1、第二节糖基转移酶及其应用糖基转移酶及其应用主要内容主要内容l糖转移酶简介 l糖基转移酶在合成中的应用 Glycosyltransferases 应用activated sugar phosphates 作为糖供体，合成 glycosidic linkage，糖受体通常为 nucleophilic group,usually an alcohol.生成的糖苷可以为 O-,N-,S-,or C-glycoside 糖转移酶简介糖转移酶简介功能：催化糖苷键的合成（功能：催化糖苷键的合成（O-,N-,S-,or C-glycoside）供体：活化的糖磷酸供体：活化的糖磷酸受体：亲核基团（蛋白、脂、核酸
2、、糖、小分子），通常为受体：亲核基团（蛋白、脂、核酸、糖、小分子），通常为-OHNon-Leloir donors：Leloir donors：糖供体底物糖供体底物糖基转移酶糖基转移酶根据根据糖供体糖供体中是否含有中是否含有核苷酸核苷酸分为分为两类两类。Leloir:阿根廷生物化学家，研究核苷酸代谢，1970，诺贝尔化学奖l 分为94 家族（distinct sequence-based families）(CAZy server,http:/rs-mrs.fr/CAZY)l 人类拥有约 270多种糖基转移酶序列，属于 33个家族。l Alpha-1,4-葡萄糖转移酶；beta-1,4-半乳糖
3、转移酶；2,3-唾液酸转移酶糖基转移酶分类糖基转移酶分类根据氨基酸序列相似性进行分类：根据氨基酸序列相似性进行分类：根据蛋白结构相似性进行分类：根据蛋白结构相似性进行分类：根据糖供体和糖苷键连接方式进行分类：根据糖供体和糖苷键连接方式进行分类：l GT-A，GT-B，其他类型Rossmann-type domains(for nucleotide binding)NDP-binding domain generally contains a conserved DXD amino acid motifGT-A fold:SpsA from Bacillus subtilusGT-B fold:
4、beta-glucosyltransferase from bacteriophage T4Leloir GTsTransglycosylase from Staphylococcus aureusOligosaccharyltransferase STT3 from Pyrococcus furiosiusNon-Leloir GTs催化机理催化机理 Glycosyltransferases catalyze the transfer of glycosyl groups to a nucleophilic acceptor with either retention or inversio
5、n of configuration at the anomeric centre.This allows the classification of glycosyltransferases as either retaining or inverting enzymes.鎓 催化机理催化机理Inverting:SN2 nucleophilic attack at the C1 atomRetaining:double displacement mechanism 糖基转移酶辅因子糖基转移酶辅因子l Many,but not all,glycosyltransferases utilize
6、divalent metal ion cofactors such as Mn2+and Mg2+.mainly in glycosyltransferases that are diphosphonucleoside-dependent.l metal ion is coordinated to an oxygen of each of the two phosphate groups,as well as to side-chain carboxylates derived from the protein.糖基转移酶抑制剂糖基转移酶抑制剂 直接抑制糖基转移酶活性直接抑制糖基转移酶活性底物
7、类似物；过渡态类似物alpha-2,6-唾液酸转移酶抑制剂Beta-1,4-半乳糖转移酶抑制剂 阻断糖供体的合成阻断糖供体的合成N-Glycan 合成过程中，首先要合成：dolichol-pp-N-acetylglucosamineUDP-GlcANc+dolichol-p-dolichol-pp-GlcNAcN-acetylglucosamine phosphorotransferase糖基转移酶在合成中的应用糖基转移酶在合成中的应用 寡糖的酶法合成寡糖的酶法合成 具有生物活性含糖天然产物的酶法合成具有生物活性含糖天然产物的酶法合成 生物制药生物制药-糖蛋白药物生产策略糖蛋白药物生产策略 生
8、物制药生物制药-糖疫苗生产策略糖疫苗生产策略OAcOAcOOAcOAcOOAcOAcOOAcOAcOHOOAcOAcOOAcOAcOOAcN3OOAcOOAcOAcOOAcOAcOOAcN3OBnOBnOBnOOBnOOHOOHOHOOHOHOOHNHAcOHOHOHOOH2679N33 steps6 stepsglycosylation4 stepsChemical Synthesis of a-Gal规模小，过程复杂，立体选择性难规模小，过程复杂，立体选择性难OOHHOHOHOO P O PONNHOOOOHHOHOHOOOHHONHAcOOROOHHOHOHOOOHHOOOHOOHHON
9、HAcOOROOOO-O-HO P O PONNHOOOOOO-O-OHHOOHHOGal1,4GlcNAc-R,acceptor-Gal epitopeUDPUDP-Gal Donor1,3-galactosyltransferaseMn2+Reaction Catalyzed by 1,3-Galactosyltransferase碳水化合物的合成碳水化合物的合成 Organic Synthesis Biosynthetic process Derivative synthesis Versatile Not very flexible Process Expensive and mult
10、istep Economic and short Environment problem Chemical waste Environment-friendly Carbohydrate polymer synthesis Not applicable Practical approach 体内糖供体合成途径体内糖供体合成途径 Glc,GlcNAc&ManSugar-6-pSugar-1-pSugar-NDPkinaseMutasepyrophorylase Gal,GalNAc&FucSugar-1-pSugar-NDPkinasepyrophorylase Neu5AcNeu5AcCMP-
11、Neu5AcpyrophorylaseCTP 糖供体相互转化:GalE:UDP-Gal UDP-Glu GalNAcE:UDP-GalNAc UDP-GlcNAc UGD(UDP-Glc dehydrogenase)：UDP-Glc UDP-GlcA UDP-GlcA decarboxylase:UDP-GlcA UDP-Xyl糖基转移酶介导的寡糖合成糖基转移酶介导的寡糖合成 PhosphorylasesGlucosyltransferasesFructosyltransferasesCyclodextrin glucanotransferasesMany微生物中的糖供体微生物中的糖供体天然糖
12、苷化合物中的糖一般为 C2,C3,C4 and/or C6 脱氧糖，而且大部分为6-deoxyhexoses family（Rhamnose）原核生物糖转移酶具有糖供体糖供体的广泛性 6-deoxyhexoses 一般通过 TDP-sugars进行底物活化（TDP-Rhamnose）优点优点:*区域、立体选择性*大量生产不足不足:*糖基转移酶在大量表达方面存在困难 糖供体比较昂贵糖供体比较昂贵.副产物抑制(i)消除副产物抑制（碱性磷酸酶）消除副产物抑制（碱性磷酸酶）(ii)原位产生糖供体原位产生糖供体.针对以上不足解决策略针对以上不足解决策略糖基转移酶在工业应用中优势与弱点糖基转移酶在工业应用
13、中优势与弱点可作为LgtC底物 碱性磷酸酶酶法再生糖供体策略酶法再生糖供体策略 UDP-sugar (A)and CMP-sugar(B)糖供体的合成糖供体的合成糖供体的合成糖供体的合成糖供体的合成糖供体的合成糖基转移酶在糖基转移酶在寡糖合成中的应用寡糖合成中的应用（一）（一）具有生物活性寡糖的酶法合成具有生物活性寡糖的酶法合成(for details visit:wang.chem.wayne.edu)OHOOHHOHOOHOOHOHOOOOHHONHACOHOOHOHOOOOHHOHOResearch on Carbohydrates in Human Immunity in Wangs
14、LaboratoryOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBlood type B antigenType B bloodType A bloodAnti-B antibody Barrier in blood transfusion Barrier in organ transplantation Cause of hyperacute rejection in animal to human xenotransplantation Human natural immunity against pathogens,tumors,etc.Non-p
15、rimate mammals(pig,mouse,dog etc.)New world monkeyHumanApeOld world monkeyAnti-Gal antibody(the most abundant natural Ab)1-2%total IgG,3-8%total IgM-Gal epitopeabundant,1 million/cellHuman-HumanHuman-AnimalOHOOHOHOHOOHHOHOOHOOHOHOOOOHHONHACOHOOHOHOOOOHHOHOThe specificity of glycosyltransferases dete
16、rmine the sugar sequenceOHOOHHOHOOHOOHOOOOOHHONHACOHOOHOHOOOOHHOHOBlood type B antigenOHOOHOH-Gal antigenOOHOHHOOOOOHHONHACOHOOHOHOOOOHHOHOOHOOHOHOOHOHHOHOOOOHHONHACOHOOHOHOOOOHHOHOhuman 1,3GalTpig 1,3GalTAnti-Gal productionmillion years ago 5-15 20-30 30-40 60-70 70-80120-130mammals-gal epitopesynt
17、hesishumansapesmonkeysOld WorldNew WorldmonkeysprosimiansmarsupialsnonprimateplacentalmammalsInactivation of 1,3 GTBiomedical use of-Galu soluble-Gal antagonist to anti-Galu -Gal conjugates in immunotherapy against bacteria,virus,and cancer cells.人工合成的必要性人工合成的必要性OOHHOHOHOO P O PONNHOOOOHHOHOHOOOHHOO
18、HOOROOHHOHOHOOOHHOOOHOOHHOOHOOROOOO-O-HO P O PONNHOOOOOO-O-OHHOOHHOReaction Catalyzed by 1,3-GalactosyltransferaseGal1,4Glc-R,acceptor-Gal epitopeUDPUDP-Gal Donor1,3-galactosyltransferaseMn2+1,3GalT 催化合成-Gal Epitopes 及其衍生物HOOOHHOHOOUDPHOOOHHOHOOUDPOHOOHHOOOOHHOR1R2HOOHOOHHOHOOHOOHOHOOOOHHOR1R2UDP-Ga
19、l4-epimeraseUDP 1,3GalTR1R2OHOHOHOHNHAcN3SPhOAllylabcdNHAcOHeNH2OHfUDP-Gal100mg/$416 UDP-Glc5g/$529.5EC 5.1.3.2Fang J,Chen X,Wang PG,et al.:J.Org.Chem.1999,64,4089-4094.OOHOHOHOOOHOHHON3OHOOOHNHAcHOOOHOHOHOOOHOHHON3OOOOHNHAcHOOOH OHOHOOOH OHOHHOOOH OHOHOOOHOHHON3OOOOHNHAcHOOOH OHOHHOHOOOHHOHOOUDPHOO
20、OHHOHOOPO3=OPO3H-COOHOCOOH2PiUTPUDP-Gal4-epimeraseUDP1,4 galactosyltransferase1,3 galactosyltransferasePPiUDP-GlcUDP-GlcPyrophospho-rylasePyruvateKinaseInorganicPyrophos-phatase通过原位再生合成 a-Gal 五糖Fang J,Li J,Chen X,Wang PG,et al:J.Am.Chem.Soc.1998,120,6635-6638.其他生物活性寡糖的合成（应用）其他生物活性寡糖的合成（应用）In Human m
21、ilkOOHOHOOHOOHOHOOHOHHOOOHOOOHOHOHOOH1,4GalTlgtC gene(a)globotriose Gb3OHOHOAcHNOHOOHOHOOHOOHOHOOHOHOHOOHOOH1,3GalNAcTlgtD gene(b)globotetrose Gb4OHOOHOOHOOHOHOOHOHHOOHOAcHNOH(c)LNT-21,3GlcNAcTlgtA geneOHOOHOOHOOHOHOOHOHOOHOAcHNOH(d)LNnT:Lacto-N-neotetraose1,4GalTlgtB geneHOOHOHOOHOHOOHOOHOOHOHOOHOH
22、OHOAcHNOH2,3transialidase(e)LSTDOHOAcHNHOOHHOHO2COH(Without the B subunits,the A subunit has no way of attaching to or entering the cell,and thus no way to exert its toxic effect.)Stx is an AB5 subunit toxin.The pentamer of(small)B subunits binding to its receptor glycosphingolipid(GSL),globotriaosy
23、l ceramide(Gb3)in glomerular endothelial cell membranes,initiates A subunit-mediated cell death leading to HUS（hemolytic uremic syndrome）,but induction of inflammatory pathways is also key.Gb3 is heterogenous in its lipid structure and membrane organization,such that different Gb3 formats are differ
24、entially recognized by Stx family members,particularly Stx2,which is more frequently associated with clinical disease.血型相关抗原酶法合成血型相关抗原酶法合成Fluorescently labeled sLex conj sLex在转移性结肠癌高表达转移性结肠癌高表达二价肿瘤抗二价肿瘤抗原酶法合成原酶法合成（用于检测肿瘤位置）（用于检测肿瘤位置）糖基转移酶应用乳腺癌、前列腺癌上高表达Globo-HP-凝集素凝集素 配体配体防治黑色素瘤防治黑色素瘤l 寡糖的合成常常需要多种糖基转
25、移酶寡糖的合成常常需要多种糖基转移酶l 为了增加产量还有用其他一些酶为了增加产量还有用其他一些酶l 分步合成费时费力分步合成费时费力？一锅多酶法一锅多酶法-Super beads-Super bugJ.Am.Chem.Soc.1995,117,5869-5870一锅多酶法合成透明质酸（hyaluronic acid）E1,hyaluronic acid synthase;E2,UDP-Glc dehydrogenase;E3,UDP-Glc pyrophosphorylase;E4,UDP-GlcNAc pyrophosphorylase;E5,pyruvate kinase;E6,lacta
26、te dehydrogenase;E7,inorganic pyrophosphatase OHOHOHOOHOOHOHOOROHOHOOHOOOOHOOROHOHOHOAcHNHOOHHOHO2CCO2HOHOAcHNHOOHHOOCMPPPiPEPE3HOOHOOHOHOHE4E5HOOHOOHOHOPHOOHOOHOGDPOHGDPPyruvateGTPHOOHOOHOPOHOGDPOOHHOCH3OHNADPH/NADHNADP/NADGlcAGlcE1E2E6E7E8OOHHOCH3OHOHOHOHOOOOHOOROHOHOOHHOCH3OHCMPSialyl Lewis X ant
27、igenLewis X antigensialyl Lewis X 酶法大量合成酶法大量合成E1:1,3-fucosyltransferase；E2:pyruvate kinase；E3:GDP-mannose pyrophosphorylaseE4:GDP-4-keto-5-deoxymannose 3,5-epimerase/GDP-4-keto-6-galactose reductase；E5:glucose dehydrogenase；E6:hexokinase；E7:phosphomannomutase；E8:a2,3-sialyltransferase激酶激酶歧化酶歧化酶Super
28、beadGalUGalTGalKPyKFRecombinant E.coli stratins overexpressingGalK,GalT,GalU or PykF1)Fermentation2)Lysis3)Add to Ni2+resinChen X,Fang J,Wang PG,et al.:J.Am.Chem.Soc.2001,123,2081-2082.Beads with:GalKGalTGalUPykFPeristaltic pumpfor circulationReservoir with:1,3GalTLacOBn 9.6 mMATP 0.96 mMPEP 19.2 mM
29、UDP 0.96 mMGlc-1-P 0.96 mMGal 12 mM MgCl2 10 mMMnCl2 10 mMKCl 100 mM HEPES 100 mM pH 7.5Liu Z,Zhang J,Chen X,Wang PG:ChemBioChem 2002,3,348-355.Production of UDP-Gal with Superbeads.Superbead 用于寡糖合成OHOOHOHOOOOHHONHAcOHOHOOHHOHOOHOOHOHOOHOOHHOHOOMeOOOHHOHOOHOOHOHOOHOOHHOOBnOOOHHOHOOHOOHHOHOOOOHHONHAc
30、OHOOOHHOHOOHOOHHOHOOBnOOOHHOHOOHOOHOHOOOOHHONHAcOOHOOHHOHOOHOOHHOOOOHHOOHN3OHOOHOHOOHOOHHOHOOBnOOOHHOHO1,3GalT85%(a72%)83%69%92%95%1,4GalT1,4GalT86%76%1,3GalT+1,4GalTa Gram scale synthesis,others are 100 mg scales从多菌种发酵到从多菌种发酵到Super bugUMPUDPUTPGlobotioseUDPUDP-GalOrotic AcidUTPPPi2PiGlc-1-PUDP-GlcU
31、DP-GalGal-1-PGalactose(100 g/L)GalKGalTGalUGlucoseFructoseC.ammoniagenes DN510E.coli NM522/pNT25/pNT32Globotriose(Gal1,4Lac)188 g/L,36 hr.LgtCE.coli NM522/pGT5PpaLactose(100 g/L)Kyowa Hakkos technology for large-scale production of UDP-Gal and globotriose utilizing metabolically engineered bacterial
32、 cells OOHOHOOHOOHOHOOHOHOHOHOHOOHglobotriose Gb3Ppa:pyrophosphataseGalU:glucose-1-phosphate uridylyltransferaseGalT:galactose-1-phosphate uridylyltransferaseGalK:galactokinaseLgtC:1,4-galactosyltransferaseCMP-NeuAc regeneration system by bacterial coupling Using this approach,sugar nucleotides incl
33、uding UDP-Gal,CMP-Neu5Ac,UDP-GlcNAc and GDP-Fuc have been successfully produced on a large scale.Superbug technologies for the synthesis of -GalBacteriaNutrientsStartingMaterialsBio-synthetic pathway engineeringa-Gal分析合成途径分析合成途径ATPADPGalK:galactokinaseGalUT:galactose-1-phosphate uridylytransferaseGa
34、lU:glucose-1-phosphate uridylyltransferasePykF:pyruvate kinase1,3GalT:1,3-galactosyltransferase1,3GalT Gal1,3LacL actoseUDP-Gal UDPPEPPyruvateGal-1-PGlc-1-PUDP-GlcUTPPykFGalUGalKGalUTGalactosePPi克隆表达验证相关酶克隆表达验证相关酶 Clone and express individual enzymes in the biosynthetic pathway of-Gal（克隆相关酶）Construc
35、tion an artificial biosynthetic gene cluster and transfer into E.coli host cell.（构建基因簇并转化）Large scale production of-Gal oligosaccharides using fermented and permeated cells.（发酵生产）EnzymeAmount(U/L)1,3GalTGalKGalTGalUPykF 6025100100501 U=1 mol/min,37oCpLDR20-KTUFgalK+galT1,3GalTgalUpykFT7 terminatorl
36、l PRpLDR20-KTUFAmprEcoR VSac IISal IXba ICla I 9 kbl cI-repressorpMB1rbcrbcrbcrbc构建多基因表达质粒构建多基因表达质粒检验多基因表达效果检验多基因表达效果66,20031,00021,50045,000LysateMIBMPure14,50097,400PykFGalKGalTGalU1,3GalTSuperbug 整个生产过程 Whole Cell ReactionAnalyzed by TLC,HPLCRt,20-36h Stop by inserting in boiling waterCentrifugat
37、ionGel filtration P21H NMR,13C NMR Analysis Culture NM522/pLDR20-KTUF CentrifugationOverexpressionResuspended inTris-HCl BufferE.coli N M 522pL D R 20-K T U F-G al7.2 g Gal1.1 g ATP8.4 g Lac3.6 g GlcTotal volume:1 L 0.3 g UDP-Glc7.2 gChen X,Liu Z,Zhang W,Fang J,Andreana P,Wang PG:ChemBioChem.2002,3,
38、47-53.大规模发酵产量大规模发酵产量Superbug-11,3GalTGal1,3Gal1,4GlcGal1,4GlcUDP-GalUDPPEPPyruvateGal-1-PGlc-1-PUDP-GlcUTPPykFGalUGalKGalTGalactoseLactoseGlycolyticpathwayGlucoseATPADPGlucosePyruvatePykFPEPGlycolytic pathwayPPiNM522添加三种三种原料：glucose,galactose,lactosePnPn-11,3GalTGal1,3Gal1,4GlcGal1,4GlcUDP-GalUDPGal
39、-1-PGlc-1-PUDP-GlcUTPGalUPpKGalTGalactoseLactoseGalactoseNM522PnPnPn-1PpKPnPPiPpK:polyphosphate kinaseSuperbug-2添加三种三种原料：多聚磷酸,galactose,lactoseHOOOHHOHOOUDPHOOOHHOHOOUDPOHOOHHOOOOHHOHOHOOHOOHHOHOOHOOHOHOOOOHHOHOOROR1,3GalTUDPSucroseFructose(energy source)SusAGalESucroseLacORE.coliChen X,Zhang J,Kowa
40、l P,Andreana P,Wang PG:J.Am.Chem.Soc.2001,123,8866-8867.SusA:sucrose synthaseGalE:UDP-Gal 4-epimerase添加两种两种原料：sucrose,lactoseSuperbug-3每次进步只有一每次进步只有一点点，积累多了点点，积累多了才会实现自己的才会实现自己的目标。目标。二二具有生物活性的含糖天然产物及其酶法合成具有生物活性的含糖天然产物及其酶法合成含糖天然产物含糖天然产物含糖天然产物含糖天然产物诺加霉素阿克拉霉素(安乐霉素)；蒽环类抗癌药，它能嵌入癌细胞的DNA上，抑制核酸的合成，特别是RNA的合成
41、，为周期非特异性药物，在G1晚期和S晚期阻断细胞周期。daunorubicin n.微道诺霉素；红比霉素；正定霉素；柔毛霉素（用作抗肿瘤药）含糖天然产物酶法合成含糖天然产物酶法合成The glycosyltransferase UrdGT2 establishes both C and O glycosidic bonds 天然抗生素竹桃霉素天然抗生素竹桃霉素(landomycin)抗菌性与红霉素类似抗菌性与红霉素类似含糖天然产物酶法合成含糖天然产物酶法合成含糖天然产物酶法合成含糖天然产物酶法合成含糖天然产物酶法合成含糖天然产物酶法合成OOHOHOObaicaleinOHOHOHOHOOOOH
42、OOOHOHluteolin 7-glucosideOHOHOHOOHOOOHOHHOOHOHOOHOOHOOHOHOHOOHquercetagetin-7-arabinosyl-galactosideOOOOHHOOHOHOMeOHOHHOquercetrinOOOHOHOOHOHOMeHOOOHOHOHOHOOHrutin 抗菌黄酮苷化合物抗菌黄酮苷化合物 OOHHOOOHOHOquercetin-3-galactosideOHOHOHOOHOOHHOOOHOHOHOmyricetin-3-O-galactosideOHOHOHOOHOOHHOOOHOHOHOmyricetin-3-O-g
43、lucosideOOHHOOHOH抗肿瘤黄酮苷化合物抗肿瘤黄酮苷化合物 OOHHOOOHOkaempferol 3-o-glucosideOOHHOOHOHOOHOOOHOOHOH3CHOOHHOHOOHOapigenin 7-O-rutinosideOOHHOOOHOHOHOmyricetin-3-galactosideOHOHOHOOHOOHHOOOHOHOHOOHOHOHOmyricetin-3-arabinosideOOHHOOOHOHOquercetin 3-galactosideOHOHOHOOHOOHHOOOHOHOquercetin 3-xylosideOOHHOOHOHHOO
44、+OHOHOcyanidin 3-galactosideOHOHOHOOH降血脂黄酮苷化合物降血脂黄酮苷化合物 研究表明，对于某些黄酮药物，糖的存在与否会对药糖的存在与否会对药效影响很大效影响很大。例如：diosmin，gossipyn和rutin对于中枢神经系统有明显的镇静作用，但相应的苷元没有类似作用。桔皮素（tangeratin）是一种非常有效的抗肿瘤细胞增生的药物，体外研究表明包括桔皮素在内的三种黄酮类药物它们的糖苷型比苷元型抗肿瘤活性更高。在对乳腺腺癌细胞抑制研究中，发现柚皮苷、芦丁、芹菜素、山奈酚、白杨素的抑制活性逐渐减弱，含有糖配体的糖苷类物质活性比其他的要高些。European
45、 journal of pharmacology,2006，539(3):168-176 A comparison of the structures among these compounds indicates the following elements are essential to the potential acceptors of UGT78D1:1)the 3-hydroxyl group,2)the double bond in the ring C,3)the 4-hydroxyl group.Guangxiang Ren,Lianwen Zhang,Glycoconj
46、J(2012)29:425432酶（酶（UGT78D1）的底物选择性）的底物选择性l 阿糖胞苷，急性白血病，糖基部分在抑制核酸代谢物与酶的结合抑制核酸代谢物与酶的结合反应中起重要作用，从而增强了其抗肿瘤效应。l 烯二炔类抗肿瘤抗生素通过糖基部分与通过糖基部分与DNA 结合结合，使药物分子嵌入DNA 双螺旋的小沟中(特别是TCCT位点)。l 蒽环类抗生素，环A 和氨基糖部分在决定药物与氨基糖部分在决定药物与DNA的选择性结合的选择性结合中起关键作用中起关键作用。该类药物与DNA作用形成复合物的稳定性主要决定于药物的糖环与DNA之间的作用。以以DNA为靶点的抗肿瘤药物为靶点的抗肿瘤药物糖配基具有协
47、助药物生物转运和特异性识别并结合协助药物生物转运和特异性识别并结合DNA 的作用的作用。l 含糖天然产物或药物大量存在l 糖分子在药物分子执行其功能时有时具有重要作用l 人工添加糖分子存在一定困难选择合适的药物分子或改造某种功能已知的糖苷药物，设计合成途径选择合适的药物分子或改造某种功能已知的糖苷药物，设计合成途径小结如下：小结如下：三：糖蛋白药物三：糖蛋白药物正在开发的蛋白质正在开发的蛋白质和抗体药物中大约和抗体药物中大约83%是糖蛋白。是糖蛋白。2000-2005(%)2005-2010(%)抗体抗体 35 25蛋白蛋白 15 12Small Molecules 8 8Revenue In
48、 Billions200020052010预计到预计到 2010年，年，糖蛋白药品市场可糖蛋白药品市场可达达680亿美元亿美元。国际生物药品市场占有率分析国际生物药品市场占有率分析 蛋白糖基化的作用Protein foldingProtein targeting/traffickingLigand recognition/bindingBiological activityStabilityRegulates protein half-lifeImmunogenicityglycocomponent of glycoproteins美国美国Amgen公司的公司的EPOGEN（EPO,红细胞生成
49、素）红细胞生成素）和和Aranesp（最新的红细胞生成素）：（最新的红细胞生成素）：EPOGEN，包含有，包含有165个氨基酸，其序列与天然的内个氨基酸，其序列与天然的内源性红细胞生成素相同，具有相等的生物活性。源性红细胞生成素相同，具有相等的生物活性。Aranesp是第二代红细胞生成蛋白，比是第二代红细胞生成蛋白，比EPOGEN多两多两条条N-糖链，包含了更多的唾液酸，其体内糖链，包含了更多的唾液酸，其体内半衰期比半衰期比EPOGEN长长3倍多倍多，从而显示出更强的生物活性。，从而显示出更强的生物活性。目前糖蛋白表达的缺点和不足非人源化的糖蛋白非人源化的糖蛋白：糖蛋白的快速清除，改变药代动力
50、学性质，补体激活，提高免疫原性和用药安全问题。现有人源化的糖蛋白现有人源化的糖蛋白：生产成本高，细胞容易污染，N-糖基化不足，糖基化程度不同（同一批次和不同批次）。Sethuraman N,Stadheim TA.Challenges in therapeutic glycoprotein production.Curr Opin Biotechnol.2006.17(4):341-6.l 掺入非天然氨基酸掺入非天然氨基酸l 特定化学反应引入糖基修饰特定化学反应引入糖基修饰l 细胞表达复杂糖修饰蛋白细胞表达复杂糖修饰蛋白 l 特定酶切（特定酶切（Endo-H）、酶连）、酶连（Endo-A/M）
