24AMINO ACIDS, PEPTIDES, AND PROTEINS, Study notes of Stereochemistry

Download 24AMINO ACIDS, PEPTIDES, AND PROTEINS and more Study notes Stereochemistry in PDF only on Docsity! Proteins are the most abundant organic molecules in animals, playing important roles in all aspects of cell structure and function. Proteins are biopolymers of acids, so named because the amino group is bonded to the carbon atom, next to the carbonyl group. The physical and chemical properties of a protein are determined by its constituent amino acids. The individual amino acid subunits are joined by amide linkages called peptide bonds. Figure 24-1 shows the general structure of an acid and a protein.a-amino a A-amino COO
a-helix NH3 C H A P T E R 24 AMINO ACIDS, PEPTIDES, AND PROTEINS 24-1 Introduction 1153
FIGURE 24-1 Structure of a general protein and its constituent amino acids. The amino acids are joined by amide linkages called peptide bonds. α carbon atom α-amino group an α -amino acid side chain H2N CH OH R C O NH CH C CH3 NH O H2N CH C CH3 OH O CH C CH2OH NH O CH C H a short section of a protein peptide bonds alanine H2N CH C CH2OH OH O serine H2N CH C H OH O glycine several individual amino acids H2N CH C CH2SH OH O cysteine H2N CH C CH(CH3)2 OH O valine NH O CH C CH2SH NH O CH C CH(CH3)2 O WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1153 1154 CHAPTER 24 Amino Acids, Peptides, and Proteins TABLE 24-1 Examples of Protein Functions Class of Protein Example Function of Example structural proteins collagen, keratin strengthen tendons, skin, hair, nails enzymes DNA polymerase replicates and repairs DNA transport proteins hemoglobin transports to the cells contractile proteins actin, myosin cause contraction of muscles protective proteins antibodies complex with foreign proteins hormones insulin regulates glucose metabolism toxins snake venoms incapacitate prey O2 The term amino acid might mean any molecule containing both an amino group and any type of acid group; however, the term is almost always used to refer to an carboxylic acid. The simplest acid is aminoacetic acid, called glycine. Other common amino acids have side chains (symbolized by R) substituted on the carbon atom. For example, alanine is the amino acid with a methyl side chain. Except for glycine, the acids are all chiral. In all of the chiral amino acids, the chirality center is the asymmetric carbon atom. Nearly all the naturally occurring amino acids are found to have the (S) configuration at the carbon atom. Figure 24-2 shows a Fischer projection of the (S) enantiomer of alanine, with the carbon chain along the vertical and the carbonyl carbon at the top. Notice that the configuration of (S)-alanine is similar to that of L- with the amino group on the left in the Fischer1-2-glyceraldehyde, a a a-amino H2N9CH29C9OH O H2N9CH9C9OH O R a substituted amino acidglycine H2N9CH9C9OH O CH3 alanine (R  CH3) a a-amino a-amino Proteins have an amazing range of structural and catalytic properties as a result of their varying amino acid composition. Because of this versatility, proteins serve an as- tonishing variety of functions in living organisms. Some of the functions of the major classes of proteins are outlined in Table 24-1. The study of proteins is one of the major branches of biochemistry, and there is no clear division between the organic chemistry of proteins and their biochemistry. In this chapter, we begin the study of proteins by learning about their constituents, the amino acids. We also discuss how amino acid monomers are linked into the protein polymer, and how the properties of a protein depend on those of its constituent amino acids. These concepts are needed for the further study of protein structure and function in a biochemistry course. H C COOH COOH H L-alanine (S )-alanine H2N H2N H2N H2N CH3 CH3 H C CHO CHO H L-(–)-glyceraldehyde (S )-glyceraldehyde HO HO CH2OH CH2OH H C COOH COOH H an L-amino acid (S) configuration R R
FIGURE 24-2 Almost all the naturally occurring amino acids have the (S) configuration. They are called L-amino acids because their stereochemistry resembles that of L-1-2-glyceraldehyde. 24-2 Structure and Stereochemistry of the Acidsa-Amino WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1154 24-2B Essential Amino Acids Humans can synthesize about half of the amino acids needed to make proteins. Other amino acids, called the essential amino acids, must be provided in the diet. The ten essential amino acids, starred in Table 24-2, are the following: arginine (Arg) valine (Val) methionine (Met) leucine (Leu) threonine (Thr) phenylalanine (Phe) histidine (His) isoleucine (Ile) lysine (Lys) tryptophan (Trp) Proteins that provide all the essential amino acids in about the right proportions for human nutrition are called complete proteins. Examples of complete proteins are those in meat, fish, milk, and eggs. About 50 g of complete protein per day is adequate for adult humans. Proteins that are severely deficient in one or more of the essential amino acids are called incomplete proteins. If the protein in a person’s diet comes mostly from one incomplete source, the amount of human protein that can be synthesized is limited by the amounts of the deficient amino acids. Plant proteins are generally incomplete. Rice, corn, and wheat are all deficient in lysine. Rice also lacks threonine, and corn also lacks tryptophan. Beans, peas, and other legumes have the most complete proteins among the common plants, but they are deficient in methionine. Vegetarians can achieve an adequate intake of the essential amino acids if they eat many different plant foods. Plant proteins can be chosen to be complementary, with some foods supplying amino acids that others lack. An alternative is to supplement the vegetarian diet with a rich source of complete protein such as milk or eggs. 1*2 24-2 Structure and Stereochemistry of the Acids 1157a-Amino chemical properties of their side chains. Each amino acid is given a three-letter abbre- viation and a one-letter symbol (green) for use in writing protein structures. Notice in Table 24-2 how proline is different from the other standard amino acids. Its amino group is fixed in a ring with its carbon atom. This cyclic structure lends additional strength and rigidity to proline-containing peptides. N H COOH H proline a-amino group a carbon a PROBLEM 24-1 Draw three-dimensional representations of the following amino acids. (a) L-phenylalanine (b) L-histidine (c) D-serine (d) L-tryptophan Gelatin is made from collagen, which is a structural protein com- posed primarily of glycine, proline, and hydroxyproline. As a result, gel- atin has low nutritional value be- cause it lacks many of the essential amino acids. PROBLEM 24-3 The herbicide glyphosate (Roundup®) kills plants by inhibiting an enzyme needed for syn- thesis of phenylalanine. Deprived of phenylalanine, the plant cannot make the proteins it needs, and it gradually weakens and dies. Although a small amount of glyphosate is deadly to a plant, its human toxicity is quite low. Suggest why this powerful herbicide has little effect on humans. PROBLEM 24-2 Most naturally occurring amino acids have chirality centers (the asymmetric carbon atoms) that are named (S) by the Cahn–Ingold–Prelog convention (Section 5-3). The common natu- rally occurring form of cysteine has a chirality center that is named (R), however. (a) What is the relationship between (R)-cysteine and (S)-alanine? Do they have the opposite three-dimensional configuration (as the names might suggest) or the same configuration? (b) (S)-alanine is an L-amino acid (Figure 24-2). Is (R)-cysteine a D-amino acid or an L-amino acid? a WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1157 1158 CHAPTER 24 Amino Acids, Peptides, and Proteins 24-2C Rare and Unusual Amino Acids In addition to the standard amino acids, other amino acids are found in protein in smaller quantities. For example, 4-hydroxyproline and 5-hydroxylysine are hydroxy- lated versions of standard amino acids. These are called rare amino acids, even though they are commonly found in collagen. Some of the less common D enantiomers of amino acids are also found in nature. For example, D-glutamic acid is found in the cell walls of many bacteria, and D-serine is found in earthworms. Some naturally occurring amino acids are not acids: acid (GABA) is one of the neurotransmitters in the brain, and is a constituent of the vitamin pantothenic acid.b-alanine g-Aminobutyric a-amino N H 4-hydroxyproline OH H H COOH 4 3 2 1 5 H2N 12345 CH2 CH CH2 CH COOH OH NH2 CH2 6 5-hydroxylysine COOH CH2CH2COOH NH H2 CH2 COOH NH2 CH2 D-glutamic acid CH2 COOH CH2OH NH H2 D-serine CH2 COOH NH2 CH2 g-aminobutyric acid a abbg b-alanine Although we commonly write amino acids with an intact carboxyl group and amino group, their actual structure is ionic and depends on the pH. The carboxyl group loses a proton, giving a carboxylate ion, and the amino group is protonated to an ammonium ion. This structure is called a dipolar ion or a zwitterion (German for “dipolar ion”). The dipolar nature of amino acids gives them some unusual properties: 1. Amino acids have high melting points, generally over 200 °C. 2. Amino acids are more soluble in water than they are in ether, dichloromethane, and other common organic solvents. 3. Amino acids have much larger dipole moments than simple amines or simple acids. 4. Amino acids are less acidic than most carboxylic acids and less basic than most amines. In fact, the acidic part of the amino acid molecule is the ¬ NH3 + H3N + ¬ CH2 ¬ COO- CH3 ¬ CH2 ¬ CH2 ¬ NH2 CH3 ¬ CH2 ¬ COOH glycine, m=14 D propylamine, m=1.4 D propionic acid, m=1.7 D 1m2 H3N + ¬ CH2 ¬ COO- glycine, mp 262 °C H2N9CH9C9OH O R uncharged structure (minor component) H3N9CH9C9O
O R dipolar ion, or zwitterion (major component)  1¬ NH22 1¬ COOH224-3 Acid–Base Properties of Amino Acids WADEMC24_1153-1199hr.qxp 16-12-2008 19:31 Page 1158 24-3 Acid–Base Properties of Amino Acids 1159 group, not a group. The basic part is the group, and not a free group. Because amino acids contain both acidic and basic groups, they are amphoteric (having both acidic and basic properties). The predomi- nant form of the amino acid depends on the pH of the solution. In an acidic solution, the group is protonated to a free group, and the molecule has an overall positive charge. As the pH is raised, the loses its proton at about pH 2. This point is called the first acid-dissociation constant. As the pH is raised further, the group loses its proton at about pH 9 or 10. This point is called the second acid-dissociation constant. Above this pH, the molecule has an overall negative charge. pKa2, ¬ NH3 + pKa1, ¬ COOH ¬ COOH¬ COO- 1¬ COO-21¬ NH3 +2 H3N9CH9COO
R9COOH R9NH2 R  pKa  5 pKb  4 pKa  10 pKb  12 ¬ NH2 ¬ COO-¬ COOH H3N9CH9COOH R  pKa1
2 pKa2
9–10cationic in acid H3N9CH9COO
R  neutral H2N9CH9COO
R anionic in base −OH H+ −OH H+ Figure 24-3 shows a titration curve for glycine. The curve starts at the bottom left, where glycine is entirely in its cationic form. Base is slowly added, and the pH is recorded. At pH 2.3, half of the cationic form has been converted to the zwitterionic form. At pH 6.0, essentially all the glycine is in the zwitterionic form. At pH 9.6, half of the zwitterionic form has been converted to the basic form. From this graph, we can see that glycine is mostly in the cationic form at pH values below 2.3, mostly in the zwitterionic form at pH values between 2.3 and 9.6, and mostly in the anionic form at pH values above 9.6. By varying the pH of the solution, we can control the charge on the molecule. This ability to control the charge of an amino acid is useful for separat- ing and identifying amino acids by electrophoresis, as described in Section 24-4.
FIGURE 24-3 A titration curve for glycine. The pH controls the charge on glycine: cationic below pH 2.3; zwitterionic between pH 2.3 and 9.6; and anionic above pH 9.6. The isoelectric pH is 6.0. 12 10 8 6 4 2 0 0.5 1.51 Equivalents of −OH added 2 0.5 1.51 2 pKa2 = 9.6 pKa1 = 2.3 Isoelectric point = 6.0 H2N CH2 C O− O H3N CH2 C zwitterionic near the isoelectric point O− O H3N CH2 C cationic below pH 2.3 OH O + + pH . . anionic above pH 9.6 WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1159 1162 CHAPTER 24 Amino Acids, Peptides, and Proteins 24-5A Reductive Amination Reductive amination of ketones and aldehydes is one of the best methods for synthe- sizing amines (Section 19-19). It also forms amino acids. When an is treated with ammonia, the ketone reacts to form an imine. The imine is reduced to an amine by hydrogen and a palladium catalyst. Under these conditions, the carboxylic acid is not reduced. a-ketoacid excess NH3 H2 Pd O R9C9COOH a-ketoacid N9H R9C9COO
NH4 imine NH2 R9CH9COO
a-amino acid This entire synthesis is accomplished in one step by treating the with ammonia and hydrogen in the presence of a palladium catalyst. The product is a racemic acid. The following reaction shows the synthesis of racemic phenyl- alanine from 3-phenyl-2-oxopropanoic acid. We call reductive amination a biomimetic (“mimicking the biological process”) synthesis because it resembles the biological synthesis of amino acids. The biosyn- thesis begins with reductive amination of acid (an intermediate in the metabolism of carbohydrates), using ammonium ion as the aminating agent and NADH as the reducing agent. The product of this enzyme-catalyzed reaction is the pure L enantiomer of glutamic acid. a-ketoglutaric NH3, H2 Pd O Ph9CH29C9COOH 3-phenyl-2-oxopropanoic acid NH2 Ph9CH29CH9COO
NH4 (D,L)-phenylalanine (ammonium salt) (30%) a-amino a-ketoacid HOOC CH2CH2 C O COO
 NH4  H N sugar H NH2  H enzyme HOOC CH2CH2 CH NH3 COO
 C H N   H2O O NH2 NAD L-glutamic acid NADH a-ketoglutaric acid C O sugar  Biosynthesis of other amino acids uses L-glutamic acid as the source of the amino group. Such a reaction, moving an amino group from one molecule to another, is called a transamination, and the enzymes that catalyze these reactions are called transaminases. For example, the following reaction shows the biosynthesis of aspartic acid using glutamic acid as the nitrogen source. Once again, the enzyme-catalyzed biosynthesis gives the pure L enantiomer of the product. O L-glutamic acid transaminase HOOC9CH2CH29CH9COO
NH3 oxaloacetic acid HOOC9CH29C9COO
 O a-ketoglutaric acid L-aspartic acid HOOC9CH2CH29C9COO
NH3 HOOC9CH29CH9COO
 WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1162 24-5 Synthesis of Amino Acids 1163 PROBLEM 24-9 Show how the following amino acids might be formed in the laboratory by reductive amination of the appropriate (a) alanine (b) leucine (c) serine (d) glutamine a-ketoacid. O (1) Br2/PBr3 (2) H2O NH3 (large excess) R9CH29C9OH OBr R9CH9C9OH ONH2 R9CH9C9O
NH4 carboxylic acid a-bromo acid (D,L)-a-amino acid (ammonium salt) In Section 19-19, we saw that direct alkylation is often a poor synthesis of amines, giving large amounts of overalkylated products. In this case, however, the reaction gives acceptable yields because a large excess of ammonia is used, making ammonia the nucleophile that is most likely to displace bromine. Also, the adjacent carboxyl- ate ion in the product reduces the nucleophilicity of the amino group. The following sequence shows bromination of 3-phenylpropanoic acid, followed by displacement of bromide ion, to form the ammonium salt of racemic phenylalanine. (1) Br2/PBr3 (2) H2O excess NH3 NH2 (D,L)-phenylalanine (salt) (30–50%) Ph9CH29CH29COOH 3-phenylpropanoic acid Br Ph9CH29CH9COOH Ph9CH29CH9COO
NH4 PROBLEM 24-10 Show how you would use bromination followed by amination to synthesize the following amino acids. (a) glycine (b) leucine (c) glutamic acid malonic ester H C H C O OEt COOEt COOEt H C R C O OEt alkylated acetic acid CO2 c H C R C O OH H (1)
OEt (2) RX H3O, heat temporary ester group To adapt this synthesis to making amino acids, we begin with a malonic ester that contains an group. The amino group is protected as a non-nucleophilic amide to prevent it from attacking the alkylating agent (RX). a-amino 24-5B Amination of an Halo Acid The Hell–Volhard–Zelinsky reaction (Section 22-4) is an effective method for introducing bromine at the position of a carboxylic acid. The racemic acid is converted to a racemic acid by direct amination, using a large excess of ammonia.a-amino a-bromoa a- 24-5C The Gabriel–Malonic Ester Synthesis One of the best methods of amino acid synthesis is a combination of the Gabriel syn- thesis of amines (Section 19-21) with the malonic ester synthesis of carboxylic acids (Section 22-16). The conventional malonic ester synthesis involves alkylation of diethyl malonate, followed by hydrolysis and decarboxylation to give an alkylated acetic acid. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1163 1164 CHAPTER 24 Amino Acids, Peptides, and Proteins The Gabriel–malonic ester synthesis begins with N-phthalimidomalonic ester. Think of N-phthalimidomalonic ester as a molecule of glycine (aminoacetic acid) with the amino group protected as an amide (a phthalimide in this case) to keep it from acting as a nucleophile. The acid is protected as an ethyl ester, and the position is further activated by the additional (temporary) ester group of diethyl malonate. Just as the malonic ester synthesis gives substituted acetic acids, the N-phthalimidoma- lonic ester synthesis gives substituted aminoacetic acids: acids. N-Phthalimido- malonic ester is alkylated in the same way as malonic ester. When the alkylated N-phthalimidomalonic ester is hydrolyzed, the phthalimido group is hydrolyzed along with the ester groups. The product is an alkylated aminomalonic acid. Decarboxylation gives a racemic acid.a-amino a-amino N H O O C COOEt COOEt N-phthalimidomalonic ester  N O O C C O O H Et protected amine protected acid glycine COOEt temporary ester group a N H O O C COOEt COOEt N O O C COOEt COOEt R H3N  C COOH COOH R N-phthalimidomalonic ester alkylated hydrolyzed heat H3N  C H COOH R -aminoa acid CO2 temporary ester group H3O+(1) base (2) R9X The Gabriel–malonic ester synthesis The Gabriel–malonic ester synthesis is used to make many amino acids that cannot be formed by direct amination of haloacids. The following example shows the synthesis of methionine, which is formed in very poor yield by direct amination. N H O O C COOEt COOEt N O O C COOEt COOEt CH2CH2SCH3 H3N  C H COOH CH2CH2SCH3 (D, L)-methionine (50%) H3O+ heat (1) NaOEt (2) Cl9CH2CH2SCH3 PROBLEM 24-11 Show how the Gabriel–malonic ester synthesis could be used to make (a) valine (b) phenylalanine (c) glutamic acid (d) leucine WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1164 24-7 Reactions of Amino Acids 1167 All the laboratory syntheses of amino acids described in Section 24-5 produce racemic products. In most cases, only the L enantiomers are biologically active. The D enan- tiomers may even be toxic. Pure L enantiomers are needed for peptide synthesis if the product is to have the activity of the natural material. Therefore, we must be able to resolve a racemic amino acid into its enantiomers. In many cases, amino acids can be resolved by the methods we have already dis- cussed (Section 5-16). If a racemic amino acid is converted to a salt with an optically pure chiral acid or base, two diastereomeric salts are formed. These salts can be sepa- rated by physical means such as selective crystallization or chromatography. Pure enantiomers are then regenerated from the separated diastereomeric salts. Strychnine and brucine are naturally occurring optically active bases, and tartaric acid is used as an optically active acid for resolving racemic mixtures. Enzymatic resolution is also used to separate the enantiomers of amino acids. Enzymes are chiral molecules with specific catalytic activities. For example, when an acylated amino acid is treated with an enzyme like hog kidney acylase or car- boxypeptidase, the enzyme cleaves the acyl group from just the molecules having the natural (L) configuration. The enzyme does not recognize D-amino acids, so they are unaffected. The resulting mixture of acylated D-amino acid and deacylated L-amino acid is easily separated. Figure 24-5 shows how this selective enzymatic deacylation is accomplished. H COOH O CH3C acylaseO2 R L-amino acid CH2N NH O COOH COOH L is deacylated acylatedracemic amino acid (easily separated mixture) C C H R CH3 NH O C C CH3 R HNH2 COOH R D-amino acid CH H COOH R CH2N CH3 D is unaffected NH COOH R C C O H ))
FIGURE 24-5 Selective enzymatic deacylation. An acylase enzyme (such as hog kidney acylase or carboxypeptidase) deacylates only the natural L-amino acid. PROBLEM 24-15 Suggest how you would separate the free L-amino acid from its acylated D enantiomer in Figure 24-5. Amino acids undergo many of the standard reactions of both amines and carboxylic acids. Conditions for some of these reactions must be carefully selected, however, so that the amino group does not interfere with a carboxyl group reaction, and vice versa. We will consider two of the most useful reactions, esterification of the carboxyl group and acylation of the amino group. These reactions are often used to protect either the carboxyl group or the amino group while the other group is being modified or coupled to another amino acid. Amino acids also undergo reactions that are specific to the acid structure. One of these unique amino acid reactions is the formation of a colored product on treatment with ninhydrin, discussed in Section 24-7C. a-amino 24-6 Resolution of Amino Acids 24-7 Reactions of Amino Acids WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1167 1168 CHAPTER 24 Amino Acids, Peptides, and Proteins O CH29Ph H3O+  phenylalanine ethyl ester phenylalanine H3N9CH9C9OCH2CH3  O CH29Ph H3N9CH9C9OH  CH3CH29OH Benzyl esters are particularly useful as protecting groups because they can be removed either by acidic hydrolysis or by neutral hydrogenolysis (“breaking apart by addition of hydrogen”). Catalytic hydrogenation cleaves the benzyl ester, converting the benzyl group to toluene and leaving the deprotected amino acid. Although the mechanism of this hydrogenolysis is not well known, it apparently hinges on the ease of formation of benzylic intermediates. CH2CHH3N  O C O CH2 Ph phenylalanine benzyl ester CHH3N  O C O
CH2 Ph phenylalanine  CH3 toluene H2, Pd PROBLEM 24-16 Propose a mechanism for the acid-catalyzed hydrolysis of phenylalanine ethyl ester. 24-7A Esterification of the Carboxyl Group Like monofunctional carboxylic acids, amino acids are esterified by treatment with a large excess of an alcohol and an acidic catalyst (often gaseous HCl). Under these acidic conditions, the amino group is present in its protonated form, so it does not interfere with esterification. The following example illustrates esterification of an amino acid. Esters of amino acids are often used as protected derivatives to prevent the car- boxyl group from reacting in some undesired manner. Methyl, ethyl, and benzyl esters are the most common protecting groups. Aqueous acid hydrolyzes the ester and regen- erates the free amino acid. proline CHH2N  O C O
CH2H2C CH2 HCl proline benzyl ester (90%) CHH2N  O C O CH2H2C CH2 CH2Ph Ph9CH29OH Cl
1¬ NH3 +2 Decarboxylation is an important reaction of amino acids in many bi- ological processes. Histamine, which causes runny noses and itchy eyes, is synthesized in the body by decarboxylation of histidine. The enzyme that catalyzes this reaction is called histidine decarboxylase. CH2CH2NH2 NH N histamine PROBLEM 24-17 Give equations for the formation and hydrogenolysis of glutamine benzyl ester. 24-7B Acylation of the Amino Group: Formation of Amides Just as an alcohol esterifies the carboxyl group of an amino acid, an acylating agent converts the amino group to an amide. Acylation of the amino group is often done to protect it from unwanted nucleophilic reactions. A wide variety of acid chlorides and anhydrides are used for acylation. Benzyl chloroformate acylates the amino group to give a benzyloxycarbonyl derivative, often used as a protecting group in peptide syn- thesis (Section 24-10). WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1168 24-7 Reactions of Amino Acids 1169 H2N CH COOH CH2 NH NH CH COOH CH2 NH N O CCH3 O O )( N-acetylhistidinehistidine H2N CH COOH CH2CH(CH3)2 NH CH COOH CH2CH(CH3)2 O CPhCH2O N-benzyloxycarbonyl leucine (90%) leucine N CH39C (acetic anhydride) PhCH2OC9Cl (benzyl chloroformate) 9O2 The amino group of the N-benzyloxycarbonyl derivative is protected as the amide half of a carbamate ester (a urethane, Section 21-16), which is more easily hydrolyzed than most other amides. In addition, the ester half of this urethane is a benzyl ester that undergoes hydrogenolysis. Catalytic hydrogenolysis of the N-benzyloxycarbonyl amino acid gives an unstable carbamic acid that quickly decarboxylates to give the deprotected amino acid. CH2 O C N H CH(CH3)2 COOH O CH CH2 CH3 HO O C N H CH COOH C CH(CH3)2 H2 CH COOH C CH(CH3)2 H2 H2N CO2 N-benzyloxycarbonyl leucine toluene a carbamic acid leucine H2, Pd PROBLEM 24-18 Give equations for the formation and hydrogenolysis of N-benzyloxycarbonyl methionine. CH COOH R H2N amino acid  2 O OH OH O pyridine O O N O
O  CO2 R CHO ninhydrin Ruhemann’s purple The reaction of amino acids with ninhydrin can detect amino acids on a wide variety of substrates. For example, if a kidnapper touches a ransom note with his fin- gers, the dermal ridges on his fingers leave traces of amino acids from skin secretions. 24-7C Reaction with Ninhydrin Ninhydrin is a common reagent for visualizing spots or bands of amino acids that have been separated by chromatography or electrophoresis. When ninhydrin reacts with an amino acid, one of the products is a deep violet, resonance-stabilized anion called Ruhemann’s purple. Ninhydrin produces this same purple dye regardless of the struc- ture of the original amino acid. The side chain of the amino acid is lost as an aldehyde. Reaction of an amino acid with ninhydrin WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1169 1172 CHAPTER 24 Amino Acids, Peptides, and Proteins The end of the peptide with the free amino group is called the N-ter- minal end or the N terminus, and the end with the free carboxyl group is called the C-terminal end or the C terminus. Peptide structures are generally drawn with the N terminus at the left and the C terminus at the right, as bradykinin is drawn in Figure 24-7. 24-8B Peptide Nomenclature The names of peptides reflect the names of the amino acid residues involved in the amide linkages, beginning at the N terminus. All except the last are given the -yl suffix of acyl groups. For example, the following dipeptide is named alanylserine. The ala- nine residue has the -yl suffix because it has acylated the nitrogen of serine. Bradykinin (Figure 24-7) is named as follows (without any spaces): arginyl prolyl prolyl glycyl phenylalanyl seryl prolyl phenylalanyl arginine This is a cumbersome and awkward name. A shorthand system is more convenient, representing each amino acid by its three-letter abbreviation. These abbreviations, given in Table 24-2, are generally the first three letters of the name. Once again, the amino acids are arranged from the N terminus at the left to the C terminus at the right. Bradykinin has the following abbreviated name: Arg-Pro-Pro-Gly-Phe-Ser-Pro-Phe-Arg Single-letter symbols (also given in Table 24-2) are becoming widely used as well. Using single letters, we symbolize bradykinin by RPPGFSPFR H3N H O C  C CH3 CH O CH2OH CNH O
alanyl serine Ala-Ser 1¬ COO-2 1¬ NH3 +2 PROBLEM 24-20 Draw the complete structures of the following peptides: (a) Thr-Phe-Met (b) serylarginylglycylphenylalanine (c) IMQDK (d) ELVIS 24-8C Disulfide Linkages Amide linkages (peptide bonds) form the backbone of the amino acid chains we call peptides and proteins. A second kind of covalent bond is possible between any cys- teine residues present. Cysteine residues can form disulfide bridges (also called disulfide linkages) which can join two chains or link a single chain into a ring. Mild oxidation joins two molecules of a thiol into a disulfide, forming a disulfide linkage between the two thiol molecules. This reaction is reversible, and a mild reduc- tion cleaves the disulfide. Similarly, two cysteine sulfhydryl groups are oxidized to give a disulfide- linked pair of amino acids. This disulfide-linked dimer of cysteine is called cystine. Figure 24-8 shows formation of a cystine disulfide bridge linking two peptide chains. 1¬ SH2 R ¬ SH + HS ¬ R IRRRJ [oxidation] [reduction] R ¬ S ¬ S ¬ R + H2O two molecules of thiol disulfide WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1172 24-8 Structure and Nomenclature of Peptides and Proteins 1173 Two cysteine residues may form a disulfide bridge within a single peptide chain, mak- ing a ring. Figure 24-9 shows the structure of human oxytocin, a peptide hormone that causes contraction of uterine smooth muscle and induces labor. Oxytocin is a nonapeptide with two cysteine residues (at positions 1 and 6) linking part of the molecule in a large ring. In drawing the structure of a complicated peptide, arrows are often used to connect the amino acids, showing the direction from N terminus to C terminus. Notice that the C ter- minus of oxytocin is a primary amide rather than a free carboxyl group.1Gly # NH22
FIGURE 24-8 Cystine, a dimer of cysteine, results when two cysteine residues are oxidized to form a disulfide bridge. CH2 CHNH peptide chain [O] (oxidize) [H] (reduce) peptide chain two cysteine residues C O SH CH2 + H2O CHNH C O S S cystine disulfide bridge CH2 CHNH C O SH CH2 CHNH C O
FIGURE 24-9 Structure of human oxytocin. A disulfide linkage holds part of the molecule in a large ring. N CH NH CH CHCH3CH2 CH2 NH2 CH2 CH2 SH2N CH2CH2C CHHO C C C O C NH NH NH NH2 CH O C O O O O O CH3 CH CH C NH NH C O O CH C O O H CH NH2CCH CH2 CH3H3C CH NHCCH2S N terminus cystine disulfide bridge C terminus (amide form) N terminus C terminus (amide form) Ile Gln Tyr S S Asn Cys Cys Pro Gly NH2Leu . WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1173 1174 CHAPTER 24 Amino Acids, Peptides, and Proteins
FIGURE 24-10 Structure of insulin. Two chains are joined at two positions by disulfide bridges, and a third disulfide bond holds the A chain in a ring. Insulin is a relatively simple protein, yet it is a complicated organic structure. How is it possible to determine the complete structure of a protein with hundreds of amino acid residues and a molecular weight of many thousands? Chemists have developed clever ways to determine the exact sequence of amino acids in a protein. We will con- sider some of the most common methods. 24-9A Cleavage of Disulfide Linkages The first step in structure determination is to break all the disulfide bonds, opening any disulfide-linked rings and separating the individual peptide chains. The individual peptide chains are then purified and analyzed separately. Cystine bridges are easily cleaved by reducing them to the thiol (cysteine) form. These reduced cysteine residues have a tendency to reoxidize and re-form disulfide bridges, however. A more permanent cleavage involves oxidizing the disulfide link- ages with peroxyformic acid (Figure 24-11). This oxidation converts the disulfide bridges to sulfonic acid groups. The oxidized cysteine units are called cysteic acid residues. 1¬ SO3H2 Figure 24-10 shows the structure of insulin, a more complex peptide hormone that regulates glucose metabolism. Insulin is composed of two separate peptide chains, the A chain, containing 21 amino acid residues, and the B chain, containing 30. The A and B chains are joined at two positions by disulfide bridges, and the A chain has an additional disulfide bond that holds six amino acid residues in a ring. The C-terminal amino acids of both chains occur as primary amides. Disulfide bridges are commonly manipulated in the process of giving hair a permanent wave. Hair is composed of protein, which is made rigid and tough partly by disulfide bonds. When hair is treated with a solution of a thiol such as 2-mercaptoethanol the disulfide bridges are reduced and cleaved. The hair is wrapped around curlers, and the disulfide bonds are allowed to re-form, either by air oxidation or by application of a neutralizer. The disulfide bonds re-form in new positions, holding the hair in the bent conformation enforced by the curlers. 1HS ¬ CH2 ¬ CH2 ¬ OH2, Gly Ile Val Glu Gln Cys Cys Ser Leu Gln Glu CysAsn Asn NH2 NH2 TyrTyrS Cys Cys S Val Ala Ser Asn Val Gln His Leu S S S S Phe N terminus Leu Gly His LeuSer Val Glu Leu Leu ValAla Tyr Gly Glu Arg Cys GlyPhePheTyrThrProLysAla . . N terminus C terminus B chain A chain C terminus Orexin A (from the Greek orexis, “appetite”) is a 33 amino acid neu- ropeptide connected by two disul- fide bridges. Orexin A is a powerful stimulant for food intake and gastric juice secretion. Scientists are study- ing orexin A to learn more about the regulation of appetite and eating, hoping to learn more about causes and potential treatments for anorexia nervosa. 24-9 Peptide Structure Determination WADEMC24_1153-1199hr.qxp 16-12-2008 19:34 Page 1174 24-9 Peptide Structure Determination 1177 Ph N C S H2N R1 CH O NH peptide H2N C Ph N C S R1 CH O NH peptideC HN Ph N C S R1 CH O NH peptideC H a phenylthiourea Step 1: Nucleophilic attack by the free amino group on phenyl isothiocyanate, followed by a proton transfer, gives a phenylthiourea.
R1 CH O NH peptideC H HN C NHPh S H C HN NHPh C CH N peptide R1 OH H2 C N NHPh C CH N peptide R1 O  H H2O C NHPh C CH H2N peptide R1 O  a thiazolinoneprotonated phenylthiourea Step 2: Treatment with HCl induces cyclization to a thiazolinone and expulsion of the shortened peptide chain.  S S N S H3O  Step 3: In acid, the thiazolinone isomerizes to the more stable phenylthiohydantoin. S OR1 N O HCl C N CC Ph H HN S a phenylthiohydantointhiazolinone R1 NHPh The phenylthiohydantoin derivative is identified by chromatography, by comparing it with phenylthiohydantoin derivatives of the standard amino acids. This gives the iden- tity of the original N-terminal amino acid. The rest of the peptide is cleaved intact, and further Edman degradations are used to identify additional amino acids in the chain. This process is well suited to automation, and several types of automatic sequencers have been developed. Figure 24-14 shows the first two steps in the sequencing of oxytocin. Before sequencing, the oxytocin sample is treated with peroxyformic acid to convert the disulfide bridge to cysteic acid residues. In theory, Edman degradations could sequence a peptide of any length. In prac- tice, however, the repeated cycles of degradation cause some internal hydrolysis of the peptide, with loss of sample and accumulation of by-products. After about 30 cycles of degradation, further accurate analysis becomes impossible. A small peptide such as bradykinin can be completely determined by Edman degradation, but larger proteins must be broken into smaller fragments (Section 24-9E) before they can be completely sequenced. PROBLEM 24-21 Draw the structure of the phenylthiohydantoin derivatives of (a) alanine (b) tryptophan (c) lysine (d) proline Second, the phenylthiourea cyclizes to a thiazolinone and expels the shortened peptide chain. Third, the thiazolinone isomerizes to the more stable phenylthiohydantoin. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1177 1178 CHAPTER 24 Amino Acids, Peptides, and Proteins
FIGURE 24-14 The first two steps in sequencing oxytocin. Each Edman degradation cleaves the N-terminal amino acid and forms its phenylthiohydantoin derivative. The shortened peptide is available for the next step. PROBLEM 24-23 The Sanger method for N-terminus determination is a less common alternative to the Edman degradation. In the Sanger method, the peptide is treated with the Sanger reagent, 2,4-dinitrofluorobenzene, and then hydrolyzed by reaction with 6 M aqueous HCl. The N-terminal amino acid is recovered as its 2,4-dinitrophenyl derivative and identified. (a) Propose a mechanism for the reaction of the N terminus of the peptide with 2,4-dinitrofluorobenzene. (b) Explain why the Edman degradation is usually preferred over the Sanger method. F NO2 O2N  H2N R1 CH C O NH peptide peptide 2,4-dinitrofluorobenzene (Sanger reagent) NO2 O2N NH CH C NH peptide O derivative amino acidsNO2 O2N NH R1 COOHCH 2,4-dinitrophenyl derivative  R1 6 M HCl, heat The Sanger method CH2 CHH2N Step 1: Cleavage and determination of the N-terminal amino acid Step 2: Cleavage and determination of the second amino acid (the new N-terminal amino acid) cysteic acid C O cysteic acid phenylthiohydantoin O SO3H CH2SO3H C CCH HN S N Ph +Tyr Ile Gln peptideNH (1) Ph Tyr Ile Gln peptide N C S (2) H3O+ . . H2N . . CH2 CHH2N C O tyrosine phenylthiohydantoin O C CCH CH2HO HN S N Ph +Ile Gln peptideNH (1) Ph Ile Gln peptide N C S (2) H3O+ . . H2N . . OH PROBLEM 24-22 Show the third and fourth steps in the sequencing of oxytocin. Use Figure 24-14 as a guide. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1178 24-9D C-Terminal Residue Analysis There is no efficient method for sequencing several amino acids of a peptide starting from the C terminus. In many cases, however, the C-terminal amino acid can be identified using the enzyme carboxypeptidase, which cleaves the C-terminal peptide bond. The products are the free C-terminal amino acid and a shortened peptide. Further reaction cleaves the second amino acid that has now become the new C terminus of the shortened peptide. Eventually, the entire peptide is hydrolyzed to its individual amino acids. Rn C O NHpeptide C O Rn
1 CH O NHpeptide C OH  Rn CH O H2N C OHHH (further cleavage) free amino acid Rn
1 C O NH CH carboxypeptidase H2O A peptide is incubated with the carboxypeptidase enzyme, and the appearance of free amino acids is monitored. In theory, the amino acid whose concentration increases first should be the C terminus, and the next amino acid to appear should be the second residue from the end. In practice, different amino acids are cleaved at different rates, making it difficult to determine amino acids past the C terminus and occasionally the second residue in the chain. 24-9E Breaking the Peptide into Shorter Chains: Partial Hydrolysis Before a large protein can be sequenced, it must be broken into smaller chains, not longer than about 30 amino acids. Each of these shortened chains is sequenced, and then the entire structure of the protein is deduced by fitting the short chains together like pieces of a jigsaw puzzle. Partial cleavage can be accomplished either by using dilute acid with a shortened reaction time or by using enzymes, such as trypsin and chymotrypsin, that break bonds between specific amino acids. The acid-catalyzed cleavage is not very selective, leading to a mixture of short fragments resulting from cleavage at various positions. Enzymes are more selective, giving cleavage at predictable points in the chain. TRYPSIN: Cleaves the chain at the carboxyl groups of the basic amino acids lysine and arginine. CHYMOTRYPSIN: Cleaves the chain at the carboxyl groups of the aromatic amino acids phenylalanine, tyrosine, and tryptophan. Let’s use oxytocin (Figure 24-9) as an example to illustrate the use of partial hydrolysis. Oxytocin could be sequenced directly by C-terminal analysis and a series of Edman degradations, but it provides a simple example of how a structure can be pieced together from fragments. Acid-catalyzed partial hydrolysis of oxytocin (after cleavage of the disulfide bridge) gives a mixture that includes the following peptides: 24-9 Peptide Structure Determination 1179 Ile-Gln-Asn-Cys Gln-Asn-Cys-Pro Pro-Leu-Gly # NH2 Cys-Tyr-Ile-Gln-Asn Cys-Pro-Leu-Gly When we match the overlapping regions of these fragments, the complete sequence of oxytocin appears: Cys-Tyr-Ile-Gln-Asn Ile-Gln-Asn-Cys Gln-Asn-Cys-Pro Cys-Pro-Leu-Gly Complete structure Cys-Tyr-Ile-Gln-Asn-Cys-Pro-Leu-Gly # NH2 Pro-Leu-Gly # NH2 The selective enzymatic cleavage of proteins is critical to many biological processes. For example, the clotting of blood depends on the enzyme thrombin cleaving fibrinogen at spe- cific points to produce fibrin, the protein that forms a clot. Proteolytic (protein-cleaving) enzymes also have applications in consumer products. For example, papain (from papaya extract) serves as a meat tenderizer. It cleaves the fibrous proteins, making the meat less tough. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1179 1182 CHAPTER 24 Amino Acids, Peptides, and Proteins O Ala-Val-Phe COCH2 NHC CH3 HC O Val Phe H2NC CH3 HC O Val Phe  CO2 Z-Ala-Val-Phe H2, Pd CH3Ph When the second amino acid (valine) is added to the protected, activated alanine, the nucleophilic amino group of valine attacks the activated carbonyl of alanine, displac- ing the anhydride and forming a peptide bond. (Some procedures use an ester of the new amino acid to avoid competing reactions from its carboxylate group.) Step 2: Form an amide bond to couple the next amino acid. Z-Ala-Val Z O NHCH C O CH3 protected, activated alanine O C OCH2CH3  H2N CH O C OH CH(CH3)2 valine Z O NHCH C CH3 N CH O C OH CH(CH3)2 H  CO2  CH3CH2OH PROBLEM 24-26 Give complete mechanisms for the formation of Z-Ala, its activation by ethyl chloroformate, and the coupling with valine. O CZ NHC CH3 H NHC CH(CH3)2 H O C OH  Cl C O OEt O CZ NHC CH3 H NHC CH(CH3)2 H O C O C O OEt  HCl Ala AlaVal Val Step 2: Form an amide bond to couple the next amino acid. Z-Ala-Val-Phe Z Ala NHC CH(CH3)2 H O C O OEt  H2N C CH2 H Ph C O OH Z Ala NHC CH H C O NH H3C CH3 C CH2 H Ph C O OH  CO2  EtOH phenylalanine C O Val To make a larger peptide, repeat these two steps for the addition of each amino acid residue: 1. Activate the C terminus of the growing peptide by reaction with ethyl chloroformate. 2. Couple the next amino acid. The final step in the solution-phase synthesis is to deprotect the N terminus of the completed peptide. The N-terminal amide bond must be cleaved without breaking any of the peptide bonds in the product. Fortunately, the benzyloxycarbonyl group is partly an amide and partly a benzyl ester, and hydrogenolysis of the benzyl ester takes place under mild conditions that do not cleave the peptide bonds. This mild cleavage is the reason for using the benzyloxycarbonyl group (as opposed to some other acyl group) to protect the N terminus. Final step: Remove the protecting group. At this point, we have the N-protected dipeptide Z-Ala-Val. Phenylalanine must be added to the C terminus to complete the Ala-Val-Phe tripeptide. Activation of the valine carboxyl group, followed by addition of phenylalanine, gives the protected tripeptide. Step 1: Activate the carboxyl group with ethyl chloroformate. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1182 24-11 Solid-Phase Peptide Synthesis 1183 PROBLEM 24-27 Show how you would synthesize Ala-Val-Phe-Gly-Leu starting with Z-Ala-Val-Phe. In 1962, Robert Bruce Merrifield of Rockefeller University developed a method for synthesizing peptides without having to purify the intermediates. He did this by at- taching the growing peptide chains to solid polystyrene beads. After each amino acid is added, the excess reagents are washed away by rinsing the beads with solvent. This ingenious method lends itself to automation, and Merrifield built a machine that can add several amino acid units while running unattended. Using this machine, Merrifield synthesized ribonuclease (124 amino acids) in just six weeks, obtaining an overall yield of 17%. Merrifield’s work in solid-phase peptide synthesis won the Nobel Prize in Chemistry in 1984. 24-11A The Individual Reactions Three reactions are crucial for solid-phase peptide synthesis. These reactions attach the first amino acid to the solid support, protect each amino group until its time to react, and form the peptide bonds between the amino acids. Attaching the Peptide to the Solid Support The greatest difference between solution-phase and solid-phase peptide synthesis is that solid-phase synthesis is done in the opposite direction: starting with the C terminus and going toward the N termi- nus, right to left as we write the peptide. The first step is to attach the last amino acid (the C terminus) to the solid support. The solid support is a special polystyrene bead in which some of the aromatic rings have chloromethyl groups. This polymer, often called the Merrifield resin, is made by copolymerizing styrene with a few percent of p-(chloromethyl)styrene. problem-solving Remember that classical (solution-phase) peptide synthesis: 1. Goes Protect the N terminus (Z group) first, deprotect last. 2. Couple each amino acid by activating the C terminus (ethyl chloroformate), then adding the new amino acid. N : C. Hint 24-11 Solid-Phase Peptide Synthesis PROBLEM 24-28 Show how the solution-phase synthesis would be used to synthesize Ile-Gly-Asn. The solution-phase method works well for small peptides, and many peptides have been synthesized by this process. A large number of chemical reactions and purifications are required even for a small peptide, however. Although the individual yields are excellent, with a large peptide, the overall yield becomes so small as to be unusable, and several months (or years) are required to complete so many steps. The large amounts of time required and the low overall yields are due largely to the purification steps. For larger peptides and proteins, solid-phase peptide synthesis is usually preferred. p-(chloromethyl)styrene C C C H HH H2Cl  styrene C C H HH CH CH2 CH CH2 CH CH2 polymer CH2Cl  P CH2Cl abbreviation Formation of the Merrifield resin WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1183 1184 CHAPTER 24 Amino Acids, Peptides, and Proteins OC O CH3 C O CH3 CH3 OC O C CH3 CH3 CH3  H2N CH R COOH amino acid C O CH3 C O CH3 CH3 CH R COOHNH  CO2  CH3 C OH CH3 CH3 Boc-amino acid Protection of the amino group as its Boc derivative di-tert-butyldicarbonate The Boc group is easily cleaved by brief treatment with trifluoroacetic acid (TFA), Loss of a relatively stable tert-butyl cation from the protonated ester gives an unstable carbamic acid. Decarboxylation of the carbamic acid gives the deprotected amino group of the amino acid. Loss of a proton from the tert-butyl cation gives isobutylene. CF3COOH. Like other benzyl halides, the chloromethyl groups on the polymer are reactive toward attack. The carboxyl group of an N-protected amino acid displaces chlo- ride, giving an amino acid ester of the polymer. In effect, the polymer serves as the alcohol part of an ester protecting group for the carboxyl end of the C-terminal amino acid. The amino group must be protected, or it would attack the chloromethyl groups. SN2 P CH2O O CHC R HN protecting group ClC HH P Cl
O
O CHC R HN protecting group Attachment of the C-terminal amino acid Once the C-terminal amino acid is fixed to the polymer, the chain is built on the amino group of this amino acid. Using the tert-Butyloxycarbonyl (Boc) Protecting Group The benzy- loxycarbonyl group (the Z group) cannot be used with the solid-phase process be- cause the Z group is removed by hydrogenolysis in contact with a solid catalyst. A polymer-bound peptide cannot achieve the intimate contact with a solid catalyst required for hydrogenolysis. The N-protecting group used in the Merrifield proce- dure is the tert-butyloxycarbonyl group, abbreviated Boc or t-Boc. The Boc group is similar to the Z group, except that it has a tert-butyl group in place of the benzyl group. Like other tert-butyl esters, the Boc protecting group is easily removed under acidic conditions. The acid chloride of the Boc group is unstable, so we use the anhydride, di-tert- butyldicarbonate, to attach the group to the amino acid. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1184 24-11 Solid-Phase Peptide Synthesis 1187 The second amino acid (valine) is added in its N-protected Boc form so that it can- not couple with itself. Addition of DCC couples the valine carboxyl group with the free group of phenylalanine.¬ NH2 Boc-Val CHBoc NH (CH3)2CH C O O
 CHH3N Ph CH2 C O O P CH2 Phe— P  DCC CHBoc NH (CH3)2CH C O CH Ph CH2 C O O P CH2 Boc-Val-Phe— P NH  DCU To couple the final amino acid (alanine), the chain is first deprotected by treatment with trifluoroacetic acid. Then the N-protected Boc-alanine and DCC are added. CF3COOH (TFA) CHBoc NH (CH3)2CH C O CH Ph CH2 C O O P CH2 Boc-Val-Phe— P NH CHH3N C O O P CH2 Val-Phe— P   CH3 C  CO2 CH3 CH2 (CH3)2CH CH Ph CH2 NH C O Step 1: Deprotection CHH3N C O O P CH2 Val-Phe— P  (CH3)2CH CH Ph CH2 NH C O CHBoc NH CH3 C O CH C O O CH2NH (CH3)2CH NH CH C O Ph CH2  DCU DCC Boc-Ala-Val-Phe— P O CH3 Boc9NH9CH9C9O− Step 2: Coupling P If we were making a longer peptide, the addition of each subsequent amino acid would require the repetition of two steps: 1. Use trifluoroacetic acid to deprotect the amino group at the end of the growing chain. 2. Add the next Boc-amino acid, using DCC as a coupling agent. Once the peptide is completed, the final Boc protecting group must be removed, and the peptide must be cleaved from the polymer. Anhydrous HF cleaves the ester linkage that bonds the peptide to the polymer, and it also removes the Boc protecting group. In our example, the following reaction occurs: Boc-Ala-Val-Phe— P CHBoc NH CH 3 C O CH C O O P CH2NH (CH3)2CH NH CH C O Ph CH2 HF CHH3N C O P CH2F Ala-Val-Phe   CH3 C CO2 CH3 CH2 (CH3)2CH CHNH C O CH3 CH Ph CH2 NH C O OH  WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1187 1188 CHAPTER 24 Amino Acids, Peptides, and Proteins PROBLEM 24-31 Show how solid-phase peptide synthesis would be used to make Ile-Gly-Asn. Proteins may be classified according to their chemical composition, their shape, or their function. Protein composition and function are treated in detail in a biochemistry course. For now, we briefly survey the types of proteins and their general classifications. Proteins are grouped into simple and conjugated proteins according to their chem- ical composition. Simple proteins are those that hydrolyze to give only amino acids. All the protein structures we have considered so far are simple proteins. Examples are insulin, ribonuclease, oxytocin, and bradykinin. Conjugated proteins are bonded to a nonprotein prosthetic group such as a sugar, a nucleic acid, a lipid, or some other group. Table 24-3 lists some examples of conjugated proteins. 24-13A Primary Structure Up to now, we have discussed the primary structure of proteins. The primary structure is the covalently bonded structure of the molecule. This definition in- cludes the sequence of amino acids, together with any disulfide bridges. All the properties of the protein are determined, directly or indirectly, by the primary struc- ture. Any folding, hydrogen bonding, or catalytic activity depends on the proper primary structure. 24-13B Secondary Structure Although we often think of peptide chains as linear structures, they tend to form orderly hydrogen-bonded arrangements. In particular, the carbonyl oxygen atoms form hydrogen TABLE 24-3 Classes of Conjugated Proteins Class Prosthetic Group Examples glycoproteins carbohydrates interferon nucleoproteins nucleic acids ribosomes, viruses lipoproteins fats, cholesterol high-density lipoprotein metalloproteins a complexed metal hemoglobin, cytochromes g-globulin, PROBLEM 24-30 Show how you would synthesize Leu-Gly-Ala-Val-Phe starting with Boc-Ala-Val-Phe— P
. Proteins are classified as fibrous or globular depending on whether they form long filaments or coil up on themselves. Fibrous proteins are stringy, tough, and usually insoluble in water. They function primarily as structural parts of the organ- ism. Examples of fibrous proteins are in hooves and fingernails, and col- lagen in tendons. Globular proteins are folded into roughly spherical shapes. They usually function as enzymes, hormones, or transport proteins. Enzymes are pro- tein-containing biological catalysts; an example is ribonuclease, which cleaves RNA. Hormones help to regulate processes in the body. An example is insulin, which regulates glucose levels in the blood and its uptake by cells. Transport pro- teins bind to specific molecules and transport them in the blood or through the cell membrane. An example is hemoglobin, which transports oxygen in the blood from the lungs to the tissues. a-keratin 24-12 Classification of Proteins 24-13 Levels of Protein Structure WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1188 24-13 Levels of Protein Structure 1189 CH CH HC C C N N R O H O C O C O C O N H N CH H HC N C O R RCH C C N N O O H H O C C O C N H N CH R HH O HR R R C = gray N = blue O = red R = green
FIGURE 24-15 The helical arrangement. The peptide chain curls into a helix so that each peptide carbonyl group is hydrogen-bonded to an hydrogen on the next turn of the helix. Side chains are symbolized by green atoms in the space-filling structure.N ¬ H a Spider web is composed mostly of fibroin, a protein with pleated-sheet secondary structure. The pleated- sheet arrangement allows for multiple hydrogen bonds between molecules, conferring great strength. Segments of peptides can also form orderly arrangements of hydrogen bonds by lining up side-by-side. In this arrangement, each carbonyl group on one chain forms a hydrogen bond with an hydrogen on an adjacent chain. This arrangement may involve many peptide molecules lined up side-by-side, resulting in a two-dimensional sheet. The bond angles between amino acid units are such that the sheet is pleated (creased), with the amino acid side chains arranged on al- ternating sides of the sheet. Silk fibroin, the principal fibrous protein in the silks of insects and arachnids, has a pleated sheet secondary structure. Figure 24-16 shows the pleated sheet structure. N ¬ H bonds with the amide hydrogens. This tendency leads to orderly patterns of hydrogen bonding: the helix and the pleated sheet. These hydrogen-bonded arrange- ments, if present, are called the secondary structure of the protein. When a peptide chain winds into a helical coil, each carbonyl oxygen can hydro- gen-bond with an hydrogen on the next turn of the coil. Many proteins wind into an helix (a helix that looks like the thread on a right-handed screw) with the side chains positioned on the outside of the helix. For example, the fibrous protein ker- atin is arranged in the structure, and most globular proteins contain segments of helix. Figure 24-15 shows the arrangement.a-helicala a-helical a a N ¬ H A 1N ¬ H2
FIGURE 24-16 The pleated sheet arrangement. Each peptide carbonyl group is hydrogen- bonded to an hydrogen on an adjacent peptide chain. N ¬ H H R N C O H CH R H N O C N CH H O C NCH C O CH H N CH H O C N CH CH CH CH CHC O C OH N H N C O H N C O N C C CH C CH N CH CH R R R R R R R R R R R R H R N C O H CH R H N O C N CH H O C NCH C O CH H N CH H O C N CH CHC OR R R R R O O OH H H N .................. .................. WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1189 1192 CHAPTER 24 Amino Acids, Peptides, and Proteins Milk turns sour because of the bacterial conversion of carbohydrates to lactic acid. When the pH becomes strongly acidic, soluble proteins in milk are denatured and precipitate. This process is called curdling. Some proteins are more resistant to acidic and basic conditions than others. For example, most digestive enzymes such as amylase and trypsin remain active under acidic conditions in the stomach, even at a pH of about 1. In many cases, denaturation is irreversible. When cooked egg white is cooled, it does not become uncooked. Curdled milk does not uncurdle when it is neutralized. Denaturation may be reversible, however, if the protein has undergone only mild denaturing conditions. For example, a protein can be salted out of solution by a high salt concentration, which denatures and precipitates the protein. When the precipitated protein is redissolved in a solution with a lower salt concentration, it usually regains its activity together with its natural conformation. 24-14B Prion Diseases Up through 1980, people thought that all infectious diseases were caused by microbes of some sort. They knew about diseases caused by viruses, bacteria, protozoa, and fungi. There were some strange diseases, however, for which no one had isolated and cultured the pathogen. Creutzfeldt–Jakob Disease (CJD) in humans, scrapie in sheep, and transmissible encephalopathy in mink (TME) all involved a slow, gradual loss of mental function and eventual death. The brains of the victims all showed unusual plaques of amyloid protein surrounded by spongelike tissue. Workers studying these diseases thought there was an infectious agent involved (as opposed to genetic or environmental causes) because they knew that scrapie and TME could be spread by feeding healthy animals the ground-up remains of sick animals. They had also studied kuru, a disease much like CJD among tribes where family members showed their respect for the dead by eating their brains. These diseases were generally attributed to “slow viruses” that were yet to be isolated. Micrograph of normal human brain tissue. The nuclei of neurons appear as dark spots. In the 1980s, neurologist Stanley B. Prusiner (of the University of California at San Francisco) made a homogenate of scrapie-infected sheep brains and systemati- cally separated out all the cell fragments, bacteria, and viruses, and found that the remaining material was still infectious. He separated out the proteins and found a protein fraction that was still infectious. He suggested that scrapie (and presumably similar diseases) is caused by a protein infectious agent that he called prion protein. This conclusion contradicted the established principle that contagious diseases require a living pathogen. Many skeptical workers repeated Prusiner’s work in hopes of finding viral contaminants in the infectious fractions, and most of them finally came to the same conclusion. Prusiner received the 1998 Nobel Prize in Medicine or Physiology for this work.Brain tissue of a patient infected with vCJD. Note the formation of (white) vacuole spaces and (dark, irregular) plaques of prion protein. (Magnification 200X) Since Prusiner’s work, prion diseases have become more important because of their threat to humans. Beginning in 1996, some cows in the United Kingdom developed “mad cow disease” and would threaten other animals, wave their heads, fall down, and eventually die. The disease, called bovine spongiform encephalopa- thy, or BSE, was probably transmitted to cattle by feeding them the remains of scrapie-infected sheep. The most frightening aspect of the BSE outbreak was that people could contract a fatal disease, called new-variant Creutzfeldt–Jakob Disease (vCJD) from eating the infected meat. Since that time, a similar disease, called chronic wasting disease, or CWD, has been found in wild deer and elk in the Rocky Mountains. All of these (presumed) prion diseases are now classified as trans- missible spongiform encephalopathies, or TSEs. The most widely accepted theory of prion diseases suggests that the infectious prion protein has the same primary structure as a normal protein found in nerve WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1192 24 Glossary 1193 cells, but it differs in its tertiary structure. In effect, it is a misfolded, denatured ver- sion of a normal protein that polymerizes to form the amyloid protein plaques seen in the brains of infected animals. When an animal ingests infected food, the polymer- ized protein resists digestion. Because it is simply a misfolded version of a normal protein, the infectious prion does not provoke the host’s immune system to attack the pathogen. When the abnormal prion interacts with the normal version of the protein on the membranes of nerve cells, the abnormal protein somehow induces the normal molecules to change their shape. This is the part of the process we know the least about. (We might think of it like crystallization, in which a seed crystal induces other molecules to crystallize in the same conformation and crystal form.) These newly misfolded protein molecules then induce more molecules to change shape. The polymerized abnormal protein cannot be broken down by the usual protease enzymes, so it builds up in the brain and causes the plaques and spongy tissue associated with TSEs. We once thought that a protein with the correct primary structure, placed in the right physiological solution, would naturally fold into the correct tertiary structure and stay that way. We were wrong. We now know that protein folding is a carefully controlled process in which enzymes and chaperone proteins promote correct fold- ing as the protein is synthesized. Prion diseases have shown that there are many factors that cause proteins to fold into natural or unnatural conformations, and that the folding of the protein can have major effects on its biological properties within an organism. active site The region of an enzyme that binds the substrate and catalyzes the reaction. (p. 1190) amino acid Literally, any molecule containing both an amino group and a carboxyl group The term usually means an with the amino group on the carbon atom next to the carboxyl group. (p. 1154) biomimetic synthesis A laboratory synthesis that is patterned after a biological synthesis. For example, the synthesis of amino acids by reductive amination resembles the biosynthesis of glutamic acid. (p. 1162) complete proteins Proteins that provide all the essential amino acids in about the right proportions for human nutrition. Examples include those in meat, fish, milk, and eggs. Incomplete proteins are severely deficient in one or more of the essential amino acids. Most plant proteins are incomplete. (p. 1157) conjugated protein A protein that contains a nonprotein prosthetic group such as a sugar, nucleic acid, lipid, or metal ion. (p. 1188) C terminus (C-terminal end) The end of the peptide chain with a free or derivatized carboxyl group. As the peptide is written, the C terminus is usually on the right. The amino group of the C-terminal amino acid links it to the rest of the peptide. (p. 1172) denaturation An unnatural alteration of the conformation or the ionic state of a protein. Denaturation generally results in precipitation of the protein and loss of its biological activity. Denaturation may be reversible, as in salting out a protein, or irreversible, as in cooking an egg. (p. 1191) disulfide linkage (disulfide bridge) A bond between two cysteine residues formed by mild oxidation of their thiol groups to a disulfide. (p. 1172) Edman degradation A method for removing and identifying the N-terminal amino acid from a peptide without destroying the rest of the peptide chain. The peptide is treated with phenyliso- thiocyanate, followed by a mild acid hydrolysis to convert the N-terminal amino acid to its phenylthiohydantoin derivative. The Edman degradation can be used repeatedly to determine the sequence of many residues beginning at the N terminus. (p. 1176) electrophoresis A procedure for separating charged molecules by their migration in a strong electric field. The direction and rate of migration are governed largely by the average charge on the molecules. (p. 1160) A-amino acid,1¬ COOH2. 1¬ NH22 24Glossary WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1193 1194 CHAPTER 24 Amino Acids, Peptides, and Proteins enzymatic resolution The use of enzymes to separate enantiomers. For example, the enan- tiomers of an amino acid can be acylated and then treated with hog kidney acylase. The enzyme hydrolyzes the acyl group from the natural L-amino acid, but it does not react with the D-amino acid. The resulting mixture of the free L-amino acid and the acylated D-amino acid is easily separated. (p. 1167) enzyme A protein-containing biological catalyst. Many enzymes also include prosthetic groups, nonprotein constituents that are essential to the enzyme’s catalytic activity. (p. 1188) essential amino acids Ten standard amino acids that are not biosynthesized by humans and must be provided in the diet. (p. 1157) fibrous proteins A class of proteins that are stringy, tough, threadlike, and usually insoluble in water. (p. 1188) globular proteins A class of proteins that are relatively spherical in shape. Globular pro- teins generally have lower molecular weights and are more soluble in water than fibrous proteins. (p. 1188) helix A helical peptide conformation in which the carbonyl groups on one turn of the helix are hydrogen-bonded to hydrogens on the next turn. Extensive hydrogen bonding stabi- lizes this helical arrangement. (p. 1189) hydrogenolysis Cleavage of a bond by the addition of hydrogen. For example, catalytic hydrogenolysis cleaves benzyl esters. (p. 1168) isoelectric point (isoelectric pH) The pH at which an amino acid (or protein) does not move under electrophoresis. This is the pH where the average charge on its molecules is zero, with most of the molecules in their zwitterionic form. (p. 1160) L-amino acid An amino acid having a stereochemical configuration similar to that of L- Most naturally occurring amino acids have the L configuration. (p. 1155) N terminus (N-terminal end) The end of the peptide chain with a free or derivatized amino group. As the peptide is written, the N terminus is usually on the left. The carboxyl group of the N-terminal amino acid links it to the rest of the peptide. (p. 1172) oligopeptide A small polypeptide, containing about four to ten amino acid residues. (p. 1171) peptide Any polymer of amino acids linked by amide bonds between the amino group of each amino acid and the carboxyl group of the neighboring amino acid. The terms dipeptide, tripep- tide, etc. may specify the number of amino acids in the peptide. (p. 1171) peptide bonds Amide linkages between amino acids. (pp. 1153, 1171) pleated sheet A two-dimensional peptide conformation with the peptide chains lined up side by side. The carbonyl groups on each peptide chain are hydrogen-bonded to hydrogens on the adjacent chain, and the side chains are arranged on alternating sides of the sheet. (p. 1189) polypeptide A peptide containing many amino acid residues. Although proteins are polypep- tides, the term polypeptide is commonly used for molecules with lower molecular weights than proteins. (p. 1171) N ¬ H COOH H L-alanine (S )-alanine H2N H2N CH3 CHO H L-(–)-glyceraldehyde (S )-glyceraldehyde HO CH2OH COOH H an L-amino acid (S) configuration R 1-2-glyceraldehyde. benzyl ester R C O CH2 H O R C O O acid toluene CH2H H2, Pd N ¬ H A WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1194 24 Study Problems 1197 24-35 Show how you would synthesize any of the standard amino acids from each starting material. You may use any necessary reagents. (a) (b) (c) (d) 24-36 Show how you would convert alanine to the following derivatives. Show the structure of the product in each case. (a) alanine isopropyl ester (b) N-benzoylalanine (c) N-benzyloxycarbonyl alanine (d) tert-butyloxycarbonyl alanine 24-37 Suggest a method for the synthesis of the unnatural D enantiomer of alanine from the readily available L enantiomer of lactic acid. lactic acid 24-38 Show how you would use the Gabriel–malonic ester synthesis to make histidine. What stereochemistry would you expect in your synthetic product? 24-39 Show how you would use the Strecker synthesis to make tryptophan. What stereochemistry would you expect in your synthetic product? 24-40 Write the complete structures for the following peptides. Tell whether each peptide is acidic, basic, or neutral. (a) methionylthreonine (b) threonylmethionine (c) arginylaspartyllysine (d) Glu-Cys-Gln 24-41 The following structure is drawn in an unconventional manner. (a) Label the N terminus and the C terminus. (b) Label the peptide bonds. (c) Identify and label each amino acid present. (d) Give the full name and the abbreviated name. 24-42 Aspartame (Nutrasweet®) is a remarkably sweet-tasting dipeptide ester. Complete hydrolysis of aspartame gives phenyl- alanine, aspartic acid, and methanol. Mild incubation with carboxypeptidase has no effect on aspartame. Treatment of aspartame with phenyl isothiocyanate, followed by mild hydrolysis, gives the phenylthiohydantoin of aspartic acid. Propose a structure for aspartame. 24-43 A molecular weight determination has shown that an unknown peptide is a pentapeptide, and an amino acid analysis shows that it contains the following residues: one Gly, two Ala, one Met, one Phe. Treatment of the original pentapeptide with carboxypeptidase gives alanine as the first free amino acid released. Sequential treatment of the pentapeptide with phenyl isothiocyanate followed by mild hydrolysis gives the following derivatives: Propose a structure for the unknown pentapeptide. 24-44 Show the steps and intermediates in the synthesis of Leu-Ala-Phe (a) by the solution-phase process. (b) by the solid-phase process. 24-45 Using classical solution-phase techniques, show how you would synthesize Ala-Val and then combine it with Ile-Leu-Phe to give Ile-Leu-Phe-Ala-Val. Ph H H S O NN CH2Ph first time Ph H H S O NN CH3 second time Ph H S O NN third time CH3CH29CH9CH9NH9C9CH9CH2CH29C9NH2 O OCH3 CONH2 NH9CO9CH2NH2 CH3 ¬ CHOH ¬ COOH CH2Br1CH322CH ¬ CH2 ¬ CHO CH2CH3 CH39CH9CH29COOH O (CH3)2CH9C9COOH WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1197 1198 CHAPTER 24 Amino Acids, Peptides, and Proteins 24-46 Peptides often have functional groups other than free amino groups at the N terminus and other than carboxyl groups at the C terminus. (a) A tetrapeptide is hydrolyzed by heating with 6 M HCl, and the hydrolysate is found to contain Ala, Phe, Val, and Glu. When the hydrolysate is neutralized, the odor of ammonia is detected. Explain where this ammonia might have been incorporated in the original peptide. (b) The tripeptide thyrotropic hormone releasing factor (TRF) has the full name pyroglutamylhistidylprolinamide. The structure appears here. Explain the functional groups at the N terminus and at the C terminus. (c) On acidic hydrolysis, an unknown pentapeptide gives glycine, alanine, valine, leucine, and isoleucine. No odor of ammonia is detected when the hydrolysate is neutralized. Reaction with phenyl isothiocyanate followed by mild hy- drolysis gives no phenylthiohydantoin derivative. Incubation with carboxypeptidase has no effect. Explain these findings. 24-47 Lipoic acid is often found near the active sites of enzymes, usually bound to the peptide by a long, flexible amide linkage with a lysine residue. (a) Is lipoic acid a mild oxidizing agent or a mild reducing agent? Draw it in both its oxidized and reduced forms. (b) Show how lipoic acid might react with two Cys residues to form a disulfide bridge. (c) Give a balanced equation for the hypothetical oxidation or reduction, as you predicted in part (a), of an aldehyde by lipoic acid. 24-48 Histidine is an important catalytic residue found at the active sites of many enzymes. In many cases, histidine appears to remove protons or to transfer protons from one location to another. (a) Show which nitrogen atom of the histidine heterocycle is basic and which is not. (b) Use resonance forms to show why the protonated form of histidine is a particularly stable cation. (c) Show the structure that results when histidine accepts a proton on the basic nitrogen of the heterocycle and then is deprotonated on the other heterocyclic nitrogen. Explain how histidine might function as a pipeline to transfer protons between sites within an enzyme and its substrate. 24-49 Metabolism of arginine produces urea and the rare amino acid ornithine. Ornithine has an isoelectric point close to 10. Propose a structure for ornithine. 24-50 Glutathione (GSH) is a tripeptide that serves as a mild reducing agent to detoxify peroxides and maintain the cysteine residues of hemoglobin and other red blood cell proteins in the reduced state. Complete hydrolysis of glutathione gives Gly, Glu, and Cys. Treatment of glutathione with carboxypeptidase gives glycine as the first free amino acid released. Treatment of glutathione with 2,4-dinitrofluorobenzene (Sanger reagent, page 1178), followed by complete hydrolysis, gives the 2,4-dinitrophenyl derivative of glutamic acid. Treatment of glutathione with phenyl isothiocyanate does not give a recognizable phenylthiohydantoin, however. * R C O H  COOH S S H2O COOH S lipoic acid SS S C O N H CH C O NH bound to lysine residue O C H2C H2 C CH N H C O N H H C CH2 N N H C O N HC CH2 CH2 H2 C C O H2N WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1198 24 Study Problems 1199 (a) Propose a structure for glutathione consistent with this information. Why would glutathione fail to give a normal product from Edman degradation, even though it gives a normal product from the Sanger reagent followed by hydrolysis? (b) Oxidation of glutathione forms glutathione disulfide (GSSG). Propose a structure for glutathione disulfide, and write a balanced equation for the reaction of glutathione with hydrogen peroxide. 24-51 Complete hydrolysis of an unknown basic decapeptide gives Gly, Ala, Leu, Ile, Phe, Tyr, Glu, Arg, Lys, and Ser. Terminal residue analysis shows that the N terminus is Ala and the C terminus is Ile. Incubation of the decapeptide with chy- motrypsin gives two tripeptides, A and B, and a tetrapeptide, C. Amino acid analysis shows that peptide A contains Gly, Glu, Tyr, and peptide B contains Ala, Phe, and Lys; and peptide C contains Leu, Ile, Ser, and Arg. Terminal residue analysis gives the following results. N terminus C terminus A Gln Tyr B Ala Phe C Arg Ile Incubation of the decapeptide with trypsin gives a dipeptide D, a pentapeptide E, and a tripeptide F. Terminal residue analysis of F shows that the N terminus is Ser, and the C terminus is Ile. Propose a structure for the decapeptide and for fragments A through F. 24-52 There are many methods for activating a carboxylic acid in preparation for coupling with an amine. The following method converts the acid to an N-hydroxysuccinimide (NHS) ester. (a) Explain why an NHS ester is much more reactive than a simple alkyl ester. (b) Propose a mechanism for the reaction shown. (c) Propose a mechanism for the reaction of the NHS ester with an amine, 24-53 Sometimes chemists need the unnatural D enantiomer of an amino acid, often as part of a drug or an insecticide. Most L-amino acids are isolated from proteins, but the D amino acids are rarely found in natural proteins. D-amino acids can be synthesized from the corresponding L amino acids. The following synthetic scheme is one of the possible methods. (a) Draw the structures of intermediates 1 and 2 in this scheme. (b) How do we know that the product is entirely the unnatural D configuration? COOH HH2N R COOH NH2H R intermediate 1 configuration configuration NaNO3 HCl Pd intermediate 2NaN3 H2 L D R ¬ NH2. N O F3CR OH O O O O F3C OH O   Et3N N R O O O O NH3; WADEMC24_1153-1199hr.qxp 16-12-2008 14:15 Page 1199