Structural Biochemistry - Study Guide - E. A. Bessolitsyna 2015

CARBOHYDRATES are widely distributed in plants and animals, where they perform both structural and metabolic Functions. In plants, carbon dioxide and Water are converted via Photosynthesis into glucose, which is subsequently stored as starch or transformed into Cellulose—the structural backbone of plant Tissues. While animals can synthesize certain carbohydrates from fats and Proteins, the majority of carbohydrates are obtained from plant-based diets.

Carbohydrates are aldehyde or ketone derivatives of polyhydric alcohols (containing more than one OH group) or compounds that yield these derivatives upon Hydrolysis.

They can be classified as follows:

Monosaccharides are carbohydrates that cannot be hydrolyzed into simpler units. Depending on the number of carbon atoms in their molecules, they are classified as trioses, tetroses, pentoses, hexoses, heptoses, or octoses; they are further divided into aldoses and ketoses based on the presence of an aldehyde or ketone group.

Disaccharides yield two monosaccharide molecules (either identical or different) upon hydrolysis.

Oligosaccharides yield 3 to 6 monosaccharides upon hydrolysis, or, more commonly, Polysaccharides comprising several dozen monosaccharide residues.

Polysaccharides yield more than 6 monosaccharide molecules upon hydrolysis. They can be linear or branched. A polysaccharide composed of identical monomer units is called a homopolysaccharide, whereas one composed of different units is termed a heteropolysaccharide. It is also worth noting that the majority of polysaccharides contain over a thousand monomer residues. Examples include starch and dextrins.

Monosaccharides are carbohydrates that cannot be hydrolyzed into simpler units. Depending on the number of carbon atoms in their molecules, they are classified as trioses, tetroses, pentoses, hexoses, heptoses, or octoses; they are further divided into aldoses and ketoses based on the presence of an aldehyde or ketone group. The carbon atoms are numbered starting either from the carbon of the aldehyde group (carbon-1) or from the one closest to the ketone group.

Compounds that share the same structural formula but differ in their spatial configuration are called isomers. The formation of such isomers is made possible by the presence of asymmetric carbon atoms within the molecule (to which four different atoms or groups are attached). The number of possible isomers for a given compound depends on the number of asymmetric carbon atoms (n) and is equal to 2ⁿ. Consequently, glucose, with its four asymmetric carbon atoms, has 16 isomers. The most important types of glucose isomers are outlined below.

Stereoisomerism or D- and L-isoforms:

Many organic molecules contain a carbon atom bonded to four different groups, and such a molecule can be visualized as a methane-like tetrahedron with the groups located at its vertices. This atom is referred to as a chiral center. In monosaccharides, assignment to the D- or L-form is determined by the spatial orientation of the hydroxyl (OH) group relative to the chiral carbon atom. If the OH group is on the left, it is the L-form; if on the right, it is the D-form. However, glyceraldehyde is the only compound that strictly follows this rule, as it contains a single chiral atom (Figure 1 A). All other carbohydrates contain multiple chiral atoms, any of which might theoretically be used to determine the stereochemical series. It was established convention that the stereochemistry-defining atom is the last chiral carbon of the molecule (the penultimate carbon atom). Nevertheless, altering THE POSITION OF the hydroxyl group exclusively at this carbon atom does not produce a true mirror image, which is a fundamental requirement for the existence of two stereoisomeric forms. This led to METABOLISM/2.html">THE CONCEPT OF carbohydrate series, meaning that all carbohydrates can be derived or synthesized from a minimal carbohydrate (a triose); thus, this triose serves as the precursor or parent compound. Since glyceraldehyde is the only triose with optical properties, this specific molecule acts as the parent compound for all others. The stereochemical assignment of the parent compound (D- or L-form) determines the configuration of all subsequent monosaccharides synthesized from it (Figure 2). During the synthesis of carbohydrates containing more than three carbon atoms (glyceraldehyde), each successive carbon atom

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Figure 1. Structural formulas of carbohydrate stereoisomers; * indicates asymmetric (chiral) atoms. A — glyceraldehyde, with * denoting the asymmetric atom that determines the D- or L-configuration; B — glucose stereoisomers, with the large * marking the asymmetric carbon atoms that determine the stereoisomeric series.

is added via the aldehyde group, causing the chiral atom of the parent compound to move further and further away from the first carbon atom, while ultimately remaining the last asymmetric atom—simply put, the penultimate carbon atom (Figure 2). The presence of asymmetric carbon atoms is responsible for the optical activity of the compound. When a beam of plane-polarized water or light passes through a solution of an optical isomer, the plane of polarization is rotated either to the right (dextrorotatory isomer, +) or to the left (levorotatory isomer, —). Compounds are designated as D(—), D(+), L(—), or L(+); this nomenclature indicates structural relationship to D- or L-glyceraldehyde, but does not necessarily imply the same sign of optical rotation. For example, the natural form of fructose is the D(—) isomer. When D- and L-isomers are present in equal amounts, the mixture exhibits no optical activity, as the rotations of the individual isomers cancel each other out. Such mixtures are called racemic (or DL-mixtures). Synthetically produced compounds are invariably racemic because the probability of forming each isomer is equal in such processes.

Figure 2. Structure OF THE D-carbohydrate series

2. Pyranose and furanose ring structures.

Aldehyde or ketone groups readily react with hydroxyl groups to form hemiacetal or hemiketal linkages. Most commonly, this reaction occurs intramolecularly, resulting in the cyclization of the monosaccharide and the formation of a modified oxygen-containing heterocycle. Five- and six-membered rings are the most stable. Five-membered carbohydrate rings resemble the furan molecule and are therefore termed the furanose form, whereas six-membered rings resemble pyran and are called the pyranose form (Figure 3). All hydroxyl groups pointing to the right in the Fischer projection end up below the ring plane, while those pointing to the left end up above it. Ketoses can also adopt ring structures (e.g., D-fructofuranose or D-fructopyranose). In a glucose solution, over 99% of the molecules exist in the pyranose form, and less than 1% in the furanose form.

Figure 3. Pyranose and furanose forms of carbohydrates

3. α- and β-anomers. Ring closure results in the formation of a hydroxyl group at the hemiacetal or hemiketal group, known as the hemiacetal or hemiketal hydroxyl. This group can be positioned below the ring, yielding the α-anomer, or above the ring, resulting in the β-anomer. The cyclic structure is maintained in solution, yet isomerization occurs regarding the positions of the aldehyde and ketone carbon atoms. This yields a mixture of 36% α-glucopyranose and 63% β-glucopyranose, with the remaining 1% consisting primarily of α- and β-anomers of glucofuranose. The equilibrium established as described above is accompanied by a phenomenon known as mutarotation: the hemiacetal ring opens and recloses, which may alter the orientation of the —H and —OH groups at carbon 1. It is hypothesized that this process proceeds via an intermediate hydrated linear (acyclic) molecule, although polarographic data indicate that the acyclic form of glucose accounts for a mere 0.0025%. In solution, glucose is dextrorotatory, which explains its alternative name—dextrose (from dextro, meaning right)—frequently used in clinical practice (Figure 4).

Figure 4. Structural formulas of glucose anomers. α- and β-anomers

4. Epimers. Isomers that differ in configuration due to the orientation of —H and —OH groups at asymmetric carbon atoms—unrelated to stereoisomer classification—are termed epimers. The biologically most significant glucose epimers are mannose and galactose, formed via epimerization at carbon atoms 2 and 4, respectively (Figure 5).

Figure 5. Epimerization of glucose

5. Aldose-ketose isomerization. Fructose shares the same chemical formula as glucose but differs in its structural formula, as fructose contains a potential ketone group at position 2, whereas glucose features a potential aldehyde group at position 1.

Six-membered rings formed by single bonds, both in cyclohexane and in sugars, typically adopt a chair conformation, as exemplified by glucose (Figure 6). Additionally, six-membered rings can assume the less stable boat conformation. They readily interconvert into one another through intermediate skew-boat Conformations.

Five-membered rings form an envelope conformation.

Figure 6. Conformation of six-membered rings

Monosaccharides are hydrophilic and, consequently, readily soluble in water. Their solubility profiles are remarkably similar, making the Separation of individual monosaccharides quite challenging. Monosaccharides do not absorb light in either the visible or ultraviolet Regions of the spectrum; hence, their concentrations cannot be determined using Spectrophotometric Methods. Because monosaccharides exist in nature either in solution or in crystalline form within physiological Temperature ranges, melting and boiling points are not applicable.

Structurally, monosaccharides contain Two Types of functional groups: alcoholic and aldehyde (or ketone) groups. Consequently, they undergo reactions characteristic of these groups. Monosaccharides participate in numerous Chemical Reactions that are critically important for both the practical utilization and investigation of carbohydrates. The reactions discussed in this section illustrate either the most prominent Properties of Carbohydrates or typical reaction pathways frequently encountered in their metabolism.

Ether formation. This reaction involves the alcoholic groups, leading to the formation of either inorganic or organic esters (ethers with alcohols or halides, or esters with acid anhydrides). The hemiacetal alcoholic group is the most reactive; therefore, it preferentially participates in ether-forming reactions. Phosphoric and acetic acid esters hold a unique position in biochemistry and are widely found in many carbohydrates and their derivatives.

Alkylation (Figure 7)

Figure 7. Alkylation

During interaction with alcohols, only the hemiacetal hydroxyl group reacts because alcohols exhibit low reactivity, resulting in the formation of an ether at the hemiacetal hydroxyl position. Halides are more reactive and thus form ethers with all free hydroxyl groups of the monosaccharide.

Acylation (Figure 8)

Figure 8. Acylation

Esterification reactions typically involve alcohol and carboxyl groups, but carboxylic acids have low reactivity and do not react directly with monosaccharides. Instead, carboxylic acid anhydrides are employed in acylation reactions; their high reactivity enables the modification of all free hydroxyl groups.

Formation of inorganic acid esters (Figure 9)

Figure 9. Formation of inorganic acid esters

Inorganic acids can be either strong or weak, which accounts for the differences in their interaction with monosaccharides. Strong acids, such as sulfuric acid, react with the hemiacetal hydroxyl group to form an ester, whereas weak acids react only after prior acylation, modifying exclusively the ester formed by the hemiacetal group.

Dehydration. In high-concentration strong mineral acids, dehydration of pentoses and hexoses takes place (Figure 10). The resulting furfural and hydroxymethylfurfural readily polymerize, yielding a brown resin. They also condense with various phenols to form characteristically colored products; many Color Reactions for carbohydrates are based on this property.

Figure 10. Dehydration

Alkaline rearrangement. In a cold dilute alkaline solution, glucose yields mannose and fructose. The reaction mechanism likely involves enolization, accompanied by the dissociation of hydrogen from the carbon atom adjacent to the carbonyl group. This is how epimers—glucose and mannose—are formed.

Oxidation of monosaccharides. Monosaccharides can be oxidized in numerous ways.

Mild oxidation to aldonic acids. In hot alkaline solutions, monosaccharides react with certain oxidizing agents, such as Cu²⁺, Ag⁺, and Fe (CN) ^-, yielding a mixture of carbohydrate oxidation products and changing the color of the oxidizing agent (Figure 11). Under these conditions, the aldehyde or ketone group is oxidized. These redox reactions serve as tests for reducing sugars, i.e., carbohydrates with a free anomeric aldehyde carbon atom. D-Gluconic acid is formed as a salt when D-glucose interacts with hypoiodites in an alkaline medium. This reaction is specific to aldoses and is used to distinguish them from ketoses.

Figure 11. Mild oxidation of monosaccharides to aldonic acids

More vigorous oxidation to aldaronic (alduronic) acids occurs in the presence of "moderate" oxidizing agents, such as low concentrations of nitric acid (Figure 12). This reaction also occurs in nature, albeit under different conditions. As a result, the hydroxyl group at the terminal carbon atom is oxidized.

Figure 12. Oxidation to alduronic acids

Harsh oxidation to aldaric acids takes place under drastic conditions, such as high concentrations of strong acids like nitric acid. Consequently, both groups—the aldehyde and the terminal alcohol group—are oxidized, yielding a dicarboxylic acid, or aldaric acid (Figure 13).

Figure 13. Oxidation to aldaric acids

Reduction of monosaccharides. Both the aldehyde group and alcohol groups can undergo reduction.

Reduction of the aldehyde group. D-Glucose and L-sorbose are reduced by hydrogen gas in the presence of a suitable metal catalyst to form sorbitol (Figure 14).

Figure 14. Reduction of the aldehyde group

Reduction of the alcohol group. Within The Cell, ribose is converted into deoxyribose (Figure 15). This reaction occurs naturally, though under somewhat different conditions. It represents one of the primary pathways for the synthesis of deoxy sugars.

Figure 15. Reduction of the alcohol group

Formation of aminosugars. The synthesis of aminosugars also occurs in nature, but the mechanism and the targeted groups differ; in the artificial system, the hemiacetal group enters the reaction as the most reactive one (Figure 16).

Figure 16. Formation of aminosugars

Chain shortening. This process is associated with oxidation under mild conditions. The carboxyl group at the terminal position is unstable and easily cleaves from the molecule, resulting in a carbohydrate that is shorter by one carbon atom (Figure 17).

Figure 17. Chain shortening

Chain elongation. This is the result of a reaction with cyanide, followed by hydrolysis to form an acid. The reduction of the carboxyl group leads to the formation of a monosaccharide that is one carbon atom longer (Figure 18).

Figure 18. Chain elongation

Hydrolysis of poly- and oligosaccharides. A hydrolysis reaction is the Cleavage of a polymer involving a water molecule. In the case of poly- and oligosaccharide hydrolysis, the glycosidic bond is attacked. As a result of the reaction, monomeric units (monosaccharides) are cleaved off. Hydrolysis reactions of poly- and oligosaccharides, followed by the Analysis of the hydrolysis products, are used for the qualitative and Quantitative determination of polysaccharide composition.

Some qualitative reactions used to identify monosaccharides.

Aldoses, ketoses

Reagent: α-naphthol (Molisch's test), Tryptophan aminoguanidine.

More sensitive for ketoses.

Ketohexoses

Reagent: resorcinol (Seliwanoff's test).

Ketohexoses, ketopentoses, methylpentoses, dihydroxyacetone

Reagent: Cysteine/carbazole.

All carbohydrates, including uronic acids and deoxypentoses

Reagent: carbazole.

Characteristic coloration with all carbohydrates.

Many carbohydrates, including polysaccharides; most commonly used for hexoses.

Reagent: cysteine/H2SO₄, anthrone.

Variable coloration with different carbohydrates.

Pentoses, heptuloses, uronic acids

Reagent: Orcinol.

Interference from other carbohydrates can be eliminated by independent methods; uronic acids are decarboxylated to pentoses prior to the reaction.

Uronic acids

Reagent: Naphthylresorcinol.

Hexosamines

Reagent: Acetylacetone-p-dimethylaminobenzaldehyde.

Hexosamines

Reagent: Nitrite/indole.

Aminosugars do not develop color without prior deamination with nitrite.

Mono- and dideoxypentoses

Reagent: Diphenylamine.

Deoxypentoses

Reagent: Tryptophan/HClO₄, indole/HCl, leucofuchsin (Feulgen reaction).

Sialic acids

Reagent: Thiobarbituric acid.

The functions of monosaccharides are highly diverse and depend on the number of carbon atoms they contain.

Trioses are intermediates in carbohydrate and Lipid Metabolism.

Tetroses are Carbohydrate Metabolism intermediates that can be components of polysaccharides.

Pentoses are carbohydrate metabolism intermediates that can be components of polysaccharides and NUCLEOTIDES.

Hexoses—glucose and fructose—are the primary sugars in carbohydrate Energy Metabolism and constituents of polysaccharides.

Heptoses are intermediates in carbohydrate metabolism.

D-ribose

Found in molecules or substances such as: nucleotides, Coenzymes, RNA.

Biological significance: a component of Nucleic Acids, coenzymes (NAD, NADP, FAD), and nucleotides; an intermediate in the Pentose Phosphate Pathway.

D-Ribulose

Found in molecules or substances such as: formed during metabolism.

Biological significance: an intermediate compound of The pentose phosphate pathway.

D-Arabinose

Found in molecules or substances such as: gum arabic, plum and cherry pulp.

Biological significance: a component of Glycoproteins.

D-Xylose

Found in molecules or substances such as: wood gum, Proteoglycans, glycosaminoglycans.

Biological significance: a component of glycoproteins.

D-Lyxose

Found in molecules or substances such as: lyxoflavin.

Biological significance: a component of lyxoflavin isolated from Heart Muscle.

L-Xylulose

Found in molecules or substances such as: an intermediate product of uronic acid metabolism.

Biological significance: an intermediate product of uronic acid metabolism.

D-Glucose

Found in molecules or substances such as: fruit juices, starch, sucrose, lactose, maltose

Biological significance: the primary bodily sugar, involved in energy metabolism and serves as a precursor for Other Compounds.

D-Fructose

Found in molecules or substances such as: honey, sucrose, lactose, inulin.

Biological significance: is converted into glucose and can be utilized in the same metabolic pathways.

D-Galactose

Found in molecules or substances such as: lactose, glycoproteins, Glycolipids.

Biological significance: it is converted into glucose and can be utilized in the same metabolic pathways.

Monosaccharide derivatives include monosaccharide esters, alduronic acids, aminosugars, deoxysugars, and Glycosides.

All monosaccharide derivatives are components of polysaccharides. Beyond this primary function, they can also perform specific roles of their own.

Alduronic acids participate in the synthesis of Vitamin C and in detoxification processes.

Deoxysugars are components of nucleotides (the monomers of DNA).

Aminosugars are constituents of Antibiotics.

Glycosides

Figure 19. Structure of certain glycosides. A—streptomycin, B—ouabain

Glycosides are compounds formed by the Condensation of a monosaccharide (or a monosaccharide residue within a more complex sugar) with the hydroxyl group of another compound, which may be another monosaccharide or a non-carbohydrate substance (referred to in this case as an aglycone). The glycosidic bond is formed as a result of a reaction between the hemiacetal (hemiketal) group of the monosaccharide and the alcoholic group of another compound; such a bond is designated as an O-glycosidic bond. Alternatively, the hemiacetal (hemiketal) hydroxyl group can react with the amino group of another compound to form an N-glycosidic bond. If the hemiacetal group belongs to glucose, the resulting compound is termed a glucoside; if to galactose, a galactoside, and so forth. Glycosides are found in many Medicinal Plants and spices, and they are also components of animal tissues. Aglycones can include methanol, glycerol, various sterols, or phenols. Glycosides of major medical importance, such as those affecting cardiac function (cardiac glycosides), contain Steroids as their aglycone component; for instance, the glycoside ouabain—an inhibitor of cell membrane Na/K-ATPase—has been isolated from foxglove (Digitalis) and Strophanthus. A number of antibiotics, notably streptomycin, also belong to glycosides (Figure 19). Nucleotides and nucleosides are classic examples of N-glycosides. However, the simplest glycosides are disaccharides, in which the aglycone is simply another monosaccharide molecule.