Chap. 7A. Carbohydrates and Glycobiology• Monosaccharides and Disaccharides

• Polysaccharides• Glycoconjugates: Proteoglycans, Glycoproteins, and Glycosphingolipids• Carbohydrates as Informational Macromolecules: the Sugar Code• Working with Carbohydrates

Fig. 7-34. Helicobacter pylori cells adhering to the gastric surface.

Intro. to CarbohydratesCarbohydrates are the most abundant biomolecules on Earth. Each year, photosynthesis converts more than 100 billion metric tons of CO2 and H20 into cellulose and other plant products. Carbohydrates are polyhydroxy aldehydes and ketones, or substances that yield such compounds on hydrolysis. Many, but not all have the empirical formula (CH2O)n, but some also contain nitrogen, phosphorus, or sulfur. Carbohydrates occur in four main size classes: monosaccharides, disaccharides, oligosaccharides, and polysaccharides. The most abundant monosaccharide in nature is D-glucose, which is also known as dextrose. A common disaccharide, sucrose, consists of the six-carbon sugars D-glucose and D-fructose. Common polysaccharides include cellulose and starches. Both of these are homopolymers of D-glucose units, but with different linkages between residues. More complex carbohydrate polymers attached to a protein or lipid moiety (glycoconjugates) are also prevalent in nature. This chapter introduces the major classes of carbohydrates and glycoconjugates and provides examples of their many structural and functional roles in biology.

Intro. to MonosaccharidesMonosaccharides are colorless, crystalline solids that are very soluble in water, but insoluble in nonpolar solvents. Most have a sweet taste. The backbones of common monosaccharides are unbranched carbon chains in which all the carbons are linked by single bonds. In this open-chain form, one of the carbon atoms is double-bonded to an oxygen atom to form a carbonyl group. Each of the other carbons has a hydroxyl group. If the carbonyl group is at an end of the carbon chain (that is, in an aldehyde group) the monosaccharide is called an aldose. If the carbonyl group is at any other position (a ketone group) the monosaccharide is a ketose. Monosaccharides with three, four, five, six, and seven carbons in their backbones are called trioses, tetroses, pentoses, hexoses, and heptoses. Many of the carbon atoms to which hydroxyl groups are attached are chiral centers. This gives rise to the many stereoisomers found in monosaccharides.

Common MonosaccharidesCommon aldoses and ketoses of three-, five-, and six-carbon lengths are shown in Fig. 7-1. The simplest monosaccharides are the two three-carbon trioses: D-glyceraldehyde, an aldotriose; and dihydroxyacetone, a ketotriose. The most common monosaccharides in nature are the aldohexose D-glucose, and the ketohexose D-fructose. The aldopentoses D-ribose and 2-deoxy-D-ribose are components of nucleotides and nucleic acids.

D & L StereoisomersAll of the monosaccharides except dihydroxyacetone contain one or more asymmetric (chiral) carbon atoms and thus occur in optically active isomeric forms. The simplest aldose, glyceraldehyde, contains one chiral center (the middle carbon atom) and therefore has two different optical isomers, or enantiomers (Fig. 7-2). One of the two enantiomers of glyceraldehyde is, by convention, designated the D isomer and the other is the L isomer. In general, a molecule with n chiral centers can have 2n stereoisomers. Glyceraldehyde has 21 = 2; the aldohexoses with four chiral centers have 24 = 16. The stereoisomers of monosaccharides of each carbon-chain length are divided into two groups that differ in the configuration about the chiral carbon that is most distant from the carbonyl carbon. Those in which the configuration of this reference carbon is the same as that of D-glyceraldehyde are designated D isomers. Those with the same configuration as L-glyceraldehyde are L isomers. Thus of the 16 possible aldohexoses, eight are D forms and 8 are L forms. The reason D forms predominate in nature is unknown.

Structures of the D MonosaccharidesThe structures of the D stereoisomers of all the aldoses

and ketoses having three to six carbon atoms are shown in Fig. 7-3 (next two slides). The carbons of a sugar are numbered beginning at the end of the chain nearest the carbonyl group. Each of the eight aldohexoses, which differ in the stereochemistry at C-2, C-3, and C-4, has its own name: D-glucose, D-mannose, D-galactose, and so forth. The four- and five-carbon ketoses are designated by inserting “ul” into the name of the corresponding aldose; for example, D-ribulose is the ketopentose corresponding to the aldopentose D-ribose. The ketohexoses are named otherwise: for example, fructose is named from the Latin fructus, “fruit”.

Structures of the D-Aldoses

Structures of the D-Ketoses

Epimers of D-AldohexosesTwo monosaccharides that differ only in the configuration around one chiral carbon atom are called epimers. D-glucose and D-mannose are epimers which differ in the configuration at C-2. D-glucose and D-galactose are epimers that differ in the configuration at C-4 (Fig. 7-4).

Common L Stereoisomers

Some sugars occur naturally in their L form. Some examples are L-arabinose (below) and the L isomers of some sugar derivatives that are common components of glycoconjugates.

Formation of Hemiacetals and HemiketalsAldotetroses and all monosaccharides with five or more

carbon atoms occur predominantly as cyclic ring structures in which the carbonyl group has formed a covalent bond with the oxygen of a hydroxyl group along the chain. The formation of these ring structures is the result of a general reaction between alcohols and aldehydes or ketones to form derivatives called hemiacetals or hemiketals (Fig. 7-5). Actually, two molecules of an alcohol can add to a carbonyl carbon. The product of the first reaction for an aldose is a hemiacetal, and the product of the first reaction for a ketose is a hemiketal. If the -OH and carbonyl groups are from the same molecule, a five- or six-membered ring results. The addition of the second alcohol molecule produces the full acetal or ketal, and the bond formed is a glycosidic linkage. When the two reacting molecules are both monosaccharides, the acetal or ketal produced is a disaccharide.

Cyclization of D-GlucoseThe reaction of the first alcohol with an aldose or ketose creates an additional chiral center at what was the carbonyl carbon. Because the alcohol can add to the carbonyl carbon by attacking either from the “front” or the “back”, the reaction can produce either of two stereoisomeric configurations, denoted and ß. For example, D-glucose (Fig. 7-6) exists in solution as an intramolecular hemiacetal in which the free hydroxyl group at C-5 has reacted with the aldehyde C-1, rendering the latter carbon asymmetric and producing two possible stereoisomers, designated and ß. These two isomeric forms, which differ only in their configuration about the hemiacetal carbon atom are called anomers, and the carbonyl carbon is called the anomeric carbon. The same

nomenclature is used to describe anomeric forms of hemiketals such as formed by fructose (see below). The and ß anomers of D-glucose interconvert via the linear form in aqueous solution by a process called mutarotation. In solution, an equilibrium mixture forms which consists of about one-third -D-glucopyranose, two-thirds ß-D-glucopyranose, and trace amounts of the linear and five-membered glucofuranose ring forms.

Pyranoses and FuranosesSix-membered monosaccharide ring compounds are called pyranoses because they resemble pyran (Fig. 7-7). Five-membered monosaccharide ring compounds are called furanoses because they resemble furan. The systematic names for the two ring forms of D-glucose are therefore -D-glucopyranose and ß-D-glucopyranose. Ketohexoses such as fructose also occur as cyclic compounds with and ß anomeric forms. In these compounds the hydroxyl group at C-5 (or C-6) reacts with the keto group at C-2 forming a furanose (or pyranose, not shown) ring containing a hemiketal linkage. D-fructose readily forms a furanose ring (Fig. 7-7). The more common anomer of this sugar in combined forms or in derivatives is ß-D-fructofuranose.

Fisher Projection & Haworth Perspective Formulas

Cyclic sugar structures are more accurately represented in Haworth perspective formulas (see below) than in Fischer projections used for linear sugar structures. In Haworth formulas the six-membered ring is tilted to make its plane almost perpendicular to that of the paper. The bonds closest to the reader are drawn thicker than those farther away. To convert the Fisher projection formula of any linear D-hexose to a Haworth perspective formula, draw the six-membered ring (five carbons, and one oxygen at the upper right), number the carbons in a clockwise direction beginning with the anomeric carbon, then add the hydroxyl groups as follows. If a hydroxyl group is to the right in the Fischer formula, it is placed pointing down in the Haworth formula. If a hydroxyl group is to the left in the Fischer formula, then it is placed pointing up in the Haworthformula. The terminal -CH2OH group projects upward for the D-enantiomer, and downward for the L-enantiomer. When the hydroxyl group on the anomeric carbon of a D-hexose is on the same side of the ring as C-6, the structure is by definition ß. When it is on the opposite side from C-6, the structure is .

Worked Example 7-1. Conversion of Fisher Projection to Haworth

Perspective Formulas

Conformational Formulas of PyranosesIt is important to keep in mind

the actual conformational structures of the ring forms of monosaccharides. For example the six-membered pyranose ring is not actually planar, as suggested by Haworth representations, but instead tends to assume either of two chair conformations (Fig. 7-8). The interconversion of the two chair forms (conformers) does not require bond breakage and does not change the configurations of substituents attached to any of the ring carbons. However, it does require a considerable input of energy. The actual three-dimensional structures of monosaccharide units are important in determining the biological properties and functions of some polysaccharides, as shown below.

Important Hexose Derivatives (I)In addition to simple hexoses such as glucose, galactose, and mannose, there are many sugar derivatives in which a hydroxyl group in the parent compound is replaced with another substituent, or a carbon atom is oxidized to a carboxyl group. In addition, hexoses in metabolic pathways commonly are phosphorylated on hydroxyl groups (Fig. 7-9).

Important Hexose Derivatives (II)In amino sugars, an -NH2 group replaces one of the -OH groups in the parent hexose. Substitution of -H for -OH produces a deoxy sugar, some of which occur in nature as L isomers. The acidic sugars contain a carboxylate group, which confers a negative charge at neutral pH. Lactones result from the formation of an ester linkage between the C-1 carboxylate group and the C-5 hydroxyl group of the sugar. Some notable functions of hexose derivatives in biology are 1) N-acetylglucosamine and N-acetylmuramic acid, components of the bacterial cell wall; and 2) N-acetylneuraminic acid (sialic acid) and fucose, components of the oligosaccharide chains of mammalian glycoproteins.

Measurement of Blood Glucose LevelMonosaccharides can be oxidized by relatively mild oxidizing agents such as cupric (Cu2+) ion which oxidizes the carbonyl carbon to a carboxyl group. Glucose and other sugars capable of this reaction therefore are called reducing sugars. This reaction (Fehling’s reaction) was used for many years to detect and monitor glucose levels in people with diabetes mellitus. Today, an immobilized enzyme (glucose oxidase) on a test strip is used to catalyze the oxidation of free glucose to D-glucono--lactone (see below). The hydrogen peroxide H2O2 produced as the second product of this reaction subsequently reacts with a colorless compound in the strip via the enzyme peroxidase to form a colored product which can be quantified using a simple photometer.

Hemoglobin Glycation in Diabetes MellitusGlucose is a reactive molecule at

high concentrations and its modification of tissue proteins is thought to be responsible for the nephropathy, neuropathy, retinopathy, and cardiovascular diseases that are common in diabetics. The nonenzymatic modification of proteins (at amino groups) by glucose is referred to as glycation, and the structures that result are called advanced glycation end products (AGEs). Hemoglobin is a protein that commonly is modified by glucose at its N-terminal valines or the -amino groups of its lysine residues (Box 7-1, Fig. 1). This reaction has little effect on hemoglobin function, but it is highly diagnostic of a patient’s long-term blood glucose level. In nondiabetics, glycated hemoglobin makes up about 5% of the total hemoglobin level. In a poorly-controlled diabetic, this value may be as high as 13%, indicating an average blood glucose level of about 300 mg/dl (about 3 times the normal level). Glucose-lowering drugs are prescribed to keep glycated hemoglobin levels at about 7%.

Disaccharides (I)A disaccharide (e.g., maltose, Fig. 7-10) is formed from two monosaccharides (two D-glucose molecules for maltose) when an -OH alcohol group of the right D-glucose condenses with the intramolecular hemiacetal of the left D-glucose. Water is eliminated, and a glycoside with a glycosidic bond is formed. The reversal of this reaction is hydrolysis by attack of a water molecule on this bond--a reaction which is readily catalyzed using dilute acid. The oxidation of a sugar by cupric ion occurs only with its linear form, which exists in equilibrium with its cyclicforms. Thus, the anomeric carbon of the D-glucose residue on the left can no longer react with Cu2+ because it is tied up in a glycosidic bond. In contrast, the hemiacetal linkage in the right D-glucose molecule can open up, and react with Cu2+. For this reason, the right end of maltose is called its reducing end. Because mutarotation interconverts the and ß forms of the right hemiacetal linkage, the bonds at this position are sometimes depicted with wavy lines to indicate that either configuration at the anomeric carbon is possible. In maltose, the configuration of the anomeric carbon atom in the glycosidic linkage is .

Disaccharides (II)The convention for formally naming disaccharides (and oligosaccharides) is as follows. 1) Start with the configuration ( or ß) at the anomeric carbon joining the first monosaccharide unit (on the left) to the second. 2) Name the nonreducing residue at the left; to distinguish five- and six-membered ring structures, insert “furano” or “pyrano” into the name. 3) Indicate in parentheses the two carbon atoms joined by the glycosidic bond, with an arrow connecting the two numbers. In maltose, (14) shows that C-1 of the first D-glucose unit is joined to C-4 of the second. 4) Name the second residue. Following this convention, maltose is -D-glucopyranosyl-(14)-D-glucopyranose. Because most sugars in the textbook are the D enantiomers and the pyranose form of hexoses predominates, a shortened version of the formal name of compounds, such as maltose, can be used which gives the configuration of the anomeric carbon and names the carbons joined by the glycosidic bond. In this abbreviated nomenclature, maltose is Glc(14)Glc. Symbols and abbreviations for common monosaccharides and some of their derivatives are listed in Table 7-1 (not covered).

Disaccharides (III)The chemical structures and full systematic names of the common disaccharides, lactose (milk sugar), sucrose (table sugar), and trehalose (a sugar occurring in insect hemolymph) are shown in Fig. 7-11. Lactose is composed of D galactose and D glucose, sucrose is composed of D fructose and D glucose, and trehalose is composed of two D glucose residues. Lactose is a reducing sugar, and its reducing end is located on the glucose unit on the right. Sucrose and trehalose are both nonreducing sugars because the anomeric carbons of both monosaccharides in these compounds are tied up in glycosidic linkages.

Intro. to PolysaccharidesMost carbohydrates found in nature occur as polysaccharides, polymers of medium to high molecular weight (Mr >20,000). Polysaccharides, also called glycans, differ from each other in the identity of their recurring monosaccharide units, in the lengths of their chains, in the types of bonds linking the monosaccharide units, and in the degree of branching. Homopolysaccharides contain only a single monomeric species, whereas heteropolysaccharides contain two or more different kinds (Fig. 7-12). Unlike proteins, polysaccharides generally do not have defined molecular weights. This is because polysaccharides are not synthesized from a template. Instead, there is no specific stopping point for the enzymes involved in their biosynthesis.

Starches (I)Starch is a storage homopolysaccharide of D glucose residues that is found in the cytoplasm of plant cells. Starch (and glycogen) is extensively hydrated because it has many exposed hydroxyl groups available to hydrogen-bond with water. Starches consist of two types of polymers called amylose and amylopectin (Fig. 7-13). Amylose (Fig. 7-13a) is a linear polymer of D glucose residues that all are connected via (14) linkages (as in maltose). The molecular weights of amylose chains vary from a few thousand to more than a million. Amylopectin is a branched polymer of D glucose residues that can weigh up to 200 million Da. The glycosidic linkages between D glucose residues in amylopectin chains are also (14); the branch point linkages between D glucose units, however, are (16) linkages (Fig. 7-13b, next slide). Branch points occur about every 24 to 30 residues.

Starches (II)A cluster of amylose and amylopectin molecules, like that believed to be present in the starch granules in plant cells, is shown in Fig. 7-13c (right). Strands of amylopectin (black) form double-helical structures with each other or with amylose strands (blue). Amylopectin has (16) branch points (red). Glucose resides at the nonreducing ends of the outer branches are removed enzymatically during the mobilization of starch for energy production. Glycogen has a structure that is similar to amylopectin, but is more highly branched and more compact.

GlycogenGlycogen is the main storage polysaccharide occurring in animal cells. Its structure is very similar to amylopectin, in that main chain linkages between D glucose units are (14) and the linkages at branch points are (16). Branch points occur more frequently in glycogen (about every 8 to 12 residues) than in amylopectin. Glycogen is especially abundant in hepatocytes of the liver where it may constitute as much as 7% of the wet weight of the tissue. Slightly less glycogen (about 2% by wet weight) is stored in skeletal muscle cells. Glycogen molecules occur in large granules that can be observed in the cytoplasm of cells by electron microscopy. A single glycogen molecule can weigh several million Da. Like amylopectin, glycogen molecules have many nonreducing ends at the ends of the branches, but only one reducing end. The enzymes of glycogen metabolism build up and break down glycogen to glucose units at the nonreducing ends of the molecule. Simultaneous reactions at the many nonreducing ends speed up the metabolism of the polysaccharide. As discussed in Chap. 2, the storage of glucose units in glycogen molecules has a much smaller osmotic effect on cells than would the storage of an equivalent amount of glucose as the free monosaccharide.

CelluloseCellulose is a linear homopolysaccharide composed exclusively of D glucose units held together in (ß14) linkages (Fig. 7-14). A single chain of cellulose can contain 10-to-15,000 residues. Due to the presence of ß linkages, cellulose chains fold quite differently than chains of D glucose in the starches and glycogen (see below). Cellulose molecules are insoluble in water and form tough fibers. Cellulose is found in the cell walls of plants, particularly in stalks, stems, trunks, and all the woody portions of the plant body. Cellulose constitutes much of the mass of wood, and cotton is almost pure cellulose. Vertebrate animals lack the hydrolytic enzymes (cellulases) that can cleave the (ß14) linkages between glucose units in cellulose. These enzymes are produced by many cellulolytic microorganisms. These microorganisms, such as Trichonympha (Fig. 7-15), a symbiotic protist that resides in the termite gut, allow the host to derive energy from the glucose units stored in cellulose. Similarly, cellulases produced by microorganisms living in the rumens of cattle, sheep, and goats allow these animals to obtain energy from cellulose present in soft grasses in the diet.

ChitinChitin is a linear homopolysaccharide composed of N-acetylglucosamine residues in (ß14) linkage (Fig. 7-16). The only chemical difference from cellulose is the replacement of the hydroxyl group at C-2 with an acetylated amino group. Chitin also forms extended fibers similar to those of cellulose. Like cellulose, chitin cannot be digested by enzymes found in vertebrates. Chitin is the principal component of the hard exoskeletons of nearly a million species of arthropods--insects, lobsters, and crabs, for example--and is probably the second most abundant polysaccharide in nature.

Folding of HomopolysaccharidesThe folding of polysaccharides in three dimensions follows the same principles as those governing the folding of polypeptides. Weak noncovalent interactions, particularly hydrogen bonds between -OH groups, are important in stabilizing structures. In addition, rotation about the and bonds adjacent to the oxygen atoms of glycosidic bonds between monosaccharide units have steric constraints as they do for the comparable bonds on either side of the carbons in the polypeptide backbone (Fig. 7-17). Analogous to polypeptides, polysaccharides can be represented as a series of rigid pyranose rings connected by an oxygen atom bridging the rings. Certain conformations are much more stable than others, as can be shown on a Ramachandran-like plot of energy as a function of and angles (Fig. 7-18, not covered).

Helical Structure of Starch (Amylose)The most stable three-dimensional structure for the (14) linked chains of starch and glycogen is a tightly coiled helix (Fig. 7-19b). The helix is stabilized by interchain hydrogen bonds. The glucose residues in the chain are also able to form hydrogen bonds to the surrounding solvent, which keep the polymer in solution. The average plane of each residue along the amylose chain forms a 60˚ angle with the average plane of the preceding residue (Fig. 7-19a), so the helical structure has six residues per turn. These tightly coiled helical structures produce the dense granules of stored starch or glycogen seen in many cells.

Interactions Between Cellulose ChainsFor cellulose, the most stable

conformation is that in which each chair is turned 180˚ relative to its neighbors, yielding a straight extended chain (Fig. 7-17, above). All -OH groups are available for hydrogen bonding with neighboring chains. With several chains lying side by side, a stabilizing network of interchain and intrachain hydrogen bonds produces straight, stable supramolecular fibers of great tensile strength (Fig. 7-20). The water content of cellulose fibers is low because extensive interchain hydrogen bonding between cellulose molecules satisfies their capacity for hydrogen bond formation.

The Extracellular MatrixThe extracellular space in the tissues of multicellular animals is filled with a gel-like material, the extracellular matrix (ECM), which holds cells together and provides a porous pathway for the diffusion of nutrients and oxygen to individual cells. The ECM that surrounds fibroblasts and other connective tissue cells is composed of an interlocking meshwork of heteropolysaccharides and fibrous proteins such as fibrillar collagens, elastins, and fibronectins. These heteropolysaccharides, the glycosaminoglycans, are a family of linear polymers composed of repeating disaccharide units (next two slides). They are unique to animals and bacteria and are not found in plants. One of the two monosaccharides is always either N-acetylglucosamine or N-acetylgalactosamine. The other monosaccharide is in most cases a uronic acid, usually D-glucuronic acid or its 5-epimer, L-iduronic acid. Some glycosaminoglycans contain sulfate groups attached to hydroxyl groups in ester linkage. The combination of sulfate groups and the carboxylate groups of the uronic acids gives glycosaminoglycans a very high density of negative charge, and an extended rod-like structure in solution. Glycosaminoglycans are specifically recognized by a number of proteins that bind them via electrostatic interactions. As discussed in the 7B lecture slide file, the sulfated glycosaminoglycans are attached to extracellular proteins to form the proteoglycans.

Glycosaminoglycans (I)The glycosaminoglycan hyaluronan (hyaluronic acid) consists of alternating residues of D-glucuronic acid and N-acetylglucosamine (Fig. 7-22). A single hyaluronan molecule contains up to 50,000 repeats of this disaccharide unit and has a molecular weight of several million. It forms clear, highly viscous solutions that serve as lubricant in the synovial fluid of joints and give the vitreous humor of the vertebrate eye its jellylike consistency. (The Greek hyalos means “glass”). Hyaluronan is also a component of the ECM of cartilage and tendons. In many species, a hyaluronidase enzyme in sperm hydrolyzes an outer glycosaminoglycan coat around the ovum, allowing sperm entry.

Other glycosaminoglycans differ from hyaluronan in three respects: they are generally much shorter polymers, they are covalently linked to specific proteins forming proteoglycans, and one or both monomeric units differ from those of hyaluronan. Chondroitin sulfate (Greek, chondros, “cartilage”) is a polymer of repeating D-glucuronic acid and sulfated N-acetylgalactosamine units (Fig. 7-22). It contributes to the tensile strength of cartilage, tendons, ligaments, and the wall of the aorta.

Glycosaminoglycans (II)Keratin sulfates (Greek keras, “horn”) lack uronic acid and their sulfate content is variable. The species shown in Fig. 7-22 is a repeating polymer of D-galactose and sulfated N-acetylglucosamine residues. Keratin sulfates are present in the cornea, cartilage, bone, and a variety of horny structures formed of dead cells: horn, hair, hoofs, nails, and claws.

Heparan sulfate (Greek hepar, “liver”) is produced by all animal cells and contains variable arrangements of sulfated and nonsulfated sugars. The species shown in Fig. 7-22 is a repeating polymer of sulfated L-iduronate and sulfated

D-glucosamine residues. The sulfated segments of the polymer allow it to interact with a large number of proteins, including growth factors and ECM components, as well as various enzymes and factors present in serum. Heparin is a fractionated form of heparan sulfate that is a therapeutic agent used to inhibit blood coagulation. Heparin binds to the protease inhibitor antithrombin, and causes it to bind to and inhibit thrombin, a protease essential to blood clotting. Heparin has the highest charge density of any known biological macromolecule.