Richard F. Daley and Sally J.

Daley
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Organic
Chemistry
Chapter 24

Carbohydrates
24.1 Classification of Carbohydrates 24.2 Monosaccharides 1253 24.3 Cyclic Forms of Monosaccharides Sidebar - The Sweet Taste 24.4 Reactions of Monosaccharides 24.5 Oxidation and Reduction Reactions 24.6 Changing the Chain Length 24.7 Fischer Proof of Glucose Structure 24.8 Glycolysis - I 1275 24.9 Glycolysis - II 1280 Sidebar - Arsenic Poisoning 24.10 Glycoside Formation 1286 24.11 Disaccharides 1291 24.12 Polysaccharides 1291 Key Ideas from Chapter 24 1250 1256 1260 1263 1265 1268 1271

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Copyright 1996-2005 by Richard F. Daley & Sally J. Daley All Rights Reserved. No part of this publication may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, recording, or otherwise, without the prior written permission of the copyright holder.

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Chapter 24

Carbohydrates

Chapter Outline
24.1 24.2 24.3 24.4 24.5 24.6 24.7 24.8 24.9 24.10 24.11 24.12 Classification of Carbohydrates
An examination of the various categories of mono- and polysaccharides

Monosaccharides
The structures of the monosaccharides

Cyclic Forms of Monosaccharides

The hemiacetal forms of monosaccharides

Reactions of Monosaccharides
Monosaccharides react much like alcohols, aldehydes, and ketones

Oxidation and Reduction Reactions

Oxidizing and reducing the terminal carbons of monosaccharides

Changing the Chain Length

Reactions that modify the length of the chain in a monosaccharide

Fischer Proof of Glucose Structure

Emil Fischers proof of the relative stereochemistry of the stereogenic centers in glucose

Glycolysis - I
A study of the mechanism for glycolysisconversion of glucose to pyruvate

Glycolysis - II
Continuing the study of the mechanism of glycolysis

Glycoside Formation
Glycosides and glycosidic bonds

Disaccharides
The structures of disaccharides

Polysaccharides
The structures of polysaccharides

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Objectives
Know the various types of carbohydrates Be able to draw the Fischer and Haworth projections of monosaccharides Recognize that the reactions of monosaccharides and simpler organic molecules follow the same pathways Understand the logic of the Fischer proof for the stereochemistry of glucose See the similarity of enzyme-catalyzed reaction pathways and the reaction pathways studied in earlier portions of this book Understand the structure of glycosides Know the structural characteristics of di- and polysaccharides

y weight, carbohydrates occur more frequently in nature than any other class of carbon-containing compounds. Carbohydrates transport and store energy in both animals and plants. They also provide the structural framework for plants. Nearly all plants and animals synthesize carbohydrates. Many carbohydrates are polymers of glucose. Glucose is converted to carbon dioxide and water to provide energy for animals. Plants gain energy by converting carbon dioxide and water to glucose. Animals store excess chemical energy as glycogen, and plants store excess energy as starch. Plants also convert glucose to cellulose, which provides the structural framework for plants. People depend on carbohydrates for a number of their activities. They consume carbohydrates and convert them to energy. They use cellulose in the form of wood for heat, housing, and furnishings. People convert wood to paper for newspapers, magazines, and this book. They make clothing from the cellulose fibers of cotton and from the cellulose derivatives rayon and cellulose acetate.

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At first glance, carbohydrate chemistry may seem complicated, but the principles of carbohydrate chemistry are the same as the chemical principles you have studied to this point. The molecules form using the same structural chemistry as do other types of compounds. Carbohydrates contain ketone, aldehyde, and alcohol functional groups. Thus, their chemistry is similar to the chemistry of those groups.

24.1 Classification of Carbohydrates

The literal definition of carbohydrate is the hydrate of carbon. Carbohydrates have the molecular formula Cm(H2O)n.

Monosaccharides are the simplest carbohydrate units. Disaccharides, trisaccharides, and polysaccharides are dimers, trimers, and polymers of monosaccharides. An aldose is a polyhydroxy aldehyde, and a ketose is a polyhydroxy ketone. Both are carbohydrates.

Early chemists used the term carbohydrate to describe a class of compounds that have the general molecular formula Cm(H2O)n. This formula suggests that carbon atoms somehow bond to water molecules to form "hydrates of carbon." Early chemists thought that carbohydrates were simply hydrated carbons because heating carbohydrates produced water and carbon. However, this view is inaccurate. Although most of the carbohydrate examples presented in this chapter have the empirical formula CH2O, their structures are more complex than suggested by the term "hydrates of carbon." Biochemists often use the term sugar synonymously with carbohydrate. Monosaccharides, or "simple sugars," are the simplest of the carbohydrate units because they cannot be hydrolyzed to other simpler carbohydrates. Disaccharides, trisaccharides, and polysaccharides are dimers, trimers, and polymers of monosaccharides. Most disaccharides and polysaccharides can be hydrolyzed readily to monosaccharides. Biochemists classify monosaccharides as either polyhydroxyaldehydes or polyhydroxyketones. Biochemists call a polyhydroxyaldehyde monosaccharide an aldose and a polyhydroxyketone a ketose. The two most familiar monosaccharides are glucose and fructose. Figure 24.1 shows the structures of these two compounds. The left structure of each type is a three-dimensional representation. The right structure is a Fischer projection of the same molecule. Glucose is an example of an aldose and fructose is an example of a ketose.

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CHO HO H H HO H H OH CHO CH2OH OH H HO H H C C C C OH H OH OH H HO H H

CHO OH H OH OH CH2OH

CH2OH Glucose

O HO H H HO H CH2OH CH2OH OH HO H H

CH2OH C C C C O H OH OH HO H H

CH2OH C O H OH OH CH2OH

CH2OH Fructose

Figure 24.1. Structures of glucose and fructose. The left structure of each group shows the all-eclipsed structure on which the other two are based. The center structure shows the bonds with wedges and dashes. Most of the time, however, biochemists draw the structures as Fischer projections, as shown on the right. See Section 11.5, page 000, for more details of the Fischer projection.

Biochemists use Fischer projections to draw monosaccharides because Fischer projections are easy to draw and clearly show the stereochemical relationships involved. Emil Fischer designed the Fischer projection to draw the chemical structures in his proof of the structure of glucose. For this proof, he received the Nobel Prize in 1902. As shown in Figure 24.1, the vertical lines in a Fischer projection are either in or behind the plane of the paper. Horizontal lines project out from the plane of the paper. For reference purposes, when drawing carbohydrate structures of an aldose, always place the aldehyde carbon on the top. When drawing a ketose, place the ketone closer to the top of the drawing.

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The Haworth projection is a standardized method, similar to the Fischer projection, that is used to represent the cyclic hemiacetal forms of monosaccharides. Haworth projections can also be linked to show di, tri, and polysaccharides.

Monosaccharides readily form cyclic hemiacetals when the sugar forms a five- or six-membered ring. The hemiacetal of glucose is particularly stable because the groups on the ring are equatorial. In the illustration of the acetal of glucose, the structure on the right is a Haworth projection. The Haworth projection is named for Sir W. N. Haworth from the University of Birmingham in England. He won the Nobel Prize in 1937 for his work in carbohydrate chemistry. The Haworth projection is equivalent to the cyclic structure on the left. Although biology and biochemistry books widely use the Haworth projection, chemists generally prefer to use the chair conformation because the chair conformation is closer to the shape of the molecule than the flat Haworth projection. In this book we will use both respresentations.
H HO HO CH2OH H OH H H OH Chair H H OH OH H O OH H H OH

CH2OH H O

Haworth projection

A hemiacetal form of D-glucose

A disaccharide is a sugar that hydrolyzes to two monosaccharide molecules per disaccharide molecule. For example, sucrose, "sugar" or "table sugar," readily hydrolyzes to one molecule of glucose and one molecule of fructose.
H3O

Sucrose

Glucose

Fructose

Both monosaccharides and disaccharides are readily soluble in water and most have a characteristic sweet flavor. Polysaccharides are carbohydrates that hydrolyze to a number of monosaccharide molecules per polysaccharide. Two common polysaccharides are starch and cellulose. Starch is more water-soluble than cellulose. A starch readily adds more monosaccharide units to store excess energy or removes monosaccharide units to provide energy to the organism. Starch easily hydrolyzes in the laboratory or in plant or animal systems. Cellulose is a larger molecule and is not water-soluble. It does not easily hydrolyze, except by specialized organisms or high temperature acid-catalyzed hydrolysis.

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Starch Cellulose

H3O H3O

~300 Glucose ~3000 Glucose

Of all the monosaccharides, glucose is by far the best known. Through the process of photosynthesis, plants form glucose in great quantities from carbon dioxide and water. Environmental chemists estimate that photosynthesis produces approximately 1011 to 1012 tons of glucose per year. This amount of glucose represents an enormous resource of stored energy. Plants use the energy formed by photosynthesis directly. Animals use that energy by consuming the plants. Exercise 24.1 Draw the wedge-and-dash and the Fischer projections of the enantiomers of glucose and fructose.

24.2 Monosaccharides
Monosaccharides usually contain from three to seven carbons. The general name for each chain length is triose (3 carbons), tetrose (4 carbons), pentose (5 carbons), hexose (6 carbons), and heptose (7 carbons). Biochemists often combine these names with the aldose or ketose designations. Thus, glucose, an aldose with six carbons, is an aldohexose, and fructose, a ketose with six carbons, is a ketohexose. Notice that all the monosaccharide illustrations used thus far in this chapter show the OH group on the chiral carbon furthest from the carbonyl group to the right side of the structure. Chemists in the late 19th century made the observation that nearly all naturally occurring monosaccharides degrade to the dextrorotatory (+) enantiomer of glyceraldehyde, the only aldotriose. Only a very few degrade to the levorotatory () enantiomer of glyceraldehyde.
CHO H HO H CH2OH CHO OH CH2OH HO H CHO HO CH2OH
L-Glyceraldehyde (S-(-)-glyceraldehyde)

CHO H CH2OH

D-Glyceraldehyde (R-(+)-glyceraldehyde)

Because degradation shortens the chain from the carbonyl end of the chain, the single chiral center in glyceraldehyde corresponds to the

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chiral carbon at the end of the carbon chain away from the carbonyl group.
CHO H HO H H OH H OH OH HO H H CHO H OH OH H H CHO OH OH H CHO OH

CH2OH D-(+)-Glucose

CH2OH D-(-)-Arabinose

CH2OH D-(-)-Erythrose

CH2OH D-(+)-Glyceraldehyde

The fact that the degradation leads to either (+) or () glyceraldehyde allows you to use the specification of the stereochemistry of the glyceraldehyde in the name of the monosaccharide. If the stereochemistry is the same as the (+)glyceraldehyde, then prefix the name by a D. If the stereochemistry is the same as ()-glyceraldehyde, then use the L prefix. The D,L system of carbohydrate nomenclature is of limited use because it specifies the conformation of only one stereogenic center. It gives no information about any other stereogenic centers that may be present in the molecule.
D

And L Notation vs Optical Activity

Note that the D and L notations refer only to the stereochemistry of the chiral carbon furthest from the carbonyl group of a monosaccharide. This notation has nothing to do with the direction that a particular sugar rotates a plane of polarized light. A sugar with the D prefix may rotate a plane of polarized light either to the right or to the left. If you want to know the direction that a particular sugar rotates polarized light, you will need to determine it experimentally.

Epimers are identical structures except for the configuration of a single stereogenic center.

A pair of structures that differ in the configuration of only one carbon atom are called epimers. For example, mannose and glucose are identical except for the configurations at C2. Generally, when referring to a pair of epimers, biochemists specify the differing carbon atom with the carbon number. For example, glucose and mannose are C2 epimers, and glucose and galactose are C4 epimers.

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C2 Epimers CHO HO HO H H H H OH OH CH2OH

D-Mannose

CHO H HO H H OH H C4 Epimers H HO OH OH CH2OH

D-Glucose

CHO OH H H OH CH2OH
D-Galactose

HO H

Exercise 24.2 Draw Fischer projections of D-allose, the C3 epimer of D-glucose, and of L-altrose, the enantiomer of the C2 epimer of D-allose. Figure 24.2 shows all of the D-aldotrioses, D-aldotetroses, Daldopentoses, and D-aldohexoses. The arrangement of these sugars in this table allows you to easily recall their structures. Below each structure are two other structures. These two lower structures are C2 epimers and have one more carbon atom than the structure above.
CHO H OH CH2OH D-Glyceraldehyde CHO H H OH OH CH2OH D-Erythrose CHO H H H OH OH OH CH2OH D-Ribose CHO H H H H OH HO OH OH OH CH2OH D-Allose H H H CHO H OH OH CH2OH D-Altrose H H H HO H H CHO H OH OH CH2OH D-Arabinose CHO OH HO H OH OH CH2OH D-Glucose HO H H CHO H H OH CH2OH D-Mannose H H H H HO H CHO OH H OH CH2OH D-Xylose CHO OH HO OH H OH CH2OH D-Gulose H HO H CHO H H OH CH2OH D-Idose H HO H HO HO H HO H CHO H OH CH2OH D-Threose CHO H H OH CH2OH D-Lyxose CHO OH HO H H OH CH2OH D-Galactose HO HO H CHO H H H OH CH2OH D-Talose

OH HO

OH HO

OH HO

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Figure 24.2. Configuration of the D aldotrioses, aldotetroses, aldopentoses, and aldohexoses. The arrangement of the structures shows the structural relationships between the various structures.

Exercise 24.3 Figure 24.2 shows only D-sugars. Draw the Fischer projections of Lxylose, L-mannose, and L-idose.

24.3 Cyclic forms of Monosaccharides

Acetals are found in Section 7.5, page 000.

An alcohol reacts with a carbonyl group producing a hemiacetal. Two moles of alcohol produce an acetal.
O C ROH OH C OR ROH C OR Hemiacetal Acetal OR

When both the alcohol and the carbonyl group are in the same compound, intramolecular nucleophilic addition can take place to form a cyclic hemiacetal. Five- and six-membered cyclic hemiacetals form particularly easily.
O HO H O OH

A pyranose ring is named for the cyclic six-membered ether pyran. Furanose is named for the cyclic fivemembered ether furan.

Many monosaccharides exist in equilibrium between the open chain form and the cyclic hemiacetal form. In aqueous solution, glucose exists almost entirely in the sixmembered pyranose ring. The ring forms by a nucleophilic addition of the hydroxyl oxygen at C5 to the aldehyde at C1. In aqueous solution, fructose exists about 20% as a five-membered furanose ring formed by the addition of the hydroxyl group oxygen at C5 to the C2 ketone. Biochemists derive the names pyranose and furanose from the names of the simple cyclic ethers pyran and furan.

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Pyran

Furan

Biochemists usually represent the pyranose and furanose rings as the Haworth projection rather than the Fischer projection. Although the Haworth projection shows the relationship between the substituents on the ring, it is misleading because, in reality, the rings are not flat. An easy method to convert from the Fischer projection of a hemiacetal to a Haworth projection starts with the Fischer projection of the acyclic, or open chain, molecule that forms the hemiacetal. For reference, lay the Fischer projection on its side with the carbonyl group to the right and toward the rear. Hydroxyl groups that are on the right in a Fischer projection are down in the Haworth projection. Conversely, hydroxyl groups that are on the left in a Fischer projection are up on the Haworth projection. The CH2OH group is always up in D sugars and always down in L sugars. Arrange the ring by placing the oxygen in the ring at the upper right. Figure 24.3 shows the conversion of the Fischer projection of D-glucose to the Haworth projection of the pyranose form of D-glucose.
C1 CHO H HO H H CH2OH C5 OH H H CH2OH H H H Rotate C4 H C1 H Turn OH C1 OH H O OH H Sideways C4 OH C4-C5 bond O C4 OH OH OH OH H C5 H OH CH2OH OH Fischer -H CH2OH CH2OH C5 C5 H O O OH H H H H C4 OH H C1 + C4 OH H C1 H OH OH OH H OH H OH
D-Glucose

H C5

D-Glucose

Haworth

Figure 24.3. Converting the Fischer projection of projection of the hemiacetal.

D-glucose

to the Haworth

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Figure 24.4 shows the conversion of the Fischer projection of D-ribose to the Haworth projection.
C1 CHO H H H C4 C3 OH OH OH CH2OH
D-Ribose

H CH2OH H HOH2C H C4 OH Rotate Turn C1 C4 H H H H C1 H sideways C3-C4 bond O O HO C3 H C3 OH OH OH OH -H HOH2C C4 H O H H C1 HOH2C + C4 H O OH H C1

Fischer

H C3 OH OH OH
D-Ribose

H C3 H OH OH Haworth

Figure 24.4. Converting the Fischer projection of D-ribose to the Haworth projection of the hemiacetal.

To name the hemiacetal form of the monosaccharides, replace the se ending from the name of the open chain form of the sugar with the pyranose or furanose ending. For example, the cyclic form of glucose is called glucopyranose and the cyclic form of ribose is called ribofuranose. Exercise 24.4 Draw the Haworth projection of the pyranose form of D-gulose. With the formation of the pyranose or furanose ring, the reaction produces a new stereogenic center at the site that was the carbonyl carbon. Because the carbonyl group is planar and can react from either side, the reaction produces a pair of diastereomers. These diastereomers are called anomers and the hemiacetal carbon atom is called an anomeric center. Glucose forms a 36:64 mixture of the two anomers designated as and anomers. With both the D and L isomers, the anomer has the OH group at C1 and the CH2OH group on C5 trans, but in the anomer both groups are cis.

Anomers are the diastereomers produced when a monosaccharide forms a hemiacetal. The former carbonyl carbon is called the anomeric center.

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CH2OH H O H H OH H OH OH H

CH2OH H OH O OH H H OH

OH H

OH H

-D-glucopyranose

-D-glucopyranose

Anomers of the cyclic forms of D-glucose

For more on diastereomers see Section 11.5, page 532.

Mutarotation is the conversion, in water, of one anomer to the other anomer.

Both anomers of glucose can be isolated and purified. As expected from diastereomers, they have different physical properties. Pure -D-glucopyranose has a melting point of 146oC and a specific rotation, []D, of +112.2o, whereas pure -D-glucopyranose has a melting point of 152oC and an []D of +18.7o. The pure anomer crystallizes from water at room temperature. The pure anomer crystallizes when the water is allowed to evaporate above 97oC. If you dissolve a sample of either anomer of D-glucose in water, the optical rotations change and ultimately converge to a constant value of +52.6o. This phenomenon, known as mutarotation, is due to the slow conversion of either anomer to the 36:64 equilibrium mixture of the two anomers. Mutarotation occurs via a reversible ring opening of either anomer to the open chain form followed by a ring closure.
H HO HO H H C2 CH2OH H OH H H C1 Rotate C1-C2 Bond H
D-Glucose

HO

CH2OH H OH O H C2 C1 H OH

HO

OH O

CH2OH H O HO C2 C1 HO H H OH H OH -D-Glucopyranose

H CH OH 2 H O HO C2 C1 HO H H OH H

OH

-D-Glucopyranose

Exercise 24.5

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Draw the Haworth projection of the two anomers of the furanose form of fructose. Exercise 24.6 Explain why there is approximately twice as much of the -Dglucopyranose at equilibrium in water as there is of the -Dglucopyranose form. Exercise 24.7 All sugars with the hemiacetal structure exhibit mutarotation. For example, -D-galactopyranose has an []D of +150.7o, and -Dgalactopyranose has an []D of +52.8o. If either anomer is dissolved in water and allowed to reach equilibrium, the []D of the resulting solution is +80.2o. Draw the pyranose forms of both anomers and calculate the percentages of each in solution.

[Sidebar] The Sweet Taste

Sugar is sweet, but many artificial sweeteners are sweeter. Sodium cyclamate is 30 times sweeter than sucrose, aspartame is 160 times sweeter, sodium saccharin is 500 times sweeter, and 5-nitro-2propoxyaniline is 4100 times sweeter than sucrose. Looking at the structures of these sweeteners, the relationship between these various structures and their sweetness is hard to see.
CH2OH HO HO O NHSO3 Na OH O CH2OH O OH Sucrose CH2OH OH Sodium cyclamate

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O O COOCH3 N Na SO2 Sodium saccharin OCH2CH2CH3 5-Nitro-2-propoxyaniline O2N NH2 NH2CHCNHCHCH2 CH2COOH Aspartame

The theory of sweet taste is called the tripartite model. Basically, the tripartite model says that a sweet tasting compound must have three parts: A, B, and . A is either a nitrogen atom or an oxygen atom bearing a hydrogen. B is any atom that can form a hydrogen bond with a hydrogen bond acceptor. The most common B atoms are nitrogen or oxygen. The component is not a specific functional group but is any hydrophobic portion of the molecule. These three components for sweetness require a specific geometric arrangement. The required distance between A and B is 260 pm, between B and is 350 pm, and between and A components is 550 pm. The sweetener binds to an active site of the glycophore (sweet tasting site) in a taste bud. This active site has the same three components that the sweet molecule has.

A H B

B H A

The tripartite model of glycophore-sweetener interaction

In -D-glucopyranose, the OH groups on C3 and C4 as well as the CH2OH group on C5 are cis to one another. This arrangement is the precise design that fits well into the tripartite model for sweetness. Both OH groups form hydrogen bonds with the A and B components. If the hydroxyl group is oriented away from the component, the CH2OH is hydrophilic.

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HO CH2 O H B O H A H O OH OH

The tripartite model applied to -D-glucose

This model also fits well with the saccharin molecule. The B component is an oxygen of the SO2 group, the NH group is the AH component, and the benzene ring is the component.

N H B

SO2 H A

The tripartite model applied to saccharin

The theory about the relationship of structure to sweetness explains the sweet flavor of many sweeteners, but no search for a sweetener based on these criteria has really turned up a new sweetener. The discovery of many sweeteners was by accident. In fact, many of these discoveries are the result of the researcher not practicing good hygiene in the lab. For example, the investigator who discovered saccharin did so while eating his lunch in the labwithout washing his hands. Likewise, the investigator who discovered cyclamate was smoking in the labagain without washing his hands. Aspartame and Acesulfame-K were discovered when the investigators licked their fingers to pick up a piece of weighing paper. A student, who was not very proficient in English, discovered sucralose when he misunderstood a request to test the compound with a request to taste the compound. If chemists today used the low-tech approach of tasting new compounds in an effort to find new artificial sweeteners, they would no doubt uncover many sweeteners. Tasting new chemical compounds was standard practice in the nineteenth century. Every new compound was tasted as a part of its characterization. Unfortunately, if tasting new compounds were part of a chemist's job description today, chemical companies would risk huge lawsuits. Many more of the

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compounds that chemists work with now are potentially more harmful than the compounds that early chemists worked with. Biochemists will continue to discover new sweeteners mostly by accident. Of course, research based on accidents is a contradiction in terms. True research on sweetness and sweeteners is impossible until someone isolates the primate sweet taste receptor. Once biochemists find this receptor, they can conduct research on hundreds or thousands of compounds by finding those compounds that bind best to the receptor.

24.4 Reactions of Monosaccharides

Monosaccharides are polyfunctional compounds that undergo any of the reactions that are typical of each of the functional groups present on the compound. Thus, monosaccharides exhibit reactivity typical of alcohols and ketones or aldehydes. However, the reagents that chemists normally use with monofunctional compounds often give undesirable side reactions with the sugars containing these functional groups. These undesirable side reactions occur because the presence of multiple functional groups tends to make them much more reactive than their monofunctional counterparts. Although many of the reactions in this section are similar to those reactions that you already know, they are adapted for carbohydrate compounds. A strong base causes two isomerization reactions in carbohydrates. Both reactions result from the formation of the enolate ion of the carbonyl group. In the first reaction, the product is the formation of the C2 epimer of the aldose. The example below shows the epimerization of D-glucose to D-mannose.
H C H HO HO H H OH H OH OH CH2OH D-Glucose HO H H O H C OH H OH OH CH 2OH HO H H O H C OH H OH OH CH2OH H OH HO HO H H O H C H H OH OH CH2OH D-Mannose O

The second reaction is the movement of the carbonyl group between C1 and C2. This reaction proceeds via a similar mechanism to the reaction above. In general, this reaction, as well as the epimerization reaction above, are equilibrium reactions. Thus, the reaction favors the most stable form of the sugar. Epimerization and

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carbonyl group movements are competitive reactions. The conversion of D-glucose to D-fructose is the most common example of this reaction.
H H C H HO HO H H OH H OH OH CH2OH D-Glucose HO H H O H C OH H OH OH CH2OH H OH HO H H O H C O H OH OH CH2OH H OH OH OH

CH2OH C HO H H O H OH OH CH2OH
D-Fructose

Exercise 24.8 Fructose epimerizes at C3. Write a mechanism for this reaction. Biochemists often prepare the ester and ether derivatives of monosaccharides because these derivatives are easier to work with than are the sugars. Monosaccharides contain so many hydroxyl groups that they are soluble in water but are soluble in only a few organic solvents. An attempt to recrystallize monosaccharides from water often forms a syrupy material. The strong hydrogen bonds that form with water make removing all the water difficult. The ester and ether derivatives of monosaccharides are similar to most other organic compounds, so they purify more easily. Biochemists normally carry out an esterification by treating the carbohydrate with either an acid chloride or an acid anhydride in the presence of a mild base. All the hydroxyl groups react, including

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the anomeric hydroxyl group. For example, -D-glucopyranose converts into the pentaacetate by reaction of acetic anhydride in pyridine solution.
H HO HO O CH2OH H O OH H H OH H O (CH3C)2O Pyridine, 0oC H O CH3CO CH3CO O CH2OCCH3 H O H H CH3CO H O O OCCH3

For more on the Williamson ether synthesis see Section 12.9, page 000.

Biochemists accomplish the synthesis of ether derivatives by a variation of the Williamson ether synthesis. Unlike the Williamson ether synthesis described earlier, silver oxide is used instead of the strong base used with simpler ethers. A strong base could cause ring opening and isomerization of the sugar. Glucose forms the pentamethyl ether by reaction with methyl iodide in the presence of silver oxide.
H HO HO CH2OH H O OH H H OH H CH3I Ag2O H CH3O CH3O H

CH2OCH3 H O OCH3 HCH O 3 H

Exercise 24.9 What is the product of the reaction of -D-(+)-xylofuranose with (CH3CO)2O in pyridine?

24.5 Oxidation and Reduction Reactions

An alditol is a monosaccharide with the carbonyl group reduced to an alcohol.

The carbonyl groups of sugars can be reduced to the corresponding alcohols. These alcohols, called alditols, form with nearly any of the reagents used to reduce a carbonyl group. The two most common reagents are sodium borohydride and catalytic hydrogenation with a nickel catalyst. Both of these reagents are useful because both borohydride and hydrogenation react under mild conditions and give higher yields of product. To name the alditols, drop the ose ending and add the itol ending to the name. For

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example, a reduction of D-glucose by sodium borohydride results in the formation of glucitol.

CHO H HO H H OH H OH OH CH2OH
D-Glucose

CH2OH H NaBH4 HO H H OH H OH OH CH2OH Glucitol (also called sorbitol)

Additionally, D-fructose can be catalytically reduced to glucitol and mannitol.

CH2OH C HO H H O H OH OH CH2OH
D-Fructose

CH2OH H H2 Ni HO H H OH H OH OH CH2OH Glucitol + HO HO H H

CH2OH H H OH OH

CH2OH Mannitol

An aldonic acid is an oxidized aldose that results when the aldehyde is oxidized to a carboxylic acid.

In water, bromine readily reacts with aldoses but not ketoses, oxidizing the aldoses to aldonic acids. Thus, this reaction provides a convenient test to differentiate between aldoses and ketoses. An aldose is present when the orange bromine solution rapidly loses its color. An example of this reaction is the formation of D-mannonic acid from D-mannose with bromine in water.

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CHO HO HO H H H H OH OH CH2OH
D-Mannose

COOH HO Br2 H2O HO H H H H OH OH CH2OH

D-Mannonic acid

An aldaric acid is the product of a reaction of an aldose oxidized to a dicarboxylic acid.

In water, nitric acid is a much stronger oxidizing agent than bromine. Nitric acid oxidizes both the aldehyde and the CH2OH of an aldose to carboxylic acids. These dicarboxylic acids are called aldaric acids. For example, D-mannose forms mannaric acid.
CHO HO HO H H H H OH OH CH2OH
D-Mannose

COOH HO HNO3 HO H H H H OH OH COOH

D-Mannaric acid

In a Tollens test, silver ions are oxidized to metallic silver by all monosaccharides.

A reducing sugar is any saccharide with either an aldehyde or ketone functional group.

The Tollens test is the process that was used years ago to form most mirrors. The Tollens test uses a solution of silver nitrate in ammonium hydroxide. The formula for the Tollens reagent is - OH. This solution oxidizes an aldehyde to a carboxylic Ag(NH3)2 c acid and forms silver metal. If the glass surface is very clean, the silver forms a beautiful though somewhat fragile mirror on the glass. If the glass is not clean, the silver forms a black precipitate. The sugars that reduce the Tollens reagent are called reducing sugars. Reducing sugars include all the monosaccharides, both aldoses as well as ketoses with a CH2OH next to the carbonyl group. In addition, any hemiacetal structure can also act as a reducing sugar.

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CHO HO HO HO H H H H OH CH2OH
D-Talose

COOH HO Ag(NH3)2 OH HO HO H H H H OH CH2OH

D-Talonic acid

+ Ag

Benedicts reagent is a solution of copper (II) sulfate with sodium citrate in aqueous base.

Benedicts reagent readily oxidizes the aldehyde functional group of reducing sugars. This reagent is a very mild oxidizing agent and has long been used as a test to screen for levels of reducing sugars in blood. An unusual concentration of glucose in the blood is an indication of diabetes. Exercise 24.10 The product of the reaction of D-glucose with nitric acid produces an optically active product, whereas the reaction with D-galactose produces an optically inactive product. Explain.

24.6 Changing the Chain Length

Chemists devoted much of their early work on carbohydrates to the understanding of the stereochemical relationships among the monosaccharides. They found that when they changed the chain length of a particular monosaccharide, they could easily see these stereochemical relationships. For example, when they degraded two different aldohexoses to the same aldopentose, they knew that the aldohexoses were C2 epimers. Section 24.7 examines this concept further. The most common method for shortening an aldose chain is the Ruff degradation. The Ruff degradation can be used to degrade a variety of natural sugars to D-glyceraldehyde. Otto Ruff, a German chemist, developed the Ruff degradation in about 1900. The Ruff degradation is a two step process. The first step is to oxidize the aldose to the corresponding aldonic acid. Second, treat the aldonic acid with hydrogen peroxide and iron(III) sulfate. The aldonic acid loses the carboxylic acid group as CO2, and the second carbon becomes the aldehyde in the new shorter chain. The remaining carbons do not change stereochemically.

Ruff degradation is a method for shortening the chain of a monosaccharide.

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CHO H HO H H OH H OH OH CH2OH
D-Glucose

COOH H Br2 H2O HO H H OH H OH OH CH2OH

D-Gluconic acid

CHO H2O2 Fe2(SO4)3 HO H H H OH OH CH2OH

D-Arabinose

The Kiliani-Fischer synthesis is a method that lengthens the chain of an aldose. See Section 7.4, page 000, for more about cyanohydrins.

The Kiliani-Fischer synthesis is effectively the reverse of the Ruff degradation. Heinrich Kiliani, of the University of Freiburg in Germany, published a paper that described his method for converting an aldose to a cyanohydrin. Emil Fischer, realizing the importance of Kiliani's method, developed it further. Fischers method took the nitrile of a cyanohydrin and converted the nitrile into an aldehyde group. The result of the Kiliani-Fischer synthesis is the formation of two new aldoses. These two aldoses are C2 epimers of an aldose one carbon longer than the starting aldose. A new stereogenic center is generated with the formation of the cyanohydrin.
CN H HCN HO H H OH H OH OH CH2OH + HO HO H H CN H H OH OH CH2OH

CHO HO H H H OH OH CH2OH
D-Arabinose

Epimeric cyanohydrins

The two cyanohydrins can be separated and converted to the corresponding aldoses. A variety of methods are used for this conversion. One commonly used method is the hydrolysis of the nitrile to a carboxylic acid. This aldonic acid rapidly forms a lactone.

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CN H HO H H OH H OH OH CH2OH The cyanohydrin H 1) Ba(OH)2 HO 2) H3O H H

COOH OH H OH OH CH2OH The aldonic acid The lactone HO H HO H H OH O CH2OH H O

The lactone can be reduced using a sodium amalgam in water at a pH of 3-5. The reduction forms both the and pyranoses.
CHO H HO HO H H OH CH2OH H O H Na(Hg), H2O pH 3 - 5 O HO HO H H Both and OH OH formed CH2OH H O H H HO H H OH H OH OH CH2OH
D-Glucose

Hydrolysis and reduction of the epimeric cyanohydrin produces Dmannose.

CN HO HO H H H H OH OH CH2OH HO 1) Ba(OH)2 HO 2) H3O H H COOH H H OH OH CH2OH HO H HO H H H O CH2OH HO O

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CHO H HO HO H H H CH2OH HO O H Na(Hg), H2O pH 3 - 5 O HO HO H H Both and H OH formed CH2OH HO O H HO HO H H H H OH OH CH2OH

D-Mannose

Exercise 24.11 What two aldohexoses would give D-xylose on Ruff degradation? What two aldohexoses result from a Kiliani-Fischer synthesis that uses Dribose as a substrate?

24.7 Fischer Proof of Glucose Structure

Emil Fischer published his proof of the structure of glucose in 1891. In 1902, he received the Nobel Prize in chemistry for his work. When you consider the state of chemistry at that time, Fischer accomplished an amazing feat with his proof. Fischer was challenged by the complexity of the structure of glucose, by the fact that many of the modern theories of chemistry were new, and by laboratory tools that were limited according to today's standards. The theory of the tetrahedral carbon, an important concept relative to his work, had only been published in the late 1870s. He conducted his laboratory work much differently than chemists do today. He had no modern means, such as chromatography, to purify his products, so his work was laborious and time consuming. His research also required that he purify the product from each reaction step and conduct numerous chemical and physical tests on it, as he had no IR, MS, or NMR to help with identification. Fischer's work consisted of a series of observations from which he made various predictions concerning glucose. His first observation was that (+)-glucose is an aldohexose. An aldohexose has four stereogenic centers, so he knew the compound had 16 possible stereoisomers. These stereoisomers make up eight pairs of enantiomers. After doing a number of chemical tests and reactions using (+)-glucose, he learned that successive Ruff degradations on (+)glucose produced (+)-glyceraldehyde. At this point, he predicted that (+)-glyceraldehyde has the OH group on the right when drawn as a Fischer projection with C1 at the top. With this assumption, he could eliminate eight of the possible structures for (+)-glucose. Thus, he excluded the eight enantiomers with the OH group on the left in the

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Fischer projection from further study. Although he knew that his prediction had only a 50/50 chance of being correct, he also knew that even if he was wrong, his logic was correct. In the early 1950s, using X-ray crystallography, other chemists showed that he had made the correct choiceindeed (+)-glyceraldehyde has the OH group on the right in a Fischer projection. Fischer's second observation was that the Kiliani-Fischer synthesis with ()-arabinose formed a mixture of (+)-glucose and (+)mannose. This observation meant that glucose and mannose were C2 epimers. The fact that the Ruff degradation on either (+)-glucose or (+)-mannose produces ()-arabinose confirmed his observation. His third observation was that nitric acid oxidizes ()-arabinose into an optically active aldaric acid. Of the four aldopentoses only two would give optically active aldaric acids. Thus, ()-arabinose is either structure B or D in Figure 24.5. Because the aldaric acids from A and C (Figure 24.5) have a plane of symmetry, they are not optically active.
CHO H H H OH OH OH CH2OH A HNO3 H H H COOH OH OH OH COOH Plane of symmetry

CHO HO H H H OH OH CH2OH B CHO H HO H OH H OH CH2OH C HNO3 H HO H HNO3 HO H H

COOH H OH OH COOH COOH OH H OH COOH Plane of symmetry

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CHO HO HO H H H OH CH2OH D HNO3 HO HO H

COOH H H OH COOH

Figure 24.5. The possible aldopentoses from Fischers structural work on glucose. Each aldopentose also shows the structure of the aldaric acid produced by reaction with nitric acid.

Fischer's fourth observation was that a Ruff degradation of ()arabinose produces ()-erythrose. Reaction of ()-erythrose with nitric acid produces optically inactive meso-tartaric acid. For ()-erythrose to be optically inactive, its structure must have both of the OH groups on the same side. Fischer's observation showed that ()-arabinose is structure B in Figure 24.5.
CHO HO H H H OH OH Ruff Degradation H H CHO OH OH HNO3 H H COOH OH OH Plane of symmetry

CH2OH (-)-Arabinose

CH2OH (-)-Erythrose

COOH meso-Tartaric acid

At this point Fischer proposed two structures (X and Y), C2 epimers, for (+)-glucose and (+)-mannose. When subjected to Ruff degradation, structures X and Y yield ()-arabinose.
CHO H HO H H X OH H OH OH CH2OH OR HO HO H H CHO H H OH OH CH2OH Y Ruff HO CHO H OH OH

Degradation H H

CH2OH (-)-Arabinose

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When reacted with nitric acid, these two structures, X and Y, both give optically active aldaric acids. Next, Fischer developed a method that converts an aldehyde group of the aldose to the CH2OH group and converts the CH2OH group to the aldehyde. The net result is to switch the two ends of the monosaccharide. With the ends switched, (+)-glucose produces a different sugar that degrades to ()-glyceraldehyde, and (+)-mannose produces (+)-mannose.
CHO H HO H H X CHO HO HO H H H H OH OH CH2OH Y Switch ends HO HO H H CH2OH H H OH OH CHO Rotate 180o HO HO H H OH H OH OH CH2OH Switch ends H HO H H CH2OH OH H OH OH CHO Rotate 180o HO HO H HO CHO H H OH H

CH2OH L-Gulose CHO H H OH OH

CH2OH D-Mannose

At that point, Fischer concluded that X is (+)-glucose and Y is (+)mannose. Using this method, Fischer determined the structures of 12 of the 16 aldohexoses. His work represents a remarkable achievement when you consider the chemical knowledge and laboratory tools available to him to work with. Exercise 24.12 An aldose, G, is optically active but, when treated with HNO3, gives an optically inactive aldaric acid. Ruff degradation of G gives H, which also gives an optically inactive aldaric acid with HNO3. Ruff degradation of H produces D-glyceraldehyde. What are the structures and names of G, H, and the aldaric acids?

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24.8 Glycolysis - I
In the summer of 1896, a German chemist, M. Hahn, was attempting to isolate some proteins from yeast. He ground the yeast in a mortar with fine sand and diatomaceous earth and filtered this mixture through cheesecloth, but the extracts were difficult to preserve. Hahn's colleague, Hans Buchner, remembering that fruit preserves were made by adding sugar, suggested this as a method of preserving the extracts. When Hahn added the sucrose to the solution, bubbles formed in the solution. Buchner had demonstrated that a life process (fermentation) occurred outside a living cell. He hypothesized that this fermentation arose from the activity of an enzyme that he called zymase. Today biochemists call this process, which actually involves 10 enzymes, glycolysis. In the process of glycolysis, glucose, and a number of other monosaccharides, form pyruvate and adenosine triphosphate (ATP). The organic portion connected to the phosphates in ATP is sometimes abbreviated as Ado.
NH2 N O O P O O O P O O O P O H OH O CH2 H N O H H OH N N

Glycolysis is the biochemical process that converts glucose to pyruvate and a small amount of energy.

Adenosine 5'-triphosphate (ATP)

One molecule of glucose forms two molecules of pyruvate and two molecules of ATP. ATP is an important molecule in biological systems because it can be used as a source of energy in various reactions. The net Go for glycolysis is 17.5 kcal/mole of glucose. Thus, glycolysis is a very exothermic process. The process of glycolysis produces pyruvate, which is needed for the citric acid cycle. The citric acid cycle produces most of the energy for the organism. This section and the next examine glycolysis from the viewpoint of an organic chemist with the emphasis on the fact that most of the steps are familiar reactions. The first step in the glycolysis process is a reaction that forms a phosphate ester at C6 of glucose. The phosphate ester forms via a nucleophilic substitution reaction involving the C6 hydroxyl group of

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glucose and the terminal phosphorus of ATP. The reaction requires the presence of a magnesium ion for the transfer to occur. Cells use the enzyme hexokinase to catalyze the reaction.
Mg O CH2OH H HO H OH O P O O
2

O P O O

O P O O Ado H HO H

CH2OPO3 H OH

O OH H H

O OH H H OH

H OH -Glucopyranose

-Glucopyranose 6-phosphate

Next, -D-glucopyranose 6-phosphate isomerizes to -Dfructofuranose 6-phosphate. The enzyme glucose 6-phosphate isomerase catalyzes this reaction. Note that the mechanism depends on having an amine and carboxyl group fixed into the active site of the enzyme. Through a series of proton transfers, the enzyme catalyzes the opening of the glucopyranose ring, the formation of the endiol intermediate, and the closing of the ring to form the fructofuranose. The curved lines in the mechanism represent those segments of the enzyme surrounding the active site.

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CH2OPO3 H HO H H OH

H2N H H O O Enzyme H HO

CH2OPO3 H OH H

H2N H O Enzyme O

O O H H OH

OH O H H OH

D-Glucose 6-phosphate

-D-Glucopyranose 6-phosphate

CH2OPO3 H HO H O H OH

H2N Enzyme O H HO

CH2OPO3 OH H OH

H CH2OH O H O

H OH

H H2N Enzyme O O

D-Fructose 6-phosphate

O H H Endiol intermediate

O3POCH2 H H OH

O
OH

OH

CH2OH H

-D-Fructofuranose 6-phosphate

The third step in glycolysis is the transfer of another phosphate group from ATP to -D-fructofuranose 6-phosphate to form -Dfructofuranose 1,6-bisphosphate. The phosphofructokinase-1 enzyme catalyzes this step of the reaction.
2

O3POCH2 H H OH

O
OH

OH ATP CH2OH H Phosphofructokinase-1

O3POCH2 H H OH

O
OH

OH

CH2OPO32 H

-D-Fructofuranose 6-phosphate

-D-Fructofuranose 1,6-bisphosphate

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The formation of -D-fructofuranose 1,6-bisphosphate is the rate-determining step in glycolysis. Thus, phosphofructokinase-1 is the critical enzyme in the regulation of glycolysis. Although the regulation of glucose is different in different organisms, a significant regulator is the presence of -D-fructofuranose 2,6-bisphosphate. This molecule signals the presence of glucose in the system and increases the activity of the phosphofructokinase-1 enzyme. An absence of -Dfructofuranose 2,6-bisphosphate signals an absence of glucose and decreases the activity of the enzyme.
2

O3POCH2 H H OH

O
OH

OPO32

CH2OH H

-D-Fructofuranose 2,6-bisphosphate

In the next step in glycolysis, the enzyme aldolase splits Dfructose 1,6-bisphosphate (a hexose) into dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate (two trioses). As its name suggests, aldolase is an enzyme that catalyzes an aldol condensation reaction. In this step, the reaction of interest is the reverse aldol reaction.
CH2OPO32 C HO H H O H OH OH CH2OPO32
D-Fructose 1,6-bisphosphate

CH2OPO32 Aldolase C O + H

CHO OH CH2OPO32
D-Glyceraldehyde

CH2OH Dihydroxyacetone phosphate

3-phosphate

The mechanism for the reverse aldol condensation reaction involves a series of four steps. In these steps the D-fructose 1,6bisphosphate interacts with several different amino acid residues at the active site of the enzyme. The first step is the nucleophilic addition to the ketone of the fructose by the -amine group of a lysine residue at the active site. This enzyme-fructose complex has the atoms all oriented properly to allow the reverse aldol condensation to occur. The next step is the actual reverse aldol producing D-glyceraldehyde 3phosphate and dihydroxyacetone phosphate. The final two steps are a

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proton transfer and hydrolysis of the enzyme-dihydroxyacetone complex.

Enzyme H3N Lys Enzyme H3N Lys

CH2OPO32 His HO HN NH H H C

CH2OPO32 His HO S Cys HN Lys NH H H

O H2N(CH2)4 H OH OH H H2N

C H O OH

N(CH2)4

Cys

CH2OPO32

CH2OPO32

H3N

Lys

CH2OPO32 C O
+

Enzyme Enzyme CH2OPO32 His H2O Enzyme H3N CHO Lys

+

H3N Lys

CH2OH HO HN N H

C H

N(CH2)4

H H3N

Cys

Lys

CH2OPO32 His HO HN N H C H

N(CH2)4

OH CH2OPO32

H H3N

Cys

Lys

The formation of D-glyceraldehyde 3-phosphate and dihydroxyacetone phosphate completes what biochemists often call the first stage of glycolysis. This first stage consumes two molecules of ATP to produce two molecules of ADP. The second stage, which is discussed in Section 24.9, produces four molecules of ATP from ADP. The Go for the first stage of glycolysis is slightly exothermic at 1.3 kcal/mole of glucose. The Go for the second stage is much more exothermic at 16.2 kcal/mole of glucose. Exercise 24.13

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What is the effect on the rate of glycolysis as the concentration of Dglucopyranose 6-phosphate changes? How is the rate affected by concentration changes of D-fructofuranose 1,6-bisphosphate? DFructofuranose 2,6-bisphosphate?

24.9 Glycolysis - II
The second stage of glycolysis involves the transformation of glyceraldehyde 3-phosphate and dihydroxyacetone phosphate to pyruvate. What then happens to the pyruvate in the metabolic process depends on the cell that generated the pyruvate and on the availability of oxygen. Under anaerobic conditions the pyruvate is reduced to lactic acid. Under aerobic conditions the pyruvate enters the citric acid cycle. Further detailed descriptions of pyruvate and the citric acid cycle are beyond the scope of this book. In the first step in the second stage of glycolysis is the conversion of dihydroxyacetone phosphate to D-glyceraldehyde 3phosphate. Triose phosphate isomerase catalyzes the reaction. The mechanism of the reaction is similar to the mechanism of glucose 6phosphate isomerase, as described in Section 24.8.

Anaerobic literally means without air or oxygen. An aerobic process requires oxygen.

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Enzyme O Glu C O H H C C O O H H NH2 Lys

CH2OPO32 Dihydroxyacetone phosphate

Enzyme O Glu C O H H C C H C H C O OH + Enzyme O OH H NH2

Lys

CH2OPO32

CH2OPO32
D-Glyceraldehyde

3-phosphate

Both halves of the glucose molecule are now the same. This makes it possible to consider the rest of the glycolysis as happening on one molecule, even though each molecule of glucose actually produces two molecules of D-glyceraldehyde 3-phosphate. The next step in glycolysis requires both an enzyme and a coenzyme to catalyze the reaction. The active site of the enzyme fits both the coenzyme and the substrate. The enzyme, glyceraldehyde 3phosphate dehydrogenase, catalyzes the oxidation of D-glyceraldehyde 3-phosphate to 1,3-bisphospho-D-glycerate.

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H O H OH CH2OPO32

Glyceraldehyde 3-phosphate dehydrogenase H

CH2OPO32 O OH CH2OPO32

D-Glyceraldehyde 3-phosphate

1,3-Bisphospho-D-glycerate

Coenzymes were introduced in Section 23.10, page 000.

The mechanism of the oxidation of D-glyceraldehyde requires that the active site of the enzyme first bind to a molecule of NAD+. NAD+ is a coenzyme and is a biological oxidizing agent. After the NAD+ binds to the enzyme, the molecule of D-glyceraldehyde 3phosphate enters the active site.
O NH2 N R NAD
+

H H N

O NH2

R NADH

The oxidation of D-glyceraldehyde means that NAD+ is reduced to NADH. In the process, NAD+ loses its aromaticity. As a result NADH - to reform NAD+. This reaction is important because readily loses a Hc of the reactivity of the hydride ion in aqueous systems. Most of the hydride sources (e.g. LiAlH4) you have studied react violently with water. There are only a limited number of reagents that can act as a hydride donor in aqueous solution. The transformation of D-glyceraldehyde 3-phosphate to 1,3bisphospho-D-glycerate is a key reaction in glycolysis. Simultaneously, an aldehyde is oxidized to a carboxylic acid and inorganic phosphate is incorporated. The product of this transformation is energy-rich. This transformation is essential to the formation of ATP from ADP. Once the NAD+ coenzyme enters the active site of the enzyme, the next step is the nucleophilic addition of the SH group of a cysteine to the carbonyl of the D-glyceraldehyde 3-phosphate. The - from the carbon bearing the sulfur. Inorganic NAD+ removes an Hc 2 c phosphate (HPO4 ) then attacks the carbonyl carbon to form the 1,3bisphospho-D-glycerate.

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Enzyme R N NH2 H
+O

Enzyme R N NH2 H O H Cys S H C O OH CH2OPO32 H

His

His

NAD

H C O OH

NH

NH

Cys

H H H

CH2OPO32
D-Glyceraldehyde

3-phosphate

Enzyme R N NH2 H H O Cys S H C OH CH2OPO32 O O H N O P O O NH Cys S

Enzyme R N NH2 H H O NADH C H O OH CH2OPO32 H

His

His

NH O

P O

OH

O C H

OPO32 OH CH2OPO32 + Enzyme

1,3-Bisphospho-D-glycerate

In the next step, the NADH dissociates from the enzyme. After the dissociation of the NADH, another molecule of NAD+ binds to the enzyme, and the catalytic cycle begins again. Phosphoglycerate kinase transfers the phosphate group introduced into the product to a molecule of ADP; thus, regenerating ATP and producing 3-phospho-Dglycerate.

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O C H

OPO32 OH CH2OPO32 + ADP Phosphoglycerate kinase

O C H

O OH + ATP

CH2OPO32 3-Phospho-D-glycerate

1,3-Bisphospho-D-glycerate

Glycolysis has three more steps. In the first step, the enzyme phosphoglycerate mutase transfers the phosphate group in 3-phosphoD-glycerate to C2 to form 2-phospho-D-glycerate.
O C H

O
OH Phosphoglycerate mutase

O C H

O
OPO32

CH2OPO32

CH2OH

3-Phospho-D-glycerate

2-Phospho-D-glycerate

In the next step, the enzyme enolase converts 2-phospho-Dglycerate to phosphoenolpyruvate. The active site of enolase contains a manganese(II) ion (Mn2), which acts as a Lewis acid accepting the electron pair from the OH group on C3 of 2-phospho-D-glycerate. This interaction forms phosphoenolpyruvate.
O C

O OPO32 OOC OPO32 CH2 Phosphoenolpyruvate

NH2 H CH2

OH Mn2

Enzyme

Phosphoenolpyruvate, an energy-rich compound, readily donates its phosphate group to ADP to form ATP and pyruvate. This step is the end of glycolysis. However, this step is not the end of the energy production from glucose. Under aerobic conditions, the pyruvate enters the citric acid cycle to produce a number of additional molecules of ATP. Under anaerobic conditions, pyruvate forms lactic acid or ethanol and CO2 and regenerates essential NAD+, thus allowing further glycolysis to occur.

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OOC ADP +

OPO32 CH2

Pyruvate kinase

OOC

O + ATP CH3 Pyruvate

Phosphoenolpyruvate

Exercise 24.14 Using a sample of glucose labeled at C1 with 14C and incubated with the Hahn's zymase extract, where would you find the label in pyruvate?

[Sidebar] Arsenic Poisoning

For centuries arsenic has had an aura of mystery and horror as an ultimate poison. Literature tells of many fictional murders in which unwitting victims had some arsenic placed in their food or drink. Arsenic (As2O3) is a white solid with an appearance similar to powdered sugar or flour. When prepared in food, arsenic is somewhat water-soluble and nearly tasteless. White arsenic has been widely used in a variety of ways from weed killer and sheep-dip to fruit spray and rat poison. In the 18th and 19th centuries physicians used arsenic to cure a variety of illnesses. Around the beginning of the 20th century, physicians used it as a treatment for Graves' disease (hyperthyroidism). The prevalent thought was if white arsenic was toxic to the people, lower concentrations might harm the disease-causing agent more than the patient. Unfortunately, some patients received the wrong dosage. The symptoms of a toxic dose of arsenic begin with gastrointestinal distress: diarrhea, vomiting, and abdominal pain. Death may occur within a few hours to a few days. However, chronic arsenic poisoning does not exhibit any gastrointestinal problems. Instead, the person experiences a gradual weight loss, a skin rash, and changes in skin pigmentation. In either case, arsenic affects the heart, kidneys, and other major organs. If death does not occur as a direct result of a toxic dose, it may occur because of the deterioration of these systems. Arsenic compounds made their way into chemical warfare as well. Phenarsazine chloride was used in World War I as a war gas. It was dispersed in the air as minute particles or "smoke." It causes a profuse watery nasal discharge; severe pain in the nose, sinuses, and

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chest; nausea; vomiting; severe depression, and weakness. Profound sensory disturbances occured later.
Cl As

N H Phenarsazine chloride

The toxicity of arsenic is attributed to its chemical similarity to - (arsenate) ion, phosphorus. White arsenic is oxidized to the AsO43c 3 c which is chemically very similar to the PO4 (phosphate) ion, and competes with the phosphate ion in for D-glyceraldehyde 3-phosphate in the reaction that forms 1,3-bisphospho-D-glycerate. Instead, 1arseno-3-phospho-D-glycerate forms, which readily hydrolyzes to 3phospho-D-glycerate and an arsenate ion.
O H OAsO32 C OH CH2OPO32 1-Arseno-3-phosphoD-glycerate H2O O H O OH CH2OPO32 3-Phospho-D-glycerate + AsO43

The formation of 3-phospho-d-glycerate from 1-arseno-3phospho-d-glycerate allows glycolysis to proceed. However, the ATP that forms in the conversion of 1,3-bisphospho-d-glycerate to 3phospho-d-glycerate is lost. Thus, the presence of arsenate stops the production of ATP by glycolysis. Moreover, the supply of ATP decreases because the earlier steps of glycolysis continue to use ATP. Cells that cannot regenerate ATP eventually die.

24.10 Glycoside Formation

Hemiacetal and acetal formation was discussed in Section 7.5, page 000.

An acetal forms when a hemiacetal reacts with an alcohol in the presence of an acid catalyst.

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OH C OR

R'OH H

OR' C OR

Hemiacetal

Acetal

A glycoside, the cyclic acetal form of a monosaccharide, converts the anomeric OH group to an ether, thus forming an acetal.

The cyclic forms of monosaccharides are hemiacetals. Treatment with an alcohol in the presence of an acid catalyst yields an acetal that replaces the anomeric hydroxyl group by an alkoxy group. The product, called a glycoside, is an acetal. For example, the reaction of -D-glucopyranose with methanol and HCl forms methyl--Dglucopyranoside along with a small amount of methyl--Dglucopyranoside.
CH2OH HO HO OH H -D-Glucopyranose O OH CH2OH HO OH O OCH3

CH3OH, HCl HO

H Methyl -D-glucopyranoside CH2OH + HO HO OH OCH3 Methyl -D-glucopyranoside O H

An aminal has a nitrogen attached to the anomeric carbon instead of an oxygen of a glycoside. A thioacetal has sulfur.

Biochemists name glycosides with a prefix of the alkyl group from the alcohol and by changing the ose suffix of the specific sugar to oside. The glycoside of glucopyranose is glucopyranoside. Glycosides include aminals and thioacetals as well as acetals. Biochemists often term glycosides as O, N, or Sglycosides depending on which atom bonds to the anomeric carbon.

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NH2 N HOCH2 H H N H H OH OH Adenosine, an N-glycoside, the "A" part of ATP. CH2OH HO HO N N HO HO CH2OH O OH NOSO3 SNCH2CH K CH2

H Sinigrin, an S-glycoside, provides the characteristic flavor of horseradish and mustard.

O OH

O HO HO H

CH2

O OCH OH H CN

Amygdalen, an O-glycoside, is from bitter almonds and the pits of peaches and apricots.

Chapter 25 covers nucleotides in detail.

O-Glycosidic bonds are important in the formation of oligomers and polymers of monosaccharides. N-Glycosidic bonds are important in the structure of ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). Nucleotides contain an N-glycosidic link to the anomeric carbon. An example of the nucleotides is the nucleotide of -Dribofuranose in RNA. In DNA, an H replaces the OH group on C2 of the ribofuranose. RNA also has uracil instead of thymine in DNA. Thymine has a CH3 group on the ring instead of the H on uracil. The various nucleotides connect together by a phosphate diester group to make either RNA or DNA. Table 24.1 lists the names of the various bases and their corresponding ribonucleotides or deoxyribonucleotides. Figure 24.6 shows a segment of a hypothetical DNA molecule. Base Structure Base Names Nucleotide

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Base Structure Base

NH2 N N H NH2 N N H O N N H O CH3 N N H O H N N H O O H N N NH2 O N N

Names Nucleotide

Adenine

Adenosine

Cytosine

Cytidine

Guanine

Guanosine

Thymine

Thymidine

Uracil

Uridine

Table 24.1. The bases and their names as well as the names of the nucleotides in RNA and DNA.

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NH2 N O CH2 H O H N N Adenosine N O H O P O O CH2 H O H N N NH2 H O P O O CH2 H O H O H O P O O CH2 H O H N O H H CH3 NH N O O H H N Cytidine O H H N NH Guanosine NH2

Thymidine O

H O P O O O H

Figure 24.6. A segment of a DNA molecule showing the N-glycosidic links with each of the four nucleotides.

Exercise 24.15 Glycosides are relatively stable under both neutral and basic conditions, but they readily hydrolyze in acidic conditions. Using methyl -D-glucopyranoside as an example, write a mechanism illustrating the acid hydrolysis of glycosides.

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24.11 Disaccharides
Disaccharides have two monosaccharides connected by a glycosidic bond.

Disaccharides contain a glycosidic acetal bond that connects two monosaccharides. The most common link is between the anomeric carbon atom of one monosaccharide and C4 of the other monosaccharide. This is called the 1,4' link. Another common link, the 1,6' link, is between the anomeric carbon and C6. A link that forms between the two anomeric carbons is called the 1,1' or 1,2' links. Each of the anomeric carbons has either the or the stereochemistry. Thus, the linkage of two anomeric carbons has six possible stereochemical variants: ', ', , , ', or '. If the two monosaccharides being linked are the same, then there are only three possibilities (, , or ). Below are some examples of disaccharides.

CH2OH HO HO

O OH O HO

CH2OH

O OH OH

Cellobiose (1,4' Glycosidic link) HO HO

CH2OH

O O OH HO HO CH2 O OH OH

CH2OH HO HO

O CH2OH OH O O OH

Gentiobiose (1,6' Glycosidic link)

CH2OH HO Su crose (Link between anomeric carbons)

24.12 Polysaccharides
A polysaccharide is a polymer made up of monosaccharide repeating units linked by glycosidic links.

Polysaccharides are polymers made up of tens, hundreds, or even thousands of monosaccharide repeating units. One example of a polysaccharide is cellulose, which is the main constituent of plant cell walls. Two common cellulose-containing plant products are wood and cotton. Wood contains 40-50% cellulose; cotton contains 90%. Cellulose is a polymer of -D-glucopyranose with 1,4' O-glycosidic links. Wood has approximately 10,000 glucose units per chain; cotton has about 15,000.

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CH2OH O HO

O OH O HO

CH2OH

O OH O HO

CH2OH

O OH O HO

CH2OH

O OH O

Segment of cellulose (A 1,4'-O-( -D-glucopyranoside) polymer)

Fibrils are groups of polysaccharide chains held together by hydrogen bonding.

The 1,4'-O-(-D-glucopyranoside) polymer structure of cellulose is very stable because all the substituents are equatorial. The cellulose fibers consist of individual cellulose molecules held together by hydrogen bonding in long parallel chains. These groups of chains are called fibrils. Fibrils are approximately 200 in diameter and account for the rigidity of plant materials. Another major component of wood is lignin. Lignin is a complex phenolic polymer that crosslinks the fibrils, thereby greatly increasing the strength of the wood. The structure of lignin is only partially understood. Fibrils account for the general chemical resistance of wood and other fibrous plant materials. Most chemical reagents cannot penetrate the surface of the fibril structures because the hydrogen bonding is so strong that it does not allow disruption by most reagents. Alkylation and acylation of some of the surface hydroxyl groups may take place, but the overall structure of the fibrils usually remains intact. Under more extreme reaction conditions, the fibrils do dissociate, which allows a number of the hydroxyl groups to react. For example, if you react cellulose with acetic acid followed by the addition of acetic anhydride with sulfuric acid, you get cellulose triacetate. Typically, the number of glucose repeating units in cellulose acetate is considerably lower than with the original cellulose. The number can be as low as 200 repeating units. Industrial chemists use cellulose acetate in plastic and fiber production to produce acetate, rayon, or acetate rayon.
CH2OAc O AcO O OAc O AcO CH2OAc

O O OAc

Segment of cellulose triacetate

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Cellulases are a group of related enzymes that hydrolyze cellulose.

Starch is a polymer made up of a few hundred monosaccharide units. Amylose is a starch that is insoluble in cold water. Amylopectin is a starch soluble in cold water.

One cellulase, the enzyme -glucosidase, is found in some species of bacteria and protozoa. -Glucosidase hydrolyzes the glycoside linkage between the glucose units of cellulose. Cud-chewing animals (ruminants) host colonies of these bacteria in their stomachs and intestines. When a cow eats hay, the bacteria convert approximately 20-30% of the cellulose to digestible carbohydrates. None of the enzymes found in mammals hydrolyze cellulose. Therefore, mammals cannot utilize the glucose found in cellulose. Starches are somewhat shorter polymers of glucose. 1,4'-O-Glycosidic bonds connect the glucose units. Starch from plant sources can be separated into two fractions. The first fraction, which is insoluble in cold water, is called amylose. The second fraction, which is soluble in cold water, is called amylopectin. A typical sample of starch consists of approximately 25% amylose and 75% amylopectin. Except for its -glycosidic linkage, amylose is a linear polymer similar to cellulose.
CH2OH O HO O OH O HO OH O HO CH2OH O CH2OH O OH O HO OH Segment of amylose (A 1,4'-O-( -D-glucopyranoside) polymer) O CH2OH O

The structure of amylopectin is more complex than the structure of amylose. Amylopectin has 1,6'--glycoside branches every 25 glucose units in the main chain. Thus, amylopectin has a complex three-dimensional structure and a more compact shape than amylose.

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OH

HO
OH O CH2OH

O CH2OH

HO
CH2OH O HO O O OH O HO

1,6'- -Glycosidic link CH2 O OH O HO CH2OH O OH

Segment of amylopectin (1,6' Branched 1,4'-O-( -D-glucopyranoside) polymer)

Animals have enzymes that hydrolyze starches. -Glycosidases begin the hydrolysis of starches in the mouth and continues it in the small intestine. These enzymes digest only the glycoside links in starch, not the glycoside links in cellulose. Thus, animals can eat grains for energy, but not wood. Exercise 24.16 How do the structural differences between amylose and amylopectin account for their different water solubilities?

Key Ideas from Chapter 24

Carbohydrates are a class of compounds with the general molecular formula Cm(H2O)n. Monosaccharides, or simple sugars, have two general forms: 1) an open chain linear polyhydroxy aldehyde or ketone and 2) a cyclic hemiacetal of the open chain molecule. Most monosaccharides prefer the cyclic hemiacetal form to the open chain form. A polyhydroxy aldehyde is called an aldose. A polyhydroxy ketone is called a ketose.

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Biochemists usually represent the stereochemistry of an open chain monosaccharide by a Fischer projection and the cyclic forms by a Haworth projection. There are two groups of monosaccharides. One group degrades to D-glyceraldehyde. The other group degrades to Lglyceraldehyde. Most naturally occurring monosaccharides degrade to D-glyceraldehyde. The D and L designations refer only to the stereochemistry of the monosaccharides and how they compare to glyceraldehyde. Except for glyceraldehyde, the designations have no connection with the direction of the rotation of a plane of polarized light. A pair of monosaccharides that are epimers differ in the stereochemistry of only one carbon atom. The reactions of monosaccharides are typical of the functional groups they contain, so they react like aldehydes, ketones, and alcohols. Bases cause an epimerization at C2 via an enolate ion. Esters form by treatment of the sugar with an acid anhydride or acyl halide. Ethers form by a variation of the Williamson ether synthesis that use methyl iodide and silver oxide as the reagents. Sodium borohydride and catalytic hydrogenations reduce the carbonyl groups of monosaccharides to alcohols, thus forming polyhydroxy alkanes called alditols. Bromine in water oxidizes the aldehyde group in an aldose to an acid, thus forming an aldonic acid. An aldonic acid is a polyhydroxy carboxylic acid. Nitric acid oxidizes both ends of an aldose to form a dicarboxylic acid called an aldaric acid. The Ruff degradation shortens the chain length of a monosaccharide by one carbon without affecting the stereochemistry of the other carbons.

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The Kiliani-Fischer synthesis lengthens the chain of an aldose by one carbon, thus forming a pair of C2 epimers one carbon longer than the original aldose. The Fischer proof of the structure of glucose used a series of reactions and logical processes to prove the relative stereochemistry of each of the stereogenic centers found in glucose. Glycolysis consists of a series of enzyme-catalyzed reactions that convert glucose to pyruvate. Glycolysis starts the process to provide heat and chemical energy to biological systems. The first steps of glycolysis involve the isomerization of glucose and the transfer of two phosphate groups from two molecules of ATP. The products of the reaction are fructofuranose 1,6bisphosphate and two molecules of ADP. The enzyme aldolase splits the fructofuranose 1,6-bisphosphate into two components. One of these components, dihydroxyacetone phosphate, is isomerized into the other Dglyceraldehyde 3-phosphate. Once one molecule of D-glucose is converted to two molecules of D-glyceraldehyde 3-phosphate, a molecule of ADP and another of D-glyceraldehyde 3-phosphate is converted to pyruvate and two molecules of ATP. A glycoside forms when an alcohol, amine, or thiol reacts with the hemiacetal form of a monosaccharide. Disaccharides have two monosaccharides linked by a glycosidic bond. These glycosidic bonds are generally between the anomeric carbon of one monosaccharide and the hydroxyl group at the anomeric site, or of C4 or C6, of another. Polysaccharides are polymers of monosaccharides (usually glucose) linked by glycosidic bonds. Cellulose and starches are two examples of polysaccharides.

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