994 CH APT E R 2 1 Amino Acids, Peptides, and Proteins

+
Amino acid pKa A-COOH pKa A@NH3 pKa Side chain

Leucine 2.36 9.60 ¬

Lysine 2.18 8.95 10.79
Methionine 2.28 9.21 ¬
Phenylalanine 1.83 9.18 ¬
Proline 1.99 10.60 ¬
Serine 2.21 9.15 ¬
Threonine 2.63 9.10 ¬
Tryptophan 2.38 9.39 ¬
Tyrosine 2.20 9.11 10.07
Valine 2.32 9.62 ¬

Notice that an amino acid can never exist as an uncharged compound, regardless of the pH of
the solution. To be uncharged, an amino acid would have to lose a proton from an +NH3 group
with a pKa of about 9 before it loses a proton from a COOH group with a pKa of about 2. This is
impossible because a weak acid (pKa = 9) cannot lose a proton easier than a strong acid (pKa = 2)
can. Therefore, at physiological pH (7.4), an amino acid exists as a dipolar ion, called a zwitterion.
A zwitterion is a compound that has a negative charge on one atom and a positive charge on a
­nonadjacent atom. (The name comes from zwitter, German for “hybrid.”)

PROBLEM 5 ♦
Alanine has pKa values of 2.34 and 9.69. Therefore, alanine exists predominately as a zwitterion in an
aqueous solution with pH 7 ____ and pH 6 ____.

A few amino acids have side chains with ionizable hydrogens (Table 21.3). The protonated
i­midazole side chain of histidine, for example, has a pKa of 6.04. Histidine, therefore, can exist in
four different forms, and the form that predominates depends on the pH of the solution.
O O O O
C C C C
CH2CH OH CH2CH O− CH2CH O− CH2CH O−
+ + +
NH3 NH3 NH3 NH2
HN NH HN NH N NH N NH
+ +
pH = 0 pH = 4 pH = 8 pH = 12
histidine

PROBLEM 6 ♦
Why are the carboxylic acid groups of the amino acids more acidic (pKa ∼ 2) than a carboxylic acid such
as acetic acid (pKa = 4.76)?

LEARN THE STRATEGY P R O B L E M 7 S O LV E D

Draw the predominant form for each of the following amino acids at physiological pH (7.4):
a. aspartate         c. glutamine            e. arginine
b. histidine           d. lysine           f. tyrosine
SOLUTION TO 7 a. Both carboxyl groups are in the basic form because the pH of the solution is
greater than their pKa values. The protonated amino group is in the acidic form because the pH of the solu-
tion is less than its pKa value.
O O
C C
−
O CH2CH O−
+
NH3
 21.4 The Isoelectric Point   995

PROBLEM 8 ♦ USE THE STRATEGY

Draw the predominant form for glutamate in a solution with the following pH:
a. 0      b. 3      c. 6      d. 11

PROBLEM 9
a. Why is the pKa of the glutamate side chain greater than the pKa of the aspartate side chain?
b. Why is the pKa of the arginine side chain greater than the pKa of the lysine side chain?

21.4 THE ISOELECTRIC POINT

The isoelectric point (pI) of an amino acid is the pH at which it has no net charge. In other words,
it is the pH at which the amount of positive charge on an amino acid exactly balances the amount
of negative charge:
pI = pH at which there is no net charge

Determining the pI of an Amino Acid without an Ionizable Side Chain

The pI of an amino acid that does not have an ionizable side chain—such as alanine—is m ­ idway LEARN THE STRATEGY
between its two pKa values. This is because at pH = 2.34 half of the molecules have a negatively
charged ­carboxyl group and half have an uncharged carboxyl group, whereas at pH = 9.69 half of
the molecules have a positively charged amino group and half have an uncharged amino group. As
the pH increases from 2.34, the carboxyl group of more molecules becomes ­negatively charged;
as the pH decreases from 9.69, the amino group of more molecules becomes positively charged.
Therefore, the number of negatively charged groups equals the number of ­positively charged groups
at the intersection (average) of the two pKa values.
O
C pKa = 2.34
CH3CH OH
+
NH3
alanine pKa = 9.69

2.34 + 9.69 12.03

pI = = = 6.02
2 2

Determining the pI of an Amino Acid with an Ionizable Side Chain

The pI of most amino acids (see Problem 12) that have an ionizable side chain is the average
of the pKa values of the similarly ionizing groups (either positively charged groups ionizing to
uncharged groups or uncharged groups ionizing to negatively charged groups). For example,
the pI of lysine is the average of the pKa values of the two groups that are positively charged in
their acidic form and uncharged in their basic form. The pI of glutamic acid is the average of
the pKa values of the two groups that are uncharged in their acidic form and negatively charged
in their basic form.

O O O
pKa = 2.18 pKa = 2.19
+ C C C
H3NCH2CH2CH2CH2CH OH HO CH2CH2CH OH
pKa = 10.79 + pKa = 4.25 +
NH3 NH3
pKa = 8.95 pKa = 9.67
lysine glutamic acid

8.95 + 10.79 19.74 2.19 + 4.25 6.44

pI = = = 9.87 pI = = = 3.22
2 2 2 2
996 CH APT E R 2 1 Amino Acids, Peptides, and Proteins

USE THE STRATEGY PROBLEM 10 ♦

Calculate the pI of each of the following amino acids:
a. asparagine      b. arginine      c. serine      d. aspartate

PROBLEM 11 ♦
a. Which amino acid has the lowest pI value?
b. Which amino acid has the highest pI value?
c. Which amino acid has the greatest amount of negative charge at pH = 6.20?
d. Which amino acid has a greater negative charge at pH = 6.20, glycine or methionine?

PROBLEM 12
Explain why the pI values of tyrosine and cysteine cannot be determined by the method described on page 995.

PROBLEM 13 ♦
a. What percentage of the a-amino group of lysine will be protonated at its pI?
6 25%      50%       7 75%
b. Answer the same question for the e-amino group of lysine.

PROBLEM 14
Explain why the pI of lysine is the average of the pKa values of its two protonated amino groups.

21.5    SEPARATING AMINO ACIDS

A mixture of amino acids can be separated by several different techniques. Electrophoresis and
­ion-exchange chromatography are two such techniques.

An amino acid will be overall positively
charged if the pH of the solution is
less than the pI of the amino acid,
and it will be negatively charged if
the pH of the solution is greater than
the pI of the amino acid.
Electrophoresis
Electrophoresis separates amino acids on the basis of their pI values. A few drops of a solution
of an amino acid mixture are applied to the middle of a piece of filter paper (or to a gel). When the
paper (or the gel) is placed in a buffered solution between two electrodes and an electric field is
applied (Figure 21.1), an amino acid with a pI greater than the pH of the solution (such as arginine)
has an overall positive charge and migrates toward the cathode (the negative electrode).

cathode anode
− +
− +

+
NH2 O O O
C C C O C
H2N NHCH2CH2CH2CH O− CH3CH O− −OCCH CH O−
2
+ + +
NH3 NH3 NH3
arginine alanine aspartate
pl = 10.76 pl = 6.02 pl = 2.98

▲ Figure 21.1
Arginine, alanine, and aspartate are separated by electrophoresis at pH = 5.

The farther an amino acid’s pI is from the pH of the solution, the more positive its overall positive
charge and the farther it migrates toward the cathode in a given amount of time. An amino acid
with a pI less than the pH of the solution (such as aspartate) has an overall negative charge
and migrates toward the anode (the positive electrode). If two molecules have the same overall
charge, the larger one will move more slowly during electrophoresis because the same charge has
to move a greater mass.
 21.6 Synthesis of Amino Acids   1001

Reductive Amination
Amino acids can also be synthesized by reductive amination of an a-keto acid (Section 16.4).
O O
1. excess NH3,
C trace acid C
RC OH RCH O−
2. H2, Pd/C
+
O NH3

PROBLEM 21 ♦
Cells can also convert a-keto acids into amino acids, but because the reagents organic chemists use for this
reaction are not available in cells, they carry out this reaction by a different mechanism (Section 24.14).
a. 
What amino acid is obtained from the reductive amination of each of the following metabolic
­intermediates in a cell by reductive amination?
O O O O
HO
OH OH HO OH
O O O O
pyruvic acid oxaloacetic acid A-ketoglutaric acid

b. 
What amino acids are obtained from the same metabolic intermediates when the amino acids are
­synthesized in the laboratory?

N-Phthalimidomalonic Ester Synthesis

Amino acids can be synthesized with better yields than those obtained by the previous two methods
via an N-phthalimidomalonic ester synthesis, a method that combines the malonic ester synthesis
(Section 17.17) and the Gabriel synthesis (Section 15.14).

THE STEPS IN THE N-PHTHALIMIDOMALONIC ESTER SYNTHESIS

O OO OO
O O OR OR
−
− + OR RO OR
RO OR + N K N N −

Br H O O ROH
O O O
A-bromomalonic ester potassium phthalimide N-phthalimidomalonic ester

R′ Br

OO
COOH O OR
+
H3N HCl, H2O OR
+ CO2 + OH N
Δ
COOH R′ R′ O Br−
O
phthalic acid amino acid

■ a-Bromomalonic ester and potassium phthalimide undergo an SN2 reaction.

■ A proton is easily removed from the a-carbon of N-phthalimidomalonic ester because it is
flanked by two carbonyl groups (Section 17.1).
■ The resulting carbanion undergoes an SN2 reaction with an alkyl halide.
■ Heating in an acidic aqueous solution hydrolyzes the two esters and the two amide bonds and
decarboxylates the 3-oxocarboxylic acid.
1002 CH APT E R 2 1 Amino Acids, Peptides, and Proteins

A variation of the N-phthalimidomalonic ester synthesis uses acetamidomalonic ester in place of

N-phthalimidomalonic ester.

O
O O O O O O O +
+ CO2 + H3N
RO− − R′ Br HCl, H2O OH
RO OR RO OR RO OR Δ
OH
R′ N R′
HN HN H acetic acid amino acid
O Br−
O O ROH
acetamidomalonic ester

Strecker Synthesis
In the Strecker synthesis, an aldehyde reacts with ammonia to form an imine. A nucleophilic
addition reaction with cyanide ion forms an intermediate, which, when hydrolyzed, forms the
amino acid (Section 15.15).

O
R trace R
acid
−
C N HCl, H2O C
C O NH3
C NH R CH C N R CH OH
HCl Δ
H H + +
NH3 NH3
aldehyde imine amino acid

PROBLEM 22 ♦
What amino acid is formed using the N-phthalimidomalonic ester synthesis when the following alkyl
­halides are used in the third step?
a. CH3CHCH2Br       b. CH3SCH2CH2Br
CH3

PROBLEM 23 ♦
What alkyl halide is used in the acetamidomalonic ester synthesis to prepare
a. lysine?          b. phenylalanine?

PROBLEM 24 ♦
What amino acid is formed when the aldehyde used in the Strecker synthesis is
a. acetaldehyde?         
b. 2-methylbutanal?       c. 3-methylbutanal?

21.7       RESOLUTION
AMINO ACIDS
OF RACEMIC MIXTURES OF

When amino acids are synthesized in nature, only the l-enantiomer is formed (Section 6.14). When
amino acids are synthesized in the laboratory, however, the product is a racemic mixture of d- and
l-amino acids. If only one isomer is desired, the enantiomers must be separated, which can be
accomplished by an enzyme-catalyzed reaction.
Because an enzyme is chiral, it reacts at a different rate with each of the enantiomers (Section 6.14).
For example, aminoacylase is an enzyme that catalyzes the hydrolysis of N-acetyl-l-amino acids, but
not N-acetyl-d-amino acids. Therefore, if the racemic mixture of amino acids is converted to a pair of
N-acetylamino acids (by a nucleophilic acyl substitution reaction) and the N-acetylated mixture is hydro-
lyzed with aminoacylase, the products will be the l-amino acid and unreacted N-acetyl-d-amino acid,
which are easily separated.
1018 CH APT E R 2 1 Amino Acids, Peptides, and Proteins

■ The nucleophilic sulfur of methionine attacks the carbon of cyanogen bromide and displaces
a bromide ion.
■ Nucleophilic attack by oxygen on the methylene group displaces the weakly basic leaving
group and forms a five-membered ring (Section 9.2).
■ Acid-catalyzed hydrolysis of the imine cleaves the protein (Section 16.8).
■ Further hydrolysis causes the lactone (a cyclic ester) to open to a carboxyl group and an alco-
hol group (Section 15.8).
The last step in determining the primary structure of a polypeptide is to figure out the location
of any disulfide bonds. This is done by hydrolyzing a sample of the polypeptide that has intact
disulfide bonds. From a determination of the amino acids in the cysteine-containing fragments, the
locations of the disulfide bonds in the polypeptide can be established (see Problem 66).

PROBLEM 40
Explain why cyanogen bromide does not cleave on the C-side of cysteine.

PROBLEM 41 ♦
Indicate the peptides produced from cleavage by the indicated reagent:
a. His-Lys-Leu-Val-Glu-Pro-Arg-Ala-Gly-Ala by trypsin
b. Leu-Gly-Ser-Met-Phe-Pro-Tyr-Gly-Val by chymotrypsin

LEARN THE STRATEGY P R O B L E M 4 2 S O LV E D

Determine the amino acid sequence of a polypeptide from the following data:
Acid-catalyzed hydrolysis gives Ala, Arg, His, 2 Lys, Leu, 2 Met, Pro, 2 Ser, Thr, and Val.
Carboxypeptidase A releases Val.
Edman’s reagent releases PTH-Leu.
Treatment with cyanogen bromide gives three peptides with the following amino acid compositions:
1. His, Lys, Met, Pro, Ser      2. Thr, Val     3. Ala, Arg, Leu, Lys, Met, Ser
Trypsin-catalyzed hydrolysis gives three peptides and a single amino acid:
1. Arg, Leu, Ser            3. Lys
2. Met, Pro, Ser, Thr, Val       
4. Ala, His, Lys, Met
S O L U T I O N Acid-catalyzed hydrolysis shows that the polypeptide has 13 amino acids. The N-terminal
amino acid is Leu (revealed by Edman’s reagent), and the C-terminal amino acid is Val (revealed by
carboxypeptidase A).

Leu Val

■ 
Because cyanogen bromide cleaves on the C-side of Met, any peptide containing Met must have Met as
its C-terminal amino acid. Thus, the peptide that does not contain Met must be the C-terminal peptide,
indicating that the twelfth amino acid is Thr. We know that peptide 3 is the N-terminal peptide because it
contains Leu. Because peptide 3 is a hexapeptide, we know that the sixth amino acid in the polypeptide
is Met. We also know that the eleventh amino acid is Met because cyanogen bromide cleavage gave the
dipeptide Thr, Val.
Ala, Arg, Lys, Ser His, Lys, Pro, Ser
Leu Met Met Thr Val

■ 
Because trypsin cleaves on the C-side of Arg and Lys, any peptide containing Arg or Lys must have that
amino acid as its C-terminal amino acid. Therefore, Arg is the C-terminal amino acid of peptide 1, so we
now know that the first three amino acids are Leu-Ser-Arg. We also know that the next two are Lys-Ala
because if they were Ala-Lys, then trypsin cleavage would give an Ala, Lys dipeptide. The trypsin data
also identify the positions of His and Lys.
Pro, Ser
Leu Ser Arg Lys Ala Met His Lys Met Thr Val
 21.14 Secondary Structure   1019

■ 
Finally, because trypsin successfully cleaves on the C-side of Lys, Pro cannot be adjacent to Lys. Thus,
the amino acid sequence of the polypeptide is

Leu Ser Arg Lys Ala Met His Lys Ser Pro Met Thr Val

PROBLEM 43 ♦ USE THE STRATEGY

Determine the primary structure of an octapeptide from the following data:
Acid-catalyzed hydrolysis gives 2 Arg, Leu, Lys, Met, Phe, Ser, and Tyr.
Carboxypeptidase A releases Ser.
Edman’s reagent releases Leu.
Treatment with cyanogen bromide forms two peptides with the following amino acid compositions:
1. Arg, Phe, Ser     2. Arg, Leu, Lys, Met, Tyr
Trypsin-catalyzed hydrolysis forms the following two amino acids and two peptides:
1. Arg     2. Ser     3. Arg, Met, Phe     4. Leu, Lys, Tyr

PROBLEM 44
Three peptides were obtained from a trypsin digestion of two different polypeptides. In each case, indicate
the possible sequences from the given data and tell what further experiment should be carried out in order
to determine the primary structure of the polypeptide.
a. polypeptide I: 1. Val-Gly-Asp-Lys    2. Leu-Glu-Pro-Ala-Arg     3. Ala-Leu-Gly-Asp
b. polypeptide II: 1. Val-Leu-Gly-Glu    2. Ala-Glu-Pro-Arg      3. Ala-Met-Gly-Lys

21.14     SECONDARY STRUCTURE

Secondary structure describes the repetitive conformations assumed by segments of the backbone
chain of a polypeptide or protein. In other words, the secondary structure describes how segments of
the backbone fold. Three factors determine the secondary structure of a segment of protein:
■ the regional planarity about each peptide bond (as a result of the partial double-bond
character of the amide bond), which limits the possible conformations of the peptide chain
(Section 21.8)
■ minimizing energy by maximizing the number of hydrogen bonds between peptide groups
(that is, hydrogen bonds between the carbonyl oxygen of one amino acid and the amide
hydrogen of another)

R
HN O
R
C O H N
R O
NH R

hydrogen bonding
between peptide
groups

■ the need for adequate separation between neighboring R groups to avoid steric strain and
repulsion of like charges

A-Helix
One type of secondary structure is an A-helix. In an a-helix, the backbone of the polypeptide coils
around the long axis of the protein molecule. The substituents on the a-carbons of the amino acids
protrude outward from the helix, thereby minimizing steric strain (Figure 21.8a). The helix is sta-
bilized by hydrogen bonds—each hydrogen attached to an amide nitrogen is hydrogen bonded to a
carbonyl oxygen of an amino acid four amino acids away (Figure 21.8b).
Section 21.10
■ To synthesize a peptide bond, the amino group of the N-terminal
amino acid must be protected (by t-Boc) and its carboxyl group
activated (with DCCD). A second amino acid is added to form a
dipeptide. Amino acids can be added to the growing C-terminal
end by repeating the same two steps: activating the carboxyl group
of the C-terminal amino acid with DCCD and then adding a new
amino acid.
Section 21.11
■ Automated solid-phase peptide synthesis allows peptides to be
synthesized more rapidly and in higher yields.
Section 21.13
■ The primary structure of a protein is the sequence of its amino
acids and the location of all its disulfide bridges.
The N-terminal amino acid can be determined with Edman’s
■
Section 21.14
■ The secondary structure of a protein describes how local
segments of the protein’s backbone fold. An A-helix and a
B-pleated sheet are two types of secondary structure.
■ A protein folds so as to maximize the number of stabilizing
interactions—namely, disulfide bonds, hydrogen bonds,
electrostatic attractions, and hydrophobic interactions.
Section 21.15
■ The tertiary structure of a protein is the three-dimensional
arrangement of all the atoms in the protein.
Section 21.16
■ The quaternary structure of a protein describes the way the
peptide chains (subunits) of a protein with more than one peptide
chain are arranged with respect to each other.
Section 21.17

reagent. The C-terminal amino acid can be identified with a

carboxypeptidase. ■ Proteins can be denatured by a change in pH, by an organic
solvent, by heat or agitation, or by certain reagents.
■ An exopeptidase catalyzes the hydrolysis of a peptide bond at the
end of a polypeptide or protein chain. An endopeptidase catalyzes
the hydrolysis of a peptide bond that is not at the end of a chain.

PROBLEMS
49. Glycine has pKa values of 2.34 and 9.60. At what pH does glycine exist in the indicated form?
O O O O O
+ + + +
a. H2N − H3N − b. H3N − c. H3N − H3N
O O O O OH
50% 50% 100% 50% 50%

50. Show the peptides that would result from cleavage by the indicated reagent:
a. Val-Arg-Gly-Met-Arg-Ala-Ser by carboxypeptidase A
b. Ser-Phe-Lys-Met-Pro-Ser-Ala-Asp by cyanogen bromide
c. Arg-Ser-Pro-Lys-Lys-Ser-Glu-Gly by trypsin

51. A titration curve is a plot of the pH of a solution as a function of added equivalents of hydroxide ion. As hydroxide ion is added to the aqueous solution,
the pH increases because hydroxide ion removes protons from the solution. The pH flattens out when hydroxide ion can remove a proton from an ioniz-
able group of an amino acid rather than a proton from the solution. Identify the amino acids that give the titration curves below.
a. 12 b. 12 c. 12
11 11 11
10 10 pH = 9.8 10 pH = 9.7
pH = 9.2
9 9 9
8 8 8
7 7 7
pH

pH

pH

6 6 6
5 pH = 6.0 5 5
4 4 4
3 3 3
2 2 2
pH = 1.8 pH = 2.3
1 1 1

1 2 3 1 2 3 1 2
equivalents HO− equivalents HO− equivalents HO−

1026
52. Which has a higher percentage of negative charge at physiological pH (7.4), leucine with pI = 5.98 or asparagine with pI = 5.43?

53. Aspartame (its structure is on page 1007) has a pI of 5.9. Draw its prevailing form at physiological pH (7.4).

54. Draw the form of aspartate that predominates at the following pH values:
a. pH = 1.0        b. pH = 2.6        c. pH = 6.0        d. pH = 11.0

55. Show how phenylalanine can be prepared by reductive amination of an a-ketocarboxylic acid.

56. A professor was preparing a manuscript for publication in which she reported that the pI of the tripeptide Lys-Lys-Lys was 10.6. One of her students
pointed out that there must be an error in her calculations because the pKa of the e-amino group of lysine is 10.8 and the pI of the tripeptide has to be
greater than any of its individual pKa values. Was the student correct?

57. What aldehydes are formed when the following amino acids are treated with ninhydrin?
a. tyrosine           b. leucine           c. arginine

58. A mixture of amino acids that do not separate sufficiently when a single technique is used can often be separated by two-dimensional c­ hromatography.
In this technique, the mixture of amino acids is applied to a piece of filter paper and separated by chromatographic techniques. The paper is then r­ otated 90°,
and the amino acids are further separated by electrophoresis, producing a type of chromatogram called a fingerprint. Identify the spots in the f­ ingerprint
­obtained from a mixture of Ser, Glu, Leu, His, Met, and Thr.
Electrophoresis −
at pH = 5

+
Chromatography

59. Determine the amino acid sequence of a polypeptide from the following data:
Complete hydrolysis of the peptide yields Arg, 2 Gly, Ile, 3 Leu, 2 Lys, 2 Met, 2 Phe, Pro, Ser, 2 Tyr, and Val.
Treatment with Edman’s reagent releases PTH-Gly.
Carboxypeptidase A releases Phe.
Treatment with cyanogen bromide yields the following three peptides:
1. Gly-Leu-Tyr-Phe-Lys-Ser-Met      2. Gly-Leu-Tyr-Lys-Val-Ile-Arg-Met      3. Leu-Pro-Phe
Treatment with trypsin yields the following four peptides:
1. Gly-Leu-Tyr-Phe-Lys          3. Val-Ile-Arg
2. Ser-Met-Gly-Leu-Tyr-Lys           4. Met-Leu-Pro-Phe

60. Explain why amino acids, unlike most amines and carboxylic acids, are insoluble in diethyl ether.

61. Explain the difference in the pKa values of the carboxyl groups of alanine, serine, and cysteine.

62. Which is the more effective buffer at physiological pH, a solution of 0.1 M glycylglycylglycylglycine or a solution of 0.2 M glycine?

63. Identify the location and type of charge on the hexapeptide Lys-Ser-Asp-Cys-His-Tyr at each of the following pH values:
a. pH = 1            b. pH = 5           c. pH = 7            d. pH = 12

64. Draw the product obtained when a lysine side chain in a polypeptide reacts with maleic anhydride.
O

NHCH NH +
O O O
(CH2)4
NH2
lysine residue maleic anhydride

1027
75. Show how valine can be prepared by
a. a Hell–Volhard–Zelinski reaction.          d. a N-phthalimidomalonic ester synthesis.
b. a Strecker synthesis.               e. an acetamidomalonic ester synthesis.
c. a reductive amination.

76. The primary structure of b-endorphin, a peptide containing 31 amino acids synthesized by the body to control pain, is shown here:
Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-
Leu-Phe-Lys-Asn-Ala-Ile-Ile-Lys-Asn-Ala-Tyr-Lys-Lys-Gly-Glu
a. What fragments are obtained as a result of treatment with each of the following?
1. trypsin      2. cyanogen bromide      3. chymotrypsin
b. How much of the primary structure can be determined if the amino acids contained in each fragment (but not their sequence) are known?

77. A chemist wanted to test his hypothesis that the disulfide bridges that form in many proteins do so after the minimum energy conformation of the protein
has been achieved. He treated a sample of an enzyme that contained four disulfide bridges with 2-mercaptoethanol and then added urea to denature the
enzyme. He slowly removed these reagents so that the enzyme could re-fold and re-form the disulfide bridges. The enzyme he recovered had 80% of its
original activity. What would be the percent activity in the recovered enzyme if disulfide bridge formation were entirely random rather than determined
by the tertiary structure? Does this experiment support his hypothesis?

78. Propose a mechanism for the rearrangement of the thiazoline obtained from the reaction of Edman’s reagent with a peptide to a PTH-amino acid
(page 1014). (Hint: Thioesters are very reactive toward nucleophiles.)

79. A normal polypeptide and a mutant of the polypeptide were hydrolyzed by an endopeptidase under the same conditions. The normal and mutant
­poly­peptide differ by one amino acid. The fingerprints of the peptides obtained from the two polypeptides are shown below. What kind of amino acid
substitution occurred as a result of the mutation? (That is, is the substituted amino acid more or less polar than the original amino acid? Is its pI lower or
higher?) (Hint: Photocopy the fingerprints, cut them out, and overlay them.)
− −
Electrophoresis at pH = 6.5

Electrophoresis at pH = 6.5

Normal Mutant

+ Paper chromatography + Paper chromatography

80.Determine the amino acid sequence of a polypeptide from the following data:
Complete hydrolysis of the peptide yields Ala, Arg, Gly, 2 Lys, Met, Phe, Pro, 2 Ser, Tyr, and Val.
Treatment with Edman’s reagent releases PTH-Val.
Carboxypeptidase A releases Ala.
Treatment with cyanogen bromide yields the following two peptides:
1. Ala, 2 Lys, Phe, Pro, Ser, Tyr       2. Arg, Gly, Met, Ser, Val
Treatment with trypsin yields the following three peptides:
1. Gly, Lys, Met, Tyr              2. Ala, Lys, Phe, Pro, Ser           3. Arg, Ser, Val
Treatment with chymotrypsin yields the following three peptides:
1. 2 Lys, Phe, Pro           2. Arg, Gly, Met, Ser, Tyr, Val      3. Ala, Ser

1029
Answers
 25.2    Structure and Properties of Amino Acids    1201

PRACTICE the skill 25.4 For each example, draw the form of the amino acid that is expected to predominate
at the stated pH.

(a) Alanine at a pH of 10 (b) Proline at a pH of 10

(c) Tyrosine at a pH of 9 (d) Asparagine at physiological pH
(e) Histidine at physiological pH (f  ) Glutamic acid at a pH of 3

APPLY the skill 25.5 At a pH of 11, arginine is a more effective proton donor than asparagine. Explain.

25.6 The OH group on the side chain of serine is not deprotonated to a significant extent
at a pH of 12. However, the OH group on the side chain of tyrosine is essentially fully depro-
tonated at a pH of 12. This can be verified by inspecting the pKa values in Table 25.2. Sug-
gest an explanation for the difference in acidity between these two OH groups.

need more PRACTICE? Try Problems 25.40, 25.47, 25.48, 25.84

Isoelectric Point
For each amino acid, there is a specific pH at which the concentration of the zwitterionic form
reaches its maximum value. This pH is called the isoelectric point (pI), and each amino acid has its
own unique pI. For amino acids that lack an acidic or basic side chain, the pI is simply the average of
the two pKa values. The following example shows the calculation for the pI of alanine.

R COOH pKa = 2.34

pKa = 9.69 + NH3

2.34 + 9.69
p = = 6.02
2

For amino acids with acidic or basic side chains, the pI is the average of the two pKa values that
correspond with the similar groups. For example, the pI of lysine is determined by the two amino
groups, while the pI of glutamic acid is determined by the two carboxylic acid groups.
Lysine Glutamic acid

+
H3N COOH HOOC COOH
pKa = 10.53 pKa = 4.25 pKa = 2.19
+ NH3 pKa = 8.95 + NH3

10.53 + 8.95 4.25 + 2.19

p = = 9.74 p = = 3.22
2 2

Separation of Amino Acids via Electrophoresis

Amino acids can be separated from each other by a variety of techniques. One method, called elec-
trophoresis, relies on a difference in pI values and can be used to determine the number of different
amino acids present in a mixture. In practice, a few drops of the mixture are applied to filter paper or
gel that is placed in a buffered solution between two electrodes. When an electric field is applied, the
amino acids separate based on their different pI values. If the pI of an amino acid is greater than the
pH of the solution, the amino acid will exist predominantly in a form that bears a positive charge
and will migrate toward the cathode. The greater the difference between pI and pH, the faster it will
migrate. An amino acid with a pI that is lower than the pH of the solution will exist predominantly
in a form that bears a negative charge and will migrate toward the anode. The greater the difference
between pI and pH, the faster it will migrate (Figure 25.2). If two amino acids have very similar pI

At pH 6

− +

FIGURE 25.2
Separation of amino acids via Lysine Alanine Glutamic acid
electrophoresis. (p = 9.74) (p = 6.02) (p = 3.22)
 25.3 Amino Acid Synthesis 1203

CONCEPTUAL CHECKPOINT
25.7 Using the data in Table 25.2, calculate the p for each of the 25.9 Identify which two of the 20 naturally occurring amino acids
following amino acids: are expected to have the same p .
(a) Aspartic acid (b) Leucine
25.10 A mixture containing phenylalanine, tryptophan, and leu-
(c) Lysine (d) Proline cine was subjected to electrophoresis. Predict which amino acid
would move the farthest, if the experiment was performed at each
25.8 For each group of amino acids, identify the amino acid
of the given pH values:
with the lowest p (try to solve this problem by inspecting their
structures, rather than performing calculations). (a) pH = 6.0 (b) pH = 5.0

(a) Alanine, aspartic acid, or lysine 25.11 Draw the aldehyde that is obtained as a by-product when
(b) Methionine, glutamic acid, or histidine l-leucine is treated with ninhydrin and NaOH.

25.3 Amino Acid Synthesis

Over the last century, many methods have been used to prepare amino acids in the laboratory. In
this section, we will explore a few of those methods.

Amino Acid Synthesis via α-Haloacids

One of the oldest methods for preparing racemic mixtures of α-amino acids involves the use
of the Hell–Volhard–Zelinsky reaction (Section 21.2) to functionalize the α position of a
carboxylic acid.

O
O
1) Br2, PBr3 R
R 2) H2O OH
OH
Br
(Racemic)

The halogen is then replaced with an amino group in an SN2 reaction.

O O
R Excess NH3 R
OH SN2 OH
Br NH2
(Racemic) (Racemic)

In Section 22.5, we saw that polyalkylation is often unavoidable when ammonia is treated with an
alkyl halide. However, in this case, polyalkylation is not a problem because the alkyl halide is fairly
large and steric hindrance prevents subsequent alkylations.

CONCEPTUAL CHECKPOINT
25.12 Identify the starting material and the reagents necessary O
to make each of the following amino acids using a Hell–Volhard–
Zelinsky reaction: (a) COOH (b) OH

(a) Leucine (b) Alanine (c) Valine

25.13 Each of the following carboxylic acids was treated with

bromine and PBr3 followed by water, and the resulting α-haloacid (c) COOH (d) Acetic acid
was then treated with excess ammonia. In each case, draw and
name the amino acid that is produced.
 25.3   Amino Acid Synthesis   1205

Analyze the structure of this alkyl halide and make sure that it will readily participate in an
SN2 reaction. If the substrate is tertiary, then an amidomalonate synthesis will fail, because
the alkylation step will not occur. In this case, the alkyl halide is primary and would be
expected to undergo an SN2 process quite rapidly.
In order to perform the amidomalonate synthesis, we must first deprotonate diethyl
acetamidomalonate with a strong base and then treat the resulting anion with the alkyl
halide. The resulting product is then heated in the presence of aqueous acid to achieve
hydrolysis and decarboxylation.
STEP 3 O O O O
OEt O
Identify the reagents
1) NaOEt H3O+
beginning with EtO OEt EtO Heat OH
acetamidomalonate. 2)
HN
N Br
H + NH3
N
O H
O N
H
NH

PRACTICE the skill 25.14 Identify the reagents necessary to make each of the following amino acids via the
amidomalonate synthesis:

(a) Isoleucine (b) Alanine (c) Valine

APPLY the skill 25.15 An amidomalonate synthesis was performed using each of the following alkyl
halides. In each case, draw and name the amino acid that was produced.

(a) Methyl iodide (b) Isopropyl chloride (c) 2-Methyl-1-chloropropane

25.16 Both leucine and isoleucine can be prepared via the amidomalonate synthesis,
although one of these amino acids can be produced in higher yields. Identify the higher
yielding process and explain your choice.

need more PRACTICE? Try Problems 25.57, 25.58, 25.59c, 25.60b, 25.89

Amino Acid Synthesis via the Strecker Synthesis

Racemic α-amino acids can also be prepared from aldehydes via a two-step process called the
Strecker synthesis.

N O
+
O H2N C H3N
NH4Cl H3O + C
OH
NaCN R H
R H R H
(Racemic) (Racemic)

The first step involves conversion of the aldehyde into an α-amino nitrile, and the second step
involves hydrolysis of the cyano group to yield a carboxylic acid group.
Formation of the α-amino nitrile likely proceeds via Mechanism 25.1. The resulting α-amino
nitrile then undergoes hydrolysis via a mechanism found in Section 20.13.
As an example, consider the synthesis of alanine via the Strecker synthesis:
O O
1) NH4Cl, NaCN
H3C
H3C H 2) H3O+ OH

+
NH3
Alanine
(racemic)

The identity of the amino acid obtained is determined by the choice of the starting aldehyde.
1206 CHAPTER 25 Amino Acids, Peptides, and Proteins

MECHANISM 25.1 FORMATION OF AN α-AMINO NITRILE

NH4Cl H Cl + NH3

Nucleophilic attack Proton transfer Proton transfer

H H H
H −
O N OH H2N OH
NH3 H +N O H Cl H + NH3

R H R H R H R H

H Cl Proton transfer

Nucleophilic attack Loss of a

leaving group
O
+ N + +
H3N H2N − NH2 H 2N OH2
C H3O + C –H2O
N C
OH
R H See Section R H R H R H
20.13

CONCEPTUAL CHECKPOINT
25.17 Identify a suitable starting material and the reagents necessary 25.18 Each of the following aldehydes was converted into an
to make each of the following amino acids using a Strecker synthesis: α-amino nitrile, and then hydrolyzed to yield an amino acid. In
each case, draw and name the amino acid that was produced.
(a) Methionine (b) Histidine
(c) Phenylalanine (d) Leucine (a) Acetaldehyde (b) 3-Methylbutanal (c) 2-Methylpropanal

Enantioselective Synthesis of l Amino Acids

Racemic mixtures of amino acids are produced from each of the three methods described thus far.
Obtaining optically active amino acids requires either resolution of the racemic mixture or enantio-
selective synthesis. Resolution is less efficient and more costly because half of the starting material
is wasted in the process. Resolution is not necessary if an enantioselective synthesis is performed. In
Section 8.9 we saw how Knowles developed a procedure for achieving asymmetric hydrogenation
and then used that procedure for the enantioselective synthesis of l-dopa.

Asymmetric hydrogenation of this π bond

COOH (S) COOH

O O (S) COOH
H2
HN Chiral
HN
O O NH2
catalyst HO
O O O O
OH

(S)-3,4-Dihydroxyphenylalanine
(L-dopa)
 25.6   Peptide Synthesis   1221

Identify the groups that must be protected (the groups that are not being coupled). The
amino group is protected by installing a Boc protecting group.

O O
H
H2N N
STEP 1 OH Boc OH
Install the appropriate
protecting groups. (Boc)2O

The carboxylic acid group is protected by converting it to an ester, such as a benzyl ester.

[H+]
OH O
H2N H2N
OH
O O

With the protecting groups in place, the next step is to form the desired peptide linkage
using DCC.

O O
H
STEP 2 N
H
N O
Couple the protected Boc OH Boc N
+ O DCC
amino acids using H2N H
O
DCC.
O

The final step is to remove the protecting groups to yield the desired product.

O O
STEP 3 H −
N O H2N O
Remove the Boc N 1) CF3COOH N
protecting groups. H 2) NaOH, H2O H
O O

PRACTICE the skill 25.34 Draw all of the steps and reagents necessary to prepare each of the following
dipeptides from their corresponding amino acids:

(a) Trp-Met (b) Ala- le (c) Leu-Val

APPLY the skill 25.35 Starting with individual amino acids, draw all of the steps and reagents necessary to
prepare a tripeptide with the sequence le-Phe-Gly.

25.36 Starting with individual amino acids, draw all of the steps and reagents necessary to
prepare a pentapeptide with the sequence Leu-Val-Phe- le-Ala.

need more PRACTICE? Try Problems 25.77–25.79, 25.88

The Merrifield Synthesis

The method outlined in this section works well for very small peptides, but it is not feasible to use it
for larger peptides, because each step requires isolation and purification due to the accumulation of
unwanted side products. Larger peptides can be prepared with the Merrifield synthesis, developed
by R. Bruce Merrifield (Rockefeller University). A protected amino acid is first tethered to beads of
an insoluble polymer. One such polymer is a polystyrene derivative with CH2Cl groups on some of
the aromatic rings.
1236 CHAPTER 25 Amino Acids, Peptides, and Proteins

ACS-STYLE PROBLEMS (Multiple Choice)

Problems 25.84–25.88 follow the style of the ACS organic 25.86 • The two pKa values for serine are given below. What is the
chemistry exam. For each of these problems, there will be isoelectric point (p ) for serine?
one correct answer and three distractors.
O
25.84 • Which of the following is the predominant form of proline at
HO OH pKa = 2.21
physiological pH (pH = 7.4)?
pKa = 9.15 + NH3
O
O
(a) 9.15 (b) 6.94 (c) 5.68 (d) 2.21
OH
OH
N
N H 24.87 • Of the following, which best describes a peptide bond and its
+
(a) H (b) H planar character?
(a) A peptide bond is within an ester group that has an sp2 hybridized
O carbonyl group.
O
− (b) A peptide bond is a CN bond with substantial double-bond
− O
O character.
N H
N + (c) A peptide bond is an amide group in which the nitrogen atom
(c) H (d) H
bears a lone pair that is localized.
(d) A peptide bond involves an amine that is rapidly inverting.
25.85 • Which of the following terms properly describes the given
peptide? 25.88 • In order to synthesize a dipeptide, a chemist prepares two
protected amino acids: a BOC-protected glycine, and the methyl ester
O
of phenylalanine. Which of the following best describes the two func-
H O CH3 H O OH tional groups that will react to form the dipeptide?
N N OH (a) The Gly carboxylic acid reacts with the Phe amino group.
H3N N N
+
(b) The Gly carboxylic acid reacts with the Phe carboxylic acid.
O H O H O
SH (c) The Gly amino group reacts with the Phe carboxylic acid.
(d) The Gly amino group reacts with the Phe ester.
(a) Tripeptide (b) Tetrapeptide
(c) Pentapeptide (d) Hexapeptide

INTEGRATED PROBLEMS
25.89 Draw the alkyl halide that would be necessary to make the
amino acid tyrosine using an amidomalonate synthesis. This same alkyl
halide is highly susceptible to polymerization. Draw a segment of the
expected polymeric material.
25.90 When leucine is prepared with an amidomalonate synthesis,
isobutylene (also called 2-methylpropene) is a gaseous by-product. Draw
a mechanism for the formation of this by-product.
25.91 The side chain of tryptophan is not considered to be basic,
despite the fact that it possesses a nitrogen atom with a lone pair.
Explain.
25.92 Consider a process that attempts to prepare tyrosine using a
Hell–Volhard–Zelinsky reaction:
(a) Identify the necessary startin g carboxylic acid.
(b) When treated with two equivalents of Br2, the starting carboxylic
acid produces a compound with the molecular formula C9H8Br2O3,
in which neither of the bromine atoms is located at the α position.
Propose a structure for the observed product, and describe the
mechanism that occurred.
 Answers
25.4. In each case, we first identify the pKa of the
carboxylic acid group and determine which form
predominates. The protonated form (RCOOH) will
predominate if pH < pKa, while the carboxylate ion will
predominate if pH > pKa. Next, we identify the pKa of
the -amino group and determine which form
predominates. The protonated form (RNH3+) will
predominate if pH < pKa, while the uncharged form
(RNH2) will predominate if pH > pKa. Finally, a similar
analysis is performed for the side chain (if necessary).
See Table 25.2 for pKa values.
(a) (b)
(c) (d)
(e) (f)
25.5. Arginine has a basic side chain, while asparagine
does not. At a pH of 11, arginine exists predominantly
in a form in which the side chain is protonated.
Therefore, it can serve as a proton donor.
25.6. Tyrosine possesses a phenolic proton which is
more readily deprotonated because deprotonation forms
a resonance-stabilized phenolate ion. In contrast,
deprotonation of the OH group of serine gives an
alkoxide ion that is not resonance-stabilized. As a result,
the OH group of tyrosine is more acidic than the OH
group of serine.
25.7.
(a) Aspartic acid has two carboxylic acid groups, so the
pI of aspartic acid is calculated using the pKa values of
the two carboxylic acid groups, as shown here (pKa
values can be found in Table 25.2):
CHAPTER 25 1161
(b) Leucine does not have an acidic side chain or a basic
side chain, so the pI of leucine is calculated using the
pKa value of the carboxylic acid group and the pKa value
of the ammonium group, as shown here (pKa values can
be found in Table 25.2):
(c) Lysine has two ammonium groups, so the pI of lysine
is calculated using the pKa values of the two ammonium
groups, as shown here (pKa values can be found in Table
25.2):
(d) Proline does not have an acidic side chain or a basic
side chain, so the pI of proline is calculated using the pKa
value of the carboxylic acid group and the pKa value of
the ammonium group, as shown here (pKa values can be
found in Table 25.2):
25.8.
(a) The pKa values for carboxylic acid groups (~2-4) are
significantly lower than the pKa values for ammonium
groups (~9-10). Aspartic acid has two carboxylic acid
groups, so it is expected to have the lowest pI.
(b) The pKa values for carboxylic acid groups (~2-4) are
significantly lower than the pKa values for ammonium
groups (~9-10). Glutamic acid has two carboxylic acid
groups, so it is expected to have the lowest pI.
25.9. Leucine and isoleucine both exhibit the same pKa
value for the carboxylic acid group. Similarly, both
leucine and isoleucine exhibit the same pKa value for the
amino group. As such, the pI value of leucine is
expected to be the same as the pI value of isoleucine.
25.10. The greater the difference between pI and pH,
the faster an amino acid will migrate. The pI of Phe =
5.48, the pI of Trp = 6.11, and the pI of Leu = 6.00.
Using these values, we make the following predictions:
(a) At pH = 6.0, Phe will travel the farthest distance.
(b) At pH = 5.0, Trp will travel the farthest distance.
25.11. The following aldehyde is expected when
L-leucine is treated with ninhydrin:
1162 CHAPTER 25

25.12.
(a) Racemic leucine can be made using a Hell- (c) (d)
Volhard-Zelinsky reaction, as shown:

25.14. In each case, we begin by identifying the side

(b) Racemic alanine can be made using a Hell-Volhard-
Zelinsky reaction, as shown:
chain connected to the  position. Then, we identify the
necessary alkyl halide and ensure that it is not tertiary
(because a tertiary alkyl halide will not undergo an SN2
reaction). An amidomalonate synthesis is performed
using acetamidomalonate as the starting material, which
is first treated with sodium ethoxide. The resulting
conjugate base (a doubly stabilized enolate) is then
treated with the alkyl halide, followed by hydrolysis with
aqueous acid and heat, to give the desired amino acid.
Note that the final step employs acidic conditions, so the
amino group of the resulting amino acid is protonated.
Highlighted below are the alpha carbon and the carbonyl
carbon in the acetamidomalonate starting material that
remain in the product (after decarboxylation and
deprotection):

(a)

(c) Racemic valine can be made using a Hell-Volhard-

Zelinsky reaction, as shown:
(b)

(c)

25.13. In each case, the process is a Hell-Volhard-

Zelinsky reaction, which installs an amino group at the
alpha position, giving the following amino acids:

(a) (b) 25.15.

(a) Alanine is obtained when methyl iodide is used as
the alkyl halide in an amidomalonate synthesis. The
methyl group (from methyl iodide) is highlighted in the
product:
 CHAPTER 25 1163

(c) Phenylalanine can be prepared from the aldehyde

below via a Strecker synthesis, as shown:

(b) Valine is obtained when isopropyl chloride is used as

the alkyl halide in an amidomalonate synthesis. The
isopropyl group (from isopropyl chloride) is highlighted
in the product:

(d) Leucine can be prepared from the aldehyde below via

a Strecker synthesis, as shown:
(c) Leucine is obtained when 2-methyl-1-chloropropane
is used as the alkyl halide in an amidomalonate
synthesis. The alkyl group (from the alkyl chloride) is
highlighted in the product:

25.18.
25.16. Leucine can be prepared via the amidomalonate
(a) Acetaldehyde is converted into a racemic mixture of
synthesis with higher yields than isoleucine, because
alanine (via a Strecker synthesis), as shown:
leucine requires an SN2 reaction with a primary alkyl
halide, while isoleucine requires an SN2 reaction with a
secondary (more hindered) alkyl halide.

25.17.
(a) Methionine can be prepared from the aldehyde
below via a Strecker synthesis, as shown:

(b) 3-Methylbutanal is converted into a racemic mixture

of leucine (via a Strecker synthesis), as shown:

(b) Histidine can be prepared from the aldehyde below

via a Strecker synthesis, as shown: (c) 2-Methylpropanal is converted into a racemic mixture
of valine (via a Strecker synthesis), as shown:
 CHAPTER 25 1167

25.34.
(a) We begin by installing the appropriate protecting groups. Then, upon treatment with DCC, the protected amino
acids are coupled. And finally, the protecting groups are removed, as shown:

(b) We begin by installing the appropriate protecting groups. Then, upon treatment with DCC, the protected amino
acids are coupled. And finally, the protecting groups are removed, as shown:

(c) We begin by installing the appropriate protecting groups. Then, upon treatment with DCC, the protected amino
acids are coupled. And finally, the protecting groups are removed, as shown:
1168 CHAPTER 25

25.35. The first two amino acid residues (in the desired peptide sequence) are Ile and Phe. So we must begin with
those amino acids. We first install the appropriate protecting groups. Then, upon treatment with DCC, the protected
amino acids are coupled. The protecting group at the C terminus is then removed and the resulting unprotected
C terminus is coupled with the appropriate protected amino acid (glycine, protected at the C terminus), using DCC.
And finally, the protecting groups are removed, as shown:

(Boc)2O
Ile Boc Ile

DCC
Boc Ile Phe OCH3

[H+]
NaOH, H2O
Phe Phe OCH3
CH3OH

Boc Ile Phe

DCC Gly OCH3

1) CF3COOH
Ile Phe Gly Boc Ile Phe Gly OCH3
2) NaOH, H2O
1180 CHAPTER 25

25.83. The amino group attacks one of the carbonyl groups of DCC, giving a tetrahedral intermediate. The carbonyl
group is then reformed upon expulsion of a resonance-stabilized leaving group. The resulting cation is then
deprotonated to give the product:

25.84. The correct answer is (d). At physiological pH, a carboxylic acid group is expected to exist predominantly as a
carboxylate ion, and the amino group is expected to exist primarily as an ammonium ion:

25.85. The correct answer is (c). The compound is constructed from five amino acid residues, as highlighted below,
and is therefore a pentapeptide:

25.86. The correct answer is (c). Serine does not have an acidic side chain or a basic side chain, so the pI of serine is
calculated by finding the average of the pKa value of the carboxylic acid group and the pKa value of the ammonium
group, as shown here:
 CHAPTER 25 1181

25.87. The correct answer is (b). Two amino acids are linked together by forming an amide. The lone pair on the
nitrogen atom of an amide is adjacent to a  bond and, therefore, is delocalized by resonance. Because of this
resonance, the peptide bond has substantial double-bond character. The carbonyl carbon and the nitrogen atom of the
amide group are both sp2 hybridized, making the six atoms highlighted below coplanar:

25.88. The correct answer is (a). The Boc group (tert-butoxycarbonyl) is a protecting group for amines, so Boc-glycine
has a protected amino group and a reactive carboxylic acid group. The phenylalanine has the opposite reactivity: the
carboxylic acid is protected as a methyl ester, and the amino group is reactive. The two protected amino acids could be
coupled with the help of DCC to prepare a protected dipeptide:

25.89. The following alkyl halide would be necessary in order to prepare tyrosine via an amidomalonate synthesis.
This starting material possesses both a nucleophilic center (the OH group) as well as an electrophilic center (it is a
primary benzylic bromide). As such, the molecules can react with each other via an SN2 process, thereby forming the
polymer shown:

25.90. The stabilized enolate ion (formed in the first step) can function as a base, rather than a nucleophile, giving an
E2 reaction:

25.91. The lone pair on that nitrogen atom is highly delocalized via resonance and is participating in aromaticity.
Accordingly, the lone pair is not available to function as a base.
 Integrated Problems 1275

ACS-STYLE PROBLEMS (Multiple Choice)

Problems 26.51–26.55 follow the style of the ACS organic 26.53 • Which of the following has the appropriate geometry for
chemistry exam. For each of these problems, there will be bilayer formation?
one correct answer and three distractors. (a) Fatty acids (b) Phospholipids

26.51 • Which of the following statements is NOT true about the (c) Triglycerides (d) All of the above
given compound?
26.54 • Which of the following compounds is a saturated fatty acid?
O O

O OH
O
(a)
O
O
O

O (b) HO

O
(a) It reacts with molecular hydrogen in the presence of Ni.
O
(b) It undergoes hydrolysis to produce glycerin. O
(c) It is a solid at room temperature.
O
(d) It is a triglyceride.
O
26.52 • Which of the following correctly identifies the isoprene units
in the given terpene? (c) O

O
O

O
(a) (b)
O
O
P
OH
(d) HO

26.55 • Which of the following are NOT lipids?

(a) Triglycerides (b) Tripeptides
(c) (d) (c) Diterpenes (d) Steroids

INTEGRATED PROBLEMS
26.56 Treatment of cholesterol with a peroxy acid (RCO3H) could
potentially produce two diastereomeric epoxides.
(a) Draw both diastereomeric epoxides.
(b) Only one of these epoxides is formed. Predict which one and
explain why the other is not formed.
26.57 Identify the products expected when estradiol is treated with
each of the following reagents:
(a) Excess Br2 (b) PCC
(c) Excess strong base followed by excess ethyl iodide
(d) Excess acetyl chloride in the presence of pyridine
26.58 Olestra (sold under the trade name Olean) is a noncaloric oil
substitute that is produced by esterifying sucrose with eight equiva-
lents of fatty acids obtained from the hydrolysis of vegetable oils. The
eight fatty acid residues give Olestra the consistency and flavor of
cooking oil, but the steric bulk of the compound prevents digestive
enzymes from hydrolyzing the ester groups. Olestra can be added
to food products to give the texture and taste of fat. But, unlike fat,
Olestra passes through the gastrointestinal (GI) tract unaltered and
unabsorbed. Hence it provides no calories. After passing through the
GI tract, it is excreted in the feces. Consumption of large amounts of
food containing Olestra (such as potato chips) can result in “loose
stools,” a polite term for an “accident.” As a result, foods made with
1198 CHAPTER 26 Answers
(c) This terpene has fifteen carbon atoms and is
therefore comprised of three isoprene units, shown here:
(d) This terpene has twenty carbon atoms and is
therefore comprised of four isoprene units, shown here:
26.50.
(a) The polar head and the two hydrophobic tails are labeled in the following structure:
Hydrophobic tails
(b) Yes, they have one polar head and two hydrophobic tails. See Figure 26.6.
26.51. The correct answer is (c). The given compound
is a triglyceride that would produce glycerin (and three
fatty acids) upon hydrolysis. The presence of double
bonds indicates that it will undergo catalytic
hydrogenation. The cis double bonds also cause kinks in
the carbon chains, thus lowering the melting point of the
compound. A polyunsaturated triglyceride is described
as an oil because it is a liquid at room temperature. It is
not a solid.
26.52. The correct answer is (a). Isoprene is a
branched, five-carbon unit. The given sesquiterpene has
three isoprene units, as highlighted below:
(e) This terpene has ten carbon atoms and is therefore
comprised of two isoprene units, shown here:
(f) This terpene has ten carbon atoms and is therefore
comprised of two isoprene units, shown here:
R
Polar head
H
O N
O
O
P N
O
OH O
26.53. The correct answer is (b). See Figure 26.6,
which illustrates that phospholipids have the appropriate
geometry for bilayer formation, while fatty acids and
triglycerides do not have the appropriate geometry for
bilayer formation.
26.54. The correct answer is (b). Fatty acids are long-
chain carboxylic acids, and structure (b) contains no
carbon-carbon double bonds, so it is described as
saturated.
26.55. The correct answer is (b). Lipids are naturally
occurring compounds that can be extracted from cells
using nonpolar organic solvents. Triglycerides, terpenes
and steroids are all lipids, but a tripeptide is not. A
tripeptide has many polar functional groups and is not
classified as a lipid.
1192 CHAPTER 24 Carbohydrates

24.71 Identify the product(s) that would be formed when each of the (a) Is salicin a reducing sugar?
following compounds is treated with aqueous acid: (b) Identify the products obtained when salicin is hydrolyzed in the
(a) Methyl α-d-glucopyranoside (b) Ethyl β-d-galactopyranoside presence of an acid.

24.72 Consider the structures of the four d-aldopentoses (Figure 24.3). (c) Is salicin an α-glycoside or a β-glycoside?

(a) When oxidized with nitric acid, which d-aldopentose produces the (d) Draw the major product expected when salicin is treated with
same aldaric acid as d-lyxose? excess acetic anhydride in the presence of pyridine.

(b) Which d-aldopentoses yield optically inactive alditols when treated (e) Would you expect salicin to exhibit mutarotation when dissolved in
with sodium borohydride? neutral water?

(c) Which d-aldopentose yields the same alditol as L-xylose? 24.77 Draw a mechanism for the following transformation:
(d) Which d-aldopentose has a β-pyranose form in which all
OH
substituents are equatorial?
O OH
24.73 Trehalose is a naturally occurring disaccharide found in bacteria, HO
insects, and many plants. It protects cells from dry conditions because HO OH HCl
OH
of its ability to retain water, thereby preventing cellular damage from
dehydration. This property of trehalose has also been exploited in the OH
preparation of food and cosmetics. Trehalose is not a reducing sugar, O
HO
it is hydrolyzed to yield two equivalents of d-glucose, and it does not O
HO
have any β-glycoside linkages. Draw the structure of trehalose.
OH
24.74 Xylitol is found in many kinds of berries. It is approximately
as sweet as sucrose but with fewer calories. It is often used in sugar-
24.78 Draw the α-N-glycoside and the β-N-glycoside formed when
less chewing gum. Xylitol is obtained upon reduction of d-xylose with
d-glucose is treated with aniline (C6H5NH2).
NaBH4. Draw a structure of xylitol.

24.75 Isomaltose is similar in structure to maltose, except that it is 24.79 Draw and name the nucleoside formed from each of the following
an alpha-1,6 glycoside, rather than an alpha-1,4 glycoside. Draw the pairs of compounds:
structure of isomaltose. (a) 2-Deoxy-d-ribose and adenine

24.76 Salicin is a natural analgesic present in the bark of willow trees, and (b) d-Ribose and guanine
it has been used for thousands of years to treat pain and reduce fevers. 24.80 Compound A is a d-aldopentose that is converted into an opti-
cally active alditol upon treatment with sodium borohydride. Draw two
OH possible structures for compound A.
OH
HO O
HO O
OH
Salicin

ACS-STYLE PROBLEMS (Multiple Choice)

Problems 24.81–24.85 follow the style of the ACS organic 24.82 • Which of the following structures represents a pyranose form
chemistry exam. For each of these problems, there will be of glucose?
one correct answer and three distractors.
CHO CH2OH
24.81 • What is the relationship between the following two carbohydrates? H OH HO H
OH OH
HO H
O
CHO CH2OH H OH
H OH C O H OH
HO H HO H CH2OH OH
(a) (b)
H OH H OH
H OH H OH
CH2OH
CH2OH CH2OH
HO O OH OH O

HO OH HO
(a) Enantiomers (b) Diastereomers H
OH
(c) Resonance forms (d) Constitutional isomers (c) H (d) OH OH
 Challenge Problems 1193

24.83 • Which of the following is NOT an example of a d sugar? 24.85 • Which of the following can be described as an epimer of the
given aldose?
CHO CHO
H OH H OH CH2OH CHO
H OH HO H CHO C O HO H
H OH H OH HO H HO H H OH
H OH HO H H OH H OH H OH

(a) CH2OH (b) CH2OH (c) CH2OH (d) CH2OH CH2OH

24.84 • Which carbon atom is described as the anomeric carbon?

CHO CHO CH2OH CH2OH
a
OH H OH H OH C O C O
O OH
HO H OH HO H H OH HO H
b OH OH d
H OH HO H H OH H OH
H H
c CH2OH CH2OH CH2OH CH2OH
H H (a) (b) (c) (d)

(a) a (b) b (c) c (d) d

INTEGRATED PROBLEMS
24.86 When d-glucose is treated with an aqueous bromine solution
(buffered to a pH of 6), an aldonic acid is formed called d-gluconic acid.
Treatment of d-gluconic acid with an acid catalyst produces a lactone
(cyclic ester) with a six-membered ring.
(a) Draw the structure of d-gluconic acid.
(b) Draw the structure of the lactone formed from d-gluconic acid,
showing the configuration at each chiral center.
(c) Would you expect this lactone to be optically active? Explain.
conformation. Explain this observation and then draw the more
stable chair conformation of L-idose in its α-pyranose form.
24.88 Draw the product that is expected when the β-pyranose form
of compound A is treated with excess ethyl iodide in the presence of
silver oxide. The following information can be used to determine the
identity of compound A:
1. The molecular formula of compound A is C6H12O6.
2. Compound A is a reducing sugar.

(d) Explain how you would distinguish between the d-gluconic acid 3. When compound A is subjected to a Wohl degradation two times
and the lactone using IR spectroscopy. sequentially, d-erythrose is obtained.
4. Compound A is epimeric with d-glucose at C3.
24.87 When each of the d-aldohexoses assumes a pyranose form, 5. The configuration at C2 is R.
the CH2OH group occupies an equatorial position in the more sta-
ble chair conformation. The one exception is d-idose, for which the 24.89 Propose an explanation for why glucose is the most common
CH2OH group occupies an axial position in the more stable chair monosaccharide observed in nature.

CHALLENGE PROBLEMS
24.90 When d-glucose is treated with aqueous sodium hydroxide, a
complex mixture of carbohydrates is formed, including d-mannose and
d-fructose. Over time, almost all aldohexoses will be present in the mix-
ture. Even L-glucose can be detected, albeit in very small concentrations.
Using the fewest number of mechanistic steps possible, draw a reason-
able mechanism showing the formation of L-glucose from d-glucose:
degradation to produce an aldopentose, which is converted into an
optically active alditol when treated with sodium borohydride. From
the information presented, there are only two possible structures for
compound X. Identify the two possibilities and then propose a chemical
test that would allow you to distinguish between the two possibilities
and thereby determine the structure of compound X.

H O H O 24.92 Compound A is a d-aldopentose. When treated with sodium

C C borohydride, compound A is converted into an alditol that exhibits three
H OH HO H signals in its 13C NMR spectrum. Compound A undergoes a Kiliani–Fischer
HO H NaOH H OH synthesis to produce two aldohexoses, compounds B and C. Upon treat-
H OH H 2O
HO H
ment with nitric acid, compound B yields compound D, while compound
C yields compound E upon treatment with nitric acid. Both D and E are
H OH HO H
optically active aldaric acids.
CH2OH CH2OH
(a) Draw the structure of compound A.
D-Glucose L-Glucose
(b) Draw the structures of compounds D and E and describe how you
24.91 Compound X is a d-aldohexose that can adopt a β-pyranose might be able to distinguish between these two compounds using
form with only one axial substituent. Compound X undergoes a Wohl 13
C NMR spectroscopy.
 Answers
1150 CHAPTER 24

24.80. There are only four D-aldopentoses, all which are shown below. Only the first two are reduced to give an
optically active alditol, as shown. The latter two are reduced to give meso compounds (not optically active):

24.81. The correct answer is (d). Both of these
compounds have the molecular formula C6H12O6, but
they have a different connectivity of atoms. The C=O
bond is located at C1 in the first compound, but located
at C2 in the second compound:
the right in the Fischer projection. Structure (b) has an
OH group in this position pointing to the left, so it is an L
sugar:

24.84. The correct answer is (d). When a carbohydrate

Therefore, these compounds are constitutional isomers
(the first is an aldose, and the second is a ketose).
Stereoisomers must have the same connectivity of atoms
(differing only in configuration), so these compounds
cannot be stereoisomers.
undergoes cyclization, the aldehydic carbon becomes the
anomeric carbon. Note that the anomeric carbon atom
still has two C–O bonds. Variation of the configuration
at the anomeric position gives rise to  and  forms of
the cyclic sugar, called anomers. The structure below
represents the β anomer:

24.82. The pyranose form of glucose has a six-

membered ring. The correct answer is (c).
24.85. The correct answer is (a). Epimers are
diastereomers that differ from each other in the
24.83. The correct answer is (b). The classification of a configuration of only one chiral center, as shown below:
sugar as D or L depends on the configuration of the
chiral center farthest from the carbonyl group. All D
sugars will have an OH group in this position pointing to