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Lecture 10

CHEM564 Lecture 10: Sec 4

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University of Alberta
Christopher Cairo

BioconjugateChemistry Cairo 4 Oligosaccharides 4.1 Reactivity of Carbohydrates 4.1.1 Classes of glycoconjugates Some definitions: glycan: mono- or oligo-saccharide structure glycoconjugate: mono- or oligo-saccaride modified protein or lipid glycolipid: glycan-modified lipid glycoprotein: glycan-modified protein glycoform: a glycoprotein isomer that differs only in its glycan structure proteoglycan: heavily O-glycosylated proteins There are three major classes of naturally occurring glycoconjugates: N-linked glycan attached to a protein via a nitrogen atom (these are currently the best understood.) O-linked glycan attached to a protein via an oxygen atom Lipid-linked glycan attached to a lipid 4.1.2 Carbohydrate structures in post-translational modification N-Link Glycans N-linked glycans are some of the most common linkages in eukaryotic glycosylation. The name derives from linkage to an asparagine (Asn, or N) residue. This linkage is part of the ‘core’ of the glycan structures. Different organisms may have different core structures. O OH O H O H2N O N OH HO NH H 2 O H2N O OH O asparagine  Asn asparagine linked 1lAc N "N-link" 141 BioconjugateChemistry Cairo O-Link Glycans O-linked glycans are less common but are routinely found in eukaryotic systems. The O-link glycan tends to have more varied forms. The basic O-link is usually an 1- GalNAc: OH HO O O HN HO O O H N OH OH 2 H2N O O serine serine linked 1-GalNAc Ser "O-link" S Prokaryotic glycosylation Bacterial systems from Camplyobacter jejuni generate glycans very similar to mammalian N- linked glycans. These glycans are linked to an asparagine, but contain a bacillosamine residue at the reducing end. The enzymes responsible are still being explored as a method for synthesizing large quantities of glycoproteins. A recent example used the following strategy: Three strains of bacteria (E coli.) were generated to product the lipid-linked oligosaccharide (LLO), target protein (AcrA), and the bacterial oligosaccharyl transferase (PglB). These components were combined in vitro, and the glycosylated protein was isolated and characterized 2 by NMR. LLO + AcrA Gly-AcrA PglB 142 BioconjugateChemistry Cairo OH O HHO AcHN OH O O HO OH AcHN O O HO O OH AcHNO O O HO OH HO OH AcHN HO O O HO AcHN O AcHN O O O AcHN P O P O O O- O- 8 OH pglB HO O HO OH acceptor protein AcHN O HO AcHN OH O O HO O OH AcHN O O HO OH HO OH AcHN HO O O HO AcHN H AcHN O N O O NHAc O O N N N H O O OH Glycan abbreviations To simplify writing complex structures, a symbolic system is often used to represent oligosaccharide structures. By convention, this often leaves out some structural information (such as glycosidic linkages). This nomenclature has some shortcomings – but it is very useful for abbreviating large structures. There are multiple versions of this nomenclature (both monochromatic and color). 143 BioconjugateChemistry Cairo Symbolic representations for Oligosaccharides HO OH glucose (Glu) O O HHO OH OH OH fucose OHO (Fuc) HO hexose O (Generic) mannose HO O HO (Man) HHO HO OH OH OH xylose (Xyl) HO OH O C galactose O 2 O (Gal) HO OH glucuronic HO OH HO OH OH acid (GlcA) HO Uronic acid N-acetyl- HO O (Generic) HO O glucosamine HO OH iduronic HO OH NHAc acid CO- OH (GlcNAc) 2 (IdoA) N-acetyl- HO OH hexosamine N-acetyl- O (Generic) galactosamine HO OH CO2 HO OH OH (GalNAc) NHAc O OH sialic acid AcHN (generic) HO Neu5Ac shown As a free glycan, the reducing end is placed at the right-hand side. For example: Full Representations   Man(1-6) GlcNAc(1-4)-GlcNAc Man(1-3)  The linkages are usually written with an assumed anomeric linkage at C1. This is certainly not always the case! More typically, the linkages are completely ignored as in the following examples of common core structures: Common Core Structures N-link core High mannose N-Link O-link A blood group antigen Linkage information is ignored here. 144 BioconjugateChemistry Cairo Note that in branched oligosaccharides, the number of terminal branch points is sometimes used to refer to the structure. For example, a saccharide with two branch points (and three non- reducing ends) would be referred to as a ‘tri-antennary’ oligosaccharide. Aoothek&w tym bols used here are from the 1994 Glycobiology Consortium. A revised version that uses color (and changes glucose to a circle) was adopted in x 2004 and may be used in some figures herein. To appreciate the role of glycans in biology, consider the central dogma: Glycosylation is a post-translational modification, and glycans can serve information carrying roles via carbohydrate-recognizing proteins (lectins). The specific glycosylation of a protein can control its locale in the cell or its interaction with other proteins. Therefore, carbohydrates have an information storing capacity – much like genetically encoded sequences. The context of most biologically interesting glycans is in a form attached to a polypeptide chain. These glycans can serve multiple roles – some are relatively passive (such as protein sorting) others are vital to protein function (recognition, or folding). Protein linked glycans can be either N-linked or O-linked. xSee the CFG nomenclature guide online at: 145 BioconjugateChemistry Cairo As a post-translational modification, glycosylation is extremely common. There are over 150 known types of post-translational modifications of proteins such as: 3 -Methylation -Sulfation -Phosphorylation -Glycosylation -Myristoylation Glycosylation is the most common of these , it is estimated that approximately 50% of all proteins are glycosylated. Phosphorylation is slightly less common, and is estimated to affect approximately 30% of all proteins. It is important to note that many glycosylated proteins exist in multiple glycoforms – species with different glycans. Glycoproteins with only minor differences (one fewer saccharide for instance) may be referred to as having microheterogeneity. 4.1.3 Cleavage of glycosidic linkages N-Link - PNGase F removes all N-linked glycans and keeps protein backbone intact, converting Asn to Asp. EndoH cleaves the N-link, and leaves a GlcNAc-Asn on the protein. Hydrazine can also be used to chemically cleave N-link glycans, but will also degrade the protein 4, 5 backbone. O-Link – There is no good enzymatic equivalent for the removal of all O-linked glycans. Usually use a series of exoglycosidases to convert the glycan to O-linked core 2 (Gal(1- 3)GalNAc), followed by cleavage using the O-glycanase (endo--N-acetylgalactosaminidase). b- elimination using strongly basic conditions can be used to chemically remove O-link glycans. 4, 5 4.1.4 Reducing end chemistry Free glycans can be easily modified at the reducing end. Chemistries are available for labeling with chromophores and for derivitization onto surfaces or other conjugates. 146 BioconjugateChemistry Cairo 4.1.5 Reductive amination OH OH R O HN O O H HO HO N AcNH O AcNH O NH Release &Purfiication OH OH R O O O HO HO AcNH AcNH OH H2N N NH 2 1) DAP 2) NaCNBH 3 OH NH2 OH R O O N HO O OH HO AcNH AcNH N H The free amine can then be used to form conjugates by amide bond formation using carboxylates: This can also be used to tag the glycan using biotin or other affinity reagents: 147 BioconjugateChemistry Cairo For protein conjugation, the amine should be converted to an orthogonal nucleophile, such as a thiol: SH NH + OH 2 NH2 +2 N NH R O OH S OH O OH N N HO OHO O OH AcNH N HO AcNH H AcNH N H The thiol can then be used to react selectively in a Michael addition to maleimide: O O RHN 2 + N N H O 2 R S RHS O O In aqueous conditions, this reaction is highly pH dependent. Between pH = 6.5 – 7.5, the reaction is selective for formation of the thioether bond. At pH = 7, thiols react about 1000X 6 faster than amines. 148 BioconjugateChemistry Cairo OH OH R O O HO O O HO AcNH AcNH OH O NH HN O H MeOH N H 2 H2NHN S HOAc O  O HN NH OH OH O R O O H HO O O N HO HN S AcNH AcNH N H O This strategy can be used in combination with other methods – both an affinity tag and a chromophore can be incorporated to ease purification or analysis. Reductive amination and coupling to a thiol-containing linker have been used to immobilize carbohydrate antigens for binding studies. 149 BioconjugateChemistry Cairo 4.1.6 Glycosylamines Conversion of the reducing end to an amine can be accomplished with ammonium carbonate. 9 The reaction can be done with mono- or oligo-saccharides, and gives the -glycosyl amine, which can be used for conjugation to peptides (vide supra). HO NH CO HO O 4 3 O HHO OH HOHO NH2 RT, 5 d OH OH 10 Improved methods are available which reduce reaction times to ~3h with >90% yields. Cummings and coworkers have developed a hybrid strategy that exploits glycosylamines to 11 immobilize glycans to surfaces with AEAB (aminoethylaminobenzamide). 4.1.7 Detection of reducing sugars A useful method for determining the presence of a reducing sugar employs 3,5-dinitrosalicylic acid (DNSA). The proposed mechanism is oxidation of the carbohydrate aldehyde to the corresponding acid, accompanied by reduction of the DNSA reagent to an amine. The reaction is usually conducted in an alkali solution and may include other additives. 575m O OH O OH OH OH O2N NO2 O N NH 2 2 DNSA O HO O H OH H OH HO H HO H H OH H OH H OH H OH OH OH 150 BioconjugateChemistry Cairo A number of other reducing end labeling strategies are known. some of these are involve fluorogenic or chromogenic reagents which can be used for detection or separation of the glycans. Examples include resorcinol, o-phenylenediamine, and arginine. 13 4.1.8 Reaction with glycosidic alcohols The hydroxyl groups of oligosaccharides are not reactive under mild reaction conditions, in part due to their high pKa values. However, if strongly alkaline conditions are suitable for the application, these can be reactive in the presence of good electrophiles. The prototypical reagent 14 used for crosslinking carbohydrates is divinyl sulfone (DVS). 4.2 Introducing reactive sites in Carbohydrates 4.2.1 Oxidation of carbohydrates by NaIO 4 Kinetic studies of the rate of cis- versus trans-diols find that unrestricted diols (such as ethylene glycol) are oxidized most rapidly. Hindered diols, such as pinnacol, are somewhat 15 slower to react. Cis-diols on a cycloalane (such as cis-1,2-cyclohexenediol) are more reactive 16, 17 that the trans-isomers. Periodate treatment can be relatively destructive at high concentrations, but at low concentrations the reagent can be extremely useful. One method is to use periodate cleavage of the glycan to generate an available aldehyde for bioconjugate strategies. This reaction is concentration dependent, and conditions can often be found that only modify the sialic acid portions of the glycan in preference to other residues.
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