26.8 Automated Peptide Synthesis: The Merrifield Solid-Phase Method
3 min read•may 7, 2024
Automated peptide synthesis revolutionized the creation of complex protein chains. By attaching to polymer beads, scientists can efficiently build peptides step-by-step, using protecting groups and coupling agents to control the process.
This method offers huge advantages over traditional techniques. It allows for longer peptides, higher yields, and easier purification. With automated synthesizers, researchers can now produce custom peptides quickly and reliably for various applications in biochemistry and medicine.
Automated Peptide Synthesis
Steps in Merrifield solid-phase synthesis
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- Attach first amino acid to insoluble polymer support (resin bead) via
- Linker chosen based on desired C-terminal functionality of peptide
- Amino acid protected at N-terminus and side chain (if necessary) prevents unwanted reactions
- Remove N-terminal protecting group (deprotection) allows coupling of next amino acid
- Activate of next amino acid and couple to free N-terminus of growing peptide chain
- Coupling reagents (, , ) facilitate formation of peptide bond
- is a crucial step in building the amino acid sequence
- Repeat deprotection and coupling steps until desired peptide sequence obtained
- Cleave completed peptide from and remove remaining side chain protecting groups
- (TFA) commonly used for cleavage and final deprotection
Advantages of polymer resin beads
- Allows use of excess reagents to drive reactions to completion
- Excess reagents and byproducts easily removed by washing resin beads, simplifies purification
- Growing peptide chain remains attached to insoluble resin throughout synthesis
- Eliminates need for intermittent purification steps required in solution-based methods
- Automated peptide synthesizers streamline process
- Synthesizer performs repetitive deprotection, coupling, and washing steps, reduces manual labor and increases efficiency
- Enables synthesis of longer peptide sequences with higher yields compared to solution-based methods
- Solid support provides a stable platform for the growing peptide chain
Resins and protecting groups
- with (9-fluorenylmethoxycarbonyl) protecting group
- Polystyrene-based resin yields peptides with free carboxylic acid at C-terminus upon cleavage
- Fmoc is base-labile protecting group for N-terminus, removed using (20% in DMF)
- Side chain protecting groups typically acid-labile (, , ), removed during final TFA cleavage step
- Fmoc/tBu strategy is orthogonal, allows selective deprotection of N-terminus while leaving side chain protecting groups intact
- PAM (4-hydroxymethylphenylacetamidomethyl) resin with Boc (tert-butyloxycarbonyl) protecting group
- Polystyrene-based resin yields peptides with C-terminal amide upon cleavage with anhydrous HF
- Boc is acid-labile protecting group for N-terminus, removed using TFA
- Side chain protecting groups also acid-labile (, ), removed during final HF cleavage step
- Boc/Bzl strategy requires graduated acidolysis, stronger acids (HF) needed for final cleavage and deprotection
- Fmoc/tBu strategy more widely used in automated peptide synthesis due to milder cleavage conditions and avoidance of hazardous HF
Considerations in Automated Peptide Synthesis
- is crucial for obtaining high-quality peptides
- strategies prevent unwanted side reactions
- must be minimized to maintain the correct stereochemistry of amino acids
- improves reproducibility and reduces human error in the peptide synthesis process
Key Terms to Review (43)
Amine Group: The amine group is a functional group consisting of a nitrogen atom covalently bonded to one or more hydrogen atoms or organic substituents. Amines are a class of organic compounds that play a crucial role in the context of automated peptide synthesis using the Merrifield solid-phase method.
Amino Acids: Amino acids are the fundamental building blocks of proteins, which are essential macromolecules that perform a vast array of functions in living organisms. These organic compounds contain an amino group (-NH2), a carboxyl group (-COOH), and a side chain (R-group) that varies among the different types of amino acids. The unique combination and sequence of amino acids give rise to the diverse structures and functions of proteins.
Automated Synthesis: Automated synthesis refers to the use of specialized equipment and computer-controlled processes to synthesize chemical compounds, particularly complex molecules like peptides, in a highly efficient and reproducible manner. This approach contrasts with traditional manual synthesis methods, offering increased speed, precision, and scalability.
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.
Bzl: Bzl, short for benzyl, is a protective group commonly used in organic chemistry, particularly in the context of peptide synthesis. It is a bulky aromatic substituent that can be selectively removed to reveal a reactive functional group, allowing for the controlled synthesis of complex peptide structures.
Capping: Capping is a critical step in the automated peptide synthesis process known as the Merrifield solid-phase method. It involves the addition of a capping agent to the growing peptide chain to prevent the formation of unwanted side products during each coupling cycle.
Carboxyl group: A carboxyl group is a functional group consisting of a carbon atom double-bonded to an oxygen atom and single-bonded to a hydroxyl group (-COOH). It is characteristic of carboxylic acids, giving these compounds their acidic properties.
Carboxyl Group: The carboxyl group is a functional group consisting of a carbon atom double-bonded to an oxygen atom and single-bonded to a hydroxyl group (-COOH). It is a key structural feature in various organic compounds, including carboxylic acids, amino acids, and proteins, and plays a crucial role in their chemical reactivity and properties.
Cbo: Cbo, or carboxybenzotriazole, is a reagent commonly used in the Merrifield solid-phase method of automated peptide synthesis. It serves as an activating agent, facilitating the formation of peptide bonds between amino acids during the stepwise assembly of peptide chains.
Coupling Efficiency: Coupling efficiency refers to the degree of success in forming the desired chemical bond between reactants during a step in a chemical synthesis process. It is a crucial concept in the context of automated peptide synthesis using the Merrifield solid-phase method.
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.
DIC: DIC, or Diisopropylcarbodiimide, is a common coupling reagent used in the Merrifield solid-phase method for automated peptide synthesis. It is a key component in the activation and coupling of amino acids during the stepwise construction of peptide chains.
Dichloromethane: Dichloromethane, also known as methylene chloride, is a colorless, volatile organic compound with a sweet, chloroform-like odor. It is widely used as a solvent, paint stripper, and in various industrial and laboratory applications.
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 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.
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.
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.
Linker: A linker is a chemical compound used in organic synthesis, particularly in the context of peptide synthesis, to covalently attach a molecule of interest to a solid support or resin. Linkers serve as the bridge between the growing peptide chain and the solid phase, enabling the automated, step-wise construction of peptides using the Merrifield solid-phase method.
Mass Spectrometry: Mass spectrometry is an analytical technique that measures the mass-to-charge ratio of ions to identify and quantify the chemical composition of a sample. It provides detailed information about the molecular structure and fragmentation patterns of compounds, making it a powerful tool in organic chemistry and various other fields.
Mass spectrometry (MS): Mass spectrometry is an analytical technique used in organic chemistry to determine the mass-to-charge ratio of ions. It helps identify the composition of a sample by generating ions and measuring their mass and charge.
Merrifield Resin: Merrifield resin is a type of solid-phase support used in automated peptide synthesis, developed by Robert Bruce Merrifield. It provides a solid matrix to which amino acids can be sequentially coupled, allowing for the efficient synthesis of peptides and proteins.
N,N-Dimethylformamide: N,N-Dimethylformamide (DMF) is a colorless, highly polar, aprotic solvent commonly used in organic synthesis, particularly in the context of automated peptide synthesis via the Merrifield solid-phase method. It is an important reagent that facilitates various chemical transformations and reactions.
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.
PAM Resin: PAM resin, or Polyacrylamide resin, is a type of solid support material commonly used in automated peptide synthesis, particularly in the Merrifield solid-phase method. It serves as an insoluble matrix to which amino acids and other reagents are attached during the step-by-step synthesis of peptide chains.
Peptide Bond Formation: A peptide bond is a covalent chemical bond formed between the carboxyl group of one amino acid and the amino group of another amino acid. This process of forming these bonds between amino acids is known as peptide bond formation, and it is a crucial step in the synthesis of peptides and proteins.
Peptide Synthesizer: A peptide synthesizer is an automated instrument used to rapidly and efficiently produce peptides, which are short chains of amino acids linked together by peptide bonds. This technology is crucial in the field of organic chemistry, particularly in the context of 26.8 Automated Peptide Synthesis: The Merrifield Solid-Phase Method.
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.
Reaction Vessel: A reaction vessel is a container used in chemical reactions to hold the reactants, products, and any necessary solvents or catalysts. It provides a controlled environment for the chemical process to take place.
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 Merrifield: Robert Bruce Merrifield was an American biochemist who developed the Merrifield solid-phase method, a groundbreaking technique for the automated synthesis of peptides and proteins. This method revolutionized the field of organic chemistry and had a significant impact on the study of 26.8 Automated Peptide Synthesis.
Solid Support: Solid support refers to an insoluble, polymeric material used as a platform for chemical reactions, particularly in the context of automated peptide synthesis. It serves as a solid foundation upon which peptides or other molecules are synthesized, allowing for efficient purification and isolation of the desired products.
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.
Solid-phase synthesis: Solid-phase synthesis is a method in which molecules (such as peptides) are chemically synthesized while attached to an insoluble material or resin, allowing for sequential addition of amino acids through repeated cycles. This technique simplifies the purification process since unwanted reagents and byproducts can be washed away without losing the target molecule.
TBu: tBu, or tert-butyl, is a bulky, branched alkyl group that is commonly used in organic chemistry, particularly in the context of automated peptide synthesis and the Merrifield solid-phase method. The tert-butyl group is known for its steric hindrance and stability, making it a useful protecting group in various synthetic reactions.
TFA Deprotection: TFA deprotection is a critical step in the Merrifield solid-phase method of automated peptide synthesis. It involves the removal of a tert-butyloxycarbonyl (Boc) protecting group from the N-terminus of the growing peptide chain using trifluoroacetic acid (TFA), allowing for the addition of the next amino acid in the sequence.
Trifluoroacetic Acid: Trifluoroacetic acid (TFA) is a strong organic acid with the chemical formula CF3COOH. It is a commonly used reagent in organic chemistry and plays a significant role in the context of substituent effects on acidity and automated peptide synthesis.
Trt: Trt, or Temporary Removal Tag, is a key concept in the context of automated peptide synthesis using the Merrifield Solid-Phase Method. It refers to the temporary removal of protecting groups from specific amino acid residues during the stepwise assembly of a peptide chain on a solid support.
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.
α-Amino acids: α-Amino acids are the building blocks of proteins, consisting of a central carbon atom (the α-carbon) to which an amino group, a carboxyl group, a hydrogen atom, and a distinctive side chain (R group) are attached. They play a crucial role in biochemistry as they polymerize to form peptides and proteins.