Carbohydrates - The Glycosidic Bond

Carbohydrates A. Definition B. Roles C. Monosaccharides 1. Chirality a. One or more asymmetric carbons b. Linear and ring forms 2. Derivatives: the chemistry of carbohydrates a. b. c. d. Oxidation Reduction Esterification amines 3. Polymerization a. The Glycosidic Bond b. Non-covalent bonds in macro-molecular structure D. Oligosaccharides 1. 2. 3. 4. Glycoproteins & glycolipids O-linked N-linked Sequence determination-ABO E. Polysaccharides 1. Polymers of glucose 2. Polymers of disaccharides Carbohydrates The Glycosidic Bond 1 Carbohydrates Hemiacetals and Hemiketals are reactive to alcohols in condensation reactions • Hemiacetals condense with alcohols to form Acetals. • Hemiketals condense with alcohols to form Ketals. • These reactions form the basis of the GLYCOSIDIC BOND. MeOH : – Glc • Two sugar molecules can be joined via a glycosidic bond between an anomeric carbon (the hemiacetal/hemiketal) and a hydroxyl carbon (the other sugar). The Glycosidic Bond • The glycosidic bond between sugars is stable and does not readily hydrolyze. – The anomeric carbon involved in the glycosidic linkage is fixed in its chirality and is therefore nonreducing. – The second monomer, with its unreacted hemiacetal, is still reducing. (b-D-adenine riboside) Carbohydrates Hemiacetals and Hemiketals are reactive to alcohols in CONDENSATION reactions • Hemiacetals condense with alcohols to form Acetals. • Hemiketals condense with alcohols to form Ketals. • These reactions form the basis of the GLYCOSIDIC BOND. • Two sugar molecules can be joined via a glycosidic bond between an anomeric carbon (the hemiacetal/hemiketal) and a hydroxyl carbon (the other sugar). The Glycosidic Bond • The glycosidic bond between sugars is stable and does not readily hydrolyze. – The anomeric carbon involved in the glycosidic linkage is fixed in its chirality and is therefore nonreducing. – The second monomer, with its unreacted hemiacetal, is still reducing. (b-D-adenineriboside) 2 Carbohydrates Disaccharides Carbohydrates Disaccharides: • Disaccharides can be named by the organization and linkage or a common name. – The disaccharide formed upon condensation of two glucose molecules via a 1 à 4 bond is described as α-D-glucopyranosyl-(1à4)-D-glucopyranose. – The common name for this disaccharide is maltose. Still undergoing If you only know the sugar and the linkage mutarotation Non-reducing à reducing If you know the exact structure of the sugar As we make sugar-polymers, the convention is to have the non-reducing sugar to the LEFT and the reducing end at the RIGHT. 3 Carbohydrates Disaccharides: Here is likely the first disaccharide you encountered in your life: Lactose. Non-reducing à reducing Carbohydrates Disaccharides: Here is likely the disaccharide you ingest the most: Sucrose. b Notice that these are drawn upside down • Nonreducing Disaccharides • • Two sugar molecules can be also joined in a glycosidic bond between two anomeric carbons. The product has two acetal groups and no hemiacetals or hemiketals. There are no reducing ends; this is a nonreducing sugar. Review of Glycosidic Bonds 4 Carbohydrates Oligosaccharides Carbohydrates Oligosaccharides: Glycoconjugates: Glycolipids Here is an oligosaccharide that we encountered with sphingoglycolipids, or for short: Glycolipids. GM1 = Gal-b(1à3)GalNAc-b(1à4)Gal-b(1à4)Glc-b(1àCeramide) Sia-b(2à3) 5 Carbohydrates Oligosaccharides: • Glycoconjugates: Glycoprotein When you attach an oligosaccharide to a protein: Glycoproteins. A protein with small oligosaccharides attached: – Carbohydrate is attached via its anomeric carbon to amino acids on the protein. • Common connections occur at Ser, Thr, and Asn. – About half of mammalian proteins are glycoproteins. – Generally, bacteria do not glycosylate their proteins. – Carbohydrates play role in protein-protein recognition. – Viral proteins are heavily glycosylated; this helps evade the immune system. • Proteins whose role is to bind specific carbohydrates/oligosaccharides = Lectins (or selectins) – Lectins important for many biological functions • Recruitment of leukocytes to sites of inflammation • Sperm-egg recognition • Virus-target cell interaction • Attachment of flora (microbiome) in gut • Nervous system development • Serum-protein turnover (sialic acid) • Targeting proteins to lysosomes for degradation (Man) How are these sugars attached? Carbohydrates Oligosaccharides: TWO classes attached to proteins, O-linked and N-linked • First, these polymers, unlike any others we have studies, are BRANCHED • Second, like glycolipids, O-linked sugars are attached via an Oglycosidic bond – Use a Ser or Thr in the sequence (recall glycophorin) – Usually smaller than N-linked – Synthesized one at a time by specific glycosyltransferases (specific for sugar, linkage, and chirality) • Third, N-linked sugars are attached via an N-glycosidic bond – Use an Asn – The Asn residues are within a 3 AA sequence context: NXS/T – All the same at the core Let’s look more closely at O-linked oligosaccharide synthesis 6 Carbohydrates Oligosaccharides: O-linked • Specific glycosyltransferases use activated sugars • Activated by attaching to nucleotides: UDP, CDP, etc. EXAMPLE: Sugar 1-P + UTP ⇌ UDP-sugar + PPi Gal b(1à3) glycosyl transferase Sia a(2à6) glycosyl transferase Fuc a(1à2) glycosyl transferase Carbohydrates Oligosaccharides: N-linked • Larger, more complex than Olinked • All have a “Core” containing GlcNAc & Man • Added as a unit, then modified before adding to Asn groups on proteins; all in the ER/Golgi • Use an isoprene, called Dolichol, to build core • Examples: Always the same linkages Let’s look at this Dolichol & its use in biosynthesis more closely…. 7 Carbohydrates Oligosaccharides: N-linked • Activated by attaching two phosphates, one using CTP (step 13) • Again, uses NDP-sugar precursors H Dolichol pyrophosphate sugar Carbohydrates Oligosaccharides: N-linked Specific glycosidases Specific glycosidases Specific glycosyl transferases 8 Carbohydrates Oligosaccharides: Determination of Sequence • Whole different problem compared to proteins and nucleic acids…. Its branched!! • Moreover, a given residue can have several (and stereo-specific) ways of attaching to a neighboring residue. • Need to use a combination of methods: – Chemical – Hydrolysis & chromatography to identify sugars – Exhaustive methylation & hydrolysis, then chromatography to identify what positions were not methylated – Biochemical – Use of enzymes that stereo-specifically hydrolyze glyosidic bonds (from the non-reducing end) EXAMPLE: First, just like protein sequencing, you need to purify glyco-protein or lipid. Lets say we isolate the glycolipid from a person’s RBC’s who is O-positive. Treat it with a ceramidase to hydrolyze the lipid from the sugar. Second, take an aliquot and just hydrolyze (like what was done for amino acid analysis). This gets the composition and stoichiometry. Carbohydrates Oligosaccharides: Determination of Sequence • Whole different problem compared to proteins and nucleic acids…. Its branched!! • Moreover, a given residue can have several (and stereo-specific) ways of attaching to a neighboring residue. • Need to use a combination of methods: – Chemical – Hydrolysis & chromatography to identify sugars – Exhaustive methylation & hydrolysis, then chromatography to identify what positions were not methylated – Biochemical – Use of enzymes that stereo-specifically hydrolyze glyosidic bonds (from the non-reducing end) EXAMPLE: First, just like protein sequencing, you need to purify glyco-protein or lipid. Lets say we isolate the glycolipid from a person’s RBC’s who is O-positive. Treat it with a ceramidase to hydrolyze the lipid from the sugar. Second, take an aliquot and just hydrolyze (like what was done for amino acid analysis). This gets the composition and stoichiometry. 9 Carbohydrates Oligosaccharides: Determination of Sequence a-glycosidase ③ Hydrolysis yielding only Fuc + Disacc. means there is an a-linkage followed by a blinkage ① Fuc b-glycosidase ② ③ Disacch. Trisacch. No reaction with the original (no reaction) trisacch means that there b-glycosidase are not sugars in b-linkage at the non-reducing end. ③ 1 Gal Gal •Methylation of C1-OH means it’s a reducing carbon at the GlcNAc reducing end •Lack of methylation of C4-OH means it was in glycosidic linkage with another sugar 1,3,6 trimethyl-GlcNAc •Lack of methylation of C1-OH means its anomeric carbon was in ① Chemical: Hydrolysis a glycosidic bond 3,4,6 trimethy-Gal •Lack② of methylation of C2-OH Chemical: Exhaustive means that it was in glycosidic methylation & hydrolysis linkage with another sugar 2,3,4 trimethyl-Fuc Biochemical: •This ③ sugar is in the middle glyosidase ① 1 GlcNAc •Again, lack of methylation of C1-OH. •That all possible hydroxyls are methylated (recall Fuc is a 6-deoxy sugar); this sugar is at a nonreducing end (no other sugars attached to it) 1 Fuc Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group 10 Carbohydrates Oligosaccharides: Determination of Sequence – In vertebrates, ganglioside carbohydrate composition determines blood groups. CH2OH OH 6CH OH 2 O–Cer OH OH OH OH OH OH OH OH L-Fuc a(1à2)-D-Gal b(1à4) D-GlcNAc b-Ceramide O blood group D-Gal a(1à3) D-GalNAc a(1à3) B blood group A blood group Glycoconjugates: Analysis 11