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27.14 The Strategy of Peptide Synthesis

27.14 The Strategy of Peptide Synthesis. General Considerations. Making peptide bonds between amino acids is not difficult. The challenge is connecting amino acids in the correct sequence.

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27.14 The Strategy of Peptide Synthesis

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  1. 27.14The Strategy of Peptide Synthesis

  2. 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—PheGly—GlyPhe—Gly Gly—Phe

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

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

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

  6. 27.15Amino Group Protection

  7. 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.

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

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

  10. 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

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

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

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

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

  15. 27.16Carboxyl Group Protection

  16. 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.

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

  18. 27.17Peptide Bond Formation

  19. 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.

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

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

  22. H O C6H11N C OCCHNHZ CH2C6H5 C6H11N 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.

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

  24. 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.

  25. O O ZNHCHCO NO2 H2NCH2COCH2CH3 CH2C6H5 The Active Ester Method +

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

  27. 27.18Solid-Phase Peptide Synthesis:The Merrifield Method

  28. 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.

  29. CH2 CH2 CH2 CH2 CH CH CH CH 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.

  30. CH2 CH2 CH2 CH2 CH CH CH CH The Solid Support • Treating the polymeric support with chloromethyl methyl ether (ClCH2OCH3) and SnCl4 places ClCH2side chains on some of the benzene rings.

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

  32. CH2 CH2 CH2 CH2 CH CH CH CH CH2Cl The Solid Support • 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.

  33. CH2 CH2 CH2 CH2 CH CH CH CH O CH2Cl – BocNHCHCO R The Merrifield Procedure

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

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

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

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

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

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

  40. 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

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