Protected Amino Acids
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With the development of biotechnology, peptides have become more and more useful in the field of biomedicine. Protected amino acids are the most basic raw materials for solid-phase synthesis of peptides. Amino acids all contain α-amino and carboxyl groups. Some of the 20 amino acids also contain side-chain active groups, such as hydroxyl, amino, guanidine and heterocyclic groups. Different amino acids need their own protecting groups, and with the different types of synthetic peptides, the selected protecting group schemes are also different. Both the amino group and the side chain active group need to be protected in the peptide reaction. After the peptide is synthesized, the protective group is removed, otherwise the misconnection of amino acids and many side reactions will occur.
Why Are Amino Acids Protected?
Amino acids are protected to ensure the precision and efficiency of peptide synthesis. During the synthesis process, amino acids can have multiple reactive sites, such as amino groups, carboxyl groups, and side chains with functional groups like hydroxyl, thiol, or amine. Without protection, these groups can participate in side reactions, leading to incorrect or incomplete peptide chains. Protecting groups are temporarily attached to these reactive sites to prevent unintended reactions. This allows chemists to:
Control the Sequence: By selectively deprotecting specific sites at precise stages, the desired sequence of amino acid incorporation can be achieved.
Improve Yield and Purity: Protection reduces the risk of side reactions, increasing the yield and purity of the final peptide product.
Facilitate Complex Syntheses: For large or complex peptides, protecting groups enable the assembly of intricate sequences that would otherwise be prone to errors and inefficiencies.
Classification of Protected Amino Acids
Protected amino acids are generally divided into single-protected amino acids and double-protected amino acids.
Single-Protected Amino Acids
Single-protected amino acids have a protective group attached only to the α-amino group (-NH₂) of the amino acid. This protection prevents the α-amino group from reacting prematurely during peptide bond formation. The carboxyl group (-COOH) and any reactive side chain groups remain unprotected, allowing the amino acid to participate in reactions involving these groups once the α-amino group is selectively deprotected. Common protective groups for α-amino protection include Boc (tert-butyloxycarbonyl), which is acid-labile and removed under acidic conditions, and Fmoc (fluorenylmethyloxycarbonyl), which is base-labile and removed under basic conditions. Single-protected amino acids are typically used in peptide synthesis methods where the main concern is preventing the α-amino group from reacting out of sequence.
Double-Protected Amino Acids
Double-protected amino acids have protective groups on both the α-amino group and the side chain active groups. This dual protection is essential for amino acids with reactive side chains that could interfere with peptide bond formation or undergo side reactions. By protecting both the α-amino group and the side chain, chemists can ensure precise control over the synthesis process. Examples include serine, threonine, cysteine, histidine, lysine, and tyrosine. For instance, serine can be protected with Boc or Fmoc on the α-amino group and tert-butyl (tBu) on the hydroxyl (-OH) side chain. Similarly, cysteine may use Boc or Fmoc for the α-amino group and trityl (Trt) or acetamidomethyl (Acm) for the thiol (-SH) side chain. This dual protection strategy is particularly important for synthesizing peptides with amino acids that have reactive side chains, ensuring that only the intended reactions occur during each step of the synthesis process, thereby obtaining high-purity peptides with the correct sequence and structure.
Protection of Amino Groups
The protection of amino groups in amino acids is a crucial step in peptide synthesis to prevent undesired reactions and ensure precise assembly. The protective groups used for amino groups can be broadly categorized into three main types: alkoxycarbonyl, acyl, and alkyl. Among these, the alkoxycarbonyl group is more commonly used, particularly the Fmoc (fluorenylmethyloxycarbonyl) protection.
Alkoxycarbonyl Protecting Groups
Alkoxycarbonyl groups, such as Boc (tert-butyloxycarbonyl) and Fmoc, are widely used in peptide synthesis for protecting the α-amino group of amino acids. The Fmoc protecting group is particularly prevalent due to its stability under acidic conditions and sensitivity to alkaline conditions. This allows for selective deprotection under mild basic conditions, which is advantageous in solid-phase peptide synthesis (SPPS).
The introduction of Fmoc protecting groups typically involves the use of reagents like Fmoc-Cl (fluorenylmethyloxycarbonyl chloride) or Fmoc-OSu (fluorenylmethyloxycarbonyl-N-hydroxysuccinimide ester). Of these, Fmoc-OSu is often preferred because it is easier to control reaction conditions and results in fewer side reactions. Under weak alkaline conditions, such as those provided by sodium carbonate or sodium bicarbonate, Fmoc-OSu reacts with the α-amino group to form the protected amino acid.
Acyl Protecting Groups
Acyl groups, such as acetyl (Ac) and benzoyl (Bz), are also used for protecting amino groups. These groups are introduced through acylation reactions and provide a different set of stability and deprotection characteristics compared to alkoxycarbonyl groups. Acyl protecting groups are typically stable under a wide range of conditions but require specific deprotection strategies, often involving hydrolysis or reduction
Alkyl Protecting Groups
Alkyl groups, such as methyl (Me) and ethyl (Et), are less commonly used for protecting amino groups compared to alkoxycarbonyl and acyl groups. Alkyl protection involves the introduction of alkyl groups via alkylation reactions. While these groups can offer robust protection, their deprotection can be more challenging and often requires harsh conditions
Combining Protecting Strategies
In peptide synthesis, it is common to use a combination of protecting groups to achieve selective and sequential deprotection. For instance, Fmoc protection is frequently used in conjunction with acid-sensitive protecting groups like Boc (tert-butyloxycarbonyl) or Z (benzyloxycarbonyl) for amino acids with active side chain groups. The Fmoc group is removed under basic conditions, while the Boc or Z groups are removed under acidic conditions, allowing for controlled deprotection steps that facilitate the sequential assembly of peptides.
Protection of Side Chain Active Groups
The active side chains of amino acids include hydroxyl, thiol, carboxyl, amino, sarcosyl, imidazole, amide, indole, and thioether groups. During peptide synthesis, these active groups are typically protected to avoid side reactions. Protecting the side chain amino groups is crucial, such as with lysine. Unlike α-amino protecting groups, side chain amino protecting groups must be selectively removable after the peptide synthesis is complete, without affecting other protecting groups.
The earliest protecting group used for this purpose was p-toluenesulfonyl (Tos), known for its stability and selective removal by Na/liquid ammonia treatment. In this process, lysine forms a ketone complex, reacts with Tos-Cl under weakly alkaline conditions to selectively protect the side chain amino group, and then the copper is removed. Using Lys (Tos) protection, researchers have successfully synthesized peptides and proteins such as oxytocin, vasopressin, and insulin.
Which Amino Acids Are Side Chain Protected?
Certain amino acids possess side chains with functional groups that are particularly reactive and prone to side reactions. To prevent this, side chain protection is employed. Some examples include:
Serine (Ser): The hydroxyl group (-OH) on the side chain is protected, often using groups like tert-butyl (tBu).
Threonine (Thr): Similar to serine, the hydroxyl group (-OH) is protected, commonly with tert-butyl (tBu).
Cysteine (Cys): The thiol group (-SH) is protected to prevent oxidation or disulfide bond formation, using groups like trityl (Trt) or acetamidomethyl (Acm).
Histidine (His): The imidazole ring is protected, often with trityl (Trt) or tert-butylcarbamate (Boc-His).
Lysine (Lys): The ε-amine group (-NH₂) on the side chain is protected, frequently with Boc or Fmoc groups.
Tyrosine (Tyr): The hydroxyl group (-OH) is protected, typically using tert-butyl (tBu).
These side chain protections are crucial for preventing side reactions that could interfere with the stepwise formation of peptide bonds.
Reference
- Isidro-Llobet A, Alvarez M, Albericio F. Amino acid-protecting groups[J]. Chemical reviews, 2009, 109(6): 2455-2504.
