27.14 The Strategy of Peptide Synthesis

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. Phe—Phe Gly—Gly Phe—Gly Gly—Phe

Amino Acids are Structurally Bifunctional

Possible Products from the Condensation of Phenylalanine and Glycine

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.

Step 2 of Peptide Synthesis: Coupling 2. Couple the two protected amino acids. PG-Phe-Gly-PG

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

Does the Requirement for Three Extra Steps Outweigh the Need to Purify a Mixture? Yes - synthesis is easier than purification

27.15 Amino Group Protection 4

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.

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.

Amine Protecting Groups: Benzyloxycarbonyl

Amine Protecting Groups: Introduction of Benzyloxycarbonyl Protecting Groups

Benzyloxycarbonyl is Abbreviated to Cbz or Z

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

Hydrogenolysis of Cbz Groups ethyl ester is stable to hydrogenolysis

Hydrogenolysis of Cbz Groups

Acid-Mediated Cleavage of Cbz Groups

Amine Protecting Groups: tert-Butyloxycarbonyl

tert-Butyloxycarbonyl is Abbreviated to Boc

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.

Acid-Mediated Cleavage of Boc Groups

27.16 Carboxyl Group Protection 4

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 by hydrogenolysis.

Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester

Simultaneous Hydrogenolysis of Cbz Group and Benzyl Ester

27.17 Peptide Bond Formation 4

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.

N,N'-Dicyclohexylcarbodiimide (DCC) is a Powerful Dehydrating Agent

Peptide Coupling is a Condensation Reaction 2. Couple the two protected amino acids. -H2O

DCC-Mediated Peptide Coupling 2. Couple the two protected amino acids.

Mechanism of DCC-Promoted Coupling

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.

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.

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.

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.

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.

Peptide Synthesis: Active Ester Method

Peptide Synthesis: Active Ester Method

27.18 Solid-Phase Peptide Synthesis: The Merrifield Method 4

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.

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.

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

Chloromethylation of Polystyrene

Solid Phase Peptide Synthesis The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).

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.

Merrifield Procedure CH2 CH CH2Cl BocNHCHCO R O –

Merrifield Procedure CH2 CH O BocNHCHCO Next, the Boc protecting group is removed with HCl.

Merrifield Procedure CH2 CH O H2NCHCO R CH2 CH O DCCI-promoted coupling adds the second amino acid

Merrifield Procedure Remove the Boc protecting group. NHCHCO R O CH2 BocNHCHC R' Remove the Boc protecting group.

Merrifield Procedure Add the next amino acid and repeat. CH2 CH NHCHCO H2NCHC R' Add the next amino acid and repeat.

Remove the peptide from the resin with HBr in CF3CO2H Merrifield Procedure CH2 CH NHCHCO R O NHCHC R' C + H3N peptide Remove the peptide from the resin with HBr in CF3CO2H

Merrifield Procedure CH2 CH CH2Br NHCHCO R O NHCHC R' C + H3N peptide –

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