糖苷键（888）.ppt

概述：基本概念,化学合成糖苷键的三个考虑因素,1）如何活化糖基分子,donor,acceptor,2）如何实现立体选择性：a or b,3）如何实现连接选择性 选择性地保护acceptor中的羟基 利用羟基活性的差别 6-OH 2,3-OH 4-OH equatorial-OH axial-OH,1,医药资料,概述：基本概念,化学合成糖苷键的基本原理,donor,acceptor,糖基化反应中， donor中间体和promoter是亲电试剂（electrophile）， acceptor是亲核试剂（nucleophile）,一般而言，糖苷键对酸敏感，对碱不敏感。,2,医药资料,3,医药资料,概述：基本概念,化学合成糖苷键的立体选择性,SN1 机制： anomeric effect，a-anomer as major product。 C-2 is non-participation group (2-OCH2Ph, 2-N3, 2-deoxy) harsh condition （strong promoter，high temperature） polar solvent SN2 机制： anchimeric effect （C-2 functional group participation effect） (-OAc, -NHAc, -I, -SR, -SeR) stable intermidiate,4,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （I） 三氯乙酰基亚胺酯和酸（BF3，TMSOTf）,强碱，利于b-TCA donor；弱碱，利于a-TCA donor。,5,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （I） 三氯乙酰基亚胺酯和酸（BF3，TMSOTf）（Schmidt）,SN2 like reaction; Stereoselectivity (SN1 vs SN2) is subject to solvent, temperature, promotor & acceptors.,BF3OEt2 CH2Cl2,TMSOTf, Et2O,6,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （II） 硫苷和DMTST, NBS, IDCP, Mn+,7,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （II） 硫苷和DMTST, NBS, IDCP, Mn+,8,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （II） 硫苷和DMTST, NBS, IDCP, Mn+,9,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （III） 溴苷和Mn+ （ Koenigs-Knorr ）,10,医药资料,概述：基本概念,化学合成糖苷键的donor和promotor （IV） Epoxide donor,11,医药资料,概述：基本概念,化学合成糖苷键的保护基策略: 常用保护基 缩写，保护和脱保护条件，选择性，稳定性,12,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（I） 酰基保护基,Acylations can often be carried out as regioselective reactions to establish selective protection patterns. In basic condition, acetyl group sometimes can shift.,13,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（II） 醚保护基,14,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（II） 醚保护基,15,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（III） 硅醚保护基,In acidic media, the relative stability is: TMS (1) TES (64) TBS (20 000) TIPS (700 000) TBDPS (5 000 000) In basic media, the relative stability is: TMS (1) TES (10-100) TBSTBDPS (20 000) TIPS (100 000) In basic condition, silyl ether sometimes can shift,16,医药资料,A: benzylidene acetals,概述：基本概念,化学合成糖苷键的保护基策略（IV） acetal,17,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（IV） acetal,18,医药资料,概述：基本概念,化学合成糖苷键的保护基策略（IV） acetal,19,医药资料,Several free monosaccharides can be converted by isopropylidenation,概述：基本概念,化学合成糖苷键的保护基策略（IV） acetal,20,医药资料,概述：基本概念,化学合成糖苷键: armed & disarmed,21,医药资料,概述：基本概念,化学合成糖苷键: armed & disarmed,22,医药资料,概述：基本概念,寡糖合成策略,固相合成法 One-pot synthesis Peter H. Seeberger Chi-Huey Wong Samuel Danishefsky Xuefei Huang,23,医药资料,天然糖苷类抗生素,合成稀有糖,24,医药资料,Activation-substitution reaction at the carbohydrate ring,天然糖苷类抗生素,合成稀有糖,25,医药资料,Tosylate as leaving group,天然糖苷类抗生素,合成稀有糖,26,医药资料,Triflate as leaving group,天然糖苷类抗生素,合成稀有糖,27,医药资料,Mesylate as leaving group,Intramolecular substitution,天然糖苷类抗生素,合成稀有糖,28,医药资料,2. Deoxyhalo derivatives,Florination with DAST,(Diethylaminosulfur trifluoride),天然糖苷类抗生素,合成稀有糖,29,医药资料,Selective florination,天然糖苷类抗生素,合成稀有糖,30,医药资料,Other halogenation,天然糖苷类抗生素,合成稀有糖,31,医药资料,3. Deoxygenation of carbohydrates derivatives,Opening of epoxides,天然糖苷类抗生素,合成稀有糖,32,医药资料,Reduction of ester,Mechanism:,天然糖苷类抗生素,合成稀有糖,33,医药资料,Examples:,Synthesis of methyl 2,4-di-O-benzoyl-6-deoxy-3-O-(1-imidazolylthiocarbonyl)-a-L-galactopyranoside,Synthesis of methyl 2,4-di-O-benzoyl-3,6-dideoxy-a-L-hexopyranoside,天然糖苷类抗生素,合成稀有糖,34,医药资料,4. Epimerizations,Misunobu reaction:,Example:,天然糖苷类抗生素,合成稀有糖,35,医药资料,Oxidation-reduction reaction:,天然糖苷类抗生素,合成稀有糖,36,医药资料,合成稀有糖,天然糖苷类抗生素,从非糖原料出发合成糖分子：,Samuel Danishefsky,George A. ODoherty,Peng G. Wang,37,医药资料,Peng George Wang Departments of Biochemistry & Chemistry The Ohio State University,Carbohydrate Chemistry & Glycobiology,L9 Enzymatic Synthesis of Oligosaccharides and glycopeptides/glycoproteins,38,医药资料,Comparison of Organic and Biosynthetic Approaches for Carbohydrate Synthesis,39,医药资料,Chemical Synthesis of a-Gal,High Cost Multiple protection, deprotection steps Tedious separation procedures,40,医药资料,Synthesis of D-Galactoside Donor,Chemical Synthesis of a-Gal,41,医药资料,Synthesis of Disaccharide Acceptor,Chemical Synthesis of a-Gal,42,医药资料,Synthesis of a-Gal Trisaccharide,Chemical Synthesis of a-Gal,W. Zhang, J. Wang, J. Li, L. Yu, P. G. Wang, J. Carbohydrate Chemistry,199918(8), 1009-1017,43,医药资料,Enzymatic Synthesis of a-Gal,44,医药资料,Enzymatic synthesis of oligosaccharides,Glycosyltransferase Superbeads and superbug technologies Glycosidase Glycosynthase,45,医药资料,Glycosyltransferase,Advantages: high regio-selectivity, the most practical method,Transfer sugar residues from an activated donor substrate to an acceptor Donor: A sugar activated by a phosphate, a lipid phosphate, or a nucleotide - usually a nucleotide-sugar Acceptor: a lipid, a protein, DNA, a small molecule acceptor, or a growing oligosaccharide.,Typically grouped into families based on the type of sugar they transfer eg. Galactosyltransferases, sialyltransferases,Glycosyltransferases are very specific enzymes, with only one particular transferase being able to catalyze one particular glycosidic linkage -One EnzymeOne Linkage,46,医药资料,Glycosyltransferase,Sugar Nucleotide,Nine Common Sugar Nucleotides,UDP-Glc, UDP-Gal UDP-GlcNAc, UDP-GalNAc UDP-GlcA, UDP-Xyl GDP-Man, GDP-Fuc CMP-NeuAc (the only monophosphate),47,医药资料,Biosynthesis of Sugar Nucleotides,48,医药资料,UDP-Glc, UDP-Gal, UDP-GlcA, UDP-Xyl,UDP-GlcNAc, UDP-GalNAc,GDP-Man and GDP-Fuc,CMP-Neu5Ac,49,医药资料,a1,3GalT Catalyzed Synthesis of a-Gal Epitopes and Their Derivatives,Fang J, Chen X, Wang PG, et al.: J. Org. Chem. 1999, 64, 4089-4094.,Examples,50,医药资料,Synthesis of a-Gal Pentasaccharide with In Situ Cofactor Regeneration,Fang J, Li J, Chen X, Wang PG, et al: J. Am. Chem. Soc. 1998, 120, 6635-6638.,Examples,51,医药资料,De Luca, C.; Wong, C.-H. J. Am. Chem. Soc. 1995, 117, 5869-5870,One-pot multi-enzyme synthesis of a hyaluronic acid polymer involving glycosyltransferases and sugar nucleotide regeneration systems,E1, hyaluronic acid synthase; E2, UDP-Glc dehydrogenase; E3, UDP-Glc pyrophosphorylase; E4, UDP-GlcNAc pyrophosphorylase; E5, pyruvate kinase; E6, lactate dehydrogenase; E7, inorganic pyrophosphatase,Examples,52,医药资料,Cytels large-scale synthesis of sialyl Lewis X antigen,E1: a1,3-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,Examples,53,医药资料,Neoses large-scale synthesis of sialyated lactose using a CMP-sialic acid synthetase/a2,3-sialyltransferse fusion enzyme with in situ regeneration of CMP-NeuAc,E1: CMP-NeuAc synthetase/a2,3-sialyltransferase fusion enzyme E2: myokinase E3: pyruvate kinase E4: GlcNAc2-epimerase E5: sialic acid aldolase,Examples,54,医药资料,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: a1,4-galactosyltransferase,Examples,55,医药资料,Examples,Synthesis of tumor-associated antigen, Globo-H hexasaccharide,Su D., Eguchi H., Yi W., Li L., Wang PG. and Xia C. Org. Lett. 2008, in press,56,医药资料,Superbug and Superbead 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,a-Gal,57,医药资料,Synthesis of oligosaccharides in biological system,OH,HO,S1,OH,HO,S2,+,O,OH,HO,S1,S2,+ H2O,OH,O,Nucleotide,HO,S1,Sugar-Nucleotide Biosynthesis & Regeneration,HO,S1,O,OH,OH,HO,S2,HO,S1,S2,*,58,医药资料,Glycosylation in Eukaryotes,Sugar Nucleotide transport,transferase,UDP,UTP,UDP,UMP,UDP,UMP,Gal,P,Gal,UDP,Gal,Gal,Acceptor,Acceptor,Cytosol Golgi,59,医药资料,Biosynthetic Pathway for UDP-Gal Regeneration and a-Gal Production,ATP,ADP,a1,3GalT,Gala1,3Lac,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,60,医药资料,UDP-Gal Superbead,GalU,GalT,GalK,PyKF,Recombinant E. coli stratins overexpressing GalK, GalT, GalU or PykF,Chen X, Fang J, Wang PG, et al.: J. Am. Chem. Soc. 2001, 123, 2081-2082.,61,医药资料,Beads with: GalK GalT GalU PykF,Peristaltic pump for circulation,Reservoir with: a1,3GalT 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 MgCl2 10 mM MnCl2 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.,62,医药资料,Synthesis of oligosaccharides with UDP-Gal Superbead,a Gram scale synthesis, others are 100 mg scales,63,医药资料,a-Gal synthesis using Superbug,a1,3GalT,Gala1,3Galb1,4Glc,Galb1,4Glc,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,NM522,64,医药资料,First Generation of a-Gal Superbug,Clone and express individual enzymes in the biosynthetic pathway of a-Gal Construction an artificial biosynthetic gene cluster and transfer into E. coli host cell. Large scale production of a-Gal oligosaccharides using fermented and permeated cells.,65,医药资料,Chen X, Liu Z, Zhang W, Fang J, Andreana P, Wang PG: ChemBioChem. 2002, 3, 47-53.,Large Scale Production of a-Gal Trisaccharide,66,医药资料,Second Generation of a-Gal Superbug,PpK: polyphosphate kinase,67,医药资料,Third Generation of a-Gal Superbug,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,68,医药资料,Application of Glycosyltransferases - Summary Pros: Excellent yield with complete regio- and stereoselectivity. No protecting groups needed. Sialyltransferases allow facile sialylation. (This is the hardest glycosylation to carry out by chemical methods.) Reactions can be carried out in water. Cons: Most requisite enzymes are not readily available, and those that are available are expensive. Regio/stereoselectivity means that substrate scope is limited and a unique enzyme is needed for almost every reaction. Nucleotide sugar donors are very expensive and/or unstable. Scale is often limited by enzyme availability/volumetric productivity. (A large amount of enzyme produces only a small amount of sugar by weight.),69,医药资料,Based on AA sequence similarity, Glycosidase are classified into 107 distinct families. (/index.html),Currently 37472 known or putative glycosidase in CAZy,433 different glycosidase crystal structures from 76 different families have been reported,Glycosidase and Transglycosidase,In vivo function: hydrolysis of glycosidic bonds,70,医药资料,Glycosidase and Transglycosidase,Advantages: available, stable, organic solvent compatible, low cost Disadvantages: low yields (competing product hydrolysis), unpredictable regioselectivity,Useful catalysts for in vitro formation of glycosidic bonds Thermodynamic control: increasing substrate concentrations decreasing product concentrations by absorption elevating reaction temperatures adding water-miscible organic co-solvents Kinetic control: using activated glycosyl donors and exogenous nucleophiles,71,医药资料,Synthetic Application of Glycosidases While glycosidases normally cleave glycosidic linkages, they can be coerced to “run backwards” (to some extent) in the presence of an appropriate glycosyl donor.,72,医药资料,The synthesis of 2 Tn-antigen epitopes is illustrative:,Application of Glycosidases,73,医药资料,Application of Glycosidases,Synthesis of glycoprotein using endo-glycosidase,Li B., Zeng Y., Hauser S., Song H., and Wang L. J. Am. Chem. Soc., 2005, 9692-93,Endo-A hydrolyze the b1,4 linkage in the core N,N-diacetylchitobiose moiety of N-glycoprotein to release the N-glycan.,74,医药资料,Application of Glycosidases - Summary Pros: Many glycosidases available (esp. in comparison to glycosyltransferases). Glycosidases are less expensive and more stable than transferases. Specificity is generally relaxed rel. to transferases, allowing broader substrate scope. Generally good regio- and stereoselectivity. No protecting groups. Cons: Enzymatic glycosylations still less scalable that chemical reactions. Volumetric productivity still low. Yields much lower than with transferases - glycosidase activity (which degrades the product) competes with glycosylation.,75,医药资料,Glycosynthase,Specifically mutated glycosidases that efficiently synthes