26.7 Peptide Synthesis
3 min read•may 7, 2024
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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- Protection
- Amino group protection prevents undesired reactions at the N-terminus using common protecting groups like (tert-butyloxycarbonyl) and (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
- is crucial for amino acids with reactive functional groups to prevent unwanted reactions during synthesis
- Coupling
- Formation of the peptide bond (also known as an ) between two amino acids occurs through activation of the carboxyl group of one amino acid, commonly done using reagents ( or ), followed by nucleophilic attack by the amino group of the other amino acid
- Deprotection
- Removal of the protecting groups after the desired peptide bond formation
- Amino group deprotection: Boc group removed using trifluoroacetic acid (), Fmoc group removed using
- 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 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 byproduct
- Addition of (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
- : A strategy using protecting groups that can be removed under different conditions, allowing selective deprotection of specific functional groups
- : Various reagents used to activate the carboxyl group for peptide bond formation, including carbodiimides, phosphonium salts, and uronium salts
- : Undesired conversion between L- and D-amino acids during synthesis, which can be minimized by using appropriate coupling conditions and reagents
Key Terms to Review (34)
Amide Bond: An amide bond is a covalent chemical bond formed between the carbonyl carbon of one molecule and the nitrogen atom of another molecule. This type of bond is crucial in the structure and function of peptides and proteins, as it serves as the linkage between individual amino acid units.
Boc: Boc, short for tert-butyloxycarbonyl, is a protective group commonly used in organic chemistry, particularly in the context of peptide synthesis and other reactions involving sensitive functional groups. It serves to temporarily mask or protect specific moieties during a reaction sequence, allowing for selective transformations to occur while preserving the integrity of the target molecule.
Boc derivative: A Boc derivative is a compound used in organic chemistry, specifically in peptide synthesis, to protect the amino group of an amino acid or peptide by attaching a t-butoxycarbonyl (Boc) group. This protection prevents unwanted reactions at the amino site during the synthesis process.
Boc-Lys(Z)-OH: Boc-Lys(Z)-OH is a protected amino acid used in the synthesis of peptides. It consists of a lysine residue with a tert-butyloxycarbonyl (Boc) protecting group on the α-amino group and a benzyloxycarbonyl (Z) protecting group on the ε-amino group of the lysine side chain. This protected amino acid is an important building block in the step-wise construction of more complex peptide sequences.
Bruce Merrifield: Bruce Merrifield was a pioneering American biochemist who developed the solid-phase peptide synthesis (SPPS) technique, revolutionizing the field of peptide chemistry and enabling the rapid and efficient synthesis of complex peptides and proteins.
Carbodiimide: A carbodiimide is a functional group with the general formula R-N=C=N-R', where R and R' are organic substituents. Carbodiimides are versatile reagents used in various organic reactions, particularly in the context of acid anhydrides and peptide synthesis.
Cbz: Cbz, short for carboxybenzyl, is a protecting group commonly used in peptide synthesis. It is a benzyl-derived group that can be attached to the amino group of an amino acid to temporarily block its reactivity during peptide coupling reactions.
Coupling Reagents: Coupling reagents are chemical compounds used in organic synthesis, particularly in the context of peptide synthesis, to facilitate the formation of covalent bonds between two molecules. These reagents play a crucial role in the controlled and efficient coupling of amino acids to construct peptide chains.
DCC: DCC, or dicyclohexylcarbodiimide, is a coupling agent commonly used in organic chemistry for the activation and coupling of carboxylic acids and alcohols, as well as in peptide synthesis and the preparation of acid anhydrides. It plays a crucial role in various reactions by facilitating the formation of new carbon-carbon or carbon-heteroatom bonds.
EDC: EDC, or Electron Deficient Coupling, is a class of reagents used in peptide synthesis to facilitate the formation of amide bonds between amino acids. These reagents help activate the carboxylic acid group of one amino acid, making it more reactive towards the nucleophilic amino group of another amino acid, thereby enabling the formation of the desired peptide bond.
Fmoc: Fmoc, or 9-fluorenylmethyloxycarbonyl, is a protecting group used in solid-phase peptide synthesis. It is a temporary protecting group that is used to selectively block the amino group of an amino acid during the stepwise assembly of a peptide chain.
Fmoc Deprotection: Fmoc deprotection is a crucial step in the solid-phase peptide synthesis (SPPS) process, where the Fmoc (fluorenylmethyloxycarbonyl) protecting group is removed from the growing peptide chain to allow for the addition of the next amino acid. This deprotection step is essential for the successful synthesis of peptides and proteins.
Fmoc derivative: An Fmoc derivative is a protective group used in peptide synthesis to temporarily protect the amino group on an amino acid, preventing it from engaging in unwanted reactions. It is easily removed under basic conditions without affecting other parts of the molecule.
Fmoc-Ala-OH: Fmoc-Ala-OH is a protected amino acid derivative used in solid-phase peptide synthesis. It consists of an alanine (Ala) residue with a fluorenylmethyloxycarbonyl (Fmoc) protecting group attached to the amino terminus, allowing for controlled, step-wise assembly of peptide chains.
HATU: HATU is a coupling reagent commonly used in peptide synthesis. It is an acronym that stands for 2-(1H-Azabenzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, and it facilitates the formation of amide bonds between amino acids during the synthesis of peptides and proteins.
HBTU: HBTU, or 2-(1H-benzotriazole-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, is a popular coupling agent used in peptide synthesis reactions. It is a versatile reagent that facilitates the formation of amide bonds between amino acids, making it a crucial tool in the field of organic chemistry and peptide chemistry.
HOBt: HOBt, or 1-Hydroxybenzotriazole, is a widely used additive in peptide synthesis reactions. It serves as an activating agent, improving the efficiency and selectivity of amide bond formation during the coupling of amino acids to construct peptide chains.
HPLC: HPLC, or High-Performance Liquid Chromatography, is an analytical technique used to separate, identify, and quantify the components of a mixture. It is a powerful tool employed in various fields, including organic chemistry, particularly in the context of peptide synthesis and automated peptide synthesis using the Merrifield solid-phase method.
Kaiser Test: The Kaiser test, also known as the ninhydrin test, is a chemical test used to detect the presence of primary amines, including amino acids and the N-terminal amino group of peptides. It is an important analytical technique in the context of peptide synthesis and automated peptide synthesis using the Merrifield solid-phase method.
MALDI-TOF: MALDI-TOF (Matrix-Assisted Laser Desorption/Ionization Time-of-Flight) is an analytical technique used for the identification and characterization of biomolecules, particularly peptides and proteins. It combines the principles of mass spectrometry with the ability to ionize and detect large, fragile molecules without significant fragmentation.
N-acylurea: N-acylurea is a reactive intermediate that can form during peptide synthesis reactions. It is an important consideration in the context of coupling reactions used to form peptide bonds between amino acids.
N-hydroxysuccinimide: N-hydroxysuccinimide (NHS) is a reactive ester compound commonly used in peptide synthesis. It serves as an efficient coupling agent, facilitating the formation of amide bonds between carboxylic acids and primary amines, a crucial step in the construction of peptide chains.
O-acylisourea: O-acylisourea is an important reactive intermediate that arises in various organic reactions, particularly in the context of alcohol reactions, acid anhydride chemistry, and peptide synthesis. It serves as a key coupling agent, facilitating the formation of new bonds and enabling the transformation of starting materials into desired products.
Orthogonal Protection: Orthogonal protection is a strategy used in organic synthesis, particularly in the context of protecting functional groups during multi-step reactions. It involves the use of two or more protecting groups that can be selectively removed without affecting the other protected functionalities, allowing for precise control over the chemical transformations.
Piperidine: Piperidine is a heterocyclic organic compound consisting of a six-membered ring with five carbon atoms and one nitrogen atom. It is a common structural motif found in many natural and synthetic compounds, and its unique properties make it an important building block in organic chemistry, particularly in the context of naming amines, peptide synthesis, and automated peptide synthesis.
PyBOP: PyBOP, or benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, is a coupling reagent commonly used in peptide synthesis. It is particularly useful in the Merrifield solid-phase method of automated peptide synthesis, where it facilitates the formation of amide bonds between amino acids to build up the desired peptide sequence.
Racemization: Racemization is the process by which a chiral molecule is converted into a racemic mixture, containing equal amounts of the two enantiomeric forms. This phenomenon is particularly relevant in the context of chirality at nitrogen, phosphorus, and sulfur, the SN1 reaction, peptide synthesis, and automated peptide synthesis using the Merrifield solid-phase method.
Rink Amide Resin: Rink amide resin is a type of solid-phase resin used in the synthesis of peptides. It is a crucial component in the Merrifield solid-phase method, which is an automated process for constructing peptide chains. The Rink amide resin serves as a solid support to which the growing peptide chain is attached during the synthesis.
Robert Bruce Woodward: Robert Bruce Woodward was a renowned American organic chemist who made significant contributions to the field of organic synthesis. He is best known for his pioneering work in the total synthesis of complex natural products, which revolutionized the understanding and development of organic chemistry.
Side Chain Protection: Side chain protection is a crucial technique used in peptide synthesis to selectively protect the reactive functional groups present on the side chains of amino acid residues, while leaving the amino and carboxyl groups available for peptide bond formation. This strategy ensures the desired peptide sequence is assembled without unwanted side reactions or crosslinking.
Solid-Phase Peptide Synthesis: Solid-phase peptide synthesis (SPPS) is a method for the chemical synthesis of peptides and proteins in which the growing peptide chain is attached to an insoluble solid support, allowing for easy purification and automation of the synthesis process.
Solution-Phase Synthesis: Solution-phase synthesis refers to the process of carrying out chemical reactions in a liquid solvent medium, as opposed to solid-phase or gas-phase methods. It is a widely used approach in organic chemistry, particularly in the context of peptide synthesis, where it allows for efficient coupling of amino acids to form peptide chains.
TFA: TFA, or Trifluoroacetic Acid, is a strong organic acid commonly used in the synthesis of peptides. It plays a crucial role in the deprotection and cleavage steps of peptide synthesis, facilitating the removal of protecting groups and the release of the final peptide product.
Wang Resin: Wang resin is a type of solid support material commonly used in peptide synthesis, particularly in the Merrifield solid-phase method. It is a polymeric resin that serves as a platform for the stepwise assembly of peptide chains.