1. Pictures from Stryer, Biochemistry (mostly)
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

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 residues.
Starch has a-1,6-linked branches every approx 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
Abcbodybuilding.com
In plant cell walls, the cellulose fibers are linked with hemicellulose (other polysaccharides) and lignin (polyphenolic plastic).

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

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,
Ishibashi et al., 2013

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

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

51. Summary
Carbohydrates and carbohydrate-active proteins play critical roles in living organisms.
Carbohydrates can be analyzed by a variety of chemical, chromatographic and spectrometric methods.
Carbohydrate structures and functions are determined by the monosaccharides present and their linkages and modifications.
Complex carbohydrates contain carbohydrates linked to other biomacromolecues (proteins, lipids)
Carbohydrates & complex carbohydrates are synthesized by a network of glycosyl transferases and transporters.