糖基转移酶与糖苷酶.ppt

单击此处编辑母版标题样式,单击此处编辑母版文本样式,第二级,第三级,第四级,第五级,*,第二节,糖基转移酶及其应用,主要内容,糖转移酶简介,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,功能：催化糖苷键的合成（,O-,N-,S-,or C-glycoside,）,供体：活化的糖磷酸,受体：亲核基团（蛋白、脂、核酸、糖、小分子），通常为,-OH,Non-Leloir donors,：,Leloir donors,：,糖供体底物,糖基转移酶,根据,糖供体中是否含有核苷酸,分为,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,分为,94,家族（,distinct sequence-based families,）,(,CAZy,server,rs-mrs.fr/CAZY,),人类拥有约,270,多种糖基转移酶序列，属于,33,个家族。,Alpha-1,4-,葡萄糖转移酶；,beta-1,4-,半乳糖转移酶；,2,3-,唾液酸转移酶,糖基转移酶分类,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：,根据糖供体和糖苷键连接方式进行分类：,GT-A,，,GT-B,，其他类型,Rossmann,-type domains(for nucleotide binding),NDP-binding domain generally contains a conserved DXD amino acid motif,GT-A fold:,SpsA,from Bacillus,subtilus,GT-B fold:beta-,glucosyltransferase,from,bacteriophage,T4,Leloir,GTs,Transglycosylase,from Staphylococcus,aureus,Oligosaccharyltransferase,STT3 from,Pyrococcus,furiosius,Non-Leloir,GTs,催化机理,Glycosyltransferases,catalyze the transfer of,glycosyl,groups,to a,nucleophilic,acceptor,with either retention or inversion of configuration at the,anomeric,centre.This allows the classification of,glycosyltransferases,as either,retaining,or,inverting,enzymes.,鎓,催化机理,Inverting:,SN2,nucleophilic,attack at the C1 atom,Retaining:,double displacement mechanism,糖基转移酶辅因子,Many,but not all,glycosyltransferases,utilize divalent metal ion cofactors such as,Mn2+and Mg2+.,mainly in,glycosyltransferases,that are,diphosphonucleoside,-dependent,.,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,直接抑制糖基转移酶活性,底物类似物；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,N-,Glycan,合成过程中，首先要合成：,dolichol-pp-N-acetylglucosamine,UDP-,GlcANc,+,dolichol-p,-,dolichol-pp-GlcNAc,N-,acetylglucosamine,phosphorotransferase,糖基转移酶在合成中的应用,寡糖的酶法合成,具有生物活性含糖天然产物的酶法合成,生物制药,-,糖蛋白药物生产策略,生物制药,-,糖疫苗生产策略,Chemical Synthesis of a-Gal,规模小，过程复杂，立体选择性难,Reaction Catalyzed by,a,1,3-Galactosyltransferase,碳水化合物的合成,体内糖供体合成途径,Glc,GlcNAc,&Man,Sugar-6-p,Sugar-1-p,Sugar-NDP,kinase,Mutase,pyrophorylase,Gal,GalNAc,&,Fuc,Sugar-1-p,Sugar-NDP,kinase,pyrophorylase,Neu5Ac,Neu5Ac,CMP-Neu5Ac,pyrophorylase,CTP,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,Phosphorylases,Glucosyltransferases,Fructosyltransferases,Cyclodextrin,glucanotransferases,Many,微生物中的糖供体,天然糖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,的广泛性,6-deoxyhexoses,一般通过,TDP-sugars,进行底物活化（,TDP-,Rhamnose,）,优点,:,区域、立体选择性,大量生产,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,.,副产物抑制,消除副产物抑制（碱性磷酸酶）,原位产生糖供体,合成廉价的糖供体类似物,.,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,可作为,LgtC,底物,碱性磷酸酶,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,糖基转移酶在,寡糖合成中的应用,（一）具有生物活性寡糖的酶法合成,Biomedical use of,a,-Gal,soluble,a,-Gal antagonist to anti-Gal,a,-Gal conjugates in immunotherapy against bacteria,virus,and cancer cells.,人工合成的必要性,a,1,3GalT,催化合成,a,-Gal,Epitopes,及其衍生物,H,O,O,O,H,H,O,H,O,O,U,D,P,H,O,O,O,H,H,O,H,O,O,U,D,P,O,H,O,O,H,H,O,O,O,O,H,H,O,R,1,R,2,H,O,O,H,O,O,H,H,O,H,O,O,H,O,O,H,O,H,O,O,O,O,H,H,O,R,1,R,2,UDP-Gal,4-epimerase,UDP,a,1,3GalT,R,1,R,2,OH,OH,OH,OH,NHAc,b,N,3,b,SPh,b,OAllyl,a,b,c,d,NHAc,OH,e,N,H,2,OH,f,UDP-Gal,100mg/$416,UDP-,Glc,5g/$529.5,EC 5.1.3.2,Fang J,Chen X,Wang PG,et al.:J.Org.Chem.1999,64,4089-4094.,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）,In Human milk,O,O,H,O,H,O,O,H,O,O,H,O,H,O,O,H,O,H,H,O,O,O,H,O,O,O,H,O,H,O,H,O,O,H,a,1,4,G,a,l,T,l,g,t,C,g,e,n,e,(,a,),g,l,o,b,o,t,r,i,o,s,e,G,b,3,O,H,O,H,O,A,c,H,N,O,H,O,O,H,O,H,O,O,H,O,O,H,O,H,O,O,H,O,H,O,H,O,O,H,O,O,H,b,1,3,G,a,l,N,A,c,T,l,g,t,D,g,e,n,e,(,b,),g,l,o,b,o,t,e,t,r,o,s,e,G,b,4,O,H,O,O,H,O,O,H,O,O,H,O,H,O,O,H,O,H,H,O,O,H,O,A,c,H,N,O,H,(,c,),L,N,T,-,2,b,1,3,G,l,c,N,A,c,T,l,g,t,A,g,e,n,e,O,H,O,O,H,O,O,H,O,O,H,O,H,O,O,H,O,H,O,O,H,O,A,c,H,N,O,H,(,d,),L,N,n,T,:,L,a,c,t,o,-,N,-,n,e,o,t,e,t,r,a,o,s,e,b,1,4,G,a,l,T,l,g,t,B,g,e,n,e,H,O,O,H,O,H,O,O,H,O,H,O,O,H,O,O,H,O,O,H,O,H,O,O,H,O,H,O,H,O,A,c,H,N,O,H,a,2,3,t,r,a,n,s,i,a,l,i,d,a,s,e,(,e,),L,S,T,D,O,H,O,A,c,H,N,H,O,O,H,H,O,H,O,2,C,O,H,(,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,),globotriaosyl,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 differentially 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,Fluorescently labeled,sLex,conj,sLex,在,转移性结肠癌高表达,二价肿瘤抗原酶法合成,（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,-H,P-,凝集素 配体,防治黑色素瘤,寡糖的合成常常需要多种糖基转移酶,为了增加产量还有用其他一些酶,分步合成费时费力,？,一锅多酶法,-Super beads-Super bug,J.Am.Chem.Soc.1995,117,5869-5870,一锅多酶法合成透明质酸（,hyaluronic,acid,）,E,1,hyaluronic,acid,synthase,;,E,2,UDP-,Glc,dehydrogenase,;,E,3,UDP-,Glc,pyrophosphorylase,;,E,4,UDP-,GlcNAc,pyrophosphorylase,;,E,5,pyruvate,kinase,;,E,6,lactate,dehydrogenase,;,E,7,inorganic,pyrophosphatase,sialyl,Lewis X,酶法大量合成,E1:,a,1,3-fucosyltransferase,；,E2:,pyruvate,kinase,；,E3:GDP-mannose,pyrophosphorylase,E4: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,歧化酶,Superbead,GalU,GalT,GalK,PyKF,Recombinant,E.coli,stratins,overexpressing,GalK,GalT,GalU,or,PykF,1)Fermentation,2),Lysis,3)Add to Ni,2+,resin,Chen X,Fang J,Wang PG,et al.:J.Am.Chem.Soc.2001,123,2081-2082.,Beads with:,GalK,GalT,GalU,PykF,Peristaltic pump,for circulation,Reservoir with:,a,1,3GalT,LacOBn,9.6,mM,ATP 0.96,mM,PEP 19.2,mM,UDP 0.96,mM,Glc-1-P 0.96,mM,Gal 12,mM,MgCl,2,10,mM,MnCl,2,10,mM,KCl,100,mM,HEPES 100,mM,pH 7.5,Liu 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,a,Gram scale synthesis,others are 100 mg scales,从多菌种发酵到,Super bug,Kyowa Hakkos technology for large-scale production of UDP-Gal and,globotriose,utilizing metabolically engineered bacterial cells,Ppa,:,pyrophosphatase,GalU,:glucose-1-phosphate,uridylyltransferase,GalT,:galactose-1-phosphate,uridylyltransferase,GalK,:,galactokinase,LgtC,:,a,1,4-galactosyltransferase,CMP-,NeuAc,regeneration system by bacterial coupling,Using this approach,sugar nucleotides including,UDP-Gal,CMP-Neu5Ac,UDP-,GlcNAc,and GDP-,Fuc,have been successfully produced on a large scale.,Superbug technologies for the synthesis of,a,-Gal,Bacteria,N,u,t,r,i,e,n,t,s,S,t,a,r,t,i,n,g,M,a,t,e,r,i,a,l,s,B,i,o,-,s,y,n,t,h,e,t,i,c,p,a,t,h,w,a,y,e,n,g,i,n,e,e,r,i,n,g,a-Gal,分析合成途径,ATP,ADP,G,a,l,K,:,g,a,l,a,c,t,o,k,i,n,a,s,e,GalUT,:galactose-1-phosphate,uridylytransferase,G,a,l,U,:,g,l,u,c,o,s,e,-,1,-,p,h,o,s,p,h,a,t,e,u,r,i,d,y,l,y,l,t,r,a,n,s,f,e,r,a,s,e,P,y,k,F,:,p,y,r,u,v,a,t,e,k,i,n,a,s,e,a,1,3,G,a,l,T,:,a,1,3,-,g,a,l,a,c,t,o,s,y,l,t,r,a,n,s,f,e,r,a,s,e,a,1,3GalT,Gal,a,1,3Lac,L,a,c,t,o,s,e,UDP-Gal,UDP,PEP,Pyruvate,Gal-1-P,Glc-1-P,UDP-,Glc,UTP,PykF,GalU,GalK,GalUT,Galactose,PPi,克隆表达验证相关酶,Clone and express individual enzymes in the biosynthetic pathway of,a,-Gal,（克隆相关酶）,Construction an artificial biosynthetic gene cluster and transfer into,E.coli,host cell.,（构建基因簇并转化）,Large scale production of,a,-Gal oligosaccharides using fermented and permeated cells.,（发酵生产）,pLDR20-,a,KTUF,galK+galT,a,1,3GalT,galU,pykF,T7 terminator,l,PR,pLDR20-,a,KTUF,Amp,r,E,c,o,R,V,S,a,c,I,I,S,a,l,I,X,b,a,I,C,l,a,I,9 kb,l,cI-repressor,pMB1,rbc,rbc,rbc,rbc,构建多基因表达质粒,检验多基因表达效果,6,6,2,0,0,3,1,0,0,0,2,1,5,0,0,4,5,0,0,0,L,y,s,a,t,e,M,I,B,M,P,u,r,e,1,4,5,0,0,9,7,4,0,0,PykF,GalK,GalT,GalU,a,1,3GalT,Superbug,整个生产过程,Whole Cell Reaction,Analyzed by TLC,HPLC,Rt,20-36h,Stop by inserting in boiling water,Centrifugation,Gel filtration P2,1,H NMR,13,C NMR Analysis,Culture,NM522/pLDR20-,a,KTUF,Centrifugation,Overexpression,Resuspended,in,Tris-HCl,Buffer,E,.,c,o,l,i,N,M,5,2,2,p,L,D,R,2,0,-,a,K,T,U,F,a,-,G,a,l,7.2 g Gal,1.1 g ATP,8.4 g Lac,3.6 g Glc,Total volume:1 L,0.3 g UDP-Glc,7.2 g,Chen X,Liu Z,Zhang W,Fang J,Andreana,P,Wang PG:,ChemBioChem,.2002,3,47-53.,大规模发酵产量,Superbug-1,a,1,3GalT,Gal,a,1,3Gal,b,1,4Glc,Gal,b,1,4Glc,UDP-Gal,UDP,PEP,Pyruvate,Gal-1-P,Glc-1-P,UDP-,Glc,UTP,PykF,GalU,GalK,GalT,Galactose,Lactose,Glycolytic,pathway,Glucose,ATP,ADP,Glucose,Pyruvate,PykF,PEP,Glycolytic,pathway,PPi,NM522,添加,三种,原料：,glucose,galactose,lactose,PpK,:polyphosphate,kinase,Superbug-2,添加,三种,原料：多聚磷酸,galactose,lactose,Chen X,Zhang J,Kowal,P,Andreana,P,Wang PG:J.Am.Chem.Soc.2001,123,8866-8867.,SusA,:sucrose,synthase,GalE,:UDP-Gal 4-epimerase,添加,两种,原料：,sucrose,lactose,Superbug-3,每次进步只有一点点，积累多了才会实现自己的目标。,二,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,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，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,The,glycosyltransferase,UrdGT2,establishes,both C and O,glycosidic,bonds,天然抗生素竹桃霉素,(,landomycin,),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,抗菌黄酮苷化合物,抗肿瘤黄酮苷化合物,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 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,J(2012)29:425432,酶（,UGT78D1,）的底物选择性,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,反应中起重要作用，从而增强了其抗肿瘤效应。,烯二炔类抗肿瘤抗生素,通过糖基部分与,DNA,结合,，使药物分子嵌入,DNA,双螺旋的小沟中,(,特别是,TCCT,位点,),。,蒽环类抗生素，环,A,和,氨基糖部分在决定药物与,DNA,的选择性结合中起关键作用,。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,DNA,为靶点的抗肿瘤药物,糖配基具有,协助药物生物转运和特异性识别并结合,DNA,的作用,。,含糖天然产物或药物大量存在,糖分子在药物分子执行其功能时有时具有重要作用,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,小结如下：,三：糖蛋白药物,正在开发的蛋白质和抗体药物中大约,83%,是糖蛋白。,2000-2005(%)2005-2010(%),抗体,35 25,蛋白,15 12,Small Molecules 8 8,糖蛋白市场发展速度,Revenue In Billions,2000,2005,2010,预期发展速度,预计到,2010,年，糖蛋白药品市场可达,680,亿美元,。,国际生物药品市场占有率分析,蛋白糖基化的作用,Protein folding,Protein targeting/trafficking,Ligand,recognition/binding,Biological activity,Stability,Regulates protein half-life,Immunogenicity,glycocomponent,of,glycoproteins,美国,Amgen,公司的,EPOGEN,（,EPO,红细胞生成素）和,Aranesp,（,最新的红细胞生成素）：,EPOGEN,，,包含有,165,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。,Aranesp,是第二代红细胞生成蛋白，比,EPOGEN,多两条,N-,糖链，包含了更多的唾液酸，其体内,半衰期比,EPOGEN,长,3,倍多,，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,：糖蛋白的快速清除，改变药代动力学性质，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,：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.,掺入非天然氨基酸,特定化学反应引入糖基修饰,细胞表达复杂糖修饰蛋白,特定酶切（,Endo-H,）、酶连（,Endo-A/M,）引入均一糖链,四：糖疫苗,病原微生物耐药性逐渐变强，需要更安全有效的防治方法,多种微生物表面成分可作为疫苗,In,principle,various types of cell surface,epitopes,characteristic,for the invading organism or related to aberrant growth of cells,can be applied to develop vaccines.,糖合成技术的出现为糖疫苗的研究应用提供了可能,The progress in establishing the structure of carbohydrate,immuno,-determinants in conjunction with improvements in carbohydrate synthesis has rendered it feasible to develop new generations of carbohydrate-based vaccines.,部分已知糖蛋白抗原,糖疫苗结构,Carrier protein,:,KLH(key hole Limpet,hemocyanin,),DT(diphtheria,toxoids,，,CRM197),TT(tetanus,Toxoids,),antigen,protein,Glycan,(,起免疫作用的只是糖链的一部分；,oligosaccharides-,有机合成或多糖降解获得,),Coupling,Bifunctional,spacer molecules(amino,carboxyl or,thiol,groups),Glycoconjugate,vaccines against bacterial infections,基于细菌荚膜多糖上的片段，如下列病原微生物：,研究内容：糖链长度与疫苗功能；糖链数目与免疫功能,Polysaccharide type-3 related,di,-,tri-and,tetrasaccharride,-,CRM197,conjugates:S.,pneumoniae,type 3 infection,oligosaccharide/,proteinratios,肺炎球菌,奈瑟氏脑膜炎球菌,流感嗜血杆菌,伤寒沙门氏菌,痢疾志贺氏菌,B,型链球菌,肺炎杆菌,偶联多条糖链更有效,病原微生物糖疫苗,抗寄生虫疫苗制备更复杂,.,Malaria(,疟疾,),toxoplasmosis,（弓形体病）,and,leishmanias,（利什曼虫）,.Malaria caused by,Plasmodiumfalciparum,（疟原虫）,靶点：对应前两者为,glycosylphosphatidylinositol,anchors(GPIs,),偶联于,KLH,获得疫苗，对疟疾有很好的防治效果。,肿瘤糖疫苗,至今,FDA,只批准了一个预防肝炎病毒的疫苗,糖相关肿瘤抗原,Muc-1,NER-2/neu,CEA,P53,STn,Globo,H,糖苷酶及其应用,Glycoside,hydrolases,(,Glycosidases,),Transglycosylases,Lyases,phosphorylases,糖链 或 糖缀合物,糖苷键天然水解时，半衰期为,470,万年；,糖苷酶可以提高催化效率,10,的,17,次方倍。,糖苷键切割相关酶,糖苷键底物及效率,糖苷酶分类,按水解机理：,保持型,&,翻转型,按糖苷键位置：,内切酶,，外切酶（,非还原端外切酶,，还原端外切酶）,包括近,13,万条糖苷酶序列，分属,130,个家族,(sequence-based families,rs-mrs.fr/CAZY,),超过,30,种糖苷酶的晶体结构已经解析,Transglycosidases,(,transglycosylases,):,在寡糖和多糖之间进行糖基化反应。,根据序列进行分类,（,EC 3.2.1.-,）,某些保持型糖苷酶（唾液酸转移酶），活性中心可以包含一个酪氨酸,某些保持型糖苷酶的酶促反应中，有邻基参与过程,Myrosinases,(,芥子苷酶,),Lyases,(,裂解酶,),通过消去反应的机制（,eg,:a-,glucan,lyases,）,Phosphorylase,&,Transglycosidases,A-R+H3PO3-A-H2PO3+RH,“,磷解”糖苷键,产物：,sugar-1-P(retention or inversion),通常为外切酶（如,glycogen,phosphorylase,),）,经过糖苷,-,酶过渡态，糖的羟基亲和进攻合成新的糖苷键,“,糖解”糖苷酶（环糊精合酶）,Transglycosidases,Phosphorylase,水解机理,都要经历鎓离子过渡态；而且活性中心包含了两个酸性氨基酸,（一般为谷氨酸和天冬氨酸）,Inverting glycosidase:A single displacement mechanism,Retaining glycosidase:A double displacement mechanism,亲和标记试剂：用于研究特定的活性位点氨基酸残基。,2-,脱氧,-2-,氟糖；,5-,氟糖。,机制：通过鎓离子过渡态去稳定化实现。,Mechanism-based,time-dependent inhibitors of retaining glycoside,hydrolases,.,糖苷酶研究工具,Time-dependent covalent,inactivators,of glycoside,hydrolases,bicyclic,inactivators,of glycoside,hydrolases,Non-covalent glycoside,hydrolase,inhibitors,Nitrogen-containing sugar-shaped,heterocycles,.,Having greater activity towards inverting,glucoside,hydrolases,rather than retaining ones,(,不可水解的底物类似物,),（杂环糖类似物）,糖苷酶在合成中的应用,可逆反应为其应用于合成提供了理论基础,热动力学控制（,hydrolysis is exothermic process,）,自身转糖基反应,反应产物与反应温度有关；,缺点：六位碳羟基产生的副反应,保持型,/,翻转型？,动力学控制（,只适用于应用活化中间体的酶，保持型水解酶,）,动力学控制：外切酶（内切酶一般具有更强的转糖基能力）,动力学控制：,aryl glycosides(,芳基,)or,glycosyl,fluorides,（氟）,通过引入,空间位阻,提高目标产物,产率,（从反应产物角度考虑）,6-oxo-galactoside,Transition state analog of the enzymatic hydrolysis reaction,(,A,)Tandem use of,galactose,oxidase,and,-galactosidase,for the synthesis of,LacNAc,.,（减少副产物的产生）,(,B,)Utilization of an,oxazoline,transition state,analog as the monomer for,chitinase,-catalyzed polymerization,（通过空间位阻影响糖苷键的构型，只能形成,beta,构型产物）,选择糖受体上的,4,位，而非,6,位,OH,避免形成,alpha,键,酶的改造：从糖苷酶到糖合成酶,3.,Glycosynthases,（糖合成酶）,Mechanism of retaining,glycosidases,:formation of a,covalent,glycosyl,-enzyme intermediate,is followed by general,base-assisted hydrolysis,or,glycosidic,bond formation,Mechanism of,glycosynthases,with,glutamate,catalytic,nucleophile,substituted by an,alanine,residue,Mechanism of,glycosynthases,with,glutamate,catalytic,nucleophile,substituted by a,serine,residue,General base-assisted displacement of,fluoride,yields a new,glycosidic,linkage with an inverted configuration,Serine forms a hydrogen bond wit