged ammonium groups on the chitosan chain is an important parameter controlling gelation in this system.A reduction in charge density on the chitosan chain appears to reduce interchain electrostatic repulsion and permit a smaller addition of thermal energy to initiate gelation.The temporal evolution of G 0and G 00,measured for a chitosan/b -GP solution composed of 2%of chitosanA.Chenite et al./Carbohydrate Polymers 46(2001)39±4743Fig.3.(continued)Fig.4.Gelation temperature as function of the pH of the chitosan solution (chitosan,2%;b -GP,0.262M).The pH differences were generated by altering the concentration of HCl solution used to dissolve a constant amount of chitosan.pH 7:2 at various temperatures,allowed the determina-tion of the dependence of gelling time on temperature (Fig.5).Gelling time appeared to display an exponential decrease with temperature varying from13min at328C to 2min at428C for this formulation of chitosan/b-GP solutions.Increasing temperature or addition of heat is therefore a factor that also accelerates the gelation process probably via the acceleration of the formation of junction zones of the polymers.Thus taking together these observa-tions of a dependence of gelation temperature on pH(Fig.4) and of gelation time on temperature(Fig.5),the gelation process of chitosan/b-GP solutions appears to be governed by a coupling between pH,temperature and the neutralisation degree of the chitosan chain in the presence of glycerol phosphate.We have also investigated the in¯uence of the concentra-tion of b-GP on gelation temperature for chitosan solutions of two different polymer concentrations(Fig.6).Gelation temperature was determined by rheology as described for the dependence of gelation temperature on pH.The result-ing sol/gel diagram(Fig.6)indicates a decrease in gelation temperature when the concentration of b-GP is increased for both1and2%(w/v)chitosan solutions.The similarity with the dependence of gelation temperature on pH(Fig.4)is expected since b-GP addition increases the pH.For both1 and2%chitosan,gelation occurs in a region of b-GPA.Chenite et al./Carbohydrate Polymers46(2001)39±4744concentration corresponding to1±2£the molar concentra-tion of amine groups of chitosan(,0.055M for1%chitosan and,0.110M for2%chitosan).The pro®le of gelation temperature versus b-GP concentration is therefore similar for the two polymer concentrations used,with a displace-ment of approximately the molar concentration difference of polymer amine groups.This is evidently the result of a charge(proton)transfer from chitosan to b-GP upon the addition of the latter,resulting in a decreasing charge density on the polymer chain at higher b-GP concentrations. The amount of b-GP required to achieve a given chain charge density is roughly proportional to the polymer concentration.It is worthwhile to point out that the results shown here were obtained on highly deacetylated chitosan(DDA of 91%)having an average medium molecular weight of about450kD.Since the properties of chitosan solutions depend greatly on these two chemical characteristics,we investigated the in¯uence of these two parameters on the formulation of chitosan/b-GP solutions at physiological pH, as well as their thermal gelation.We found that the temperature of incipient gelation increases as the degree of deacetylation decreases,while the molecular weight showed no signi®cant effect on the temperature of gelation (Chenite et al.,2000).Moreover,for chitosan deacetylated to about95%,the gelation temperature has been lowered to around348C.3.2.Potential mechanisms of gelationOur current study has revealed several important conse-quences of b-GP addition to chitosan solutions,in particular the maintenance of chitosan solubility at physiological pH and the temperature-sensitive character of these chitosan/ b-GP solutions allowing for rapid hydrogel formation upon heating.We have furthermore demonstrated a clear interdependence of pH,b-GP concentration,and tempera-ture,all of which signi®cantly affect gel formation.This study does not provide suf®cient data to allow a complete molecular description of the mechanism of gelation to be proposed,however some indications of important para-meters and molecular interactions can be gleaned from our results and from published literature for other thermally sensitive polymer systems.In considering molecular mechanisms of gelation for these systems it is important to keep in mind the broad range of molecular interactions which can occur in aqueous solutions of the cationic poly-electrolyte chitosan and the divalent anionic base glycerol phosphate including:(1)electrostatic repulsion between like-charged chitosan chains;(2)electrostatic attraction between oppositely charged chitosan and the phosphate moiety of b-GP;(3)attractive hydrophobic and hydrogen bonding between chitosan chains;and(4)the hydrophobic or water-structuring character of the glycerol moiety of b-GP.The precipitation of chitosan upon increasing the pH above a critical value(such as,6.2when using a strong base)can,for example,be explained by a reduction of charge density along the polymer backbone reducing inter-chain electrostatic repulsion and allowing the attractive hydrophobic and hydrogen-bonding forces to predominate and precipitate chitosan.Addition of b-GP rather than a strong base maintains solubility of chitosan to a much higher pH(,7.2)probably due to its mild basic character and potentially due to an attraction of phosphate moieties of b-GP to remaining charged amine groups(NH31)of chito-san,and thereby exposing the glycerol moiety to separate chitosan chains in solution and maintain its solubility at low temperature.Upon heating of these chitosan/b-GP solu-tions,physical junction zones of chitosan chain segments throughout the solution occur to form a hydrogel,necessa-rily by inducing a sudden preponderance of attractive hydro-phobic and hydrogen bonding forces over interchain electrostatic repulsion.This thermally induced shift in attractive versus repulsive interchain forces could arise from many sources including:(1)reduced chitosan chain polarity and increased hydrophobicity upon heating;(2) reduced polarity and increased structuring of free water by the glycerol moeity of b-GP thus dehydrating chitosan chains and also causing increased interchain hydrophobic attraction;and(3)a thermally induced transfer of protons from chitosan amine groups to the phosphate moeity of b-GP thereby further reducing both chain charge density and chitosan attraction to b-GP and allowing for preponderance of attractive interchain hydrophobic and hydrogen-bonding forces between chains.Importantly we may exclude the purely ionic cross-linking that is responsible for the gelation of chitosan aqueous solutions with other divalent anions including oxalate(Hirano,Yamaguchi,Fukui,&Iwata, 1990b),molybdate(Draget,VaÊrum,Moen,Gynnild,& Smidsréd,1992),sulphate or phosphate ions,since such ions induce immediate temperature-insensitive precipitation and at a relatively acid pH where the chitosan retains it positive charge.Furthermore,we have also found that b-GP is freely diffusible after gelation and is not retained in the physically cross-linked network(Filion et al.,in prepara-tion).Finally,the contribution of hydrogen bonding versus hydrophobic forces could be pH-dependent.Unlike hydro-gen bonds,hydrophobic forces are known to be tempera-ture-dependent and were suggested to be a source of the thermoreversibility found previously in chitosan/b-GP gels (Chenite et al.,2000).Evidence can be found in the literature to support the occurrence of these temperature-induced alterations of molecular interactions in chitosan/b-GP solutions listed above.First,considering the polarity of the chitosan chain,poly(ethylene oxide)(PEO)solutions are known to become less soluble and precipitate at higher temperatures in aqueous solutions,potentially due to a conformational transition to a less-polar form(Saeki,Kuwahara,Nakata, &Kaneko,1976).Polysaccharides can also be considered as ethylene oxide containing polymers,as well as some cellulose derivatives which have demonstrated reducedA.Chenite et al./Carbohydrate Polymers46(2001)39±4745solubility in aqueous media upon heating(KarlstroÈm, Carlsson,&Lindman,1990;Sarkar,1979).Moreover, Park,Choi,and Park(1983)have suggested that at the precipitating pH of chitosan,a conformational change occurs allowing NH2groups to form intermolecular hydrogen bonds aiding the formation of a hydrated precipi-tate.Secondly,concerning the role of the glycerol moiety of glycerol phosphate,it has been previously shown that polyols and sugars can stabilise proteins against denatura-tion due to their structuring effect on water molecules, thereby strengthening protein±protein hydrophobic interac-tions(Back,Oakenfull,&Smith,1979;Gekko&Koga, 1983;Gekko,Mugishima,&Koga,1987;Gekko& Timasheff,1981;Na,Butz,Bailey,&Carroll,1986). Gekko and Koga(1983)and Gekko and Timasheff(1981) have additionally shown that the addition of polyols to aqueous solutions of collagen or carrageenan raise the tran-sition temperature from gel to sol upon heating indicating that the initial gel structure was reinforced by the presence of polyols,requiring more thermal energy to disrupt it. Finally,modulation of electrostatic repulsion between chains by controlling chain charge density is the mechanism by which the solution pH controls the state of chitosan.It is thus probable that one effect of temperature on chitosan/b-GP solutions is also to alter the chitosan chain charge density,potentially by reducing it upon heating via intrinsic temperature dependence of the various p K a s or via conformation-charge coupling(conformational dependence of the polyelectrolyte electrostatic free-energy).The presence of divalent ions in solution adds yet another inter-esting aspect due to the strong pair correlations between divalent ions(compared to monovalent ions)also reducing interchain electrostatic repulsion(Svensson,Jonsson,& Wodward,1990).Ongoing studies are underway to ascertain the relative importance of each of these potential gelation mechanisms.4.ConclusionsA thermally gelling chitosan system was prepared by neutralising highly deacetylated chitosan solutions with b-glycerol phosphate to retain chitosan in solution at physio-logical pH.Upon heating to moderate temperatures,these solutions quickly transformed into a hydrogel structure as demonstrated by rheological measurements.Furthermore, the sol/gel transition temperature was pH-sensitive and gelling time was shown to be temperature-dependent.The molecular mechanism of gelation may involve multiple interactions between chitosan,glycerol phosphate,and water,several of which may be thermally modulated.The ability to prepare these low concentration(1±2%w/v) polymer solutions which gel upon mild heating,for example from4to378C,and which are biocompatible,biodegradable and adhesive to human tissues,provides for new opportunities in the delivery of sensitive therapeutics.Further studies are now being pursued to elucidate the physicochemical mechanism of gelation as well as to investigate the potential of this system for speci®c bio-medical applications including tissue repair and regenera-tion,and in the delivery of protein-and gene-based therapeutics.ReferencesBack,J.F.,Oakenfull,D.,&Smith,M.B.(1979).Increased thermal stability of proteins in the presence of sugars and polyols.Biochemistry, 18(23),5191±5196.Chambon,F.,&Winter,H.H.(1987).Linear viscoelasticity at the gel point of a crosslinking PDMS with imbalanced stoichiometry.Journal of Rheology,31,683±697.Chandy,T.,&Sharma, C.P.(1990).Chitosan as a biomaterial.Biomaterials Arti®cial Cells and Arti®cial Organs,18,1±24. Chenite,A.,Chaput,C.,Combes,C.,Jalal,F.,&Selmani,A.(1999).Patent WO09907416A1.Chenite,A.,Chaput,C.,Wang,D.,Combes,C.,Buschmann,M.D., Hoemann,C.D.,Leroux,J.C.,Atkinson,B.L.,Binette,F.,&Selmani,A.(2000).Novel injectable neutral solutions of chitosan formbiodegradable gels in situ.Biomaterials,21,2155±2161.Clark,A.H.,Richardson,R.K.,Ross-Murphy,S.B.,&Stubbs,J.M.(1983).Structural and mechanical properties of agar/gelatin co-gels.Small-deformation studies.Macromolecules,16,1367±1374. Draget,K.I.,VaÊrum,K.M.,Moen,E.,Gynnild,H.,&Smidsrùd,O.(1992).Chitosan cross-linked with Mo(VI)polyoxyanions:a new gelling system.Biomaterials,13,635±638.Felt,O.,Buri,P.,&Gurny,R.(1998).Chitosan:a unique polysaccharide for drug delivery.Drug Development and Industrial Pharmacy,24, 979±993.Filion,D.,Lavertu,M.,Chenite,A.,&Buschmann,M.,in preparation. Gekko,K.,&Koga,S.(1983).Increased thermal stability of collagen in the presence of sugars and polyols.Journal of Biochemistry,94,199±205. Gekko,K.,Mugishima,H.,&Koga,S.(1987).Effects of sugars and polyols on the sol±gel transition of k-carrageenan:calorimetric study.International Journal of Biological Macromolecules,9,146±152. Gekko,K.,&Timasheff,S.N.(1981).Mechanism of protein stabilization by glycerol:preferential hydration in glycerol±water mixtures.Biochemistry,20,4667±4676.He,P.,Davis,S.S.,&Illum,L.(1998).In vitro evaluation of the muco-adhesive properties of chitosan microspheres.International Journal of Pharmaceutics,166,75±88.Henriksen,I.,Green,K.L.,Smart,J.D.,Smistad,G.,&Karlsen,J.(1996).Bioadhesion of hydrated chitosans:an in vitro study.International Journal of Pharmaceutics,145,231±240.Hirano,S.,Seino,H.,Akiyama,Y.,&Nonaka,I.(1990a).In C.G.Gebelein &R.L.Dunn,Progress in biomedical polymers(p.283).New York: Plenum Press.Hirano,S.,Yamaguchi,R.,Fukui,N.,&Iwata,M.(1990b).A chitosan oxalate gel:its conversion to an N-acetylchitosan gel via a chitosan gel.Carbohydrate Research,201,145±149.Hoamann,C.D.,Sun,J.,Binette F.,McKee,M.D.,&Buschmann,M.D., (2000).In Federation of European Connective Tissue Societies,1±5 July,Patras,Greece.Illum,L.(1998).Chitosan and its use as a pharmaceutical excipient.Pharmaceutical Research,15,1326±1331.KarlstroÈm,G.,Carlsson,A.,&Lindman,B.(1990).Phase diagrams of nonionic polymer±water systems.Experimental and theoreitical studies of the effects of surfactants and other cosolutes.Journal of Physical Chemistry,94,5005±5015.Lehr,C-M.,Bouwstra,J.A.,Schacht,E.H.,&Junginger,H.E.(1992).InA.Chenite et al./Carbohydrate Polymers46(2001)39±47 46。

Carbohydrate Research, 230 (1992) 327-342

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established by the ‘H- (Table I) and ‘“C-NMR data (Table II). The chemical shift (61.5 ppm) of the C-6 resonance of 20 is increased to 77.3 ppm in 23 due to the iminium group. When water was added to this mixture, the corresponding 6-O-formy1 derivative 24 (6 67.7 for C-6) was formed. On heating the solution of 23, the 6-bromo-6-deoxy derivative 22 (6 3 1.8 for C-6) was formed. When the addition of the components was reversed, i.e., 20 was dissolved in N,N-dimethylformamidc, and N-bromosuccinimide and triphenylphosphine were added. the reaction followed the mechanism suggested first by Rydon’” , i.e., the alkoxyphosphonium salt 21 was formed (6 71.3 for C-6) (for NMR data, see Tables 1 and II). On heating the solution, 21 was converted into the 6-bromo-6-deoxy derivative” 22 (6 3 1.8 for C-6). The t,,, values of 21 and 23 at 70” were 40 and 120 min, respectively; consequently, the phosphonium group in 21 is a better leaving group than the iminium group in 23. Thus, in the case of 2, depending on the sequence of the addition of the reagents, either the iminium intermediate 12 or the phosphonium intermediate 10 should be formed. Independent of the sequence, the mixture had to be heated to 100” to yield the 6-bromo-6-deoxy derivative 8. When the mixture was quenched with water before heating to loo”, only the 3-0-formyl derivative 3 could be detected; consequently, the iminium salt 12, and not the phosphonium salt 10, must have been present as an intermediate. However, when the phosphonium salt 10” was dissolved in N,N-dimethylformamide, it remained unchanged on heating to 120” and no reaction took place when the amidinium salt 16 was added. Thus, 10 was ruled out as a possible intermediate. A further possibility was the rearrangement of 2 into I ,2: 3,.5-di-O-isopropylidene-a-o-glucofuranose (25) before or during the reaction. Compound 25 is well known”-.12, but no data have been published for its stability and on the rearrangement reaction of 2 and 25. Conformer 25 was synthesised by the protection of HO-6 of 1,2-0-isopropylidene-cu_D-glucofuranose (29) by silylation ( --f 30). acidcatalysed reaction with 2-methoxypropcne in acetone t--f 31) and treatment with tetrabutylammonium fluoride in tetrahydrofuran to afford 25. The isomerisation of 25 into 2 in N,N-dimethylformamide catalysed by p-toluenesulfonic acid was monitored by NMR spectroscopy, and the results are depicted in Fig. 1. Isomerisation and hydrolysis occurred and, after 3.5 h at 45”, a mixture of 2 (59%), 25 (2%) and 29 (39%) was obtained. A similar mixture resulted after 6 h at 45” when 2 was the starting material. Thus, some 25 will always be formed in a solution of 2 in N,N-dimethylformamide when an acid is present. As the primary hydroxyl group of 25 reacts more rapidly than the secondary hydroxyl group of 2, the 2 + 25 equilibrium might be shifted towards 25 during the reaction, yielding the &bromo6-deoxy derivative 8 via activation of HO-6. When N-bromosuccinimidc and triphenylphosphine were added to a solution of 25 in N,N-dimethylformamide. the 6-0-phosphonium salt 28 was formed as an intermediate. On the other hand, when 25 was added after the reagents, the preformed 16 reacted with 25 to yield the h-0-iminium salt 27. Both the intermedi-

虾肝肠孢子虫病及其防治手段的研究进展

广　西　农　学　报Journal of Guangxi Agriculture 第38卷　第2期Vol.38，No.2452023年4月April，2023动物科学虾肝肠孢子虫病及其防治手段的研究进展韦秀颖1,2 杜泓明2 覃绍敏1 刘金凤1 吴健敏1*（1.广西壮族自治区兽医研究所广西兽医生物技术重点实验室，广西南宁 530001；2.广西大学动物科学技术学院，广西南宁 530004）摘要：虾肝肠孢子虫病（Hepatopancreatic microsporidiaosis ，HPM ）是由虾肝肠孢子虫（Enterocytozoon hepatopenaei ， EHP ）感染引起的一种虾类专性细胞内寄生的寄生虫病。

近年来该病在全世界对虾养殖区域内广泛流行，造成对虾生长缓慢综合症（MSGS ），损失堪比白斑综合症病毒病（WSSV ），严重影响对虾养殖业的健康与持续发展，引起了广泛关注。

本文就HPM 的病原、流行情况、传播途径、预防与检测手段及相关药物研究进展进行综述，以期为HPM 防控提供参考。

关键词：对虾；虾肝肠孢子虫；预防与检测；药物研究中图分类号：S851.34+7.34 文献标识号：A 文章编号：1003-4374（2023）02-0045-05Research Progress on Hepatopancreatic Microsporidiaosis and Its Control MethodsWei Xiu-ying 1,2, Du Hong-ming 2, Qin Shao-min 1, Liu Jin-feng 1, Wu Jian-min 1*（1. Guangxi Key Laboratory of Veterinary Biotechnology, Guangxi Veterinary Research Institute, Nanning, Guangxi 530001, China； 2. College of Animal Science and Technology, Guangxi University, Nanning,Guangxi 530004, China）Abstract: Hepatopancreatic microsporidiaosis is an obligate intracellular parasitic disease of penaeidcaused by Enterocytozoon hepatopenaei infection. In recent years, the disease has been widely prevalent inimportant penaeid farming areas around the world, causing Monodon Slow Growth Syndrome （MSGS）, and the loss has been comparable to with White Spot Syndrome Virus （WSSV）, seriously affecting the healthand sustainable development of the penaeid farming industry, which has caused widespread concern. In this paper, the pathogen, epidemic situation, transmission route, prevention and detection methods, and related drug research progress of HPM are reviewed in order to provide a reference for the prevention and control of HPM.Key words: penaeid, enterocytozoon hepatopenaei , prevention and detection, drug research 虾肝肠孢子虫（EHP）是近年来发现的一种对对虾养殖行业危害较大的寄生虫病病原，最早于2003年在泰国生长缓慢的斑节对虾（Penaeus monodon ）中被发现，直至2009 年才被成功分离，并被定义为孢子虫属的一个新物种。

英语资料

Preparation and characterisation of methylated hemicelluloses fromwheat strawJ.M.Fang a,*,P.Fowler a ,J.Tomkinson a ,C.A.S.Hill baThe BioComposites Centre,University of Wales,Bangor,Gwynedd LL572UW,UKbSchool of Agricultural and Forest Sciences,University of Wales,Bangor,Gwynedd LL572UW,UKReceived 19May 2000;accepted 17January 2001AbstractWheat straw hemicellulose ether was prepared by methylation with methyl iodide using sodium hydride as a catalyst reacted in dimethyl sulphoxide.The degree of substitution (DS)was determined by elemental analysis.According to the elemental analysis data,the DS-value can be calculated;it was 1.7.The structure of the methylated hemicellulose formed was characterised by nuclear magnetic resonance (NMR)and Fourier transform infrared (FT-IR)spectroscopy.The thermal properties of the prepared ether were studied by simultaneous thermal analyser (STA 625).Differential scanning calorimetry (DSC)was also used to determine the thermal pro®le of the material degradation.It was found that the thermal stability of wheat straw hemicellulose ether increased by methylation.q 2001Elsevier Science Ltd.All rights reserved.Keywords :Xylose;Methylation;Hemicelluloses1.IntroductionOver 3000million tonnes of cereal straw is produced in the world per annum.The agricultural crop residues repre-sent an enormous under-utilised resource,especially wheat straw,of which approximately 170million tones are produced yearly in Europe.These amounts are signi®cant enough to consider wheat straw as a generic source of renewable materials,particularly for the production of chemical derivatives from cellulose,hemicelluloses and lignin (Montane,Farriol,Salvado,Jollez &Chornet,1998).Wheat straw contains 14±15%lignin,35±40%cellulose,and 30±35%hemicelluloses.The hemicelluloses are made up of a (1!4)linked b -d -xylan main chain with l -arabi-nofuranosyl and d -xylopyranosyl side chains attached at position O±C(3),and d -glucopyranosyluronic acid (or 4-O -methyl-d -glucopyranosyluronic acid)groups attached at position O±C(2)(Sun,Lawther &Banks,1996;Wilkie,1979).The backbone contains b -d -xylopyranose units,each of which has two hydroxyl groups available for modi-®cation.The hydroxyl groups allow the potential for ester-i®cation,etheri®cation,oxidation and other reactions such as hydrolysis and reduction.Some hemicellulose deriva-tives have been prepared by using these reactions and various reagents.For example,Croon and Timell(1960a,b)reported the methylation of a 4-O -methyl glucur-onoxylan with dimethyl sulphate,®nding that HO±C(2)was more reactive than HO±C(3).A series of carboxymethyl ethers with DS varying from 0.13to 0.92was prepared bySchmorak and Adams (1957)from beechwood xylan.Sjo Ès-troÈm (1989)showed in the carboxymethylation of hardwood pulps that xylan was carboxymethylated to a higher DS than cellulose.Also,the HO±C(2)was much more reactive than the HO±C(3)by a factor of 2.4and 3.3for the xylan and cellulose components,respectively (Lai,1996).Manzi and Cerezo (1986)concluded from the methylation of galacto-mannans in organic media,that the extent of reaction was signi®cantly in¯uenced by the orderly structures of the poly-mer.The chemical and physical properties of the derivatives are in¯uenced by the types of substituent,the degree of substitution (DS),the uniformity of substitution,the degree of polymerisation (DP)and the distribution of molecular weight (Ishizu,1991).The DS is de®ned as the average number of hydroxyl groups substituted in an anhydroxylose unit,the maximum DS is 2.The nature of substituents in¯uences both the physi-cal and chemical properties.Physical properties such as solubility and swelling are strongly affected by changing the DS.The uniformity of substitution depends upon equal derivatisation along the hemicellulose chain.This is mediated by the relative reactivities of hydroxyl groups,which depend on a steric factor and by the homogeneityCarbohydrate Polymers 47(2002)285±2930144-8617/01/$-see front matter q 2001Elsevier Science Ltd.All rights reserved.PII:S0144-8617(01)00186-2/locate/carbpol*Corresponding author.of the reaction.Since the derivatisation of hemicelluloses is generally conducted under homogeneous conditions,all hydroxyl groups along a hemicellulose chain are equally accessible to reagents.In order to realise this aim,the following method has been proposed especially for etheri-®cation and for methylation:(1)use of the strongly basic methylsul®nyl carbanion in a non-aqueous medium to generate the polysaccharide alkoxide;(2)use of hemicellu-loses,which are soluble in an organic solvent,such as dimethyl sulphoxide (DMSO),as a starting material for etheri®cation performed under alkaline conditions;(3)use of non-aqueous hemicellulose solvents as a reaction medium.Wheat straw hemicelluloses were methylated by the above procedure.The product was examined using various analytical techniques.2.Experimental 2.1.MaterialsWheat straw was obtained from Compak Co.(Gainsbor-ough,England).The straw was ®rst ground in a Christie laboratory mill to pass a 0.7mm size screen,then stored at 58C until use.Sodium hydride (NaH),dimethyl sulphox-ide (DMSO)and methyl iodide (CH 3I)were purchased from Aldrich Chemical Company.2.2.Preparation and characterisation of hemicelluloses Isolation of hemicelluloses was performed using a new alkaline hydrogen peroxide method,as follows:to 100g of ®nely powered wheat straw was added 1l of de-ionised water with 0.2%(w/v)Na 2±EDTA´2H 2O (ethylenediamine tetraacetic acid,disodium salt,dihydrate)at pH6,heated to 908C for 1h.The straw was ®ltered,and washed with de-ionised water.The treatment was repeated three times to remove transition metal ions (e.g.manganese,copper and iron).The straw was extracted with aqueous 10%potassium hydroxide and stirred at room temperature for 16h.The extract was ®ltered,70ml of hydrogen peroxide (30%H 202)was added into the ®ltrate,the pHadjusted to 11.5with acetic acid and stirred at 608C for 16h.After the reac-tion,the temperature was allowed to decrease to 208C,the mixed solution was acidi®ed to pH6.0with acetic acid,and triple volume of 95%ethanol (IMS)added,the mixture was left to stand for hours until the supernatant became clear,then the precipitate was ®ltered and washed with 70%IMS.The pellet was dried in a stream of air (Fig.1).The dried hemicelluloses were ground in a mill,to pass a 200m m sieve and stored in a desiccator until required for analysis and methylation.Hemicelluloses were analysed for neutral sugars and uronic acids after hydrolysis of 10mg samples for 2h at 1208C in 7ml of 2.0M tri¯uoroacetic acid (sealed vials).J.M.Fang et al./Carbohydrate Polymers 47(2002)285±293286Fig.1.Scheme for extraction of wheat straw hemicelluloses by H 2O 2.Samples were evaporated to dryness and the sugars were then converted to their alditol acetates.The sugar deriva-tives in dichloromethane were analysed by gas chromato-graphy(GC),and the relative percentages were calculated (Blakeney,Harris,Henry&Stone,1983).Alkaline nitro-benzene oxidation of residual lignin from the hemicellulosic preparation was performed at1708C for3h.The lignin content in hemicelluloses was calculated to be2.40by multiplying the yield of phenolic,obtained by nitrobenzene oxidation(Sun,Fang,Rowlands&Bolton,1998).Methods of uronic acid analysis,determination of phenolic acids and aldehydes in nitrobenzene oxidation mixtures with high-performance liquid chromatography(HPLC),and measure-ment of the native hemicellulosic molecular weights have been described in previous papers(Lawther,Sun&Banks, 1995;Sun et al.,1996)IR spectra were obtained on an FT-IR(Nicolet750)spec-trophotometer using a KBr disc containing1%(w/w)of ®nely ground sample.The liquid13C NMR spectrum was obtained on a Bruker250AC spectrometer operating at 62.8MHz,it was recorded at258C from200mg of sample dissolved in1.0ml D2O.Standard acquisition and proces-sing software were used.2.3.Methylation of hemicellulosesThe methylation procedure was essentially that described by Hakomori(1964),which is a modi®cation of the method of Sandford and Conrad(1966),which involves the use of the dimethyl sul®nyl anion(Chaykovsky&Corey,1962)as a base.This was prepared by dissolving sodium hydride (NaH)in dimethyl sulphoxide(DMSO)at608C for 45min.The base was then added to a solution of the poly-saccharide in DMSO followed by the addition of methyl iodide(Collins&Ferrier,1995).The methylsul®nyl anion was prepared as follows.Into a dry,100ml two-necked round-bottom¯ask®tted with a condenser and a nitrogen(N2)line containing a magnetic stirring bar was added1.5g of hexane-washed sodium hydride.The¯ask was placed in an oil bath with a thermo-meter,15ml of dried DMSO(Perrin,Armarego&Perrin, 1980;Riddick&Bunger,1970)was added to the¯ask and then sodium hydride was added.The¯ask was heated with stirring under N2at65±708C(Corey&Chaykovsky,1962) for1h(Furniss,Hannaford,Smith&Tatchell,1989),until the solution became clear and green and the evolution of H2 gas ceased.The methylation method is as follows:wheat straw hemi-celluloses were ground to pass through a200mesh sieve and dried overnight at608C in an oven.Of the dried material,1g was added to50ml of dry dimethyl sulfoxide in a100ml two-necked round-bottom¯ask containing a magnetic stir-ring bar,®tted with condenser and N2line.The suspension was heated at1208C and stirred with a magnetic stirrer until all of the hemicellulose dissolved(about1.5h)and,after cooling to room temperature,10ml of methyl sul®nyl anion was added.The amount of base was a35%excess over the number of equivalents of hydroxyl plus carboxyl groups present,calculated on the basis of a hemicellulose composed of78.6%xylose,14.2%arabinose,4.8%glucuro-nic acid and a few percent of other sugars.Upon addition of the anion,a gel formed immediately but gradually lique®ed and,after stirring at room temperature for a few minutes (about10min),the reaction mixture became homogeneous. The minimum time for complete alkoxide formation after the addition of base was1±2h.For the methylation reaction,the hemicellulose alkoxide solution was maintained at208C,and3ml of methyl iodide was added to the stirred solution at a rate such that the temperature did not rise above258C.Within a few minutes after addition of methyl iodide,heat evolution ceased,the solution became clear and the viscosity was markedly reduced.At this stage,the reaction was deemed complete. The reaction mixture was left overnight with stirring.The reaction mixture was then poured into a250ml separator funnel,using100ml water for washing,and the mixture was extracted three times with chloroform(1£200ml, 2£100ml).The combined extract was washed with water (100ml).The chloroform extract was concentrated to give a yellow solid(Asensio,1987)under reduced pressure at 408C,and the dried sample(1.03g)was kept in a desiccator for further analysis.2.4.Determination of methylated hemicelluloses Elemental analysis was used to calculate the DS-value of methylated hemicelluloses.The sample was ground to a powder and dried,then analysed on a Carlo Erba EA1108 CHN S-O instrument,using theªsquare to linear®t methodºto measure the carbon and hydrogen content in the substi-tuted and native hemicellulose samples.From the carbon and hydrogen contents,the DS-value was calculated. The thermogravimetry experiments of native and methy-lated hemicelluloses were conducted using a simultaneous thermal analyser(STA625).The sample of approximately 10mg was heated with a heating rate of108C/min up to 6008C.Prior to thermal analysis,the samples were dried in a vacuum over808C for24h.3.Results and discussion3.1.Analysis of the isolated hemicellulosesThe isolated hemicelluloses were analysed using GC, GPC,HPLC,FT-IR,and NMR.The GC sugar analysis showed that xylose was present as a predominant sugar component,comprising78.6%of the total sugars.The second major sugar was arabinose(14.2%);glucose (3.1%),galactose(2.3%),rhamnose(1.4%),and mannose (0.5%)were other minor constituents.The uronic acids, mainly MeGlcA were present in a noticeable amount (4.8%).Gel permeation chromatography(GPC)analysisJ.M.Fang et al./Carbohydrate Polymers47(2002)285±293287showed that the native hemicelluloses had an average mole-cular weight of 21,790g mol 21with a 1.95polydispersity,corresponding to a degree of polymerisation of 165.From HPLC analysis,alkaline nitrobenzene oxidation of the lignin content in the isolated wheat straw hemicelluloses indicated a lignin content of 3.86%.The FT-IR and 13C NMR results further con®rmed the structural features of the native hemicelluloses,with a backbone of b -(1!4)-linked d -Xyl p units,side chains with l -Ara f ,d -Xyl p and 4-O -d -GlcpA (or d -GlcpA)(Sun et al.,1996).3.2.Yield and the degree of substitutionThe percentage yield of the methylated hemicelluloses was calculated from the mass of product obtained based on the mass of starting material used,and using the follow-ing relationship:%Weight of methylated hemicelluloses Weight of native hemicelluloses =132£160where 132is the main chain of xylose unit molecularweight;160is the molecular weight of methylated xylose.The weight of the native hemicelluloses was 1g before methylation.After the reaction,the weight of methylated hemicelluloses was 1.03g,the theoretical maximum weight of the product was 1.21g,from these data and the above yield (%)formula calculation,the yield of the product was 85.1%.The DS of the methylated hemicelluloses is calculated from elemental analysis data,and according to the relation-ship below:DSC% C 5H 8O 4 2C 5C 2C% CH 3±Hwhere C%is from elemental analysis;C 5H 8O 4is the anhy-droxylose unit of hemicellulose backbone;CH 3is the substi-tuent from methyl iodide.Elemental analysis of methylated hemicelluloses gave the following results:C,51.474%;H,7.703%;O,40.823%(the values as calculated from theory are C,52.49%;H,7.55%;O,39.96%).According to the elemental analysis data,the DS-value can be calculated;it was 1.7.This corresponds to an approximate molecularJ.M.Fang et al./Carbohydrate Polymers 47(2002)285±293288Fig.2.FT-IR spectra of:(a)wheat straw hemicelluloses and (b)methylated hemicelluloses.formula(MF)of C6.7H11.9O4(the theoretical MF is C7H12O4) for the prepared methylated hemicelluloses.As can be seen,the yield and DS are satisfactory,which con®rms that the method of methylation was successful, over90%of the free hydroxyl groups in the native hemi-celluloses were methylated under the reaction conditions given,but the yield and DS were lower than the theoretical maximum of100%yield and DS of2.The incomplete reac-tion is presumed to be because:(1)some of the hemicellu-loses are degraded in the reaction;(2)the hemicelluloses in DMSO may not be fully swollen,in which case the DMSO cannot completely penetrate between the hemicellulose chains and,therefore,some of the reagents do not attack the OHgroups of the hemicelluloses.If so,prolongation of the reaction time may be required to ensure complete dissolution of the hemicelluloses in DMSO.3.3.FT-IR spectraThe methylation procedure afforded new material. Scheme1(schematic diagram for methylation of wheat straw hemicelluloses)and Fig.2show typical analyses of products from methylated and native hemicelluloses.The data illustrate several points:(1)the initial methylation yielded a product with99%of the theoretical methoxyl content;(2)in the hemicellulosic framework,which contains b-d-xylopyranose units,most of the monomer units bear two hydroxyl groups.Therefore,the maximum theoretical DS-value for the methylated hemicelluloses is2 (Scheme1);(3)the uronic acid residues are esteri®ed in the reaction as indicated by the presence of strong carbonyl stretching frequencies identi®ed in the infrared spectrum (b)at1752cm21(ester C y O).The FT-IR spectra of native hemicelluloses as compared with methylated hemicelluloses are shown in Fig.2.As can be seen from spectrum(a),the wheat straw hemicelluloses were typical arabinoxylans.The absorbances at1580,1467, 1414,1340,1255,1165,1090,1043,990and897cm21are associated with native hemicelluloses.A sharp band at 897cm21is characteristic of b-glucosidic linkages between the sugar units(Gupta,Madan&Bansal,1987).The low intensity of the band at990cm21suggests the presence of arabinosyl units,which are attached only at position3of the xylopyranosyl constituents(Ebringerova,Hromadkova, Alfoldi&Berth,1992).As the vibrational mode of xylans at1165cm21has been assigned to C±O and C±O±C stretching with some contribution of OHbending mode (Kalutskaya,1988),the band at1043cm21may be assigned to COHbending modes.The appearance of two other prominent bands at1414and1467cm21is attributed to the C±H,OH and CH2bending(Kacurakova,Ebringerova, Hirsch&Hromadkova,1994),respectively.The absorption at1580cm21in spectrum(a)is principally associated with the C y O stretch of carboxylic anion for GlcA in native hemicelluloses.A strong broad band due to hydrogen bond hydroxyl groups appears at3430cm21and the symmetric C±Hvibration band appears at2866and 2920cm21(Aburto,Thiebaud,Alric,Borredon,Bikiaris, Prinos et al.,1997).From the FT-IR spectra of Fig.2,there are obvious changes between methylated and native hemicelluloses.In the spectrum of methylated hemicelluloses(b),an absorp-tion band at1752cm21,is attributable to the stretching deformation of the ester carbonyl group.This originates from the4-O-methoxyl group of a glucuronic acid residue in the xylan,which is a small peak in accord with the low uronic acid content,indicating esteri®cation of the uronic acid residues during the reaction.The almost complete disappearance of a peak at1580cm21in spectrum(b)indi-cates that all of the uronic acid residues have been esteri®ed. In addition,the signi®cant broad band associated with hydroxyl groups(OH)of the native hemicelluloses at 3430cm21has clearly reduced in intensity in spectrum (b),after the etheri®cation reaction,owing to ether forma-tion.It thus indicates that the etheri®cation takes place with high DS owing to the OHgroup peak decreasing substan-tially.The symmetric C±Hvibration band at2933and 2840cm21has increased,which implies that CH3groups have been introduced.In the infrared pattern(b),the bands for CH3-stretching,CH3-deformation,and stretching vibrations for the ether bond,particularly for CH3±O±C at 1169cm21were pronounced.Hakomori(1964)assigned bands at1169,1103and1076cm21,due to ether bonds as in spectrum(b)herein.Another change was a pronounced decrease in intensity of the band at1414cm21(CH,OH bending),which indicates that methyl group substitution of the hydroxyl groups present in native hemicelluloses has occurred.3.4.Methylation mechanismClassical methylation methods have relied upon the initial conversion of the hydroxyl groups to alkoxides by reacting the polysaccharide with base in aqueous solution (Sandford&Conrad,1966).The added methylating reagent then reacts with the alkoxides to yield methyl ether.Any free base in the reaction mixture competes with the alkoxide for the alkylating reagent.In the initial reaction,ROH1 B2$RO21BHan equilibrium is established at a point dependent on the strength and concentration of the base,B2 complete conversion to the alkoxide requires a base stronger than OH2.However,the strongest base that can exist in aqueous solution is the OH2ion,since stronger bases will react with water to form this ion.Thus,in the standard methylation procedures the extent of ether formation is in¯uenced by the point of equilibrium in the base-catalysed reaction,which in turn is limited by the base strength of OH2.In the method applied here,these limitations are over-come by use of the strongly basic methylsul®nyl carbanion (Corey&Chaykovsky,1962)in a non-aqueous medium to generate the polysaccharide alkoxide.It is apparent that the equilibrium in this reaction lies almost completely in theJ.M.Fang et al./Carbohydrate Polymers47(2002)285±293289direction of alkoxide formation since,upon addition of the alkylating reagent,formation of the methyl ether is complete within a few minutes.The competing reaction between the excess methylsul®nyl anion and methyl iodide does not interfere as long as methyl iodide is added in excess of total base.Generation of the alkoxide requires about 1±2h.The overall chemical reaction was divided into two steps,as shown below:(1)Hemicelluloses±OH 1CH 3±SO±CH 22Na 1!Hemicelluloses±O±Na 11CH 3±SO±CH 3(2)Hemicellulose±O±Na 11CH 3I !Hemicellulose±O±CH 31NaIThe reaction mechanism involves the methyl sul®nyl anion abstracting a proton from the hemicelluloses to afford the polyalcoxide (Scheme 2Ðmechanism of methylation of wheat straw hemicelluloses).The stoichiometric methy-lation reaction requires complete dissolution of the hemi-cellulose prior to addition of the methylsul®nyl anion,a condition which is greatly facilitated by lyophilisation and sieving of the hemicellulose before attempting to dissolve it in dimethyl sulfoxide.3.5.13C NMR spectraIn order to characterise the structural features of hemi-celluloses,the isolated hemicelluloses were analysed by 13C NMR spectroscopy in D 2O.The spectrum is shown in Fig.3,it was interpreted on the basis of reported data for structu-rally de®ned l -arabino-(4-O -methyl-d -glucurono)-d -xylan (Sun et al.,1996).This d -xylan type of polysaccharide is generally found in the cell walls of wheat straw.Such poly-saccharides are based on the structure comprising b -(1!4)-linked d -xylopyranosyl residues with some hydro-xyl groups substituted with carbohydrate moieties.TheseJ.M.Fang et al./Carbohydrate Polymers 47(2002)285±293290Fig.3.13C NMR spectrum of wheat straw hemicelluloses in D 2O.are represented mostly by d-glucuronic acid and its4-O-methyl derivative and l-arabinofuranosyl units.A greater variety of glycosyl substituents is typical of heteroxylans (Kacurakova et al.,1994).In native hemicelluloses Fig.3, the main b-(1!4)-linked d-Xyl p units are characterised by the signals at104.9,78.4,77.6,75.9and65.8ppm,which correspond to C-1,C-4,C-3,C-2and C-5of the b-d-Xyl p units,respectively(Fidalgo,Terron,Martinez,Gonzalez, Gonzalez-Vila&Galletti,1993).The signals at112.2, 89.1,83.0,81.2and64.3ppm correspond to C-1,C-4,C-2,C-3and C-5of a-l-Ara f residue,respectively.Three signals at176.1,85.0(data not shown in the spectrum), and58.0ppm originate from the C-6,C-4and4-O-methoxyl group of glucuronic acid residue in the xylan,which are very weak and in accord with the low uronic acid content. The signal at26.8ppm is most likely due to±CH3from acetic acid from the extraction process,and the correspond-ing signal at179.2ppm is probably due to the carbonyl group of CH3COOHpresent within the sample. Methylation of the hemicelluloses gave a product,which was analysed using solution1HNMR and13C NMR spectro-scopy.30mg of sample was dissolved in1.0ml D6-DMSO. The spectrum is shown in Fig.4,from13C NMR data,the chemical shifts for the main(1!4)-linked b-d-Xyl p units are characterised by the signals at101.0,75.5,82.6,83.3 and62.1ppm,which correspond to C-1,C-2,C-3,C-4and C-5of the b-d-Xyl p units,respectively.The strong signals at59.7and59.9ppm are possibly due to the methyl group (±CH3)substituted on the C-2and C-3positions.3.6.Thermal propertiesThermal characteristics of the native and methylated hemicelluloses obtained were studied using thermogravi-metric analysis(TG)and differential scanning calorimetry (DSC).As can be seen from the TG plot of native hemi-celluloses in Fig.5a,there is a very slight mass loss until a temperature of1908C is reached.On further heating there is a sharp weight loss,to give a residue of40%at6008C. The DSC(Fig.5a)curve shows two peaks,the main peak at2858C,and a small peak at3508C for this degradation to a carbonaceous residue.In the case of the methylated hemicelluloses,decomposition commences at2308C with a steady loss continuing with an increase in temperature up to6008C,when almost88%of the material is lost in Fig. 5b,and the DSC plot showed a small peak at2408C together with the main peak shifted towards higher temperature3608C.Such behaviour might be expected, because the major part of the original material has been converted into a new form(Aggarwal&Dollimore,1998). Compared with TG plots from Fig.5,the plots clearly show the presence of the methylated material distinct from the native one by degradation at higher temperature. The greater thermal stability of ether is probably due to the lower amount of remaining hydroxyl groups after methy-lation.4.ConclusionsThe development of the methylation reaction makes feasible the use of methyl iodide as the alkylating reagent. The present method has obvious advantages in that the reaction is more rapid and complete when catalysed by the carbanion,can be controlled by the amount of the reagent added and can be carried out at room temperatureJ.M.Fang et al./Carbohydrate Polymers47(2002)285±293291in one continuous process without the use of complicated apparatus.The hemicelluloses studied were successfully etheri®ed using methyl iodide in DMSO with NaH.The structure of the methylated hemicelluloses formed was determined by FT-IR,and further con®rmed using solution-state NMR spectroscopy.The carbon content obtained from elemental analysis data permitted calculation of the DS.The native and etheri®ed hemicelluloses were then treated to thermal analysis,the TG-DSC plots clearly show that the etheri®edJ.M.Fang et al./Carbohydrate Polymers47(2002)285±293292hemicelluloses differ from the native one by the occurrence of the peaks and different decomposition temperatures.In all cases,the TG-DSC shows an endothermic degradation to carbon but,generally,the carbon residue decreases after etheri®cation.The TG plot also demonstrates a marked increase in thermal stability because large amounts of methyl group substituents remain by methylation.AcknowledgementsThe authors are grateful for the®nancial support of this research from the European Community under the Industrial &Technologies Programme(Brite-EuRam III)ÐDepoly-merisation,Polymerisation and Applications of Biosustain-able Raw Materials for Industrial End Uses.ReferencesAburto,J.,Thiebaud,S.,Alric,I.,Borredon,E.,Bikiaris,D.,Prinos,J.,& Panayiotou,C.(1997).Properties of octanoated starch and its blends with polyethylene.Carbohydrate Polymers,34,101±112. 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NoteConversion of diacetyl-C -(b -D -glucopyranosyl)phloroglucinolto spiroketal compoundsShingo Sato,*Toshihiro Kumazawa,Ko-ichi Watanabe,Shigeru Matsuba and Jun-ichi OnoderaDepartment of Chemistry and Chemical Engineering,Faculty of Engineering,Yamagata University,3-5-6Jonan,Yonezawa,Yamagata 992-8510,JapanReceived 13August 2003;accepted 30September 2003Abstract—Diacetyl-C -(b -D -glucopyranosyl)phloroglucinol was converted by reﬂuxing in water to spiro(benzofuran-[2H ]furan)a new compound,along with spiro(benzofuran-[2H ]pyran).The stereochemistry of the quaternary carbon of both spiro compounds had an S -conﬁguration.Ó2003Elsevier Ltd.All rights reserved.Keywords:Diacetyl-C -(b -D -glucopyranosyl)phloroglucinol;Spiro[benzofuran-2(3H ),20-[2H ]pyran];Spiro[benzofuran-2(3H ),20-[2H ]furan];Quater-nary carbon;Stereochemistry;PinnatiﬁnosideWe previously reported on the conversion of the C -(b -D -glycopyranosyl)phloroacetophenone to spiro de-rivatives by reﬂuxing in water in the presence of p -toluenesulfonic acid (p -TsOH);C -(b -D -glucopyr-anosyl)phloroacetophenone and C -(b -D -galactopyr-anosyl)phloroacetophenone to (2S ,30S ,40R ,50R )-7-acetyl-spiro[benzofuran-2(3H ),20-[2H ]pyran]-30,4,40,50,6-pentaol (1)1and (2R ,30S ,40S ,50R )-7-acetyl-spiro[benzofuran-2(3H ),20-[2H ]pyran]-30,4,40,50,6-pentaol (2),2respec-tively.OOHOHOHO OHOHCOMe 1OOOHOHCOMeHO HO HO2At the time of our reports,these spiroketal com-pounds were not known to be naturally occurring.3However,in 2001,Zhang and Xu 4reported on the iso-lation of four ketohexose furanosides from the leaves of Crataegus pinnatiﬁda Bge.var.major N.E.Br.(Rosa-ceae),which is used as a medicinal plant to improve digestion,inhibit the retention of food,promote blood circulation,and resolve blood stasis both in traditional and folk medicine.5Pinnatiﬁnosides A and B are ﬂav-ones (see structures),containing a spiro(benzofuran-furan)ring in which the stereochemistry at C-30,C-40,and C-50is analogous to that of D -arabinose and the stereochemistry of the spiro-quaternary carbon is R .Pinnatiﬁnosides C and D are also ﬂavones that contain a spiro(benzofuran-furan)ring,in which the stereochem-istry at C-30,C-40,and C-50is analogous to that of*Corresponding author.Tel.:+81-238-26-3121;fax:+81-238-26-3413;e-mail:shingo-s@yz.yamagata-u.ac.jp0008-6215/$-see front matter Ó2003Elsevier Ltd.All rights reserved.doi:10.1016/j.carres.2003.09.035Carbohydrate Research 339(2004)429–433CarbohydrateRESEARCHD -ribose,and the stereochemistry of the spiro-quater-nary carbons is R and S ,respectively.However,while the naturally occurring spiroketal ﬂavones all contain a spiro(benzofuran-[2H ]furan)ring,both of the spiroke-tals synthesized by us also contain a spiro(benzofuran-[2H ]pyran)ring.The spiroketal skeletons of pinnatiﬁ-nosides A and B,and C and D could be constructed from C -b -D -gluco-and -allopyranoside,respectively,based on the reactions which we developed.OOHOOCH 2OR H HO H HO H OH OR = H: Pinnatifinoside A R = acetyl: Pinnatifinoside BOOHOOCH 2OCCH 3H H H HO HOOPinnatifinoside DOOOHO O OHHHH 32COHOPinnatifinoside CO OHH In an ongoing study of the conversion of C -glyco-pyranosylphloroacetophenone to the spiroketal,we ex-amined the conversion of diacetyl-C -(b -D -glucopyrano-syl)phloroglucinol (7)to the corresponding spiro com-pound in a similar pound 7could not be obtained by the direct O ﬁC glycoside rearrangement of diacetylphloroglucinol (3)(Scheme 1).However,an O ﬁC glycoside rearrangement of the phloroacetophe-none,6–8followed by acetylation of the hydroxyl group,and C -acetylation using BF 3Æ2AcOH and O-deacetyl-ation gave 7in good yield (Scheme 2).Since the reﬂuxing of 7in water in the presence of a catalytic amount of p -TsOH caused deacetylation,resulting in the formation of C -b -D -glucopyranosylphloroacetophenone,7was reﬂuxed in water in the absence of any catalyst.The conversion,as expected,proceeded slowly.After re-ﬂuxing for 1day,the resulting product was acetylated by treatment with acetic anhydride,pyridine,and a cata-lytic amount of DMAP,giving two acetates,which were separated and isolated by silica-gel column chromato-graphy (n -hexane–EtOAc).A detailed spectroscopic study of both acetates indicated that a new product,spiro(benzofuran-[2H ]furan)(9),was produced along with spiro(benzofuran-[2H ]pyran)(8)in 9.8%and 26.0%yield,respectively.The 1H NMR spectrum of 8was analogous to that of 1except for the presence of another C -acetyl group.However,that of 9was diﬀerent from any spiroketal synthesized thus far.The H-50signal at 4.36ppm (1H,ddd,J 4.0,6.0,and 7.5Hz)was shifted upﬁeld (D d 1.11),and the H-60a at 4.21ppm and the H-60b at 4.43ppm downﬁeld (D d 0.29and 0.25)compared to that of 8,respectively.The above ﬁndings suggest that 9does not contain a pyran ring linked between the C-6oxygen and a quaternary carbon (C-2)of the benzofu-ran like 8,which contains a spiro[benzofuran-2(3H ),20-[2H ]furan]ring linked between the C-5oxygen and a quaternary carbon (C-2).The following data point to the presence of a spiro[benzofuran-3(2H ),20-[2H ]furan]ring;4;9the coupling constants for H-30,-40,and -50(J 3;4¼7:0,J 4;5¼6:0Hz)are not consistent with a pyran ring like 8(J 3;4¼10:5,J 4;5¼3:5Hz).Further,the dif-ference in chemical shifts between the methylene protons on C-3of the 2H -benzofuran (Dd 0.136)is apparently larger than those for spiro(benzofuran-[2H ]pyran)[1(D d 0.01),2(D d 0.00),and 8(D d 0.04)].To determine the stereochemistry of 9more precisely,nuclear Overhauser and exchange spectroscopy (NOESY)and correlation spectroscopy via long-range coupling spectrum (CO-LOC)experiments were carried out(see Figs.1and2). In the NOESY spectrum,a correlation was found be-tween H-60a and H-40,and between H-60a and one of the two acetyl groups on the benzene ring,as well as be-tween H-30and H-3a,respectively.These correlations indicate that the stereochemistry of the quaternary car-bon is an S-conﬁguration and opposite to that of the natural products,pinnatiﬁnosides A and B.If the qua-ternary carbon has an R-conﬁguration,the above cor-relation between H-60a and one of the two acetyl groups, and between H-30and H-3a would not exist.Thus,the stereochemistry of the quaternary carbon is of the S-conﬁguration.The stereochemistry at C-3,C-4,and C-5 is the same,as that of D-glucose as was found for8.In the COLOC correlation of9,H-3a and-3b showed acorrelation with the quaternary carbon(C-2: 117.4ppm).Further,H-3b showed a correlation with C-30[77.7p-4(146.8ppm),and C-7a(158.3ppm)].H-3a also showed a correlation with the C-4and C-7a.H-30 showed a correlation with C-40(74.7ppm),H-40showed a correlation with C-30and C-50(79.5ppm),H-6a showed a correlation with C-50.From the above struc-tural data,we conclude that the hydrolysis of the di-acetyl-C-(b-D-glucopyranosyl)phloroglucinol produced mainly a spiro[benzofuran-2(3H),20-[2H]pyran]and a new spiro[benzofuran-2(3H),20-[2H]furan].Flavones having a spiro[benzofuran(2H)furan]skeleton,pinn-atiﬁnosides A,B,C,and D might be also formed by the hydrolysis of the corresponding C-(b-D-glycopyrano-syl)ﬂavones in nature.We are currently attempting the synthesis of pinnatiﬁnoside A using the above approach.1.Experimental1.1.GeneralThe anhydrous CH2Cl2used in this reaction was pre-pared in situ by distillation from CaH2.For separation and puriﬁcation,ﬂash column chromatography was performed on silica gel(230–400mesh,Fuji-Silysia Co., Ltd.,BW-300).HPLC was performed using an Inertsil ODS-3column(GL Science;5l m,4.6·250mm mobile phase,MeOH–water).Melting points were determined on a Yanagimoto micro-melting point apparatus and are uncorrected.Mass spectral data were obtained byfast-atom bombardment(FAB)using3-nitrobenzyl al-cohol(NBA)or glycerol as a matrix on a JEOL JMS-AX505HA instrument.Optical rotations were recorded on a JASCO DIP-370polarimeter.Elemental analyses were performed on a Perkin–Elmer PE2400II instru-ment.NMR spectra were recorded on a Varian Inova 500spectrometer using Me4Si as an internal standard.1.2.1,3-Diacetyl-2,6-O-benzylphloroglucinol(3) Compound3was synthesized via the diacetylation of phloroglucinol,followed by mono-O-methoxymethyl-ation,di-O-benzylation,and the O-demethoxymethyl-ation of phloroglucinol in an overall yield of40%,as shown in Scheme3.Colorless needles(from n-hexane–EtOAc):mp137°C. IR(KBr)m3444,2945,2884,1699,1612,1585,1367, 1259,1219,1190,and1099cmÀ1.1H NMR(CDCl3)d 2.49and2.60(each3H,s,ArAc·2),4.93and5.13(each 2H,s,benzylic CH2),6.36(1H,s,ArH),7.34–7.42(10H, m,ArH),13.47(1H,ArOH).FABMS(NBA,m=z)391 (M+H)þ.Calcd for C24H22O5:C,73.83;H, 5.68. Found:C,73.78;H,5.75.1.3.b-C-(20,30,40,60-Tetra-O-acetyl-D-glucopyranosyl)-diacetylphloroglucinol(6)Compound5(1.62g,2.60mmol)was stirred at50°C for 1h in10mL of boron triﬂuoride–acetic acid complex (BF3Æ2AcOH).The reaction mixture was poured into water,and the solution was extracted with toluene twice. The organic layer was washed with water and brine and dried over anhydrous Na2SO4.After removing the sol-vent,the residue was recrystallized from EtOH to give6 (881mg,62.8%)as colorless prisms:mp206–206.5°C.½a 25D +18.8(c0.50,CHCl3).IR(KBr)m3440,3132,2927,1755,1633,1365,1236,and1045cmÀ1.1H NMR (CDCl3)d1.84,2.02,2.08,2.14(each3H,s,OAc·4), 2.71(6H,s,ArAc·2),3.94(1H,ddd,J 2.4,3.5,and 10.2Hz,H-50),4.19(1H,dd,J2.4and12.6Hz,H-60a), 5.24(1H,dd,J3.5and12.6Hz,H-60b),5.26(1H,d,J 9.4Hz,H-10),5.28(1H,dd,J9.4and10.2Hz,H-40),5.33(1H,t,J9.4Hz,H-30),5.40(1H,t,J9.4Hz,H-20), 9.25(1H,s,OH),16.17(1H,s,chelated OH).FABMS (NBA,m=z)541(M+H)þ.Calcd for C24H28O14:C, 53.33;H,5.22.Found:C,53.36;H,5.04.1.4.Diacetyl-C-(b-D-glucopyranosyl)phloroglucinol(7) To a stirred solution of6(600mg,1.11mmol)in MeOH (5mL),0.5mL of a25%NaOMe solution was added, followed by stirring at room temperature for0.5h. Dowex50W(Hþ)resin was added to the resulting mixture until the reaction mixture reached neutrality. Afterﬁltering,theﬁltrate was evaporated and recrys-tallized from EtOH to give7(397mg,95%)as colorless prisms:mp150–151°C.½a 25D+115(c0.52,MeOH).IR (KBr)m3430,2931,1616,1365,and1292cmÀ1.1H NMR(DMSO-d6)d2.62(6H,s,Ac·2),3.24–3.40(5H, m,H-30,-40,-50,-60a,b),3.44(1H,t,J10.0Hz,H-20), 4.74(1H,d,J10.0Hz,H-10).FABMS(glycerol,m=z) 373(M+H)þ.Calcd for C16H20O10Æ0.25H2O:C,50.99; H,5.48.Found:C,50.90;H,5.44.Compound7(550mg, 1.46mmol)was reﬂuxed in water(220mL)for1day.After removing the water in vaccuo,the residual syrup was acetylated by stirring with a solution of acetic anhydride(20mL),pyridine (5mL),and4-dimethylaminopyridine(5mg)at room temperature for1day.After the usual workup,the res-idue was column chromatographed on silica gel(1:1 n-hexane–EtOAc)to give8(218.6mg,26.4%)as color-less prisms and9(85.2mg,9.8%)as a colorless oil.1.5.(2S,30S,40R,50R)-30,4,40,50,6-Pentakis-acetoxy-5,7-diacetyl-30,40,50,60-tetrahydrospiro[benzofuran-2(3H),20-[2H]pyran](8)Colorless prisms:mp171–172°C.½a 25D)144(c1.025, CHCl3).IR(KBr)m1776,1747,1697,1620,1371,1224, and1184cmÀ1.1H NMR(CDCl3)d2.05,2.07,and2.11 (each3H,s,OAc·3), 2.26and 2.27(each3H,s, ArOAc·2),2.42and2.62(each3H,s,Ar Ac·2),3.13 and3.14(each1H,d,J17.0Hz,3-CH2),3.92(1H,dd,J 2.0and13.0Hz,H-6a),5.47(1H,dd,J 2.0,3.5,and1.5Hz,H-50),4.18(1H,dd,J1.5and13.0Hz,H-6b), 5.30(1H,dd,J3.5and10.5Hz,H-40),5.63(1H,d,J 10.5Hz,H-30).13C NMR(CDCl3)d20.6(OAc·2), 20.6,20.9,20.9(OAc·3),31.4,31.9(Ar Ac·2),36.3(C-3),63.9(C-60),68.40,68.46,68.56(C-30,C-40,C-50), 112.9(C-2),114.4(C-5*),117.9(C-7*),123.1(C-3a*), 145.7(C-4**),146.8(C-6**),158.7,167.0,169.0,169.9, 170.1,170.4(OAc·5),195.2,197.2(Ar Ac·2).*,**: interchangeable.FABMS(glycerol,m=z)565(M+H)þ, 523,481.Calcd for C26H28O14:C,55.32;H,5.00.Found: C,55.44;H,4.97.1.6.(2S,30S,40R,50R)-30,4,40,6,60-Pentakis-acetoxy-5,7-diacetyl-50-acetoxymethylspiro[benzofuran-2(3H),20-[2H]furan](9)Colorless amorphous powder.½a 25D )15.3(c 1.035,CHCl3).IR(KBr)m2925,1778,1749,1697,1624,1371, and1180cmÀ1.1H NMR(CDCl3)d2.05,2.06,and2.07 (each3H,s,OAc·3), 2.26and 2.27(each3H,s, ArOAc·2),2.42and2.61(each3H,s,Ar Ac·2),3.25 and3.38(each1H,3-CH2),4.21(1H,dd,J7.5and 12.0Hz,H-6a),4.36(1H,ddd,J4.0,6.0,and7.5Hz,H-50),4.43(1H,dd,J4.0and12.0Hz,H-6b),5.44(1H,dd, J6.0and7.0Hz,H-40),5.59(1H,d,J7.0Hz,H-30).13C NMR(CDCl3)d20.7and21.0(ArOAc·2),31.3and 31.9(Ar Ac·2),34.8(C-3),64.4(C-60),74.7(C-40),77.7 (C-30),79.5(C-50),114.2(C-2),116.4(C-3a),117.38(C-7),117.39(C-5),145.5(C-6),146.8(C-4),158.3(C-7a), 195.5and197.4(Ar Ac·2).FABMS(glycerol m=z)565 (M+H)þ.Calcd for C26H28O14:C,55.32;H, 5.00. 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