1. Carbohydrates James R. Ketudat Cairns Aj. Jim Pictures from Stryer, Biochemistry (mostly)

2. What are Carbohydrates?

3. (CH2O)n • Aldehydes (aldose sugars) • Ketones (ketose sugars) • 3 or more carbons.

4. Fischer Projections D and L isostereomers depend on the configuration of the chiral carbon furthest from the carbonyl.

5. D-Triose to D-Hexose L-sugars are the mirror image of the D-sugar. Sugars that differ in stereochemistry at one position are called epimers.

6. D-Ketoses

7. Carbonyl reactions with alcohols Note: Similar reactions can occur with amines and other nucleophiles.

8. Monosaccharide cyclization • Formation of an internal hemiacyl or hemiketal is favorable, if it forms a 5 or 6 member ring. • Furanose = 5 member ring • Pyranose = 6 member ring D-Glucopyranose

9. Anomeric Configuration • Sugars can have two anomeric configurations for each type of ring. • In solution, there are a mix of linear and ring forms that depends on the stability of each.

10. Sugars are not flat and can form different puckered shapes • Furanose envelopes are most stable. • Pyranose chairs & boats are stable. • Most stable depends on steric interactions. • Axial OH tend to bump, while equatorial do not. Ribose envelopes Glucose chair & boat

11. Pyranoses can move through many structures, only a few are stable B = boat C = chair H = half chair S = skew boat Vocadlo & Davies, 2008

12. Glycosides • Reaction at the anomeric carbon (hemiacyl or hemiketal position) form glycosides. • The sugar is trapped in one anomeric configuration. • The bond between the sugar and aglycone is called a glycosidic bond • The product is a glycoside.

13. Glucosides in nature Glucosides are glycosides with glucose for a sugar. The compounds shown are properly called b-D-glucopyranosides. Ketudat Cairns & Esen, 2010, Cell. Mol. Life Sci.

14. Modified & branched monosaccharides • Many modified monosaccharides exist in nature. • There are also branched monosaccharides, • e.g. apiose

15. Oligosaccharides • If two or more monosaccharides polymerize through glycosidic bonds, the are oligosaccharides. • The number of monosaccharides is designated by di-, tri-, tetra-, penta-, hexa- • They can be explicitly described as shown for the common disaccharides to the right. • Often, they are given names like cellobiose, cellotriose or (1,4)-b-D-mannobiose to simply indicate their size and linkage.

16. Reducing & nonreducing sugars • Sugars that have a free anomeric carbon can undergo redox reactions with Cu2+ • (Fehling’s reagent). • They are called reducing sugars, since they reduce the copper,while they are oxidized.

17. Redox products of sugars • Sugars can be oxidized at the anomeric carbon to form aldonic acids. • E.g. D-gluconic acid, the produce of a Fehling reagent reaction. • Sugars can be oxidized at a primary alcohol to form a uronic acid. • E.g. D-glucuronic acid, D-galacturonic acid, etc. • These carboxylic acids can form 5 or 6 member rings, such as in L-ascorbic acid. • Sugars can also be reduced to alditols (polyalcohols).

18. Carbohydrate Composition Analysis • Carbohydrate sugar composition can be tested by hydrolysis (acid or base with heat to break glycosidic bonds), TLC, HPLC, IC or modification and GC/MS. • Compare to standard sugars. • HPLC, IC and GC can potentially quantify sugars. • Modification by acetylation, methylation or trimethyl silanation can make sugars volatile for GC (and acetylation can make them detectable by UV for HPLC).

19. Carbohydrate Linkage analysis • Carbohydrate linkages can be determined by Nuclear Magnetic Resonance, if the polymer is not too complex. • Methylation analysis can determine which hydroxyls are linked. • First methylate all free hydroxyls • Then hydrolyze glycosidic bonds • Reduce and acetylate the linkage positions. • Run methyl acetyl alditols on GC/MS and compare elution positions to standards. • Does not tell anomeric configuration, just linkage.

20. Methylization analysis chemistry

21. Carbohydrate sequencing • Can see the loss of sugars (hexose, pentose, etc.) by mass spectrometry (MS) • Can see fragmentation of sugars in MS spectrum. • Can use specific enzymes to cut off sugars one at a time and look at mass differences. • E.g. neuraminidase to cut off sialic acid, • a-mannosidase to cut off a-linked mannosyl residues. • These enzymes are called glycosidases or glycoside hydrolases (GH).

22. MS sequencing of N-linked polysaccharide.

23. Positive ion MALDI-TOF mass spectra of derivatized N-linked glycans from bovine fetuin Derivatized with MeI Derivatized with methanol/DMT-MM

24. Polysaccharides are important structural and storage molecules • Polysaccharides can be grouped by the kinds of monosaccharides they contain • Glucans contain glucose • Mannans contain mannose • Arabinoxyloglucans contain arabinose, xylose and glucose. • Cellulose, a b-glucan is the most abundant polymer on earth. • Chitin/chitosan, a similar structural polysaccharide is also very abundant. • Starch and glycogen represent storage polysaccharides • Alpha-linked glucose polymers

25. Comparison of Cellulose with Glycogen and Starch • Cellulose is a straight chain, made from alternating orientations of b-1,4-linked glucosyl residues. • Starch and glycogen are coiled a-1,4-linked glucosyl polymers. Amylose coil www.agrana.com/en/1761.asp

26. Glycogen vs. Starch (Amylopectin) • Glycogen and starch (amylopectin) differ in how many 1,6-linked branches they contain. • Glycogen has an a-1,6-linkage every approx. 8-14 residues. • Starch has a-1,6-linked branches every approx. 24-30 a-1,4-linked residues.

27. Cellulose in cell wall structure • Cellulose fibers are semicrystalline due to regular hydrogen bonding • Therefore, they are hard to break down.

28. Plant cell wall polysaccharides • In plant cell walls, the cellulose fibers are linked with hemicellulose (other polysaccharides) and lignin (polyphenolic plastic). Abcbodybuilding.com

29. Other structural polysaccharides dalwoo.tripod.com/structure.htm

30. Complex Carbohydrates • Complex carbohydrates are complexes of carbohydrates with other macromolecules • Glycoproteins – found in all domains of life • Proteoglycans • Peptidoglycans (bacterial cell walls) • Glycolipids

31. Types of Eukaryotic glycoproteins • Cytosolic: single N-acetylglucosamine residues on Ser or Thr hydroxyls. • Likely a regulatory function, like phosphorylation or acetylation. • Secreted: • N-linked: bound to asparagine (Asn, N) • Initial core oligosaccharide added in ER. • O-linked: bound to hydroxyl groups (Ser, Thr, HyPro, HyLys). • Mucin-like added in Golgi • Alpha-mannose linked started in ER • Others

32. Secretory pathway

33. Adding of monosaccharides to molecules • Glycosyl transferases transfer sugars from nucleotidyl glycosides to other molecules in nature. • In the lab, we can also use glycosidases to reverse hydrolyze or transfer glycoside sugars.

34. Synthesis of core oligosaccharides for N-linked glycosylation • A core oligosaccharide is synthesized on dolichol in the ER membrane for transfer to a glycoprotein Asn in the N-X-S/T sequence. Cytosol ER matrix Dolichol phosphate

35. Core oligosaccharide addition to proteins • The core oligosaccharide is added to proteins in the ER. • The three Glc residues must be cleaved off before the protein can leave the ER. • Glucose-binding lectins prevent proteins from escaping the ER unfolded. • The alpha-glucosidases that cut off the Glc will not cut off the last until the protein is folded. • If the last Glc is not removed, glc transferase adds another to retain the protein in the ER. • Abnormal O-mannosylation marks for them to be removed from the cycle and degraded.

36. Calnexin, Glucosidase & Glucosyltransferase ensure secretory protein folding.

37. C-type lectins like Calnexin use Ca to bind sugars • Lectins are proteins that bind specific sugars • C-type lectins are animal lectins that use bind calcium to help bind the sugar.

38. Glycosylation is further modified in the Golgi apparatus • In the Golgi glycosidases cut off more of the core oligosaccharides. • Glycosyl transferases add other sugars after the trimming. • The exact carbohydrate varies with the type of organism, cell and protein. • Variation in the amount of carbohydrate added to one protein: microheterogeneity.

39. Elastase, a simple glycoprotein

40. Phosphorylation and sulfonation also happen in the Golgi • Phosphomannose is important for sorting of several glycolipid & proteoglycan degrading enzymes to the lysosome. • Lack of the enzymes to transfer the phosphate to mannose results in I-cell disease, where inclusions of undigested glycolipids and proteoglycan develop. • First GlcNAc-Phosphate is added to the Mannose 6-hydroxyl, then GlcNAc is cut off.

41. O-linked glycoproteins • Secreted O-linked glycoproteins can have from one to thousands of sugars added. • Dystroglycans and some other proteins have alpha-O-Man added in ER. • The first sugar added is usually GalNAc or Gal in mucin-like glycosylation in the Golgi apparatus. • Further sugars added in the Golgi. • The sugars added can contain important information, such as the blood group.

42. Glycosamino Glycans • Glycosamino glycans are carbohydrates that are usually bound to proteoglycans. • Play important roles in connective tissues. • Also called mucopolysaccharides.

43. Proteoglycans • Core proteins can have many times their weight in glycosaminoglycan carbohydrate attached. • They hydrate and form a compressible component to give cushioning to joints and related tissues. • They also form a part of the intracellular matrix between cells. Jeffrey & Watt bjr.bjrjournals.org www.histo-moleculaire.com/siteconj/images/037...

44. Bacterial Peptidoglycans • Peptidoglycans are major components of bacterial cell walls • Thick coating on outside of Gram positive bacteria • Thinner layer between membranes of Gram negative bacteria • Cut by Lysozyme. Gram positive bacteria cell wall structure

45. Peptidoglycan cell walls Staphylococcus aureus peptidoglycan

46. Glycolipids Glycolipids: Glycosphingolipids Glycoglycerol lipids: Plant galactolipids Animal PtdGlc (below) Cholesterol glucoside Wennekes et al., 2009, Angew. Chem. Int. Ed. 48, 8848-8869 Ishibashi et al., 2013

47. Glycosphingolipid synthesis & catabolism Wennekes et al., 2009, Angew. Chem. Int. Ed. 48, 8848-8869

48. Carbohydrate Active proteins • Carbohydrate binding proteins/domains • Carbohydrate binding modules (CBM) can bind simple sugars or more extensive regions. • Lectins bind simple sugars. • Carbohydrate Active Enzymes (CAZy) • Glycoside Hydrolases (GH, glycosidases) • Transglycosidases (TG) catalyze transfer rather than hydrolysis. • Glycosyl Transferases (GT) • Polysaccharide Lyases- nonhydrolytic cleavage of glycosyl linkages • Carbohydrate Esterases • Other carbohydrate modifying enzymes

49. Viral Carbohydrate-Active Proteins • The flu virus strains are distinguished by forms of carbohydrate active proteins. • Hemaglutanin (H in virus name) binds to sialic acid on cell surface to invade. • Neuraminidase (N in virus name) cuts off the sialic acid to free the virus, once inside the cell.

50. Neuraminidase