Protecting Groups in Peptide Synthesis: A Detailed Guide

Peptide synthesis is an important technology in the fields of biochemistry and drug development. The use of protecting groups is crucial for synthesizing peptides with high purity and activity. Protecting groups temporarily block the reactive groups of amino acids to prevent side reactions, ensuring specificity and efficiency in synthesis. This article provides a comprehensive overview of the selection, application, and deprotection strategies of protecting groups in peptide synthesis, offering references for related research.

Classification and Application of Protecting Groups

(1) α-Amino Protecting Groups

Z (Benzyloxycarbonyl): Z is a well-established amino protecting group that is still widely used today. Its advantages include easy preparation, stable Z-amino acids that can crystallize, and resistance to racemization during activation. It can be removed under conditions such as H2/Pd, HBr/AcOH, or Na/liquid ammonia.

Boc (tert-Butoxycarbonyl): Boc is commonly used in the coupling processes of peptide synthesis. It can be removed under acidic conditions while avoiding catalytic hydrogenation, alkaline hydrolysis, and sodium/liquid ammonia reduction. Boc is typically deprotected in anhydrous TFA at 0°C.

Fmoc (9-Fluorenylmethyloxycarbonyl): Fmoc is the only widely used amino acid protecting group in the carbamate class that can be removed under mild basic conditions. Fmoc deprotection can be achieved with a dilute piperidine solution or diethylamine in DMF at room temperature.

(2) Acyl-Type Protecting Groups

Tfa (Trifluoroacetyl): Tfa was introduced by Weygand in 1952 for peptide synthesis. It is removed under relatively mild conditions and can be easily cleaved using piperidine or sodium hydroxide. However, it is prone to racemization during activation and can cause chain cleavage during alkaline hydrolysis, which limits its use.

Tos (p-Toluenesulfonyl): Tos is an early protective group known for its excellent stability. It can only be removed by Na/liquid ammonia treatment, which is unaffected by acid or base conditions and catalytic hydrogenation. However, the complexity of the Na/liquid ammonia treatment and potential peptide bond cleavage have led to its decreased use today.

oNbs (o-Nitrobenzenesulfonyl): The introduction of an o-nitro group on the benzene ring of Tos results in oNbs, significantly improving the situation. The sulfonamide protecting group allows N-acylation of amino acids without racemization caused by the diketone mechanism. This group can be removed using phenylthiol or alkylthiol, overcoming the removal difficulties of Tfa and Tos.

(3) Alkyl-Type Protecting Groups

Trt (Triphenylmethyl): Trt is a commonly used alkyl-type protecting group that can be removed under mild conditions, such as with anhydrous HCl/MeOH, TFA, or HBr/AcOH. Due to its large steric hindrance, Trt is generally difficult to introduce onto the α-amino group and is more stable on side-chain amino acids, making it more commonly used for protecting side-chain amino acids rather than the α-amino group.

Dde (N-1-(4,4-Dimethyl-2,6-dioxohexyl)ethyl): Dde is stable to TFA and piperidine, and can be removed with a 2% (v/v) hydrazine/DMF solution. This allows the retention of the main chain amino protection while removing side-chain amino protection, facilitating the synthesis of branched peptides.

Nde (N-1-(4-Nitro-1,3-dioxane-2-yl)ethyl): Nde is a new alkyl-type amino protecting group that is stable to both acids and bases. It can be used in Fmoc and Boc strategies and is relatively easy to prepare. Deprotection can be achieved with nucleophilic reagents and palladium.

(4) Protection of Side Chain Active Groups

ω-Carboxyl Protection: Aspartic acid (Asp) and glutamic acid (Glu) contain two carboxyl groups, and under alkaline or strongly acidic conditions, the ω-carboxyl group on the side chain is prone to side reactions. Commonly used protecting groups include tert-butyl ester (OtBu) in the Fmoc strategy and β-cyclohexyl ester (OcHex) in the Boc strategy.

Hydroxyl Protection: The phenolic hydroxyl group in tyrosine (Tyr), and the hydroxyl groups in serine (Ser) and threonine (Thr) are active groups that must be protected during peptide solid-phase synthesis. Common protecting groups include 2-bromobenzyloxycarbonyl (BrZ) and benzyl (Bzl) in the Boc strategy, and tert-butyl (tBu) in the Fmoc strategy.

Indole Protection: Nπ-allyl (All) and Nπ-allyloxy-methyl (Alom) are new types of protecting groups. All and Alom act on the π-nitrogen of the imidazole ring, inhibiting racemization. They are stable to both acids and bases, and can be used in Fmoc and Boc strategies. They are easy to prepare and can be removed with nucleophilic reagents and palladium.

Thiol Protection: The thiol group in cysteine has strong nucleophilicity, oxidizing potential, and acidity, and must be selectively masked during all stages of synthesis. Common protecting groups include Trt, tBu, and acetamidomethyl (Acm), with different deprotection conditions.

Imidazole Protection: Histidine (His) is one of the most problematic amino acids in peptide synthesis. When the imidazole ring in histidine is unprotected, two main issues arise: N-acylation and racemization. Reversible masking of the imidazole functionality can avoid these problems.

ε-Amino Protection: Lysine (Lys) has a strong basic ε-amino group with nucleophilic properties. In the Boc strategy, 2-chlorobenzyl (Z(2−Cl)) is often used to protect the ε-amino group. It is stable to acid and can prevent premature removal of side-chain protection during Boc group deprotection. In the Fmoc strategy, the Boc group is a good protecting group with strong stability to bases, effectively suppressing side reactions.

Guamidine Protection: Guanidine protection groups are typically used to protect the guanidine group of arginine (Arg). Common protecting groups include nitrobenzyloxycarbonyl (NPys) and p-toluenesulfonyl (Tos). NPys is unstable under acidic conditions and easily removed, while Tos is unstable under basic conditions and readily cleaved.

Strategy for Selecting Protecting Groups

Stability and Reactivity: The selection of protecting groups must consider both their stability and reactivity. α-amino and α-carboxyl protecting groups are temporary, needing to be easily removed once the amino acid is incorporated into the peptide chain. In contrast, side-chain protecting groups are considered "permanent" as they do not interfere with reactions during peptide synthesis and are removed at the end of the synthesis process.

Deprotection Conditions: When selecting a protecting group, the mildness and selectivity of the deprotection conditions should be considered. For example, Fmoc groups are removed under basic conditions, while Boc groups are removed under acidic conditions. Choosing the appropriate deprotection method can effectively reduce side reactions, increasing the synthesis efficiency and product purity.

Compatibility with Synthesis Strategies: Different protecting groups are suitable for different synthesis strategies (such as the Boc or Fmoc strategy). Choosing the right protecting group can optimize the synthesis conditions, enhancing feasibility and efficiency.

Strategies for Removing Protecting Groups

Acidic Deprotection: This method is suitable for removing Boc protecting groups, typically using TFA or HF under acidic conditions. Care should be taken, as amino acids or peptide segments sensitive to acids may not be suitable for this method.

Basic Deprotection: This method is suitable for Fmoc protecting groups, typically using piperidine or ammonium hydroxide under basic conditions. Basic deprotection is widely used in Fmoc solid-phase peptide synthesis because it does not readily induce racemization.

Selective Deprotection: Selective deprotection can be achieved by exploiting differences in the removal conditions of various protecting groups. For example, Dde and Nde protecting groups are stable under TFA and piperidine conditions but can be removed with hydrazine/DMF. Side-chain protecting groups (such as Trt and tBu) are usually removed at the end of the synthesis process.

Research Progress of Novel Protecting Groups

With the ongoing exploration of peptide synthesis technologies, the development of novel protecting groups has become a research hotspot. For example, Nsc is considered a better protecting group than Fmoc, as it is more stable under alkaline conditions, less sensitive to weakly alkaline solvents, and has stronger hydrophilicity, which can effectively reduce the occurrence of side reactions. Additionally, the emergence of new, alkali-labile amino acid protecting groups, such as Bsmoc, Mspoc, and Bspoct, has provided more options for peptide synthesis.

Applications of Protecting Groups in Drug Development

Protecting groups play a crucial role in the synthesis of peptide drugs. By selecting and using protecting groups rationally, peptides with specific biological activities can be synthesized for the treatment of various diseases. For example, in the synthesis of antitumor peptides and antimicrobial peptides, protecting groups help ensure the structural integrity of the peptides, enhancing the drug's efficacy and stability.

Conclusion

Protecting groups are essential in peptide synthesis. They temporarily shield active groups, preventing side reactions, ensuring the sequence and directionality of synthesis, improving synthesis efficiency and product purity, and reducing the likelihood of racemization and degradation. The selection of appropriate protecting groups and their deprotection conditions is key to successful peptide synthesis. With the continuous development and application of novel protecting groups, peptide synthesis technology will continue to evolve and improve, providing stronger support for biochemical research and drug development.

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