Reactions of Monosaccharides Because monosaccharides have only two kinds of functional groups, hydroxyls and carbonyls, most of the chemistry of monosaccharides is the familiar chemistry of these two groups. As we’ve seen, alcohols can be converted into esters and ethers and can be oxidized; carbonyl compounds can react with nucleophiles and can be reduced. Ester and Ether Formation Monosaccharides behave as simple alcohols in much of their chemistry. For example, carbohydrate –OH groups can be converted into esters and ethers, which are often easier to work with than the free sugars. Because of their many hydroxyl groups, monosaccharides are usually soluble in water but insoluble in organic solvents such as ether. They are also difficult to purify and have a tendency to form syrups rather than crystals when water is removed. Ester and ether derivatives, however, are soluble in organic solvents and are easily purified and crystallized. Esterification is normally carried out by treating a carbohydrate with an acid chloride or acid anhydride in the presence of a base ( and ). All the –OH groups react, including the anomeric one. For example, β - D -glucopyranose is converted into its pentaacetate by treatment with acetic anhydride in pyridine solution. Carbohydrates are converted into ethers by treatment with an alkyl halide in the presence of base—the Williamson ether synthesis ( ). Standard Williamson conditions using a strong base tend to degrade sensitive sugar molecules, but silver oxide works well as a mild base and gives high yields of ethers. For example, α - D -glucopyranose is converted into its pentamethyl ether in 85% yield on reaction with iodomethane and Ag 2 O. Problems Glycoside Formation We saw in that treatment of a hemiacetal with an alcohol and an acid catalyst yields an acetal. In the same way, treatment of a monosaccharide hemiacetal with an alcohol and an acid catalyst yields an acetal called a glycoside , in which the anomeric –OH has been replaced by an –OR group. For example, reaction of β - D -glucopyranose with methanol gives a mixture of α and β methyl D -glucopyranosides. (Note that a gly coside is the functional group name for any sugar, whereas a glu coside is formed specifically from glucose.) Glycosides are named by first citing the alkyl group and then replacing the - ose ending of the sugar with - oside. Like all acetals, glycosides are stable in neutral water. They aren’t in equilibrium with an open-chain form, and they don’t show mutarotation. They can, however, be hydrolyzed to give back the free monosaccharide plus alcohol on treatment with aqueous acid ( ). Glycosides are abundant in nature, and many biologically important molecules contain glycosidic linkages. For example, digitoxin, the active component of the digitalis preparations used for treatment of heart disease, is a glycoside consisting of a steroid alcohol linked to a trisaccharide. Note also that the three sugars are linked to one another by glycoside bonds. The laboratory synthesis of glycosides can be difficult because of the numerous –OH groups on the sugar molecule. One method that is particularly suitable for preparing glucose β -glycosides involves treatment of glucose pentaacetate with HBr, followed by addition of the appropriate alcohol in the presence of silver oxide. Called the Koenigs–Knorr reaction , the sequence involves formation of a pyranosyl bromide, followed by nucleophilic substitution. For example, methylarbutin, a glycoside found in pears, has been prepared by reaction of tetraacetyl- α - D -glucopyranosyl bromide with p -methoxyphenol. Although the Koenigs–Knorr reaction appears to involve a simple backside S N 2 displacement of bromide ion by alkoxide ion, the situation is actually more complex. Both α and β anomers of tetraacetyl- D -glucopyranosyl bromide give the same β -glycoside product, implying that they react by a common pathway. This result can be understood by assuming that tetraacetyl- D -glucopyranosyl bromide (either α or β anomer) undergoes a spontaneous S N 1-like loss of Br – , followed by internal reaction with the ester group at C2 to form an oxonium ion. Since the acetate at C2 is on the bottom of the glucose ring, the C–O bond also forms from the bottom. Backside S N 2 displacement of the oxonium ion then occurs with the usual inversion of configuration, yielding a β -glycoside and regenerating the acetate at C2 ( ). Mechanism of the Koenigs–Knorr reaction, showing the neighboring-group effect of a nearby acetate. The participation shown by the nearby acetate group in the Koenigs–Knorr reaction is referred to as a neighboring-group effect and is a common occurrence in organic chemistry. Neighboring-group effects are usually noticeable only because they affect the rate or stereochemistry of a reaction; the nearby group itself does not undergo any evident change during the reaction. Biological Ester Formation: Phosphorylation In living organisms, carbohydrates occur not only in the free form but also linked through their anomeric center to other molecules such as lipids (glycolipids) or proteins (glycoproteins). Collectively called glycoconjugates, these sugar-linked molecules are components of cell walls that are crucial to the mechanism by which different cell types recognize one another. Glycoconjugate formation occurs by reaction of the lipid or protein with a glycosyl nucleoside diphosphate. This diphosphate is itself formed by initial reaction of a monosaccharide with adenosine triphosphate (ATP) to give a glycosyl monophosphate, followed by reaction with uridine triphosphate (UTP), to give a glycosyl uridine diphosphate. (We’ll see the structures of nucleoside phosphates in Section 28.1 .) The purpose of the phosphorylation is to activate the anomeric –OH group of the sugar and make it a better leaving group in a nucleophilic substitution reaction by a protein or lipid ( ). Glycoprotein formation occurs by initial phosphorylation of the starting carbohydrate with ATP to a glycosyl monophosphate, followed by reaction with UTP to form a glycosyl uridine 5′-diphosphate. Nucleophilic substitution by an –OH (or –NH 2 ) group on a protein then gives the glycoprotein. Reduction of Monosaccharides Treatment of an aldose or ketose with NaBH 4 reduces it to a polyalcohol called an alditol . The reduction occurs by reaction of the open-chain form present in the aldehyde/ketone ⇄ hemiacetal equilibrium. Although only a small amount of the open-chain form is present at any given time, that small amount is reduced, more is produced by opening of the pyranose form, that additional amount is reduced, and so on, until the entire sample has undergone reaction. D -Glucitol, the alditol produced by reduction of D -glucose, is itself a naturally occurring substance found in many fruits and berries. It is used under the name D -sorbitol as a sweetener and sugar substitute in many foods. Problems Oxidation of Monosaccharides Like other aldehydes, aldoses are easily oxidized to yield the corresponding carboxylic acids, called aldonic acids . A buffered solution of aqueous Br 2 is often used for this purpose. Historically, the oxidation of an aldose with either Ag + in aqueous ammonia (called Tollens’ reagent ) or Cu 2+ with aqueous sodium citrate ( Benedict’s reagent ) formed the basis of simple tests for what are called reducing sugars . ( Reducing because the aldose reduces the metal oxidizing agent.) Some simple diabetes self-test kits sold in drugstores still use Benedict’s reagent to detect glucose in urine, though more modern methods have largely replaced it. All aldoses are reducing sugars because they contain an aldehyde group, but some ketoses are reducing sugars as well. Fructose reduces Tollens’ reagent, for example, even though it contains no aldehyde group. Reduction occurs because fructose is readily isomerized to a mixture of aldoses (glucose and mannose) in basic solution by a series of keto–enol tautomeric shifts ( ). Glycosides, however, are nonreducing because the acetal group is not hydrolyzed to an aldehyde under basic conditions. Fructose, a ketose, is a reducing sugar because it undergoes two base-catalyzed keto–enol tautomerizations that result in conversion to a mixture of aldoses. If warm, dilute HNO 3 (nitric acid) is used as the oxidizing agent, an aldose is oxidized to a dicarboxylic acid called an aldaric acid . Both the aldehyde carbonyl and the terminal –CH 2 OH group are oxidized in this reaction. Finally, if only the –CH 2 OH end of the aldose is oxidized without affecting the –CHO group, the product is a monocarboxylic acid called a uronic acid . The reaction can only be done enzymatically; no chemical reagent is known that can accomplish this selective oxidation in the laboratory. Problems Chain Lengthening: The Kiliani–Fischer Synthesis Much early activity in carbohydrate chemistry was devoted to unraveling the stereochemical relationships among monosaccharides. One of the most important methods used was the Kiliani–Fischer synthesis , which results in the lengthening of an aldose chain by one carbon atom. The C1 aldehyde group of the starting sugar becomes C2 of the chain-lengthened sugar, and a new C1 carbon is added. For example, an aldopentose is converted by Kiliani–Fischer synthesis into two aldohexoses. Discovery of the chain-lengthening sequence was initiated by the observation of Heinrich Kiliani in 1886 that aldoses react with HCN to form cyanohydrins ( ). Emil Fischer immediately realized the importance of Kiliani’s discovery and devised a method for converting the cyanohydrin nitrile group into an aldehyde. Fischer’s original method for conversion of the nitrile into an aldehyde involved hydrolysis to a carboxylic acid, ring closure to a cyclic ester (lactone), and subsequent reduction. A modern improvement involves reducing the nitrile over a palladium catalyst, yielding an imine intermediate that is hydrolyzed to an aldehyde. Note that the cyanohydrin is formed as a mixture of stereoisomers at the new chirality center, so two new aldoses, differing only in their stereochemistry at C2, result from Kiliani–Fischer synthesis. Chain extension of D -arabinose, for example, yields a mixture of D -glucose and D -mannose. Problems Chain Shortening: The Wohl Degradation Just as the Kiliani–Fischer synthesis lengthens an aldose chain by one carbon, the Wohl degradation shortens an aldose chain by one carbon. Wohl degradation is almost the exact opposite of the Kiliani–Fischer sequence. That is, the aldose aldehyde carbonyl group is first converted into a nitrile, and the resulting cyanohydrin loses HCN under basic conditions—the reverse of a nucleophilic addition reaction. Conversion of the aldehyde into a nitrile is accomplished by treatment of an aldose with hydroxylamine to give an imine called an oxime ( ), followed by dehydration of the oxime with acetic anhydride. The Wohl degradation does not give particularly high yields of chain-shortened aldoses, but the reaction is general for all aldopentoses and aldohexoses. For example, D -galactose is converted by Wohl degradation into D -lyxose. Problems

Science


The Eight Essential Monosaccharides Humans need to obtain eight monosaccharides for proper functioning. Although all eight can be biosynthesized from simpler precursors if necessary, it’s more energetically efficient to obtain them from the diet. The eight are L -fucose (6-deoxy- L -galactose), D -galactose, D -glucose, D -mannose, N -acetyl- D -glucosamine, N -acetyl- D -galactosamine, D -xylose, and N -acetyl- D -neuraminic acid ( ). All are used for the synthesis of the glycoconjugate components of cell membranes, and glucose is also the body’s primary source of energy. Structures of the eight monosaccharides essential to humans. Of the eight essential monosaccharides, galactose, glucose, and mannose are simple aldohexoses, while xylose is an aldopentose. Fucose is a deoxy sugar , meaning that it has an oxygen atom “missing.” That is, an –OH group (the one at C6) is replaced by an –H. N -Acetylglucosamine and N -acetylgalactosamine are amide derivatives of amino sugars in which an –OH (the one at C2) is replaced by an –NH 2 group. N -Acetylneuraminic acid is the parent compound of the sialic acids, a group of more than 30 compounds with different modifications, including various oxidations, acetylations, sulfations, and methylations. Note that neuraminic acid has nine carbons and is an aldol reaction product of N -acetylmannosamine with pyruvate (CH 3 COCO 2 – ). Neuraminic acid is crucial to the mechanism by which an influenza virus spreads. All the essential monosaccharides arise from glucose, by the conversions summarized in . We’ll not look specifically at these conversions, but might note that Problems 25-30 through 25-32 and 25-35 at the end of the chapter lead you through several of the biosynthetic pathways. An overview of biosynthetic pathways for the eight essential monosaccharides. Problems

Science


Disaccharides We saw in that reaction of a monosaccharide with an alcohol yields a glycoside in which the anomeric –OH group is replaced by an –OR substituent. If the alcohol is itself a sugar, the glycosidic product is a disaccharide . Maltose and Cellobiose Disaccharides contain a glycosidic acetal bond between the anomeric carbon of one sugar and an –OH group at any position on the other sugar. A glycosidic bond between C1 of the first sugar and the –OH at C4 of the second sugar is particularly common. Such a bond is called a 1→4 link. The glycosidic bond to an anomeric carbon can be either α or β. Maltose, the disaccharide obtained by enzyme-catalyzed hydrolysis of starch, consists of two α - D -glucopyranose units joined by a 1→4- α -glycoside bond. Cellobiose, the disaccharide obtained by partial hydrolysis of cellulose, consists of two β - D -glucopyranose units joined by a 1→4- β -glycoside bond. Maltose and cellobiose are both reducing sugars because the anomeric carbons on their right-hand glucopyranose units have hemiacetal groups and are in equilibrium with aldehyde forms. For a similar reason, both maltose and cellobiose exhibit mutarotation of α and β anomers of the glucopyranose unit on the right. Despite the similarities of their structures, cellobiose and maltose have dramatically different biological properties. Cellobiose can’t be digested by humans and can’t be fermented by yeast. Maltose, however, is digested without difficulty and is fermented readily. Problems Lactose Lactose is a disaccharide that occurs naturally in both human and cow’s milk. It is widely used in baking and in commercial milk formulas for infants. Like maltose and cellobiose, lactose is a reducing sugar. It exhibits mutarotation and is a 1→4- β -linked glycoside. Unlike maltose and cellobiose, however, lactose contains two different monosaccharides— D -glucose and D -galactose—joined by a β -glycosidic bond between C1 of galactose and C4 of glucose. Sucrose Sucrose, or ordinary table sugar, is probably the most abundant pure organic chemical in the world. Whether from sugar cane (20% sucrose by weight) or sugar beets (15% by weight), and whether raw or refined, all table sugar is sucrose. Sucrose is a disaccharide that yields 1 equivalent of glucose and 1 equivalent of fructose on hydrolysis. This 1 : 1 mixture of glucose and fructose is often referred to as invert sugar because the sign of optical rotation changes, or inverts, during the hydrolysis of sucrose ([ α ] D = +66.5) to a glucose/ fructose mixture ([ α ] D = –22.0). Some insects, such as honeybees, have enzymes called invertases that catalyze the sucrose hydrolysis. Honey, in fact, is primarily a mixture of glucose, fructose, and sucrose. Unlike most other disaccharides, sucrose is not a reducing sugar and does not undergo mutarotation. These observations imply that sucrose is not a hemiacetal and that glucose and fructose must both be glycosides. This can happen only if the two sugars are joined by a glycoside link between the anomeric carbons of both sugars—C1 of glucose and C2 of fructose.

Science


Polysaccharides and Their Synthesis Polysaccharides are complex carbohydrates in which tens, hundreds, or even thousands of simple sugars are linked together through glycoside bonds. Because they have only the one free anomeric –OH group at the end of a very long chain, polysaccharides aren’t reducing sugars and don’t show noticeable mutarotation. Cellulose and starch are the two most widely occurring polysaccharides. Cellulose Cellulose consists of several thousand D -glucose units linked by 1→4- β -glycoside bonds like those in cellobiose. Different cellulose molecules then interact to form a large aggregate structure held together by hydrogen bonds. Nature uses cellulose primarily as a structural material to impart strength and rigidity to plants. Leaves, grasses, and cotton, for instance, are primarily cellulose. Cellulose also serves as raw material for the manufacture of cellulose acetate, known commercially as acetate rayon, and cellulose nitrate, known as guncotton, which is the major ingredient in smokeless powder, the explosive propellant used in artillery shells and in ammunition for firearms. Starch and Glycogen Potatoes, corn, and cereal grains contain large amounts of starch, a polymer of glucose in which the monosaccharide units are linked by 1→4- α -glycoside bonds like those in maltose. Starch can be separated into two fractions: amylose and amylopectin. Amylose accounts for about 20% by weight of starch and consists of several hundred glucose molecules linked together by 1→4- α -glycoside bonds. Amylopectin accounts for the remaining 80% of starch and is more complex in structure than amylose. Unlike cellulose and amylose, which are linear polymers, amylopectin contains 1→6- α -glycoside branches approximately every 25 glucose units. Starch is digested in the mouth and stomach by α -glycosidases, which catalyze the hydrolysis of glycoside bonds and release individual molecules of glucose. Like most enzymes, α -glycosidases are highly selective in their action. They hydrolyze only the α -glycoside links in starch and leave the β -glycoside links in cellulose untouched. Thus, humans can digest potatoes and grains but not grass and leaves. Glycogen is a polysaccharide that serves the same energy storage function in animals that starch serves in plants. Dietary carbohydrates not needed for immediate energy are converted by the body into glycogen for long-term storage. Like the amylopectin found in starch, glycogen contains a complex branching structure with both 1→4 and 1→6 links ( ). Glycogen molecules are larger than those of amylopectin—up to 100,000 glucose units—and contain even more branches. A representation of the structure of glycogen. The hexagons represent glucose units linked by 1→4 and 1→6 glycoside bonds. Polysaccharide Synthesis With numerous –OH groups of similar reactivity, polysaccharides are so structurally complex that their laboratory synthesis has long been a particularly difficult problem. Several methods have recently been devised, however, that have greatly simplified the problem. Among these approaches is the glycal assembly method . Easily prepared from the appropriate monosaccharide, a glycal is an unsaturated sugar with a C1–C2 double bond. To ready it for use in polysaccharide synthesis, the glycal is first protected at its primary –OH group by formation of a silyl ether ( ) and at its two adjacent secondary –OH groups by formation of a cyclic carbonate ester. Then, the protected glycal is epoxidized. Treatment of the protected glycal epoxide in the presence of ZnCl 2 as a Lewis acid with a second glycal having a free –OH group causes acid-catalyzed opening of the epoxide ring by S N 2 backside attack ( ) and yields a disaccharide. The disaccharide is itself a glycal, so it can be epoxidized and coupled again to yield a trisaccharide, and so on. Using the appropriate sugars at each step, a great variety of polysaccharides can be prepared. After these sugars are linked, the silyl ethers and cyclic carbonate protecting groups are removed by hydrolysis. Among the numerous complex polysaccharides that have been synthesized in the laboratory are several tumor-associated carbohydrate antigens (TACAs) that are currently being tested as potential vaccines for breast, prostate, lung, colon, and ovarian cancers.

Science


Some Other Important Carbohydrates In addition to the common carbohydrates mentioned in previous sections, there are a variety of important carbohydrate-derived materials. Their structural resemblance to sugars is clear, but they aren’t simple aldoses or ketoses. Deoxy sugars, as we saw in , have an oxygen atom “missing.” That is, an –OH group is replaced by an –H. The most common deoxy sugar is 2-deoxyribose, a monosaccharide found in DNA (deoxyribonucleic acid). Note that 2-deoxyribose exists in water solution as a complex equilibrium mixture of both furanose and pyranose forms. Amino sugars, such as D -glucosamine, have an –OH group replaced by an –NH 2 . The N -acetyl amide derived from D -glucosamine is the monosaccharide unit from which chitin, the hard crust that protects insects and shellfish, is made. Still other amino sugars are found in antibiotics such as streptomycin and gentamicin. Sweetness Say the word sugar and most people immediately think of sweet-tasting candies, desserts, and such. In fact, most simple carbohydrates do taste sweet but the degree of sweetness varies greatly from one sugar to another. With sucrose (table sugar) as a reference point, fructose is nearly twice as sweet, but lactose is only about one-sixth as sweet. Comparisons are difficult, though, because perceived sweetness varies depending on the concentration of the solution being tasted and on personal opinion. Nevertheless, the ordering in is generally accepted. The desire of many people to cut their caloric intake has led to the development of synthetic sweeteners such as saccharin, aspartame, acesulfame, and sucralose. All are far sweeter than natural sugars, so the choice of one or another depends on personal taste, government regulations, and (for baked goods) heat stability. Saccharin, the oldest synthetic sweetener, has been used for more than a century, although it has a somewhat metallic aftertaste. Doubts about its safety and potential carcinogenicity were raised in the early 1970s, but it has now been cleared of suspicion for human consumption. The real thing comes from sugarcane fields like this one. (credit: modification of work “Flowering sugarcane field” by “carrotmadman6”/Flickr, CC BY 2.0) Sweetness of Some Sugars and Sugar Substitutes Name Type Sweetness Lactose Disaccharide 0.16 Glucose Monosaccharide 0.75 Sucrose Disaccharide 1.00 Fructose Monosaccharide 1.75 Cyclamate Synthetic 40 Aspartame Semisynthetic 180 Acesulfame-K Synthetic 200 Saccharin Synthetic 350 Sucralose Semisynthetic 600 Alitame Semisynthetic 2,000 Neotame Semisynthetic 6,000 Advantame Semisynthetic 20,000 Acesulfame potassium, one of the more recently approved sweeteners, is proving to be extremely popular in soft drinks because it has little aftertaste. Sucralose, another recent sweetener, is particularly useful in baked goods because of its stability at high temperatures. Alitame, marketed in some countries under the name Aclame, is not approved for sale in the United States. It is some 2000 times as sweet as sucrose and, like acesulfame-K, has no aftertaste. Of the eight synthetic sweeteners listed in , only sucralose has clear structural resemblance to a carbohydrate, although it differs dramatically in containing three chlorine atoms. Aspartame, Alitame, and Advantame are dipeptides. aldaric acid alditol aldonic acid aldose amino sugar α anomer , β anomer anomeric center anomer carbohydrate complex carbohydrate D sugar deoxy sugar disaccharide Fischer projection furanose glycoside ketose Kiliani–Fischer synthesis Koenigs–Knorr reaction L sugar monosaccharide mutarotation polysaccharide pyranose reducing sugar simple sugar uronic acid Wohl degradation Summary Now that we’ve now seen all the common functional groups and reaction types, our focus has changed to looking at the major classes of biological molecules. Carbohydrates are polyhydroxy aldehydes and ketones. They are classified according to the number of carbon atoms and the kind of carbonyl group they contain. Glucose, for example, is an aldohexose, a six-carbon aldehydo sugar. Monosaccharides are further classified as either D sugars or L sugars , depending on the stereochemistry of the chirality center farthest from the carbonyl group. Carbohydrate stereochemistry is frequently depicted using Fischer projections , which represent a chirality center as the intersection of two crossed lines. Monosaccharides normally exist as cyclic hemiacetals rather than as open-chain aldehydes or ketones. The hemiacetal linkage results from reaction of the carbonyl group with an –OH group three or four carbon atoms away. A five-membered cyclic hemiacetal is called a furanose , and a six-membered cyclic hemiacetal is called a pyranose . Cyclization leads to the formation of a new chirality center called the anomeric center and the production of two diastereomeric hemiacetals called alpha ( α ) and beta ( β ) anomers . Much of the chemistry of monosaccharides is the familiar chemistry of alcohols and aldehydes/ketones. Thus, the hydroxyl groups of carbohydrates form esters and ethers. The carbonyl group of a monosaccharide can be reduced with NaBH 4 to form an alditol , oxidized with aqueous Br 2 to form an aldonic acid , oxidized with HNO 3 to form an aldaric acid , oxidized enzymatically to form a uronic acid , or treated with an alcohol in the presence of acid to form a glycoside . Monosaccharides can also be chain-lengthened by the multistep Kiliani–Fischer synthesis and can be chain-shortened by Wohl degradation . Disaccharides are complex carbohydrates in which simple sugars are linked by a glycoside bond between the anomeric center of one unit and a hydroxyl of the second unit. The sugars can be the same, as in maltose and cellobiose, or different, as in lactose and sucrose. The glycosidic bond can be either α (maltose) or β (cellobiose, lactose) and can involve any hydroxyl of the second sugar. A 1→4 link is most common (cellobiose, maltose), but others such as 1→2 (sucrose) are also known. Polysaccharides , such as cellulose, starch, and glycogen, are used in nature as structural materials, as a means of long-term energy storage, and as cell-surface markers. Summary of Reactions Additional Problems Visualizing Chemistry Mechanism Problems Carbohydrate Structures Carbohydrate Reactions General Problems

Science


Why This Chapter? The building blocks of life that we call proteins are aptly named after Proteus, the early Greek sea-god whose name means “first” or “primordial.” (credit: modification of work by Coyau/Wikimedia Commons, CC BY-SA 3.0) Continuing our look at the main classes of biomolecules, we’ll focus in this chapter on amino acids, the fundamental building blocks from which up to 150,000 or so different proteins in our bodies are made. We’ll then see how amino acids are incorporated into proteins and examine the structures of those proteins. Any understanding of biological chemistry would be impossible without this knowledge. Proteins occur in every living organism, are of many different types, and have many different biological functions. The keratin of skin and fingernails, the fibroin of silk and spider webs, and the estimated 50,000 or so enzymes that catalyze the biological reactions in our bodies are all proteins. Regardless of their function, all proteins have a fundamentally similar structure and are made up of many amino acids linked together in a long chain. Amino acids, as their name implies, are difunctional. They contain both a basic amino group and an acidic carboxyl group. Their value as building blocks to make proteins stems from the fact that amino acids can join together into long chains by forming amide bonds between the –NH 2 of one amino acid and the –CO 2 H of another. For classification purposes, chains with fewer than 50 amino acids are often called peptides , while the term protein is generally used for larger chains.

Science


Structures of Amino Acids We saw in and that a carboxyl group is deprotonated and exists as the carboxylate anion at a physiological pH of 7.3, while an amino group is protonated and exists as the ammonium cation. Thus, amino acids exist in aqueous solution primarily in the form of a dipolar ion, or zwitterion (from the German zwitter, meaning “hybrid”). Amino acid zwitterions are internal salts and therefore have many of the physical properties associated with salts. They have large dipole moments, are relatively soluble in water but insoluble in hydrocarbons, and are crystalline with relatively high melting points. In addition, amino acids are amphiprotic; they can react either as acids or as bases, depending on the circumstances. In aqueous acid solution, an amino acid zwitterion is a base that accepts a proton onto its –CO 2 – group to yield a cation. In aqueous base solution, the zwitterion is an acid that loses a proton from its –NH 3 + group to form an anion. The structures, abbreviations (both three- and one-letter), and p K a values of the 20 amino acids commonly found in proteins are shown in . All are α -amino acids , meaning that the amino group in each is a substituent on the α carbon—the one next to the carbonyl group. Nineteen of the twenty amino acids are primary amines, RNH 2 , and differ only in the nature of their side chain —the substituent attached to the α carbon. Proline is a secondary amine whose nitrogen and α carbon atoms are part of a five-membered pyrrolidine ring. The 20 Common Amino Acids in Proteins Name Abbreviations MW Structure p K a α -CO 2 H p K a α -NH 3 + p K a side chain p I Neutral Amino Acids Alanine Ala A 89 2.34 9.69 — 6.01 Asparagine Asn N 132 2.02 8.80 — 5.41 Cysteine Cys C 121 1.96 10.28 8.18 5.07 Glutamine Gln Q 146 2.17 9.13 — 5.65 Glycine Gly G 75 2.34 9.60 — 5.97 Isoleucine Ile I 131 2.36 9.60 — 6.02 Leucine Leu L 131 2.36 9.60 — 5.98 Methionine Met M 149 2.28 9.21 — 5.74 Phenylalanine Phe F 165 1.83 9.13 — 5.48 Proline Pro P 115 1.99 10.60 — 6.30 Serine Ser S 105 2.21 9.15 — 5.68 Threonine Thr T 119 2.09 9.10 — 5.60 Tryptophan Trp W 204 2.83 9.39 — 5.89 Tyrosine Tyr Y 181 2.20 9.11 10.07 5.66 Valine Val V 117 2.32 9.62 — 5.96 Acidic Amino Acids Aspartic acid Asp D 133 1.88 9.60 3.65 2.77 Glutamic acid Glu E 147 2.19 9.67 4.25 3.22 Basic Amino Acids Arginine Arg R 174 2.17 9.04 12.48 10.76 Histidine His H 155 1.82 9.17 6.00 7.59 Lysine Lys K 146 2.18 8.95 10.53 9.74 In addition to the 20 amino acids commonly found in proteins, 2 others—selenocysteine and pyrrolysine—are found in some organisms, and more than 700 nonprotein amino acids are also found in nature. γ -Aminobutyric acid (GABA), for instance, is found in the brain and acts as a neurotransmitter; homocysteine is found in blood and is linked to coronary heart disease; and thyroxine is found in the thyroid gland, where it acts as a hormone. Except for glycine, H 2 NCH 2 CO 2 H, the α carbons of amino acids are chirality centers. Two enantiomers of each are therefore possible, but nature uses only one to build proteins. In Fischer projections, naturally occurring amino acids are represented by placing the –CO 2 – group at the top and pointing the side chain downwards, as if drawing a carbohydrate ( ) and then placing the –NH 3 + group on the left. Because of their stereochemical similarity to L sugars ( ), the naturally occurring α -amino acids are often referred to as L amino acids. The nonnaturally occurring enantiomers are called D amino acids. The 20 common amino acids can be further classified as neutral, acidic, or basic, depending on the structure of their side chains. Fifteen of the twenty have neutral side chains, two (aspartic acid and glutamic acid) have an extra carboxylic acid function in their side chains, and three (lysine, arginine, and histidine) have basic amino groups in their side chains. Note that both cysteine (a thiol) and tyrosine (a phenol), although usually classified as neutral amino acids, nevertheless have weakly acidic side chains that can be deprotonated in a sufficiently basic solution. At the physiological pH of 7.3, the side-chain carboxyl groups of aspartic acid and glutamic acid are deprotonated and the basic side-chain nitrogens of lysine and arginine are protonated. Histidine, however, which contains a heterocyclic imidazole ring in its side chain, is not quite basic enough to be protonated at pH 7.3. Note that only the pyridine-like, doubly bonded nitrogen in histidine is basic. The pyrrole-like singly bonded nitrogen is nonbasic because its lone pair of electrons is part of the six- π -electron aromatic imidazole ring ( ). Humans are able to biosynthesize only 11 of the 20 protein amino acids, called nonessential amino acids. The other 9, called essential amino acids, are biosynthesized only in plants and microorganisms and must be obtained in our diet. The division between essential and nonessential amino acids is not clear-cut, however. Tyrosine, for instance, is sometimes considered nonessential because humans can produce it from phenylalanine, but phenylalanine itself is essential and must be obtained in the diet. Arginine can be synthesized by humans, but much of the arginine we need also comes from our diet. Problems

Science


Amino Acids and the Henderson–Hasselbalch Equation: Isoelectric Points According to the Henderson–Hasselbalch equation ( and ), if we know both the pH of a solution and the p K a of an acid HA, we can calculate the ratio of [A – ] to [HA] in the solution. Furthermore, when pH = p K a , the two forms A – and HA are present in equal amounts because log 1 = 0. pH = p K a + log [A – ] [HA] or log [A – ] [HA] = pH – p K a To apply the Henderson–Hasselbalch equation to an amino acid, let’s find out what species are present in a 1.00 M solution of alanine at pH = 9.00. According to , protonated alanine [ + H 3 NCH(CH 3 )CO 2 H] has p K a1 = 2.34 and neutral zwitterionic alanine [ + H 3 NCH(CH 3 )CO 2 – ] has p K a2 = 9.69: Because the pH of the solution is much closer to p K a2 than to p K a1 , we need to use p K a2 for the calculation. From the Henderson–Hasselbalch equation, we have: log [A – ] [HA] = pH – p K a = 9 .00 – 9 .69 = –0 .69 So [A – ] [HA] = antilog(–0 .69) = 0 .20 and [A – ] = 0 .20[HA] In addition, we know that [A – ] + [HA] = 1 .00 M Solving the two simultaneous equations gives [HA] = 0.83 and [A – ] = 0.17. In other words, at pH = 9.00, 83% of alanine molecules in a 1.00 M solution are neutral (zwitterionic) and 17% are deprotonated. Similar calculations can be done at other pH values and the results plotted to give the titration curve shown in . Each leg of the titration curve is calculated separately. The first leg, from pH 1 to 6, corresponds to the dissociation of protonated alanine, H 2 A + . The second leg, from pH 6 to 11, corresponds to the dissociation of zwitterionic alanine, HA. It’s as if we started with H 2 A + at low pH and then titrated with NaOH. When 0.5 equivalent of NaOH is added, the deprotonation of H 2 A + is 50% complete; when 1.0 equivalent of NaOH is added, the deprotonation of H 2 A + is finished and HA predominates; when 1.5 equivalents of NaOH is added, the deprotonation of HA is 50% complete; and when 2.0 equivalents of NaOH is added, the deprotonation of HA is finished. A titration curve for alanine, plotted using the Henderson–Hasselbalch equation . Each of the two legs is plotted separately. At pH < 1, alanine is entirely protonated; at pH = 2.34, alanine is a 50 : 50 mix of protonated and neutral forms; at pH = 6.01, alanine is entirely neutral; at pH = 9.69, alanine is a 50 : 50 mix of neutral and deprotonated forms; at pH > 11.5, alanine is entirely deprotonated. Look carefully at the titration curve in . In acid solution, the amino acid is protonated and exists primarily as a cation. In basic solution, the amino acid is deprotonated and exists primarily as an anion. In between the two is an intermediate pH at which the amino acid is exactly balanced between anionic and cationic forms, existing primarily as the neutral, dipolar zwitterion. This pH is called the amino acid’s isoelectric point (p I ) and has a value of 6.01 for alanine. The isoelectric point of an amino acid depends on its structure, with values for the 20 common amino acids given previously in . The 15 neutral amino acids have isoelectric points near neutrality, in the pH range 5.0 to 6.5. The two acidic amino acids have isoelectric points at lower pH so that deprotonation of the side-chain −CO 2 H does not occur at their p I, and the three basic amino acids have isoelectric points at higher pH so that protonation of the side-chain amino group does not occur at their p I . More specifically, the p I of any amino acid is the average of the two acid-dissociation constants that involve the neutral zwitterion. For the 13 amino acids with a neutral side chain, p I is the average of p K a1 and p K a2 . For the four amino acids with either a strongly or weakly acidic side chain, p I is the average of the two lowest p K a values. For the three amino acids with a basic side chain, p I is the average of the two highest p K a values. Just as individual amino acids have isoelectric points, proteins have an overall p I due to the cumulative effect of all the acidic or basic amino acids they may contain. The enzyme lysozyme, for instance, has a preponderance of basic amino acids and thus has a high isoelectric point (p I = 11.0). Pepsin, however, has a preponderance of acidic amino acids and a low isoelectric point (p I ~ 1.0). Not surprisingly, the solubilities and properties of proteins with different p I ’s are strongly affected by the pH of the medium. Solubility in water is usually lowest at the isoelectric point, where the protein has no net charge, and is higher both above and below the p I, where the protein is charged. We can take advantage of the differences in isoelectric points to separate a mixture of proteins into its pure components. Using a technique known as electrophoresis , a mixture of proteins is placed near the center of a strip of paper or gel. The paper or gel is moistened with an aqueous buffer of a given pH, and electrodes are connected to the ends of the strip. When an electric potential is applied, the proteins with negative charges (those that are deprotonated because the pH of the buffer is above their isoelectric point) migrate slowly toward the positive electrode. At the same time, those amino acids with positive charges (those that are protonated because the pH of the buffer is below their isoelectric point) migrate toward the negative electrode. Different proteins migrate at different rates, depending on their isoelectric points and on the pH of the aqueous buffer, thereby effecting a separation of the mixture into its components. illustrates this process for a mixture containing basic, neutral, and acidic components. Separation of a protein mixture by electrophoresis . At pH = 6.00, a neutral protein does not migrate, a basic protein is protonated and migrates toward the negative electrode, and an acidic protein is deprotonated and migrates toward the positive electrode. Problems

Science


Synthesis of Amino Acids α -Amino acids can be synthesized in the laboratory using some of the reactions discussed in previous chapters. One of the oldest methods of α -amino acid synthesis begins with α bromination of a carboxylic acid by treatment with Br 2 and PBr 3 (the Hell–Volhard–Zelinskii reaction; ). S N 2 substitution of the α -bromo acid with ammonia then yields an α -amino acid. Problems The Amidomalonate Synthesis A more general method for preparation of α -amino acids is the amidomalonate synthesis, a straightforward extension of the malonic ester synthesis ( ). The reaction begins with the conversion of diethyl acetamidomalonate into an enolate ion by treatment with base, followed by S N 2 alkylation with a primary alkyl halide. Hydrolysis of both the amide protecting group and the esters occurs when the alkylated product is warmed with aqueous acid, and decarboxylation then takes place to yield an α -amino acid. For example, aspartic acid can be prepared from ethyl bromoacetate, BrCH 2 CO 2 Et: Problems Reductive Amination of α -Keto Acids Yet another method for the synthesis of α -amino acids is by reductive amination of an α -keto acid with ammonia and a reducing agent. Alanine, for instance, is prepared by treatment of pyruvic acid with ammonia in the presence of NaBH 4 . As described in , the reaction proceeds through formation of an intermediate imine which is then reduced. Enantioselective Synthesis The synthesis of an α -amino acid from an achiral precursor by any of the methods just described yields a racemic mixture, with equal amounts of S and R enantiomers. To use an amino acid in the laboratory synthesis of a naturally occurring protein, however, the pure S enantiomer must be obtained. Two methods are used in practice to obtain enantiomerically pure amino acids. One way requires resolving the racemic mixture into its pure enantiomers ( ). A more direct approach, however, is to use an enantioselective synthesis to prepare only the desired S enantiomer directly. As discussed in the Chapter 19 Chemistry Matters , the idea behind enantioselective synthesis is to find a chiral reaction catalyst that will temporarily hold a substrate molecule in an unsymmetrical, chiral environment. While in that chiral environment, the substrate may be more open to reaction on one side than on another, leading to an excess of one enantiomeric product. William Knowles at the Monsanto Company discovered in 1968 that α -amino acids can be prepared enantioselectively by hydrogenation of a Z enamido acid with a chiral hydrogenation catalyst. ( S )-Phenylalanine, for instance, is prepared at 98.7% purity, contaminated by only 1.3% of the ( R ) enantiomer, when using a chiral rhodium catalyst. For this discovery, Knowles shared the 2001 Nobel Prize in Chemistry. The most effective catalysts for enantioselective amino acid synthesis are coordination complexes of rhodium(I) with 1,5-cyclooctadiene (COD) and a chiral diphosphine such as ( R , R )-1,2-bis( o -anisylphenylphosphino)ethane, the so-called DiPAMP ligand. This complex owes its chirality to the presence of trisubstituted phosphorus atoms ( ). Problems

Science


Peptides and Proteins Proteins and peptides are amino acid polymers in which the individual amino acids, called residues , are linked together by amide bonds, or peptide bonds. An amino group from one residue forms an amide bond with the carboxyl of a second residue, the amino group of the second forms an amide bond with the carboxyl of a third, and so on. For example, alanylserine is the dipeptide that results when an amide bond forms between the alanine carboxyl and the serine amino group. Note that two dipeptides can result from reaction between alanine and serine, depending on which carboxyl group reacts with which amino group. If the alanine amino group reacts with the serine carboxyl, serylalanine results. The long, repetitive sequence of –N–CH–CO– atoms that makes up a continuous chain is called the protein’s backbone . By convention, peptides are written with the N-terminal amino acid (the one with the free –NH 3 + group) on the left and the C-terminal amino acid (the one with the free –CO 2 – group) on the right. The name of a peptide is denoted by the abbreviations listed in for each amino acid. Thus, alanylserine is abbreviated Ala-Ser or A-S, and serylalanine is abbreviated Ser-Ala or S-A. The one-letter abbreviations are more convenient, though less immediately recognizable, than the three-letter abbreviations. The amide bond that links amino acids together in peptides is no different from any other amide bond ( ). An amide nitrogen is nonbasic because its unshared electron pair is delocalized by resonance with the carbonyl group. This overlap of the nitrogen p orbital with the p orbitals of the carbonyl group imparts a certain amount of double-bond character to the C–N bond and restricts rotation around it. The amide bond is therefore planar, and the N–H is oriented 180° to the C═O . A second kind of covalent bonding in peptides occurs when a disulfide linkage, RS–SR, is formed between two cysteine residues. As we saw in , a disulfide is formed by mild oxidation of a thiol, RSH, and is cleaved by mild reduction. A disulfide bond between cysteine residues in different peptide chains links the otherwise separate chains together, whereas a disulfide bond between cysteine residues in the same chain forms a loop. Insulin, for instance, is composed of two chains that total 51 amino acids and are linked by two cysteine disulfide bridges. Problems

Science


Amino Acid Analysis of Peptides To determine the structure of a protein or peptide, we need to answer three questions: What amino acids are present? How much of each is present? In what sequence do the amino acids occur in the peptide chain? The answers to the first two questions are provided by an automated instrument called an amino acid analyzer. An amino acid analyzer is based on techniques worked out in the 1950s by William Stein and Stanford Moore at the Rockefeller University, who shared the 1972 Nobel Prize in Chemistry for their work. In preparation for analysis, the peptide is broken into its constituent amino acids by reducing all disulfide bonds, capping the –SH groups of cysteine residues by S N 2 reaction with iodoacetic acid, and hydrolyzing the amide bonds by heating with aqueous 6 M HCl at 110 °C for 24 hours. The resultant amino acid mixture is then separated into its components by a technique called chromatography, either high-pressure liquid chromatography (HPLC) or ion-exchange chromatography. In both HPLC and ion-exchange chromatography, the mixture to be separated is dissolved in a solvent, called the mobile phase, and passed through a metal tube or glass column that contains an adsorbent material, called the stationary phase. Because different compounds adsorb to the stationary phase to different extents, they migrate through the chromatography column at different rates and are separated as they emerge (elute) from the end. In the ion-exchange technique, separated amino acids eluting from the chromatography column mix with a solution of a substance called ninhydrin and undergo a rapid reaction that produces an intense purple color. The color is measured by a spectrometer, and a plot of elution time versus spectrometer absorbance is obtained. Because the time required for a given amino acid to elute from a standard column is reproducible, the identities of the amino acids in a peptide can be determined. The amount of each amino acid in the sample is determined by measuring the intensity of the purple color resulting from its reaction with ninhydrin. shows the results of amino acid analysis of a standard equimolar mixture of 17 α -amino acids. Typically, amino acid analysis requires about 100 picomoles (2–3 μ g) of sample for a protein containing about 200 residues. Amino acid analysis of an equimolar mixture of 17 amino acids . Problems

Science


Peptide Sequencing: The Edman Degradation With the identities and relative amounts of amino acids known, a peptide can then be sequenced to find out in what order the amino acids are linked. Much peptide sequencing is now done by mass spectrometry, using either electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) linked to a time-of-flight (TOF) mass analyzer, as described in . Also in common use is a chemical method of peptide sequencing called the Edman degradation . The general idea of peptide sequencing by Edman degradation is to cleave one amino acid at a time from an end of the peptide chain. That terminal amino acid is then separated and identified, and the cleavage reactions are repeated on the chain-shortened peptide until the entire peptide sequence is known. Automated protein sequencers are available that allow as many as 50 repetitive sequencing cycles to be carried out before a buildup of unwanted by-products interferes with the results. So efficient are these instruments that sequence information can be obtained from as little as 1 to 5 picomoles of sample—less than 0.1 μ g. As shown in , Edman degradation involves treatment of a peptide with phenyl isothiocyanate (PITC), C 6 H 5 –N═C═S , followed by reaction with trifluoroacetic acid. The first step attaches the PITC to the –NH 2 group of the N-terminal amino acid, and the second step splits the N-terminal residue from the peptide chain, yielding an anilinothiazolinone (ATZ) derivative plus the chain-shortened peptide. Further acid-catalyzed rearrangement of the ATZ derivative with aqueous acid converts it into a phenylthiohydantoin (PTH), which is identified by comparison of its elution time with the known elution times of PTH derivatives of the 20 common amino acids. The chain-shortened peptide is then automatically resubmitted for another round of Edman degradation. MECHANISM Mechanism of the Edman degradation for N-terminal analysis of peptides . Complete sequencing of large proteins by Edman degradation is impractical because of the buildup of unwanted by-products. To get around this problem, a large peptide chain is first cleaved by partial hydrolysis into a number of smaller fragments, the sequence of each fragment is determined, and the individual fragments are fitted together by matching their overlapping ends. In this way, protein chains with more than 400 amino acids have been sequenced. Partial hydrolysis of a peptide can be carried out either chemically with aqueous acid or enzymatically. Acid hydrolysis is unselective and gives a more-or-less random mixture of small fragments, but enzymatic hydrolysis is quite specific. The enzyme trypsin, for instance, catalyzes hydrolysis of peptides only at the carboxyl side of the basic amino acids arginine and lysine; chymotrypsin cleaves only at the carboxyl side of the aryl-substituted amino acids phenylalanine, tyrosine, and tryptophan. Problems

Science


Peptide Synthesis Once the structure of a peptide is known, its synthesis can be undertaken—perhaps to obtain a larger amount for biological evaluation. A simple amide might be formed by treating an amine and a carboxylic acid with a carbodiimide (either DCC or EDC; ), but peptide synthesis is a more difficult problem because many different amide bonds must be formed in a specific order, rather than at random. The solution to the specificity problem is protection ( ). If we want to couple alanine with leucine to synthesize Ala-Leu, for instance, we could protect the –NH 2 group of alanine and the –CO 2 H group of leucine to shield them from reacting, then form the desired Ala-Leu amide bond by reaction with EDC or DCC, and then remove the protecting groups. A number of different amino- and carboxyl-protecting groups have been devised, but only a few are used in peptide synthesis. Carboxyl groups are often protected simply by converting them into methyl or benzyl esters. Both groups are easily introduced by standard methods of ester formation ( ) and are easily removed by mild hydrolysis with aqueous NaOH. Benzyl esters can also be cleaved by catalytic hydrogenolysis of the weak benzylic C–O bond ( RCO 2 –CH 2 Ph + H 2 → RCO 2 H + PhCH 3 ). Amino groups are sometimes protected as their tert -butyloxycarbonyl amide (Boc) or, more commonly, as their fluorenylmethyloxycarbonyl amide (Fmoc). The Boc protecting group is introduced by reaction of the amino acid with di- tert -butyl dicarbonate in a nucleophilic acyl substitution reaction and is removed by brief treatment with a strong acid such as trifluoroacetic acid, CF 3 CO 2 H. The Fmoc protecting group is introduced by reaction with fluorenylmethyloxycarbonyl chloride and is removed by treatment with a 20% solution of the amine piperidine in dimethylformamide as solvent. Thus, five steps are needed to synthesize a dipeptide such as Ala-Leu: These steps can be repeated to add one amino acid at a time to the growing chain or to link two peptide chains together. Many remarkable achievements in peptide synthesis have been reported, including a complete synthesis of human insulin. Insulin is composed of two chains totaling 51 amino acids linked by two disulfide bridges. The three-dimensional structure of insulin, shown previously, was determined by Dorothy Crowfoot Hodgkin, a British chemist who received the 1964 Nobel Prize in Chemistry for her work on this and other complex biological molecules. Problems

Science


Automated Peptide Synthesis: The Merrifield Solid-Phase Method As you might imagine, the synthesis of a large peptide chain by sequential addition of one amino acid at a time is a long and arduous process. An immense simplification is possible, however, using methods introduced by R. Bruce Merrifield, who received the 1984 Nobel Prize in Chemistry. In the Merrifield solid-phase method, peptide synthesis is carried out with the growing amino acid chain covalently bonded to small beads of a polymer resin, rather than in solution. In the original procedure, polystyrene resin was used, prepared so that one of every hundred or so benzene rings contained a chloromethyl (−CH 2 Cl) group. A Boc-protected C-terminal amino acid was then attached to the resin through an ester bond formed by S N 2 reaction. With the first amino acid bonded to the resin, a repeating series of four steps is then carried out to build a peptide. The steps in the solid-phase procedure have been improved substantially over the years, but the fundamental idea remains the same. A common procedure is use of the Wang resin, developed by Su-Sun Wang, with the Fmoc protecting group. Less common is using the PAM (phenylacetamidomethyl) resin with Boc as the N-protecting group. Robotic peptide synthesizers are now used to automatically repeat the coupling, washing, and deprotection steps with different amino acids. Each step occurs in high yield, and mechanical losses are minimized because the peptide intermediates are never removed from the insoluble polymer until the final step. Peptides with 20 amino acids can be routinely prepared in a few hours.

Science


Protein Structure Proteins are usually classified as either fibrous or globular, according to their three-dimensional shape. Fibrous proteins , such as the collagen in tendons and connective tissue and the myosin in muscle tissue, consist of polypeptide chains arranged side by side in long filaments. Because these proteins are tough and insoluble in water, they are used in nature for structural materials. Globular proteins , by contrast, are usually coiled into compact, roughly spherical shapes. These proteins are generally soluble in water and are mobile within cells. Most of the more than 3000 or so enzymes that have been characterized are globular proteins, including the 1300 different enzymes in the human body. Proteins are so large that the word structure takes on a broader meaning than with simpler organic compounds. In fact, chemists speak of four different levels of structure when describing proteins. The primary structure of a protein is simply the amino acid sequence. The secondary structure of a protein describes how segments of the peptide backbone orient into a regular pattern. The tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape. The quaternary structure describes how different protein molecules come together to yield large aggregate structures. Primary structure is determined, as we’ve seen, by sequencing the protein. Secondary, tertiary, and quaternary structures are determined either by NMR or by X-ray crystallography ( Chapter 12 Chemistry Matters ). The most common secondary structures are the α helix and the β -pleated sheet. An α helix is a right-handed coil of the protein backbone, much like the coil of a spiral staircase ( a). Each turn of the helix contains 3.6 amino acid residues, with a distance between coils of 540 pm, or 5.4 Å. The structure is stabilized by hydrogen bonds between amide N–H groups and C═O groups four residues away, with an N–H····O distance of 2.8 Å. The α helix is an extremely common secondary structure, and nearly all globular proteins contain many helical segments. Myoglobin, a small globular protein containing 153 amino acid residues in a single chain, is one example ( b). A β -pleated sheet differs from an α helix in that the peptide chain is fully extended rather than coiled and the hydrogen bonds occur between residues in adjacent chains ( a). The neighboring chains can run either in the same direction (parallel) or in opposite directions (antiparallel), although the antiparallel arrangement is more common and somewhat more energetically favorable. Concanavalin A, for instance, consists of two identical chains of 237 residues, with extensive regions of antiparallel β sheets ( b). (a) The α -helical secondary structure of proteins is stabilized by hydrogen bonds between the N–H group of one residue and the C═O group four residues away. (b) The structure of myoglobin, a globular protein with extensive helical regions that are shown as coiled ribbons in this representation. (a) The β -pleated sheet secondary structure of proteins is stabilized by hydrogen bonds between parallel or antiparallel chains. (b) The structure of concanavalin A, a protein with extensive regions of antiparallel β sheets, shown as flat ribbons. What about tertiary structure? Why does a protein adopt the shape it does? The forces that determine the tertiary structure of a protein are the same forces that act on all molecules, regardless of size, to provide maximum stability. Particularly important are the hydrophilic (water-loving; ) interactions of the polar side chains on acidic or basic amino acids and the hydrophobic (water-fearing) interactions of nonpolar side chains. These acidic or basic amino acids with charged side chains tend to congregate on the exterior of the protein, where they can be solvated by water. Amino acids with neutral, nonpolar side chains tend to congregate on the hydrocarbon-like interior of a protein molecule, away from the aqueous medium. Also important for stabilizing a protein’s tertiary structure are the formation of disulfide bridges between cysteine residues, the formation of hydrogen bonds between nearby amino acid residues, and the presence of ionic attractions, called salt bridges , between positively and negatively charged sites on various amino acid side chains within the protein. Because the tertiary structure of a globular protein is delicately maintained by weak intramolecular attractions, a modest change in temperature or pH is often enough to disrupt that structure and cause the protein to become denatured . Denaturation occurs under such mild conditions that the primary structure remains intact, but the tertiary structure unfolds from a specific globular shape to a randomly looped chain ( ). A representation of protein denaturation . A globular protein loses its specific three-dimensional shape and becomes randomly looped. Denaturation is accompanied by changes in both physical and biological properties. Solubility is drastically decreased, as occurs when egg white is cooked and the albumins unfold and coagulate. Most enzymes lose all catalytic activity when denatured, since a precisely defined tertiary structure is required for their action. Although most denaturation is irreversible, some cases are known where spontaneous renaturation of an unfolded protein to its stable tertiary structure occurs, accompanied by a full recovery of biological activity.

Science


Enzymes and Coenzymes An enzyme is a substance—usually a large protein—that acts as a catalyst for a biological reaction. Like all catalysts, an enzyme doesn’t affect the equilibrium constant of a reaction and can’t bring about a chemical change that is otherwise unfavorable. An enzyme acts only to lower the activation energy for a reaction, thereby making the reaction take place more rapidly than it otherwise would. Sometimes, in fact, the rate acceleration brought about by enzymes is extraordinary. Millionfold rate increases are common, and the glycosidase enzymes that hydrolyze polysaccharides increase the reaction rate by a factor of more than 10 17 , changing the time required for the reaction from millions of years to milliseconds! Unlike many of the catalysts that chemists use in the laboratory, enzymes are usually specific in their action. Often, in fact, an enzyme will catalyze only a single reaction of a single compound, called the enzyme’s substrate. For example, the enzyme amylase, found in the human digestive tract, catalyzes only the hydrolysis of starch to yield glucose; cellulose and other polysaccharides are untouched by amylase. Different enzymes have different specificities. Some, such as amylase, are specific for a single substrate, but others operate on a range of substrates. Papain, for instance, a globular protein of 212 amino acids isolated from papaya fruit, catalyzes the hydrolysis of many kinds of peptide bonds. In fact, it’s this ability to hydrolyze peptide bonds that makes papain useful as a cleaner for contact lenses. Enzymes function through a pathway that involves initial formation of an enzyme–substrate complex (E · S), followed by a multistep chemical conversion of the enzyme-bound substrate into enzyme-bound product (E · P) and final release of product from the complex. E + S ⇄ E ⋅ S ⇄ E ⋅ P ⇄ E + P The overall rate constant for conversion of the E · S complex to products E · P is called the turnover number because it represents the number of substrate molecules a single enzyme molecule turns over into product per unit time. A value of about 10 3 per second is typical, although carbonic anhydrase can reach a value of up to 600,000. The extraordinary rate accelerations achieved by enzymes are due to a combination of several factors. One important factor is simple geometry: an enzyme will adjust its shape to hold the substrate, other reactants, and various catalytic sites on acidic or basic residues in the precise geometry needed for reaction. In addition, the wrapping of the enzyme around the substrate can create specialized microenvironments that protect the substrate from the aqueous medium and can dramatically change the behavior of acid–base catalytic residues in the active site. But perhaps most important is that the enzyme stabilizes and thus lowers the energy of the rate-limiting transition state for reaction. That is, it’s not the ability of the enzyme to bind the substrate that matters but rather its ability to bind and stabilize the transition state. Often, in fact, the enzyme binds the transition structure as much as 10 12 times more tightly than it binds the substrate or products. An energy diagram for an enzyme-catalyzed process might resemble that in . Energy diagrams for uncatalyzed and enzyme-catalyzed processes . The enzyme makes available an alternative, lower-energy pathway. Rate enhancement is due to the ability of the enzyme to bind to the transition state for product formation, thereby lowering its energy. Enzymes are classified into six categories depending on the kind of reaction they catalyze, as shown in . Oxidoreductases catalyze oxidations and reductions; transferases catalyze the transfer of a group from one substrate to another; hydrolases catalyze hydrolysis reactions of esters, amides, and related substrates; lyases catalyze the elimination or addition of a small molecule such as H 2 O from or to a substrate; isomerases catalyze isomerizations; and ligases catalyze the bonding of two molecules, often coupled with the hydrolysis of ATP. The systematic name of an enzyme has two parts, ending with - ase. The first part identifies the enzyme’s substrate, and the second part identifies its class. Hexose kinase, for example, is a transferase that catalyzes the transfer of a phosphate group from ATP to a hexose sugar. Classification of Enzymes Class Some subclasses Function Oxidoreductases Dehydrogenases Oxidases Reductases Introduction of double bond Oxidation Reduction Transferases Kinases Transaminases Transfer of phosphate group Transfer of amino group Hydrolases Lipases Nucleases Proteases Hydrolysis of ester Hydrolysis of phosphate Hydrolysis of amide Lyases Decarboxylases Dehydrases Loss of CO 2 Loss of H 2 O Isomerases Epimerases Isomerization of chirality center Ligases Carboxylases Synthetases Addition of CO 2 Formation of a new bond In addition to their protein part, most enzymes also contain a small non-protein part called a cofactor . A cofactor can be either an inorganic ion, such as Zn 2+ , or a small organic molecule, called a coenzyme . A coenzyme is not a catalyst but is a reactant that undergoes chemical change during the reaction and requires an additional step to return to its initial state. Many coenzymes are derived from vitamins—substances that an organism requires in small amounts for growth but is unable to synthesize and must receive in its diet ( Chapter 20 Chemistry Matters ). Coenzyme A from pantothenate (vitamin B 5 ), NAD + from niacin (vitamin B 3 ), FAD from riboflavin (vitamin B 2 ), tetrahydrofolate from folic acid, pyridoxal phosphate from pyridoxine (vitamin B 6 ), and thiamin diphosphate from thiamin (vitamin B 1 ) are examples. shows the structures of some common coenzymes. Problems

Science


How Do Enzymes Work? Citrate Synthase As we saw in the previous section, enzymes work by bringing substrate and other reactant molecules together, holding them in the orientation necessary for reaction, providing any necessary acidic or basic sites to catalyze specific steps, and stabilizing the transition state for reaction. As an example, let’s look at citrate synthase, an enzyme that catalyzes the aldol-like addition of acetyl CoA to oxaloacetate to give citrate. This reaction is the first step in the citric acid cycle, in which acetyl groups produced by degradation of food molecules are metabolized to yield CO 2 and H 2 O. We’ll look at the details of the citric acid cycle in Section 29.7 . Citrate synthase is a globular protein of 433 amino acids with a deep cleft lined by an array of functional groups that can bind to the substrate, oxaloacetate. On binding oxaloacetate, the original cleft closes and another opens up nearby to bind acetyl CoA. This second cleft is also lined by appropriate functional groups, including a histidine at position 274 and an aspartic acid at position 375. The two reactants are now held by the enzyme in close proximity and with a suitable orientation for reaction. shows the structure of citrate synthase as determined by X-ray crystallography, along with a close-up of the active site. Structures and Functions of Some Common Coenzymes Coenzymes X-ray crystal structure of citrate synthase . Part (a) is a space-filling model and part (b) is a ribbon model, which emphasizes the α -helical segments of the protein chain and indicates that the enzyme is dimeric; that is, it consists of two identical chains held together by hydrogen bonds and other intermolecular attractions. Part (c) is a close-up of the active site in which oxaloacetate and an unreactive acetyl CoA mimic are bound. As shown in , the first step in the aldol reaction of acetyl CoA and oxaloacetate is generation of the enol of acetyl CoA. The side-chain carboxyl of an aspartate residue acts as base to abstract an acidic α proton, while at the same time the side-chain imidazole ring of a histidine donates H + to the carbonyl oxygen. The enol thus produced then performs a nucleophilic addition to the ketone carbonyl group of oxaloacetate. The first histidine acts as a base to remove the –OH hydrogen from the enol, while a second histidine residue simultaneously donates a proton to the oxaloacetate carbonyl group, giving citryl CoA. Water then hydrolyzes the thiol ester group in citryl CoA in a nucleophilic acyl substitution reaction, releasing citrate and coenzyme A as the final products. MECHANISM Mechanism of the addition of acetyl CoA to oxaloacetate to give ( S )-citryl CoA, catalyzed by citrate synthase . The Protein Data Bank Enzymes are so large, so structurally complex, and so numerous that the use of computer databases and molecular visualization programs has become an essential tool for studying biological chemistry. Of the various databases available online, the Kyoto Encyclopedia of Genes and Genomes (KEGG) database (http://www.genome.jp/kegg/pathway.html), maintained by the Kanehisa Laboratory of Kyoto University Bioinformatics Center, is useful for obtaining information on biosynthetic pathways of the sort we’ll be describing in Chapter 29. For obtaining information on a specific enzyme, the BRENDA database (http://www.brenda-enzymes.org), maintained by the Institute of Biochemistry at the University of Cologne, Germany, is particularly valuable. Perhaps the most useful of all biological databases is the Protein Data Bank (PDB), operated by the Research Collaboratory for Structural Bioinformatics (RCSB). The PDB is a worldwide repository of X-ray and NMR structural data for biological macromolecules. In mid-2022, data for more than 192,888 structures were available, and more than 7000 new ones were being added yearly. To access the Protein Data Bank, go to www.rcsb.org and a home page like that shown in will appear. As with much that is available online, however, the PDB site is changing rapidly, so you may not see quite the same thing. The Protein Data Bank home page . (credit: RCSB Protein Data Bank) To learn how to use the PDB, begin by running the short tutorial listed under Learn on the left side column. After that introduction, start exploring. Let’s say you want to view citrate synthase, the enzyme that catalyzes the addition of acetyl CoA to oxaloacetate to give citrate. Type “citrate synthase” (with quotation marks) into the small search box on the top line, click on “Search,” and a list of 2,400 or so structures will appear. Scroll down near the end of the list until you find the entry with a PDB code of 5CTS and the title “Proposed Mechanism for the Condensation Reaction of Citrate Synthase: 1.9-Angstroms Structure of the Ternary Complex with Oxaloacetate and Carboxymethyl Coenzyme A.” Alternatively, if you know the code of the enzyme you want, you can enter it directly into the search box. Click on the PDB code of entry 5CTS, and a new page containing information about the enzyme will open. If you choose, you can download the structure file to your computer and open it with any of numerous molecular graphics programs to see an image like that in . The biologically active molecule is a dimer of two identical subunits consisting primarily of α -helical regions displayed as coiled ribbons. For now, just select “View in Jmol” under the enzyme image on the right side of the screen to see some of the options for visualizing and further exploring the enzyme. An image of citrate synthase, downloaded from the Protein Data Bank . (credit: Protein Data Bank, 1AL6 B. PDB ID: 1AL6 B. Schwartz, K.W. Vogel, K.C. Usher, C. Narasimhan, H.M. Miziorko, S.J. Remington, D.G. Drueckhammer. Mechanisms of Enzyme-Catalyzed Deprotonation of Acetyl-Coenzyme A, CC BY 1.0) α -amino acids α helix backbone β -pleated sheet C-terminal amino acid coenzyme cofactor denatured Edman degradation enzyme fibrous proteins globular proteins isoelectric point (p I ) N-terminal amino acid peptides primary structure proteins quaternary structure residues secondary structure side chain tertiary structure turnover number zwitterion Key Concepts and Summary Proteins and peptides are large biomolecules made of α -amino acid residues linked together by amide, or peptide, bonds. Twenty amino acids are commonly found in proteins, and all except glycine have stereochemistry similar to that of L sugars. In neutral solution, amino acids exist as dipolar zwitterions . Amino acids can be synthesized in racemic form by several methods, including ammonolysis of an α -bromo acid, alkylation of diethyl acetamidomalonate, and reductive amination of an α -keto acid. Alternatively, an enantioselective synthesis of amino acids can be carried out using a chiral hydrogenation catalyst. Determining the structure of a peptide or protein begins with amino acid analysis. The peptide is hydrolyzed to its constituent α -amino acids, which are separated and identified. Next, the peptide is sequenced. Edman degradation by treatment with phenyl isothiocyanate (PITC) cleaves one residue from the N terminus of the peptide and forms an easily identifiable phenylthiohydantoin (PTH) derivative of the N-terminal amino acid . An automated series of Edman degradations can sequence peptide chains up to 50 residues in length. Peptide synthesis involves the use of protecting groups. An N-protected amino acid with a free –CO 2 H group is coupled using DCC or EDC to an O-protected amino acid with a free –NH 2 group. Amide formation occurs, the protecting groups are removed, and the sequence is repeated. Amines are usually protected as their tert -butyloxycarbonyl (Boc) or fluorenylmethyloxycarbonyl (Fmoc) derivatives; acids are usually protected as esters. The synthesis is often carried out by the Merrifield solid-phase method, in which the peptide is bonded to insoluble polymer beads. Proteins have four levels of structure. Primary structure describes a protein’s amino acid sequence; secondary structure describes how segments of the protein chain orient into regular patterns—either α helix or β -pleated sheet ; tertiary structure describes how the entire protein molecule coils into an overall three-dimensional shape; and quaternary structure describes how individual protein molecules aggregate into larger structures. Proteins are classified as either globular or fibrous. Fibrous proteins such as α -keratin are tough, rigid, and water-insoluble; globular proteins such as myoglobin are water-soluble and roughly spherical in shape. Many globular proteins are enzymes —substances that act as catalysts for biological reactions. Enzymes are grouped into six classes according to the kind of reaction they catalyze. In addition to their protein part, many enzymes contain cofactors , which can be either metal ions or small organic molecules called coenzymes . Summary of Reactions Amino acid synthesis ( ) From α -bromo acids Diethyl acetamidomalonate synthesis Reductive amination of an α -keto acid Enantioselective synthesis Peptide sequencing by Edman degradation ( ) Peptide synthesis ( ) Amine protection Carboxyl protection Additional Problems Visualizing Chemistry Mechanism Problems Amino Acid Structures and Chirality Amino Acid Synthesis and Reactions Peptides and Enzymes General Problems

Science


Why This Chapter? Soap bubbles, so common yet so beautiful, are made from animal fat, a lipid. (credit: “Reflections in soap bubbles” by Image Picture Photography/Flickr, CC BY 2.0) We’ve now covered two of the four major classes of biomolecules—proteins and carbohydrates—and have two remaining. In this chapter, we’ll cover lipids, the largest and most diverse class of biomolecules, looking both at their structure and function and at their metabolism. Lipids are naturally occurring organic molecules that have limited solubility in water and can be isolated from organisms by extraction with nonpolar organic solvents. Fats, oils, waxes, many vitamins and hormones, and most nonprotein cell-membrane components are some examples. Note that this definition differs from the sort used for carbohydrates and proteins in that lipids are defined by a physical property (solubility) rather than by structure. Of the many kinds of lipids, we’ll be concerned in this chapter with only a few: triacylglycerols, eicosanoids, terpenoids, and steroids. Lipids are classified into two broad types: those like fats and waxes, which contain ester linkages and can be hydrolyzed, and those like cholesterol and other steroids, which don’t have ester linkages and can’t be hydrolyzed.

Science


Waxes, Fats, and Oils Waxes are mixtures of esters of long-chain carboxylic acids with long-chain alcohols. The carboxylic acid usually has an even number of carbons from 16 to 36, while the alcohol has an even number of carbons from 24 to 36. One of the major components of beeswax, for instance, is triacontyl hexadecanoate, the ester of the C 30 alcohol (1-triacontanol) and the C 16 acid (hexadecanoic acid). The waxy protective coatings on most fruits, berries, leaves, and animal furs have similar structures. Animal fats and vegetable oils are the most widely occurring lipids. Although they appear different—animal fats like butter and lard are solids, whereas vegetable oils like corn and peanut oil are liquid—their structures are closely related. Chemically, fats and oils are triglycerides, or triacylglycerols —triesters of glycerol with three long-chain carboxylic acids called fatty acids . Animals use fats for long-term energy storage because they are far less highly oxidized than carbohydrates and provide about six times as much energy as an equal weight of stored, hydrated glycogen. Hydrolysis of a fat or oil with aqueous NaOH yields glycerol and three fatty acids. The fatty acids are generally unbranched and contain an even number of carbon atoms between 12 and 20. If double bonds are present, they have mostly, although not entirely, Z, or cis, geometry. The three fatty acids of a specific triacylglycerol molecule need not be the same, and the fat or oil from a given source is likely to be a complex mixture of many different triacylglycerols. lists some of the commonly occurring fatty acids, and lists the approximate composition of fats and oils from different sources. Structures of Some Common Fatty Acids Name No. of carbons Melting point (°C) Structure Saturated Lauric 12 43.2 CH 3 (CH 2 ) 10 CO 2 H Myristic 14 53.9 CH 3 (CH 2 ) 12 CO 2 H Palmitic 16 63.1 CH 3 (CH 2 ) 14 CO 2 H Stearic 18 68.8 CH 3 (CH 2 ) 16 CO 2 H Arachidic 20 76.5 CH 3 (CH 2 ) 18 CO 2 H Unsaturated Palmitoleic 16 –0.1 ( Z ) -CH 3 ( CH 2 ) 5 CH═ CH(CH 2 ) 7 CO 2 H Oleic 18 13.4 ( Z ) -CH 3 ( CH 2 ) 7 CH═ CH(CH 2 ) 7 CO 2 H Linoleic 18 –12 ( Z , Z ) -CH 3 ( CH 2 ) 4 (CH═ CHCH 2 ) 2 (CH 2 ) 6 CO 2 H Linolenic 18 –11 ( all Z ) -CH 3 CH 2 (CH═ CHCH 2 ) 3 (CH 2 ) 6 CO 2 H Arachidonic 20 –49.5 ( all Z ) -CH 3 (CH 2 ) 4 (CH═ CHCH 2 ) 4 CH 2 CH 2 CO 2 H Composition of Some Fats and Oils Saturated fatty acids (%) Unsaturated fatty acids (%) Source C 12 lauric C 14 myristic C 16 palmitic C 18 stearic C 18 oleic C 18 linoleic Animal fat Lard — 1 25 15 50 6 Butter 2 10 25 10 25 5 Human fat 1 3 25 8 46 10 Whale blubber — 8 12 3 35 10 Vegetable oil Coconut 50 18 8 2 6 1 Corn — 1 10 4 35 45 Olive — 1 5 5 80 7 Peanut — — 7 5 60 20 More than 100 different fatty acids are known and about 40 occur widely. Palmitic acid (C 16 ) and stearic acid (C 18 ) are the most abundant saturated fatty acids; oleic and linoleic acids (both C 18 ) are the most abundant unsaturated ones. Oleic acid is monounsaturated because it has only one double bond, whereas linoleic, linolenic, and arachidonic acids are polyunsaturated fatty acids because they have more than one double bond. Linoleic and linolenic acids occur in cream and are essential in the human diet; infants grow poorly and develop skin lesions if fed a diet of nonfat milk for prolonged periods. Linolenic acid, in particular, is an example of an omega-3 fatty acid , which has been found to lower blood triglyceride levels and reduce the risk of heart attack. The name omega-3 means that there is a double bond three carbons in from the noncarboxyl end of the chain. The data in show that unsaturated fatty acids generally have lower melting points than their saturated counterparts, a trend that is also true for triacylglycerols. Since vegetable oils generally have a higher proportion of unsaturated to saturated fatty acids than animal fats ( ), they have lower melting points. The difference is a consequence of structure. Saturated fats have a uniform shape that allows them to pack together efficiently in a crystal lattice. In unsaturated vegetable oils, however, the C ═ C bonds introduce bends and kinks into the hydrocarbon chains, making crystal formation more difficult. The more double bonds there are, the harder it is for the molecules to crystallize and the lower the melting point of the oil. The C ═ C bonds in vegetable oils can be reduced by catalytic hydrogenation, typically carried out at high temperature using a nickel catalyst, to produce saturated solid or semisolid fats. Prior to 2015, industrially produced margarine and shortening were produced by hydrogenating soybean, peanut, or cottonseed oil until the proper consistency is obtained. As of 2015, trans fats are no longer considered safe by the FDA and have since been phased out of industrially produced foods. Unfortunately, the hydrogenation reaction is accompanied by some cis–trans isomerization of the remaining double bonds, producing fats with about 10% to 15% trans unsaturated fatty acids. Dietary intake of trans fatty acids increases cholesterol levels in the blood, thereby increasing the risk of heart problems. The conversion of linoleic acid into elaidic acid is an example. Problems

Science


Soap Soap has been in use for nearly 5000 years. As early as 2800 BC , the Babylonians boiled fats with ashes to create a soap-like substance. Ancient Egyptian medical papyri dating from 1550 BC reveals that Egyptians bathed regularly with soap made from a mixture of animal fats, vegetable oils, and alkaline salts. Chemically, soap is a mixture of the sodium or potassium salts of the long-chain fatty acids produced by hydrolysis ( saponification ) of animal fat with alkali. Wood ash was used as a source of alkali until the early 1800s, when the LeBlanc process for making Na 2 CO 3 by heating sodium sulfate with limestone became available. Crude soap curds contain glycerol and excess alkali as well as soap but can be purified by boiling with water and adding NaCl or KCl to precipitate the pure carboxylate salts. The smooth soap that precipitates is dried, perfumed, and pressed into bars for household use. Dyes are added to make colored soaps, antiseptics are added for medicated soaps, pumice is added for scouring soaps, and air is blown in for soaps that float. Regardless of these extra treatments and regardless of price, though, all soaps are basically the same. Soaps act as cleansers because the two ends of a soap molecule are so different. The carboxylate end of the long-chain molecule is ionic and therefore hydrophilic ( ), or attracted to water. The long hydrocarbon portion of the molecule, however, is nonpolar and hydrophobic, avoiding water and therefore more soluble in oils. The net effect of these two opposing tendencies is that soaps are attracted to both oils and water and are therefore useful as cleansers. When soaps are dispersed in water, the long hydrocarbon tails cluster together on the inside of a tangled, hydrophobic ball, while the ionic heads on the surface of the cluster protrude into the water layer. These spherical clusters, called micelles , are shown schematically in . Grease and oil droplets are solubilized in water when they are coated by the nonpolar, hydrophobic tails of soap molecules in the center of micelles. Once solubilized, the grease and dirt can be rinsed away. A soap micelle solubilizing a grease particle in water. An electrostatic potential map of a fatty acid carboxylate shows how the negative charge is located in the head group. As useful as they are, soaps also have drawbacks. In hard water, which contains metal ions, soluble sodium carboxylates are converted into insoluble magnesium and calcium salts, leaving the familiar ring of scum around bathtubs and a gray tinge on white clothes. Chemists have circumvented this problem by synthesizing a class of synthetic detergents based on salts of long-chain alkylbenzenesulfonic acids. The mechanism of synthetic detergents is the same as that of soaps: the alkylbenzene end of the molecule is attracted to grease, while the anionic sulfonate end is attracted to water. Unlike soaps, though, sulfonate detergents don’t form insoluble metal salts in hard water and don’t leave an unpleasant scum. Problems

Science


Phospholipids Just as waxes, fats, and oils are esters of carboxylic acids, phospholipids are esters of phosphoric acid, H 3 PO 4 . Phospholipids are of two general types: glycerophospholipids and sphingomyelins . Glycerophospholipids are based on phosphatidic acid, which contains a glycerol backbone linked by ester bonds to two fatty acids and one phosphoric acid. Although the fatty-acid residues can be any of the C 12 –C 20 units typically present in fats, the acyl group at C1 is usually saturated and the one at C2 is usually unsaturated. The phosphate group at C3 is also bonded to an amino alcohol such as choline [HOCH 2 CH 2 N(CH 3 ) 3 ] + , ethanolamine (HOCH 2 CH 2 NH 2 ), or serine [HOCH 2 CH(NH 2 )CO 2 H]. The compounds are chiral and have an L, or R, configuration at C2. Sphingomyelins are the second major group of phospholipids. These compounds have sphingosine or a related dihydroxyamine as their backbone and are particularly abundant in brain and nerve tissue, where they are a major constituent of the coating around nerve fibers. Phospholipids are found widely in both plant and animal tissues and make up approximately 50% to 60% of cell membranes. Because they are like soaps in having a long, nonpolar hydrocarbon tail bound to a polar ionic head, phospholipids in the cell membrane organize into a lipid bilayer about 5.0 nm (50 Å) thick. As shown in , the nonpolar tails aggregate in the center of the bilayer in much the same way that soap tails aggregate in the center of a micelle. This bilayer serves as an effective barrier to the passage of water, ions, and other components into and out of cells. Aggregation of glycerophospholipids into the lipid bilayer that composes cell membranes.

Science


Prostaglandins and Other Eicosanoids Prostaglandins are a group of C 20 lipids that contain a five-membered ring with two long side chains. First isolated in the 1930s by Ulf von Euler at the Karolinska Institute in Sweden, much of the structural and chemical work on prostaglandins was carried out by Sune Bergström and Bengt Samuelsson. All three shared Nobel Prizes for their work. The name prostaglandin derives from the fact that the compounds were first isolated from sheep prostate glands, but they have subsequently been found to be present in small amounts in all body tissues and fluids. The several dozen known prostaglandins have an extraordinarily wide range of biological effects. Among their many properties, they can lower blood pressure, affect blood platelet aggregation during clotting, lower gastric secretions, control inflammation, affect kidney function, affect reproductive systems, and stimulate uterine contractions during childbirth. Prostaglandins, together with related compounds called thromboxanes and leukotrienes, make up a class of compounds called eicosanoids because they are derived biologically from 5,8,11,14-eicosatetraenoic acid, or arachidonic acid ( ). Prostaglandins (PG) have a cyclopentane ring with two long side chains; thromboxanes (TX) have a six-membered, oxygen-containing ring; and leukotrienes (LT) are acyclic. Structures of some representative eicosanoids. All are derived biologically from arachidonic acid . Eicosanoids are named based on their ring system (PG, TX, or LT), substitution pattern, and number of double bonds. The various substitution patterns on the ring are indicated by letter as in , and the number of double bonds is indicated by a subscript. Thus, PGE 1 is a prostaglandin with the “E” substitution pattern and one double bond. The numbering of the atoms in the various eicosanoids is the same as in arachidonic acid, starting with the –CO 2 H carbon as C1, continuing around the ring, and ending with the –CH 3 carbon at the other end of the chain as C20. The naming system for eicosanoids. Eicosanoid biosynthesis begins with the conversion of arachidonic acid to PGH 2 , catalyzed by the multifunctional PGH synthase (PGHS), also called cyclooxygenase (COX). There are two distinct enzymes, PGHS-1 and PGHS-2 (or COX-1 and COX-2), both of which accomplish the same reaction but appear to function independently. COX-1 carries out the normal physiological production of prostaglandins, and COX-2 produces additional prostaglandin in response to arthritis or other inflammatory conditions. Vioxx, Bextra, and several other drugs selectively inhibit the COX-2 enzyme but cause potentially serious heart and gastrointestinal problems in weakened patients. (See the Chapter 15 Chemistry Matters .) PGHS accomplishes two transformations, an initial reaction of arachidonic acid with O 2 to yield PGG 2 and a subsequent reduction of the hydroperoxide group (–OOH) to the alcohol PGH 2 . The sequence of steps involved in these transformations was shown in . Further processing of PGH 2 leads to other eicosanoids. PGE 2 , for instance, arises by an isomerization of PGH 2 catalyzed by PGE synthase (PGES) ( ). Mechanism of the conversion of PGH 2 into PGE 2 . Problems

Science


Terpenoids We saw in the Chapter 8 Chemistry Matters that terpenoids are a vast and diverse group of lipids found in all living organisms. Despite their apparent structural differences, all terpenoids contain a multiple of five carbons and are derived biosynthetically from the five-carbon precursor isopentenyl diphosphate ( ). Although formally a terpenoid contains oxygen, while a hydrocarbon is called a terpene, we’ll use the term terpenoid to refer to both for simplicity. Structures of some representative terpenoids. You might recall from the chapter on Alkenes: Reactions and Synthesis that terpenoids are classified according to the number of five-carbon multiples they contain. Monoterpenoids contain 10 carbons and are derived from two isopentenyl diphosphates, sesquiterpenoids contain 15 carbons and are derived from three isopentenyl diphosphates, diterpenoids contain 20 carbons and are derived from four isopentenyl diphosphates, and so on, up to triterpenoids (C 30 ) and tetraterpenoids (C 40 ). Lanosterol, for instance, is a triterpenoid from which steroid hormones are made, and β -carotene is a tetraterpenoid that serves as a dietary source of vitamin A ( ). The terpenoid precursor isopentenyl diphosphate, formerly called isopentenyl pyrophosphate and thus abbreviated IPP, is biosynthesized by two different pathways, depending on the organism and the structure of the final product. In animals and higher plants, sesquiterpenoids and triterpenoids arise primarily from the mevalonate pathway, whereas monoterpenoids, diterpenoids, and tetraterpenoids are biosynthesized by the 1-deoxyxylulose 5-phosphate (DXP) pathway, also called the methylerithritol phosphate, or MEP, pathway. In bacteria, both pathways are used. We’ll look only at the mevalonate pathway, which is more common and better understood at present. The Mevalonate Pathway to Isopentenyl Diphosphate As shown in , the mevalonate pathway begins with the conversion of acetate to acetyl CoA, followed by Claisen condensation to yield acetoacetyl CoA. A second carbonyl condensation reaction with a third molecule of acetyl CoA, this one an aldol-like process, then yields the six-carbon compound 3-hydroxy-3-methylglutaryl CoA, which is reduced to give mevalonate. Phosphorylation, followed by loss of CO 2 and phosphate ion, completes the process. Step 1 of : Claisen Condensation The first step in mevalonate biosynthesis is a Claisen condensation to yield acetoacetyl CoA, a reaction catalyzed by acetoacetyl-CoA acetyltransferase. An acetyl group is first bound to the enzyme by a nucleophilic acyl substitution reaction with a cysteine –SH group. Formation of an enolate ion from a second molecule of acetyl CoA, followed by Claisen condensation, then yields the product. Step 2 of : Aldol Condensation Acetoacetyl CoA next undergoes an aldol-like addition of an acetyl CoA enolate ion in a reaction catalyzed by 3-hydroxy-3-methylglutaryl-CoA synthase. The reaction occurs by initial binding of the substrate to a cysteine –SH group in the enzyme, followed by enolate-ion addition and subsequent hydrolysis to give (3 S )-3-hydroxy-3-methylglutaryl CoA (HMG-CoA). MECHANISM The mevalonate pathway for the biosynthesis of isopentenyl diphosphate from three molecules of acetyl CoA. Individual steps are explained in the text. Step 3 of : Reduction Reduction of HMG-CoA to give ( R )-mevalonate is catalyzed by 3-hydroxy-3-methylglutaryl-CoA reductase and requires 2 equivalents of reduced nicotinamide adenine dinucleotide phosphate (NADPH), a close relative of NADH ( ). The reaction occurs in two steps and proceeds through an aldehyde intermediate. The first step is a nucleophilic acyl substitution reaction involving hydride transfer from NADPH to the thioester carbonyl group of HMG-CoA. Following expulsion of HSCoA as leaving group, the aldehyde intermediate undergoes a second hydride addition to give mevalonate. Step 4 of : Phosphorylation and Decarboxylation Three additional reactions are needed to convert mevalonate to isopentenyl diphosphate. The first two are straightforward phosphorylations by ATP that occur through nucleophilic substitution reactions on the terminal phosphorus. Mevalonate is first converted to mevalonate 5-phosphate (phosphomevalonate) by reaction with ATP; mevalonate 5-phosphate then reacts with a second ATP to give mevalonate 5-diphosphate (diphosphomevalonate). The third reaction results in phosphorylation of the tertiary hydroxyl group, followed by decarboxylation and loss of phosphate ion. The final decarboxylation of mevalonate 5-diphosphate seems unusual because decarboxylations of acids do not typically occur except in β -keto acids and malonic acids, in which the carboxylate group is two atoms away from an additional carbonyl group. As discussed in , the function of this second carbonyl group is to act as an electron acceptor and stabilize the charge resulting from loss of CO 2 . In fact, though, the decarboxylation of a β -keto acid and the decarboxylation of mevalonate 5-diphosphate are closely related. Catalyzed by mevalonate-5-diphosphate decarboxylase, the substrate is first phosphorylated on the free –OH group by reaction with ATP to give a tertiary phosphate, which undergoes spontaneous S N 1-like dissociation to give a tertiary carbocation. The positive charge then acts as an electron acceptor to facilitate decarboxylation in the same way a β carbonyl group does, giving isopentenyl diphosphate. (In the following structures, the diphosphate group is abbreviated OPP.) Problems Conversion of Isopentenyl Diphosphate to Terpenoids The conversion of isopentenyl diphosphate (IPP) to terpenoids begins with its isomerization to dimethylallyl diphosphate, abbreviated DMAPP and formerly called dimethylallyl pyrophosphate. These two C 5 building blocks then combine to give the C 10 unit geranyl diphosphate (GPP). The corresponding alcohol, geraniol, is itself a fragrant terpenoid that occurs in rose oil. Further combination of GPP with another IPP gives the C 15 unit farnesyl diphosphate (FPP), and so on, up to C 25 . Terpenoids with more than 25 carbons—that is, triterpenoids (C 30 ) and tetraterpenoids (C 40 )—are synthesized by dimerization of C 15 and C 20 units, respectively ( ). Triterpenoids and steroids, in particular, arise from dimerization of farnesyl diphosphate to give squalene. An overview of terpenoid biosynthesis from isopentenyl diphosphate. The isomerization of isopentenyl diphosphate to dimethylallyl diphosphate is catalyzed by IPP isomerase and occurs through a carbocation pathway. Protonation of the IPP double bond by a hydrogen-bonded cysteine residue in the enzyme gives a tertiary carbocation intermediate, which is deprotonated by a glutamate residue as base to yield DMAPP. X-ray structural studies on the enzyme show that it holds the substrate in an unusually deep, well-protected pocket to shield the highly reactive carbocation from reaction with solvent or other external substances. Both the initial coupling of DMAPP with IPP to give geranyl diphosphate and the subsequent coupling of GPP with a second molecule of IPP to give farnesyl diphosphate are catalyzed by farnesyl diphosphate synthase. The process requires Mg 2+ ion, and the key step is a nucleophilic substitution reaction in which the double bond of IPP behaves as a nucleophile in displacing diphosphate ion leaving group (PP i ) or DMAPP. Evidence suggests that the DMAPP develops a considerable cationic character and that spontaneous dissociation of the allylic diphosphate ion in an S N 1-like pathway probably occurs ( ). Mechanism of the coupling reaction of dimethylallyl diphosphate (DMAPP) and isopentenyl diphosphate (IPP), to give geranyl diphosphate (GPP). Further conversion of geranyl diphosphate into monoterpenoids typically involves carbocation intermediates and multistep reaction pathways that are catalyzed by terpene cyclases. Monoterpene cyclases function by first isomerizing geranyl diphosphate to its allylic isomer linalyl diphosphate (LPP), a process that occurs by spontaneous S N 1-like dissociation to an allylic carbocation, followed by recombination. The effect of this isomerization is to convert the C2–C3 double bond of GPP into a single bond, thereby making cyclization possible and allowing E / Z isomerization of the double bond. Further dissociation and cyclization by electrophilic addition of the cationic carbon to the terminal double bond then gives a cyclic cation, which might either rearrange, undergo a hydride shift, be captured by a nucleophile, or be deprotonated to give any of the several hundred known monoterpenoids. As just one example, limonene, a monoterpenoid found in many citrus oils, arises by the biosynthetic pathway shown in . Mechanism for the formation of the monoterpenoid limonene from geranyl diphosphate. Proposing a Terpenoid Biosynthesis Pathway Propose a mechanistic pathway for the biosynthesis of α -terpineol from geranyl diphosphate. Strategy α -Terpineol, a monoterpenoid, must be derived biologically from geranyl diphosphate through its isomer linalyl diphosphate. Draw the precursor in a conformation that approximates the structure of the target molecule, and then carry out a cationic cyclization, using the appropriate double bond to displace the diphosphate leaving group. Since the target is an alcohol, the carbocation resulting from cyclization evidently reacts with water. Solution Problems

Science