Peptide synthesis, particularly solid-phase peptide synthesis (SPPS), is a widely employed method for creating peptides of varying lengths and sequences. The process has revolutionized the ability to produce synthetic peptides for a broad range of applications, from pharmaceutical development to structural biology. A critical step in the synthesis is the peptide bond formation, which is catalyzed by condensation agents. These agents activate the carboxyl group of an amino acid, facilitating its reaction with the amine group of another amino acid to form a peptide bond.
What is solid-phase peptide synthesis (SPPS)?
Solid-phase peptide synthesis (SPPS) is an important method for synthesizing peptides. In SPPS, peptides are synthesized on an insoluble resin that serves as the solid support. The process begins by attaching the C-terminal amino acid to the resin, and subsequent amino acids are added step by step in a sequence dictated by the desired peptide. Each elongation cycle typically consists of deprotection (removing a protecting group from the amino acid) and condensation (forming a peptide bond between the activated amino acid and the growing chain).
The most challenging part of peptide synthesis is the formation of the peptide bond, which requires an efficient activation of the carboxyl group to promote its reaction with the amino group. This activation is achieved through the use of condensation agents, which are chosen based on factors such as compatibility with the resin, reaction efficiency, and minimization of side reactions.
General process of synthesis
- First, the initial amino acid (the C-terminal residue) is attached to the resin (loading).
- After the amino acid binds to the resin, the resin is washed to remove by-products and excess reagents.
- Next, the amino protecting group is removed, and the resin is washed again. Under the action of a coupling agent, the next amino acid is coupled to the peptide-resin complex. This coupling and washing cycle is repeated until the entire peptide sequence is completed.
- Finally, all protecting groups are removed, the peptide-resin is washed, and the peptide is cleaved from the resin.
Key Considerations in Choosing a Condensation Agent
The selection of a condensation agent is crucial for ensuring efficient peptide bond formation. Key factors influencing the choice of a condensation agent include:
Activation Efficiency: The reagent must activate the carboxyl group of the amino acid efficiently to form the reactive intermediate, which will then react with the amine group of the next amino acid.
Minimization of Side Reactions: The condensation agent should ideally minimize side reactions such as racemization, hydrolysis, and degradation of sensitive amino acids or functional groups.
Compatibility with the Resin: The reagent must not interfere with the resin or other components of the reaction mixture, including solvents and protecting groups.
Solubility: Condensation agents should be sufficiently soluble in the solvent system used during SPPS to achieve optimal reaction conditions.
“Orthogonal” protection in solid-phase peptide synthesis
Many amino acid side chains are reactive and, if not protected, can form by-products. To successfully synthesize peptides, side chains need to be protected, and these protecting groups must remain stable throughout repeated deprotection of the amino group. Ideally, the amino-protecting group and side-chain-protecting group can be removed under completely different conditions-such as removing the amino-protecting group under basic conditions and the side-chain-protecting group under acidic conditions. This approach is known as “orthogonal” protection. Common combinations include Boc/Bzl protection and Fmoc/tBu protection. In the Boc/Bzl protection scheme, the Boc group temporarily protects the amino group, while the benzyl group provides more permanent protection for side chains. Although both Boc and benzyl protecting groups are acid-sensitive, making Boc/Bzl not a truly orthogonal protection strategy, it is still widely used. This is because the Boc group can be removed under mild acidic conditions (e.g., 50% TFA in DCM solution), whereas the benzyl group requires a very strong acid (such as HF or TFMSA) for removal.
Condensing agents in solid-phase synthesis
Carbodiimides
Carbodiimides, such as dicyclohexylcarbodiimide (DCC) and 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDC), are among the most widely used condensation agents in peptide synthesis. DCC, DIC, EDCI. Dicyclohexylcarbodiimide (DCC) and diisopropylcarbodiimide (DIC) are commonly used to convert carboxylic acids into amides, esters, and anhydrides. They can also convert primary amides into nitriles, which may lead to side reactions when aspartic acid and glutamic acid residues are present in peptides. DCC generates dicyclohexylurea as a by-product, which is nearly insoluble in most organic solvents and precipitates out of the reaction mixture as the reaction progresses. Therefore, DCC is highly useful in solution-phase reactions but less suitable for reactions on resin. In solid-phase synthesis, DIC is used because its urea by-product is more soluble and remains in solution. In some applications, such as protein modification, ethyl-(N’,N’-dimethylaminopropyl) carbodiimide hydrochloride (EDCI) is used. This carbodiimide reagent and its urea by-product are water-soluble, allowing by-products and excess reagent to be removed with water washes.
In peptide synthesis, to prevent racemization of amino acids during carbodiimide activation, an equivalent amount of 1-hydroxybenzotriazole (HOBt) is commonly added along with the carbodiimide.
Mechanism of Action: Carbodiimides activate the carboxyl group of an amino acid by forming an acyl-urea intermediate. This intermediate then reacts with the amino group of another amino acid, resulting in the formation of a peptide bond.
Advantages: Carbodiimides are relatively inexpensive and easy to handle. They can be used for a wide variety of amino acids and peptides, and they are effective under mild conditions, minimizing the risk of side reactions such as racemization.
Limitations: One of the significant drawbacks of carbodiimides is the formation of urea byproducts, such as dicyclohexylurea (DCU) when DCC is used. These byproducts can precipitate out of solution and require removal by filtration, which can reduce the yield. Moreover, carbodiimides can also lead to the formation of side products due to incomplete activation.
Benzotriazole Derivatives
Benzotriazole derivatives, such as 1-hydroxybenzotriazole (HOBt), benzotriazole-1-yl-oxy-tris-pyrrolidino-phosphonium hexafluorophosphate (BOP), and Oxyma, are frequently used in peptide synthesis to improve the efficiency of carbodiimide-based activation.
To avoid racemization and side reactions associated with carbodiimide condensing agents, many alternative reagents have been developed to generate OBt esters in situ. Benzotriazol-1-yloxytris(dimethylamino)phosphonium hexafluorophosphate (BOP) was one of the first reagents developed. BOP does not lead to dehydration of asparagine and glutamine side-chain amides to form nitrile by-products, and it causes minimal racemization of amino acids. However, BOP produces highly carcinogenic hexamethylphosphoramide as a by-product in condensation reactions, so it must be handled carefully. PyBOP offers comparable condensation efficiency to BOP but with a lower risk from its by-products. PyAOP is also an effective condensing agent, especially for coupling between N-methyl-protected amino acids. PyBrOP is often used for coupling sterically hindered amino acids, such as N-methyl amino acids or α,α-dialkylglycines, where other condensing agents are less efficient. BOP-Cl is commonly used to couple Fmoc-α,α-dialkyl amino acids to tritylphenyl resins.
Mechanism of Action: These reagents work by stabilizing the active ester intermediate that is formed during the activation process. HOBt, for example, reacts with the carbodiimide to produce a highly reactive ester intermediate, enhancing the efficiency and reducing side reactions.
Advantages: The addition of HOBt or related compounds can significantly improve the efficiency of peptide bond formation, reduce side reactions, and suppress racemization, especially for sterically hindered or sensitive amino acids.
Limitations: While these reagents are effective, their cost can be higher compared to carbodiimides alone, and their use requires additional care in handling and disposal due to their chemical reactivity.
Uronium Salts
Uronium salts, such as ethyl(2-dimethylamino-ethyl)-phosphonium tetramethylfluorophosphate (EEDQ) and t-butylphosphonium hexafluorophosphate (TBTU), are another class of reagents used in peptide bond formation. These agents activate carboxyl groups by forming reactive phosphonium or uronium species.
Mechanism of Action: Uronium salts generate reactive species that facilitate the coupling of the carboxyl group with the amine group of the incoming amino acid. These species are highly electrophilic, promoting efficient peptide bond formation.
Advantages: Uronium salts are highly effective in promoting fast and efficient peptide bond formation. They are particularly useful for synthesizing peptides with low solubility or challenging sequences. They also tend to produce fewer side products.
Limitations: Despite their efficiency, uronium salts can be expensive and may lead to contamination with byproducts if not carefully controlled. Their use in SPPS may also be limited by the compatibility with the resin or other reagents.
Phosgene Derivatives
Phosgene derivatives, such as chlorophosgene and triphosgene, are potent agents used for activating carboxyl groups in peptide synthesis.
Mechanism of Action: Phosgene derivatives react with the carboxyl group of amino acids to form an acylated intermediate. This intermediate then reacts with the amine group of the next amino acid, facilitating the peptide bond formation.
Advantages: Phosgene derivatives are highly efficient in peptide bond formation and can work well with challenging amino acids. They offer high yields and can help minimize racemization and side reactions.
Limitations: The major disadvantage of phosgene derivatives is their toxicity and safety concerns, as phosgene is a highly hazardous substance. Additionally, the reagents can be expensive, and their use requires specialized equipment for handling and disposal.
Activation with Mixed Anhydrides
Mixed anhydrides are formed by reacting an amino acid with an acid chloride or an acylating agent. This reaction produces an acylated amino acid intermediate that is then used for peptide bond formation.
Mechanism of Action: The mixed anhydride is a reactive intermediate that can readily react with the amino group of another amino acid, resulting in the formation of a peptide bond.
Advantages: Mixed anhydrides can be highly effective, especially when used in combination with reagents like HOBt or pyridine. The method is versatile and applicable to a broad range of amino acids.
Limitations: One challenge with this approach is that mixed anhydrides are highly reactive, which can sometimes lead to side reactions if the reaction conditions are not carefully controlled. Additionally, the preparation of mixed anhydrides can be more complex than other methods.
New Developments in Condensation Agents
Recent advances in peptide synthesis have led to the development of newer, more efficient condensation agents. These agents aim to address the limitations of traditional reagents by enhancing reaction specificity, improving yields, and reducing side reactions.
One promising area is the development of green reagents, which are designed to be environmentally friendly and non-toxic. For example, Oxyma Pure, a derivative of HOBt, is one such reagent that has gained popularity due to its improved efficiency and lower toxicity profile.
Additionally, solid-phase activated reagents (SPARs) are being explored as a way to streamline the synthesis process by coupling the activation step directly to the solid-phase resin. These reagents eliminate the need for liquid-phase activation, which can reduce solvent consumption and simplify the overall process.