Jones M., Fleming S.A. Organic Chemistry

23.4 Peptide Chemistry 1199

A peptide will react with isothiocyanate at the amino terminus to form a

thiourea. The thiourea can be cleaved in acid without breaking the other amide

bonds in the peptide. As we saw in Chapter 21 (p. 1088), sulfur is an excellent

neighboring group, and the introduction of the thiourea places a sulfur atom

in just the right position to aid in cleavage of the amino terminus amino acid

from the rest of the peptide (Fig. 23.36). The process involves initial protona-

tion of the amino terminus carbonyl. The neighboring sulfur then adds to

the carbonyl carbon, triggering the cleavage reaction to give a molecule called

a thiazolinone.

sulfur acts as

neighboring

group

Side chain of

amino terminus

amino acid

Good leaving

group

Intact peptide chain,

one amino acid shorter,

can be used for

further analysis

S

N

OH

..

H

N

..

+

O

Ph NH

proton

transfers

cleavage

OH

..

H

2

N

..

SN

..

Ph NH

Rest of

peptide

chain

..

H

2

N

SN

..

Ph NH

deprotonation

A thiazolinone

A phenylthiohydantoin

R

R R

N

O

H

3

O/H

2

O

+

S

N

Ph

R

+

Rest of

peptide

chain

OH

..

S

..

Ph NH

R

..

H

N

HN

..

Rest of

peptide

chain

Rest of

peptide

chain

..

H

WEB 3D

FIGURE 23.36 The Edman degradation procedure for sequencing polyamino acids uses

phenylisothiocyanate.The amino terminus is identified by the structure of the

phenylthiohydantoin formed. Notice that this method does not destroy the peptide

chain as the Sanger procedure does.

Further acid treatment converts the thiazolinone into a phenylthiohydantoin

containing the R group of the amino terminus amino acid, which can now be

determined based on the identity of the R group. The critical point is that

the rest of the peptide remains intact and the Edman procedure can now be

1200 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

N

H

3

N

O

..

–

+

Edman

..

Edman

R

H

N

H

O

S

N

Ph

R

O

..

O

H

3

N

..

+

O

H

3

N

O

..

–

R

H

..

N

H

O

S

N

Ph

R

..

O

..

–

R

FIGURE 23.37 The

primary structures of

polyamino acids can be

determined through

repeated applications of

the Edman degradation

procedure, which is one

method of sequencing.

repeated, in principle, as many times as are needed to sequence the entire pep-

tide (Fig. 23.37).

N

O

..

BrCN

Methionine Methionine

R

H

..

H

O

N

..

R

H

..

H

O

SCH

3

..

N

O

..

H

N

..

H

O

..

N

..

H

++

O

CH

3

S

Lactone

..

O

N

..

H

2

N

..

O

H

2

N

..

O

N

..

R

O

..

O

..

O

..

Lactone

FIGURE 23.38 The carboxy

end of each methionine is

cleaved on treatment of a

polypeptide with cyanogen

bromide. A lactone is formed

in place of a methionine.

PROBLEM 23.20 Write a mechanism for the formation of the phenylthiohydan-

toin from the thiazolinone (Fig. 23.36).

But the practical world intrudes, and even the Edman degradation procedure,

good as it is, becomes ineffective for polypeptides longer than about 20–30 amino

acids. Impurities build up at each stage in the sequence of determinations and even-

tually the reaction mixture becomes too complex to yield unequivocal results.

Accordingly, methods have been developed to cut proteins into smaller pieces at

known positions in the amino acid sequence.These methods take advantage of both

biological reactions in which enzymes break peptides only at certain amino acids,

and a very few specific cleavages induced by small-molecule laboratory reagents.

Perhaps the best of these “chemical”(enzymes are chemicals, too) cleavages uses

cyanogen bromide (BrCN) and takes advantage of the nucleophilicity of sulfur and

the neighboring group effect to induce cleavage at the carboxy side of each methio-

nine in a polypeptide (Fig. 23.38).

23.4 Peptide Chemistry 1201

The sulfur atom of methionine first displaces bromide from cyanogen bro-

mide to form a sulfonium ion (Fig. 23.39).Then attack by the proximate carbonyl

group of methionine displaces the excellent leaving group, methylthiocyanate, and

forms a five-membered ring called a homoserine lactone. Hydrolysis steps follow

to produce two smaller peptide fragments, one containing a lactone at its car-

boxy terminus.

C

N

O

..

BrCN

HCl

Methionine

R

H

N

NBr

..

H

O

H

3

CS

..

N

..

R

H

N

..

H

O

+

intramolecular

S

N

2

addition

elimination deprotonation

proton

transfers

Sulfonium ion

(an excellent

leaving group)

addition–elimination

Homoserine lactone

N

O

..

R

H

N

..

O

..

N

..

Br

..

R

H

N

..

H

O

CH

3

S

+

–

C

N

O

..

R

H

N

..

O

..

N

..

R

H

..

N

H

O

..

CH

3

S

+

NC

..

H

2

O

of H

2

O

..

N

..

R

H

N

..

O

N

..

R

H

..

N

H

O

+

..

H

2

O

..

N

..

R

H

N

..

O

N

..

R

H

..

N

H

2

O

+

..

HO

N

O

..

R

N

..

H

O

N

..

R

H

..

H

2

N

O

..

O

+

O

..

O

..

FIGURE 23.39 The mechanism of cleavage using BrCN.

1202 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Most methods used to chop large peptides into smaller fragments take advan-

tage of enzymatic reactions. Chymotrypsin, for example, is an enzyme that

cleaves peptides at the carboxyl groups of amino acids containing aromatic side

chains (phenylalanine, tryptophan, or tyrosine).Trypsin cleaves only at the car-

boxy end of lysine and arginine. There are several other examples of this kind

of specificity. Long peptide chains can be degraded into shorter fragments that

can be effectively sequenced by the Edman procedure. A little detective work suf-

fices to put together the entire sequence. Let’s look at a simple example and then

do a real problem.

Consider the decapeptide in Figure 23.40. We can determine the amino termi-

nus through the Sanger procedure to be Phe. Next, we use the enzyme trypsin in a

separate experiment to cleave the decapeptide into three fragments.As we know the

peptide must start with Phe, the first four amino acids are . The

only other cleavage point is at Arg, so the fragment must be the mid-

dle piece. If this tripeptide were at the end, with Arg as the carboxy terminus, no

cleavage would be possible. The sequence must be

.Arg

.

Glu

.

Leu

.

Val

Phe

.

Tyr

.

Trp

.

Lys

.

Asp

.

Ile

.

Asp

.

Ile

.

Arg

Phe

.

Tyr

.

Trp

.

Lys

Sanger

Phe must be the amino terminus

trypsin

(cleaves only after

Lys or Arg)

Can be sequenced

using Edman

Must start with Phe

Contains a cleavage

point (Arg)—must be

middle fragment

N

H

Ph

COOH

Phe

.

Tyr

.

Trp

.

Lys

.

Asp

.

Ile

.

Arg

.

Glu

.

Leu

.

Val

Phe

.

Tyr

.

Trp

.

Lys

Asp

.

Ile

.

Arg

+

Glu

.

Leu

.

Val

O

2

N

NO

2

FIGURE 23.40 An example of peptide sequencing using Sanger and Edman procedures as well as enzymatic

cleavage reactions.

PROBLEM 23.21 The nonapeptide bradykinin can be completely hydrolyzed in

acid to give three molecules of Pro, two molecules each of Arg and Phe, one mol-

ecule of Ser, and one molecule of Gly. Treatment with chymotrypsin gives the

pentapeptide , the tripeptide . End-

group analysis shows that the amino acids on both the amino and carboxy termini

are the same. Provide the sequence for bradykinin.

23.4c Synthesis of Peptides Now that we know how to sequence proteins,

how about the reverse process—how can we put amino acids together in any

sequence we want? Can we synthesize proteins? It is simply a matter of forming

amide bonds in the proper order, and this task might seem easy. However, two dif-

ficulties surface as soon as we begin to think hard about the problem. First, there’s

a lot of work to do in order to make even a small protein. Imagine that we only want

Ser

.

Pro

.

Phe, and ArgArg

.

Pro

.

Pro

.

Gly

.

Phe

23.4 Peptide Chemistry 1203

COO

–

Ala

.

Ala

.

Leu

Further

reactions

Leu

.

Ala

Leu

.

Leu

H

3

N

+

COO

–

Leu

H

3

N

+

H

3

N

+

O

..

COO

–

N

..

H

3

N

+

O

..

COO

–

N

..

H

3

N

+

O

..

COO

–

N

..

H

3

N

+

O

..

COO

–

N

..

H

FIGURE 23.41 The possible combinations of two unprotected amino acids.

to create the nine amide linkages of the decapeptide in Figure 23.40. Even if we can

make each amide bond in 95% yield, we are in trouble because a series of nine

reactions each proceeding in 95% yield, which probably seems quite good to any-

one who has spent some time in an organic lab, gives an overall yield of only

(0.95)

9

 63%. Obviously, to get anywhere in the real world of proteins, in which

chain lengths of hundreds of amino acids are common, we are going to have to

do much better than this.

Second, there is a problem of specificity. Suppose we want to make the triv-

ial dipeptide . Even if we can avoid simple acid–base chemistry, ran-

dom reaction between these two amino acids will give us at least the four

possible dimeric molecules, and in practice, other larger peptides will be pro-

duced as well (Fig. 23.41).

Ala

.

Leu

1204 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Ala

SOCl

2

H

3

N

+

–

Leu

H

3

N

+

O

–

..

O

..

–

O

..

Acid chloride

of Ala

H

3

N

+

O

..

O

..

Cl

..

Ala

•

Leu

H

3

N

+

O

..

N

..

H

..

FIGURE 23.42 Some specificity in the reaction between two amino acids might be obtained by allowing the

acid chloride of one amino acid to react with the other amino acid.

COO

H

3

N

+

COO

–

H

3

N

+

–

H

3

N

+

O

..

Cl

..

H

3

N

+

O

..

COO

–

N

..

H

3

N

+

O

..

COO

–

N

..

H

Ala

.

Ala

Leu

Ala

.

Leu

Acid chloride

of Ala

(activated acid)

FIGURE 23.43 Here, too, more than

one product is inevitable, because as

the acid chloride is formed it will

react with any amine in solution.

However, even this procedure is hopeless! What is to prevent the amino group

of the original alanine from reacting with the acid chloride? Nothing. Again, a mix-

ture is certain to result (Fig. 23.43). Remember, we ultimately will have to make

dozens, perhaps hundreds of amide bonds. We can tolerate no mediocre yields or

even worse, mixtures, if we hope to make useful amounts of product. We need to

find the most efficient way of making the amide bond specifically at the point we

want it, and at the same time avoiding side reactions.

One simple strategy for avoiding undesired reactions between alanine and

leucine would be to activate the carbonyl group of alanine by transforming the

acid group of alanine into the acid chloride, and then treating it with leucine

(Fig. 23.42).

In this trivial case, not only do we need to activate the carboxyl group of ala-

nine, but we need to block the reaction at the amino group of alanine and at the

carboxyl group of leucine. Three positions must be modified (the carboxyl groups

of alanine and leucine and the amino group of alanine). Left free for reaction are

the two positions we hope to join, the activated carboxyl group of alanine and the

23.4 Peptide Chemistry 1205

Di-tert-butyl dicarbonate

O

..

O

..

O

..

Benzyl chloroformate

(benzyloxycarbonyl chloride)

Ph

Cl

O

..

O

..

(CH

3

)

3

CO OC(CH

3

)

3

H

3

N

+

O

=

..

COOH

New amide links

COO

–

N

..

H

O

..

COOH

H

Cbz

COOH

H

tBoc

COOH

N

..

H

Ph

O

..

N

..

N

..

(CH

3

)

3

CO

..

FIGURE 23.45 Two protecting or blocking groups for the amine ends of amino acids.

So now we have two ways of blocking one amino group, in this case the amino

group of alanine,so it cannot participate in the formation of the peptide amide bond.

Now we need to block the carboxy end of the other amino acid, leucine. This pro-

tection can be accomplished by simply transforming the acid into an

ester (Fig. 23.46).

We need to do two more things. First, we still have to do the

actual joining of the unprotected amino group of leucine to the unpro-

tected carboxyl group of alanine, and we need to do this very efficient-

ly and under the mildest possible conditions. Second, we need to

deprotect the blocked amino and carboxyl groups after the amide link-

age has been formed.

Dicyclohexylcarbodiimide (DCC) is the reagent of choice for

peptide bond making. Formation of the amide bond is a dehydration

reaction (water is the other product), and DCC is a powerful dehy-

unmodified amino group of leucine. Remember also that eventually the blocking,

or protecting, groups will have to be removed (Fig. 23.44).

COO

–

Ala

Block Activate

Only these two

positions can react

Block

H

3

N

+

COO

–

Leu

H

3

N

+

FIGURE 23.44 In any successful

strategy, we must activate the carboxy

end and block the amino end of

one amino acid while blocking

reaction at the carboxy end of the

other amino acid.

Amino groups can be blocked by converting them into carbamates through sim-

ple addition–elimination reactions (p.901).Two popular methods involve the trans-

formation of the free amine into a carbamate by reaction with either di-tert-butyl

dicarbonate or benzyl chloroformate. Biochemists are even more addicted to

acronyms than are organic chemists,and these protecting groups are called tBoc and

Cbz, respectively (Fig. 23.45).

FIGURE 23.46 A carboxy end can be protected by

converting it into an ester.

COO

–

H

3

N

+

H

2

N

H

2

OEt

O

OEt

..

HOEt

..

+

Leucine Leucine ester

1206 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

COOH

N

H

2

N

H

+

C

O

OR

..

N

NN

H

+

HH

O

OR

..

O

..

Dicyclohexylurea

(DCU)

New peptide bond

O

..

(DCC)

tBoc

N

..

N

..

WEB 3D

FIGURE 23.47 Peptide bond

formation can be achieved using

dicyclohexylcarbodiimide (DCC).

drating agent. The hydrated form of DCC is dicyclohexylurea (DCU), which is a

product of the reaction (Fig. 23.47).

C

A ketene

O

..

Amides

O

..

C

Isocyanates

O

..

Ureas

O

..

R

H

N

C

Isothiocyanates

S

..

Thioureas

..

R

H

N

C

NH

3

..

NH

3

..

NH

3

..

NH

3

..

R

H

N

Carbodiimides

Guanidines

NH

2

..

NH

2

..

NH

2

..

NH

2

..

R

N

..

R

N

..

R

N

..

WEB 3D

FIGURE 23.48 Cumulated double

bonds (DCC in this case) react with

all manner of nucleophiles, including

amines and carboxylate anions.

What is the mechanism of the coupling reaction of Figure 23.47? Remember

that other cumulated double bonds (p. 512) such as ketenes (p. 515), isocyanates

(p. 918), and isothiocyanates (p.1198) all react rapidly with nucleophiles.The relat-

ed DCC is no exception,and it is attacked by amines to give molecules called guani-

dines. Carboxylates are nucleophiles too, and will also add to DCC (Fig. 23.48).

23.4 Peptide Chemistry 1207

So in the coupling reaction of Figure 23.47 the unprotected carboxylic acid group

of alanine adds to DCC to give an anhydride-like intermediate that can react with

a second alanine carboxylic acid to give the anhydride.The amino acid leucine then

adds,via an addition–elimination mechanism,to give the dipeptide (Fig. 23.49). Note

that the dipeptide is still protected in two places.

tBoc

N

R

N

H

Protected alanine

O

OH

(DCC)

C

R=

..

Anhydride

(two steps)

addition–

elimination

N

H

O

..

N

R

..

tBoc

..

N

HO

OH

..

R

..

N

H

R

..

N

H

O

..

N

H

..

N

H

O

..

O

..

tBoc

..

N

H

tBoc

+

..

N

..

elimination

deprotonation

Dipeptide

(protecting groups still present)

..

N

H

O

..

O

..

–

O

..

Protected

leucine

Protected

alanine

addition

NH

2

H

2

N

O

EtO

..

EtO

..

N

..

N

H

+

O

..

O

..

OEt

+

O

H

tBoc

O

..

–

..

N

FIGURE 23.49 The mechanism of

amino acid coupling using DCC.

Now all we have to do is remove these protecting groups and,at last, we will have

made our dipeptide, .The tBoc blocking group is removed by treatment with

very mild acid, which does not cleave amide bonds. If the Cbz protecting group is

Ala

.

Leu

..

R

N

..

R

N

C

Carbodiimides

An anhydride-like

intermediate

..

O

R

H

N

1. R

2. H

3

O / H

2

O

+

O

–

O

..

R

N

..

FIGURE 23.48 (continued)

1208 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Deprotect tBoc

H

O

+

1. mild acid

CF

3

COOH

at 25 ⬚C

2. NaOH/H

2

O

3. neutralize

Ala

.

Leu

Ala

.

Leu

Ala

.

Leu

Deprotect Cbz

H

O

H

2

Pd/C

N

O

PhCH

2

O

OEt

Deprotect ester

1. NaOH

H

2

O

2. neutralize

H

N

O

OEt

O

N

OEt

H

O

N

tBoc

H

3

N

H

2

N

O

–

FIGURE 23.50 Methods for removing the protecting groups used in peptide synthesis.

used, it can be easily removed by catalytic hydrogenation (Fig. 23.50).The carboxylic

acid on the carboxy terminus can be regenerated by treatment of the ester with base.

The ester will hydrolyze faster than the amide.

What about the yield for these reactions? Remember, this process, with all its

protection and deprotection steps, must be repeated many, many times in the syn-

thesis of a large peptide or a protein.These steps are efficient—they proceed in high

yield because there are essentially no side

reactions.The problem of tedious repetition

has been solved in an old-fashioned way—

by automation.Polypeptides can be synthe-

sized by a machine invented by R. Bruce

Merrifield (1921–2006). In Merrifield’s

procedure, the carboxy end of a tBoc-pro-

tected amino acid is first anchored to a

material constructed of polystyrene in

which some phenyl rings have been substi-

tuted with chloromethyl groups.The amino

acid displaces the chlorine through an S

N

2

reaction (Fig. 23.51).

tBoc

H

N

R

O

Modified polystyrene

Ph Ph Ph

Cl

Ph Ph Ph

O

H

N

R

O

tBoc

O

N

H

R

tBoc

O

–

FIGURE 23.51 Polystyrene can be

chloromethylated in a Friedel–Crafts

procedure, and the chlorines replaced

through S

N

2 reaction with the unprotected

carboxy end of an amino acid. This

technique anchors an amino acid to the

polymer chain.

Jones M., Fleming S.A. Organic Chemistry

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