26.7 Peptide Synthesis

Peptide synthesis is a crucial process in creating chains of amino acids. It involves three main steps: protection, coupling, and deprotection. These steps ensure that amino acids link up correctly, forming the desired peptide sequence.

Understanding peptide synthesis is key to grasping how proteins are made. Protecting groups, coupling mechanisms, and deprotection methods are essential concepts. Mastering these techniques allows scientists to create custom peptides for various applications in research and medicine.

Peptide Synthesis

Steps in peptide synthesis

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1. Protection
   • Amino group protection prevents undesired reactions at the N-terminus using common protecting groups like Boc (tert-butyloxycarbonyl) and Fmoc (fluorenylmethyloxycarbonyl)
   • Carboxyl group protection prevents undesired reactions at the C-terminus using common protecting groups such as methyl ester, benzyl ester, or t-butyl ester
   • Side chain protection is crucial for amino acids with reactive functional groups to prevent unwanted reactions during synthesis
2. Coupling
   • Formation of the peptide bond (also known as an amide bond) between two amino acids occurs through activation of the carboxyl group of one amino acid, commonly done using carbodiimide reagents (EDC or DCC), followed by nucleophilic attack by the amino group of the other amino acid
3. Deprotection
   • Removal of the protecting groups after the desired peptide bond formation
   • Amino group deprotection: Boc group removed using trifluoroacetic acid (TFA), Fmoc group removed using piperidine
   • Carboxyl group deprotection: Methyl or benzyl esters removed by hydrolysis, t-butyl esters removed using TFA

Protecting groups for peptide synthesis

• Amino protecting groups
  • Boc (tert-butyloxycarbonyl) is an acid-labile protecting group removed using trifluoroacetic acid (TFA)
  • Fmoc (fluorenylmethyloxycarbonyl) is a base-labile protecting group removed using piperidine
• Carboxyl protecting groups
  • Methyl ester is removed by hydrolysis using sodium hydroxide or lithium hydroxide
  • Benzyl ester is removed by catalytic hydrogenation or hydrolysis
  • t-Butyl ester is an acid-labile protecting group removed using trifluoroacetic acid (TFA)

Mechanism of peptide bond formation

• Activation of the carboxyl group: Carbodiimide reagent (EDC or DCC) reacts with the carboxyl group of an amino acid to form an O-acylisourea intermediate
• Nucleophilic attack by the amino group: The amino group of the other amino acid attacks the electrophilic carbon of the O-acylisourea intermediate, leading to the formation of the peptide bond
• Rearrangement of the O-acylisourea intermediate
  • Can undergo rearrangement to form an unreactive N-acylurea byproduct
  • Addition of HOBt (1-hydroxybenzotriazole) suppresses this rearrangement by reacting with the O-acylisourea to form a more stable active ester intermediate less prone to rearrangement
• Regeneration of the carbodiimide: The carbodiimide reagent is regenerated after the peptide bond formation and can participate in further coupling reactions

Advanced concepts in peptide synthesis

• Orthogonal protection: A strategy using protecting groups that can be removed under different conditions, allowing selective deprotection of specific functional groups
• Coupling reagents: Various reagents used to activate the carboxyl group for peptide bond formation, including carbodiimides, phosphonium salts, and uronium salts
• Racemization: Undesired conversion between L- and D-amino acids during synthesis, which can be minimized by using appropriate coupling conditions and reagents