The Strategy of Peptide Synthesis 4

General Considerations Making peptide bonds between amino acids is not difficult. 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

N-Protected phenylalanine General Strategy 1. Limit the number of possibilities by "protecting" the nitrogen of one amino acid and the carboxyl group of the other. N-Protected phenylalanine C-Protected glycine NHCHCOH CH2C6H5 O X H2NCH2C O Y

General Strategy 2. Couple the two protected amino acids. NHCH2C O Y NHCHC CH2C6H5 X NHCHCOH CH2C6H5 O X H2NCH2C O Y

General Strategy 3. Deprotect the amino group at the N-terminus and the carboxyl group at the C-terminus. NHCH2C O Y NHCHC CH2C6H5 X NHCH2CO O H3NCHC CH2C6H5 + – Phe-Gly

Amino Group Protection 4

Protect Amino Groups as Amides Amino groups are normally protected by converting them to amides. Benzyloxycarbonyl (C6H5CH2O—) is a common protecting group. It is abbreviated as Z. Z-protection is carried out by treating an amino acid with benzyloxycarbonyl chloride.

Protect Amino Groups as Amides CH2OCCl O H3NCHCO CH2C6H5 O – + + 1. NaOH, H2O 2. H+ NHCHCOH CH2C6H5 O CH2OC (82-87%)

Protect Amino Groups as Amides NHCHCOH CH2C6H5 O CH2OC is abbreviated as: ZNHCHCOH CH2C6H5 O or Z-Phe

Removing Z-Protection An advantage of the benzyloxycarbonyl protecting group is that it is easily removed by: a) hydrogenolysis b) cleavage with HBr in acetic acid

Hydrogenolysis of Z-Protecting Group NHCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2OC H2, Pd H2NCHCNHCH2CO2CH2CH3 CH2C6H5 O CH3 CO2 (100%)

HBr Cleavage of Z-Protecting Group NHCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2OC HBr H3NCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2Br + CO2 Br – (82%)

The tert-Butoxycarbonyl Protecting Group NHCHCOH CH2C6H5 O (CH3)3COC is abbreviated as: BocNHCHCOH CH2C6H5 O or Boc-Phe

HBr Cleavage of Boc-Protecting Group (CH3)3COC NHCHCNHCH2CO2CH2CH3 CH2C6H5 HBr H3NCHCNHCH2CO2CH2CH3 CH2C6H5 O CH2 C H3C + CO2 Br – (86%)

Carboxyl Group Protection 4

Protect Carboxyl Groups as Esters 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.

Hydrogenolysis of Benzyl Esters NHCHCNHCH2COCH2C6H5 CH2C6H5 O C6H5CH2OC H2, Pd H3NCHCNHCH2CO CH2C6H5 O + – C6H5CH3 CO2 CH3C6H5 (87%)

Peptide Bond Formation 4

Forming Peptide Bonds The two major methods are: 1. coupling of suitably protected amino acids using N,N'-dicyclohexylcarbodiimide (DCCI) 2. via an active ester of the N-terminal amino acid.

DCCI-Promoted Coupling ZNHCHCOH CH2C6H5 O H2NCH2COCH2CH3 O + DCCI, chloroform ZNHCHC CH2C6H5 O NHCH2COCH2CH3 (83%)

Mechanism of DCCI-Promoted Coupling ZNHCHCOH CH2C6H5 O + C6H11N C NC6H11 CH2C6H5 O C6H11N C H OCCHNHZ

Mechanism of DCCI-Promoted Coupling The species formed by addition of the Z-protected amino acid to DCCI is similar in structure to an acid anhydride and acts as an acylating agent. Attack by the amine function of the carboxyl-protected amino acid on the carbonyl group leads to nucleophilic acyl substitution. CH2C6H5 O C6H11N C H OCCHNHZ

Mechanism of DCCI-Promoted Coupling C6H11N C C6H11NH H O ZNHCHC CH2C6H5 O NHCH2COCH2CH3 + H2NCH2COCH2CH3 O CH2C6H5 O C6H11N C H OCCHNHZ

The 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.

The Active Ester Method ZNHCHCO CH2C6H5 O H2NCH2COCH2CH3 O NO2 +

The Active Ester Method ZNHCHCO CH2C6H5 O H2NCH2COCH2CH3 O NO2 + chloroform ZNHCHC CH2C6H5 O NHCH2COCH2CH3 HO NO2 + (78%)

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.

The Solid Support CH2 CH 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.

The Solid Support CH2 CH Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2 side chains on some of the benzene rings.

The Solid Support CH2 CH CH2Cl The side chain chloromethyl group is a benzylic halide, reactive toward nucleophilic substitution (SN2).

The Solid Support CH2 CH CH2Cl 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.

The Merrifield Procedure CH2 CH CH2Cl BocNHCHCO R O –

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

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

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

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

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

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

Peptide Synthesis

The Merrifield Method Merrifield also 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