Jones M., Fleming S.A. Organic Chemistry

Подождите немного. Документ загружается.

23.4 Peptide Chemistry 1189

–

Amide

bonds

Amide

bond

FIGURE 23.19 Peptides are

polyamino acids linked through

amide bonds.

By far, the most important reaction of amino acids is their polymerization to

peptides and proteins through the formation of new amide bonds (Fig. 23.19).The

following sections are devoted to this reaction and some of its consequences.

Alanine

A tripeptide: Alanylserylvaline

Amino

terminus

Carboxy

terminus

Ala Ser

Val

ASV

Serine Valine

–

Amide links to be made

WEB 3D

FIGURE 23.20 The tripeptide alanylserylvaline, .The amino acid at the amino terminus starts

the name, and the amino acid at the carboxy terminus ends it.

Ala

Ser

Val

Summary

Many of the reactions of carboxylic acids and amines appear in the chemistry of

amino acids.Acylation and Fischer esterification are two examples.There are some

new reactions, however, that involve both the amino and the acid groups. Reaction

with ninhydrin is an example.

23.4 Peptide Chemistry

23.4a Nomenclature and Structure Figure 23.20 shows the schematic

construction of a three amino acid peptide (a tripeptide) made from alanine, serine,

and valine. Don’t confuse this picture with real synthetic procedures—we will get to

them soon enough. Peptides are usually written with the amino terminus on the

left, proceeding toward the carboxy terminus on the right. They are always named

from the amino terminus to the carboxy terminus. So the tripeptide in Figure 23.20

is alanylserylvaline, or .Ala

Ser

Val, or A

COO

–

COO

–

SCH

PROBLEM 23.15 Draw the structures for

PROBLEM 23.16 Name the peptides drawn below.

Thr

PheGlu

andAsp

His

Cys,Pro

Gly

Tyr,

1190 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

A cross-linked pair of chains—

the link is a disulfide (S

S) bridge

Cys

oxidation

Disulfide

Cys

FIGURE 23.21 Formation of disulfide

bonds can link one polyamino acid

chain to another.

The sequence of amino acids in a peptide or protein constitutes its primary struc-

ture.These chains of amino acids are linked in another way.Connections between chains

or within the same chain are often formed through disulfide bridges (Fig. 23.21).

Cysteine is usually the amino acid involved in these bridges, which are made through

oxidation of the thiol groups (p. 809).

There are other important structural features of polyamino acids. The amide

groups prefer planarity and the hydrogen bonds between the NH hydrogens and

amide carbonyl groups, along with the structural constraints imposed by the disul-

fide links, lead to regions of order in most large peptides.

23.4 Peptide Chemistry 1191

WORKED PROBLEM 23.17 Explain why amides prefer planarity.

ANSWER Amides are stabilized by resonance.

–

This resonance stabilization is maximized when the 2p orbitals on N, C, and O

overlap as much as possible, which requires sp

hybridization and planarity.

α-Helix

Axis of helix

β-Pleated sheet

FIGURE 23.22 Two examples

of ordered secondary structure

in a polypeptide, the α-helix and

β-pleated sheet.

There are even higher structural orders for these molecules. It might have been

that polypeptides were best described as ordered regions of secondary structure

(α-helix or β-pleated sheets) connected by sections of random coil. In such a case,

Especially common are regions arranged in an ␣-helix and repeating folded sec-

tions known as ␤-pleated sheets (Fig. 23.22). Both of these regions allow regular

patterns of hydrogen bonding to develop, thereby imposing order on the sequence

of amino acids. In the regions that appear as an α-helix the hydrogen bonds are

between the coils of the helix, whereas in the pleated sheets they hold lengths of the

chains in roughly parallel lines. Other regions consist of a disordered, random coil,

series of amino acids. The combination of these structural features determines the

folding pattern, or secondary structure of the polypeptide.

a protein would have no fixed overall structure beyond the sections defined by

secondary structure. But this idea has been shown not to be correct. X-Ray

structure determinations of crystallized proteins have revealed these species to

have specific, three-dimensional structures.

This tertiary structure of the pro-

tein is determined partly by hydrogen bonding, but also by van der Waals and

electrostatic forces.These giant molecules are composed of a carbon backbone

to which the amino acid side chains,or R groups,are attached.These R groups

are either hydrocarbon-like, nonpolar groups such as methyl,isopropyl,or ben-

zyl, or highly polar amino, hydroxyl, or sulfhydryl groups. In the natural envi-

ronment of a protein, water, it should be no surprise to find that the protein

adjusts its shape so that the polar, hydrophilic groups are aimed outward toward

the polar solvent, whereas the nonpolar,“greasy”hydrophobic hydrocarbon por-

tions cluster inside the molecule, maximally protected from the hostile aque-

ous environment (Fig. 23.23).

Many proteins adopt globular shapes such as that shown in Figure 23.23, which

maximizes interior hydrophobic and exterior hydrophilic interactions. Other proteins

are fibrous, taking the form of a superhelix composed of ropelike coils of α-helices.

Figure 23.24 shows a typical protein, the enzyme lactate dehydrogenase,which incor-

porates five α-helices and six β-sheets.

1192 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

It is a general problem that until recently the structures of proteins could only be determined by X-ray

diffraction studies on crystallized materials. Work was limited by the availability of crystals, and crystal-

lization of these molecules is usually difficult. Successful formation of X-ray quality crystals of a protein is

cause for celebration (and publication) even before the work of structure determination begins. But how do

we know that the structure of the molecule in solution is the same as that in the solid state crystal?

Remember, biological activity is tied intimately to the detailed structure of these molecules—might we not

be led astray by a structure that owed its shape to crystal packing forces in the solid state,and that was quite

different in solution? Indeed we might. These days, it is becoming possible to use very high-field NMR

spectrometers,along with increasingly sophisticated NMR pulse techniques,to determine structures in solu-

tion. Remember that high-field spectrometers are anything but cheap; the cost is about $10

per megahertz,

often of your tax dollars.

α-Helix

β-Sheets

FIGURE 23.24 Lactate dehydrogenase is a large protein that has well-defined tertiary

structure.

R = Hydrocarbon-like,

nonpolar side chains

R = Polar side chains

FIGURE 23.23 A globular protein,

ordered so as to put the nonpolar side

chains in the inside of the “glob” and

the polar side chains outside

interacting with the polar solvent

medium.

23.4 Peptide Chemistry 1193

This tertiary structure of a protein is extraordinarily important because it deter-

mines the shape of these huge molecules and biological activity depends intimately

on these shapes. Evolution has produced proteins that have pockets into which spe-

cific molecules called substrates fit. This enzyme–substrate binding allows for the

proper orientation for reaction of the substrate.The reaction could be complicated,

or as simple as the hydrolysis of an ester. In other molecules, substrates are bound

only for transportation purposes.These binding sites are a direct consequence of the

primary structure that determines the secondary and tertiary structures. Figure 23.25

gives a schematic representation of this process.

FIGURE 23.25 A binding site in a

protein captures (binds) a small

molecule for transport, or reaction

with appropriately located reactive

groups.

How do we know that the same tertiary structure is adopted by every pro-

tein molecule of a given amino acid sequence? One clue is that synthetic pro-

teins have the same biological activity as the natural proteins. If we reproduce

the primary sequence of a protein correctly, the proper biological activity

appears. Because this biological activity depends directly on the details of shape,

we can conclude that the molecule must be folding properly into the appropri-

ate tertiary structure.

Another clue comes from experiments in which the tertiary structure of a pro-

tein is deliberately disrupted, which is called denaturing the protein. Many ways

of denaturing a protein are irreversible.When an egg is cooked, the thermal ener-

gy denatures the proteins in the egg white in an irreversible way. Once the egg is

fried, there is no unfrying it. Similar chemistry can be induced by pH change.The

curdling of milk products is an example. But some denaturing reactions are

reversible. For example, disulfide bonds in a protein can be broken through treat-

ment with thiols (Fig. 23.26). This disruption of the secondary structure trans-

forms the ordered sections of the protein into the random coil arrangements.

Active globular protein

oxidize

Denatured (random coil)

FIGURE 23.26 A reversible method of denaturing proteins, of destroying the secondary and

tertiary structure, is to break the disulfide bonds. Air oxidization re-forms the disulfides;

the higher order structures are regenerated, and biological activity is reestablished.

1194 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Similar reversible disruptions of order can be achieved through treatment with urea

(in a reaction whose precise mechanism remains unknown), or, sometimes, through

heating.If these denatured proteins are allowed to stand,the thiol groups are reox-

idized by air to the appropriate disulfide linkages and the original tertiary struc-

ture with its biological activity returns!

There is even higher order to some proteins. Particularly large proteins are

collections of smaller polypeptides held together by intramolecular attractions—

van der Waals and electrostatic forces—into superstructures with quaternary

structure. The classic example of quaternary structure is hemoglobin, the pro-

tein responsible for oxygen absorption and transport in humans. To accomplish

these biological functions the protein incorporates four heme units,each consist-

ing of an iron atom surrounded by heterocyclic rings which coordinate with the

metal (Fig. 23.27).

Imidazole

from histidine

HOOC

Heme Bound heme unit

Protein

Coordinated O

HOOC

FIGURE 23.27 The heme unit and an oxygen-coordinated heme structure of hemoglobin.

The protein surrounding the heme is called globin, and in each globin subunit

a histidine side chain (an imidazole) is poised to hold the heme in position. There

are four subunits in hemoglobin, two pairs of slightly different peptide chains

(Fig.23.28).These are held together by electrostatic and van der Waals forces as well

as hydrogen bonding, and take the shape of a giant tetrahedron.

Given all this general information about structure,we still face two difficult prob-

lems. First, how do we determine the amino acid sequence of an unknown protein,

and second, once we know that structure, how can we synthesize it?

23.4b Determination of Protein Structure If suitable crystals can be

obtained, and if the X-ray diffraction pattern for the crystal can be solved in a rea-

sonable amount of time, the structure of the protein is apparent. However, this

process is not yet a general procedure. Crystallization of these molecules remains

difficult, and although the determination of the structure of small molecules by

X-ray is now routine and rapid, the determination of the structures of proteins

remains difficult and time consuming.

In principle, we can get an overall picture of the composition of a protein by first

destroying all disulfide links in the molecule, and then hydrolyzing all the peptide

FIGURE 23.28 The quaternary

structure of hemoglobin. Each heme

unit is attached to a globin subunit.

23.4 Peptide Chemistry 1195

bonds. Remember: These peptide bonds are amides which means acid hydrolysis

should convert them into acids (p. 902). This procedure regenerates all the amino

acids of which the molecule is built. Figure 23.29 shows a hypothetical example. Of

course, this technique does nothing to reveal either primary structure or any higher

structure.

In practice, the fragments formed when the disulfide bonds are broken are first

separated from one another and then hydrolyzed. This separation is no trivial task,

but several methods are now available,including electrophoresis (p.1180) which works

for peptides as well as amino acids. Various kinds of chromatography are also effec-

tive.In gel-filtration chromatography the mixture is passed over a column of poly-

mer beads which,on the molecular level, contain holes into which the smaller peptide

fragments fit more easily than the larger pieces. Accordingly, the larger fragments

pass more rapidly down the column, and a separation is achieved. Ion-exchange

chromatography uses electrostatic attractions to hold more highly charged frag-

ments on the column longer than more nearly neutral molecules. As in any chro-

matographic technique, the more tightly held molecules move more slowly than

those less tightly held by the medium (Fig. 23.30).

1. RSH

2 Cys Ser2 Phe

Lys

2.H

Lys

Cys

Phe

Ser

Cys

FIGURE 23.29 Cleavage of a

hexapeptide to its constituent amino

acids.The disulfide bonds are first

broken to produce two fragments,

which are hydrolyzed to break the

amide bonds.

Mix of peptide

fragments

Solutions of separated peptide

fragments ready for sequencing

Individual peptide

fragments move down the

column at different rates

FIGURE 23.30 A schematic

description of chromatographic

separation of peptide fragments.

1196 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Once the peptide fragments are separated, each can be hydrolyzed to its con-

stituent amino acids. Now we need methods for determining how much of each

amino acid is present. Again chromatography is used. The amino acids are separated

on an ion-exchange column.As each amino acid is eluted from the column,it emerges

into a chamber in which it reacts with ninhydrin (p. 1187) to form a purple solution.

The intensity of the purple color is proportional to the amount of amino acid pres-

ent, and can be plotted against time or amount of solvent used for elution from the

column.This procedure produces a chromatogram consisting of a series of peaks of

various sizes. How do we tell which peak corresponds to which amino acid? This

identification is done by running samples of known amino acids through the col-

umn, determining their retention times, and matching these with those of the

unknown amino acids (Fig. 23.31).

But this technique only gives us the coarsest picture of the structure of the pro-

tein: We know the constituent amino acids and their relative amounts, but we have

no idea of their order, the primary structure. An important task is to find a way to

determine the sequence of amino acids in a protein. The first step in making this

determination is again to destroy the disulfide links and separate the constituent

polypeptides. A simple method of determining the amino acid at the amino termi-

nus is called the Sanger degradation, after Frederick Sanger (b. 1918). A peptide is

allowed to react with 2,4-dinitrofluorobenzene, which undergoes a nucleophilic aro-

matic substitution reaction (p.675).A product is formed that labels the terminal amino

Polypeptide

Mixture of

individual

amino acids

in here

Amino acid 2

Amino acid 1

Intensity of purple color =

amount of amino acid

Time

(volume of solvent)

Ion-exchange

column separates

amino acids

Ninhydrin chamber—

a spectrometer

detects the purple

color

Amino

acid 1

Amino

acid 2

Amino

acid 3

FIGURE 23.31 Peptide fragments can be hydrolyzed to constituent amino acids, which can then be separated

by chromatography. Reaction of each amino acid with ninhydrin gives a purple color that can be quantitatively

analyzed spectroscopically.The intensity of the purple color is proportional to the amount of amino acid

formed.

23.4 Peptide Chemistry 1197

Identified as the amino terminus

(hydrolysis

of the amide

links)

(adduct)

R R

FIGURE 23.32 The amino acid at the

amino terminus can be identified by

reaction with 2,4-dinitrofluorobenzene,

followed by hydrolysis. Only the amino

acid at the amino terminus of the chain

will be labeled.

It is also possible to determine the amino acid at the other end of a peptide chain,

the carboxy terminus. The method of choice uses an enzymatic reaction in which one

of a number of carboxypeptidases specifically cleaves the terminal amino acid at the

carboxy end of the chain. As each carboxy terminal amino acid reacts with the enzyme,

a new amino acid is revealed to be cleaved in turn by more carboxypeptidase. Careful

monitoring of the production of amino acids as a function of time can give a good idea

of the sequence (Fig. 23.33). Still, it is clear that things will get messy as long peptides

are turned into a soup containing an increasingly complex mixture of amino acids.

First to appear

Second to appear

enzyme

(carboxypeptidase)

enzyme

Last to appear

–

FIGURE 23.33 Carboxypeptidase is

an enzyme that cleaves only at the

carboxy terminus of a polypeptide.

group with a 2,4-dinitrobenzene group (Fig. 23.32). Hydrolysis now generates a num-

ber of amino acids, but only the amino acid terminus is labeled! Unfortunately, we

have had to destroy the entire peptide to make this determination!

PROBLEM 23.18 Write a mechanism for adduct formation in Figure 23.32. What

is the function of the nitro groups?

A much better method would be one that included no overall hydrolysis step

and that could be more controlled than the cleavage reactions set in motion by treat-

ment with a carboxypeptidase. We need a method that unzips the protein from one

end or the other, revealing at each step the terminal amino acid.

Methods have now been developed that allow just such a step-by-step sequenc-

ing of peptides. One effective way is called the Edman degradation, after Pehr

Edman (1916–1977), and uses the molecule phenylisothiocyanate (Fig. 23.34).

Isocyanates have appeared before (p. 918), and an isothiocyanate is just the sulfur

analogue of an isocyanate.

1198 CHAPTER 23 Amino Acids and Polyamino Acids (Peptides and Proteins)

Curtius

rearrangement

Acyl azide

h ν

An isocyanate

Phenylisothiocyanate

NCS

WEB 3D

FIGURE 23.34 An isothiocyanate

is just the sulfur version of an

isocyanate, the product of a Curtius

rearrangement.

PROBLEM 23.19 Write a mechanism for the isocyanate formation in Figure 23.34.

Like isocyanates, isothiocyanates react rapidly with nucleophiles. Reactions

of isocyanates with ammonia or amines yield ureas. In a similar fashion,

isothio

cyanates give a thiourea as the first formed intermediate (Fig. 23.35).

WEB 3D

A urea

Phenylisothiocyanate

A thiourea

NCS

A thiourea

FIGURE 23.35 Isothiocyanates react

with amines to give thioureas.

Phenylisothiocyanate can be used

to label the amino terminus of a

polypeptide.