27.14 The Strategy of Peptide Synthesis

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27.14The Strategy of Peptide Synthesis
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The Challenge of Peptide Synthesis

• Making peptide bonds between amino acids is not
  difficult there are numerous methods to make
  amides from amines and carboxylic acids.
• The challenge is connecting amino acids in the
  correct sequence.
• Random peptide bond formation in a mixture of
  phenylalanine and glycine, for example, will give
  four dipeptides.
• PhePhe GlyGly PheGly GlyPhe

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Amino Acids are Structurally Bifunctional
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Possible Products from the Condensation of
Phenylalanine and Glycine
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Step 1 of Peptide Synthesis Protection

• 1. Limit the number of possible reactions by
  "protecting" the nitrogen of one amino acid and
  the carboxyl group of the other.

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Step 2 of Peptide Synthesis Coupling

• 2. Couple the two protected amino acids.

PG-Phe-Gly-PG
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Peptide Synthesis, Step 3 Global Deprotection

• 3. Deprotect the amino group at the N-terminus
  and the carboxyl group at the C-terminus.

Phe-Gly
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Does the Requirement for Three Extra Steps
Outweigh the Need to Purify a Mixture?
Yes - synthesis is easier than purification
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27.15Amino Group Protection
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What are we Trying to Achieve by Protecting the
Amino Group?
Amino groups can behave as nucleophiles and
undergo reaction with carboxylic acid
derivatives. The nitrogen atom in amides is much
less nucleophilic. As a result, amide derivatives
of amines can be viewed as protecting groups.
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Peptide Synthesis Amine Protecting Groups

• Amino groups are normally protected by converting
  them to amides. The nitrogen atom in an amide
  does not behave as a nucleophile and will not
  react with carboxyl groups.
• Benzyloxycarbonyl (C6H5CH2O) is a common
  protecting group. It is abbreviated as Z or Cbz.
• Cbz-protection is carried out by treating an
  amino acid with benzyloxycarbonyl chloride.

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Amine Protecting Groups Benzyloxycarbonyl
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Amine Protecting Groups Introduction of
Benzyloxycarbonyl Protecting Groups
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Benzyloxycarbonyl is Abbreviated to Cbz or Z
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Cleavage of Cbz Groups

• An advantage of the benzyloxycarbonyl protecting
  group is that it is easily removed by
• a) catalytic hydrogenolysis under extremely mild
  conditions
• b) cleavage with HBr in acetic acid
• Both reagents cleave the relatively weak benzylic
  carbon-oxygen ether bond, albeit by different
  mechanisms

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Hydrogenolysis of Cbz Groups
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Hydrogenolysis of Cbz Groups
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Acid-Mediated Cleavage of Cbz Groups
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Amine Protecting Groups tert-Butyloxycarbonyl
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tert-Butyloxycarbonyl is Abbreviated to Boc
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Cleavage of Boc Groups

• The tert-butyloxycabonyl protecting group is
  readily removed by treatment wit strong,
  anhydrous BrØnsted acids
• cleavage with trifluoroacetic acid in methylene
  chloride
• cleavage with HBr in acetic acid
• Both reagents cleave the quaternary
  carbon-oxygen ether bond by an acid-mediated
  elimination reaction.

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Acid-Mediated Cleavage of Boc Groups
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27.16Carboxyl Group Protection
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Peptide Synthesis Carboxyl Protecting Groups
Carboxyl groups are normally protected as
esters. Deprotection of methyl and ethyl esters
is by hydrolysis in base. Benzyl esters can be
cleaved byhydrogenolysis.
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Simultaneous Hydrogenolysis of Cbz Group and
Benzyl Ester
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Simultaneous Hydrogenolysis of Cbz Group and
Benzyl Ester
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27.17Peptide Bond Formation
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Peptide Synthesis Forming Peptide Bonds

• The two major methods are
• coupling of suitably protected amino acids using
  N,N'-dicyclohexylcarbodiimide (DCC)
• via an active ester of the N-terminal amino acid.

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N,N'-Dicyclohexylcarbodiimide (DCC) is a Powerful
Dehydrating Agent
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Peptide Coupling is a Condensation Reaction

• 2. Couple the two protected amino acids.

-H2O
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DCC-Mediated Peptide Coupling

• 2. Couple the two protected amino acids.

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Mechanism of DCC-Promoted Coupling
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O-Acylisoureas are Powerful Acylating Agents
The O-acylisourea intermediate formed by addition
of the Cbz-protected amino acid to DCCI is
similar in structure to an acid anhydride and
acts as an acylating agent.
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Mechanism of DCC-Promoted Coupling
Attack by the amine function of the
carboxyl-protected amino acid on the carbonyl
group leads to nucleophilic acyl substitution.
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Mechanism of DCC-Promoted Coupling
Attack by the amine function of the
carboxyl-protected amino acid on the carbonyl
group leads to nucleophilic acyl substitution.
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Peptide Synthesis Forming Peptide Bonds

• The two major methods are
• coupling of suitably protected amino acids using
  N,N'-dicyclohexylcarbodiimide (DCCI)
• via an active ester of the N-terminal amino acid.

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Peptide Synthesis Active Ester Method
A p-nitrophenyl ester is an example of an "active
ester. p-Nitrophenyl is a better leaving group
than methyl or ethyl, and p-nitrophenyl esters
are more reactive in nucleophilic acyl
substitution.
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Peptide Synthesis Active Ester Method
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Peptide Synthesis Active Ester Method
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27.18Solid-Phase Peptide SynthesisThe
Merrifield Method
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Solid Phase Peptide Synthesis
In solid-phase synthesis, the starting material
is bonded to an inert solid support. Reactants
are added in solution. Reaction occurs at the
interface between the solid and the solution.
Because the starting material is bonded to the
solid, any product from the starting material
remains bonded as well. Purification involves
simply washing the byproducts from the solid
support.
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Polystyrene is the Basis for the Solid Support

• The solid support is a copolymer of styrene and
  divinylbenzene. It is represented above as if
  it were polystyrene. Cross-linking with
  divinylbenzene simply provides a more rigid
  polymer.

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Functionalization of Polystyrene

• Treating the polymeric support with chloromethyl
  methyl ether (ClCH2OCH3) and SnCl4 places ClCH2
  side chains on some of the benzene rings.

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Chloromethylation of Polystyrene
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Solid Phase Peptide Synthesis
The side chain chloromethyl group is a benzylic
halide, reactive toward nucleophilic substitution
(SN2).
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Solid Phase Peptide Synthesis
The chloromethylated resin is treated with the
Boc-protected C-terminal amino acid.
Nucleophilic substitution occurs, and the
Boc-protected amino acid is bound to the resin as
an ester.
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Merrifield Procedure
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Merrifield Procedure
Next, the Boc protecting group is removed with
HCl.
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Merrifield Procedure
DCCI-promoted coupling adds the second amino acid
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Merrifield Procedure
Remove the Boc protecting group.
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Merrifield Procedure
Add the next amino acid and repeat.
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Merrifield Procedure

• Remove the peptide from the resin with HBr in
  CF3CO2H

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Merrifield Procedure

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Merrifield Procedure

• Merrifield automated his solid-phase method.
• Synthesized a nonapeptide (bradykinin) in 1962 in
  8 days in 68 yield.
• Synthesized ribonuclease (124 amino acids) in
  1969.369 reactions 11,391 steps
• Nobel Prize in chemistry 1984